17-benzimidazolyl-10α-methyl-steroidal derivatives, process for their preparation, use and pharmaceutical compositions

Abstract

The present application relates to a kind of compound, its preparation method, its purposes, and its pharmaceutical composition.The methyl of C-10 position of the steroid derivative of the present application is the α configuration of new compound, and it has stronger inhibitory effect on prostate cancer, colon cancer, lung cancer cell.In order to prepare the above-mentioned steroid derivative, a series of intermediates corresponding to the steroid derivative are also synthesized.The steroid derivative of the present application can be used as medicine to effectively treat cancer, especially including prostate cancer, colon cancer or lung cancer and the like.The methyl of C-10 position of the steroid derivative of the present application is α configuration, compared with β configuration, and the reactivity with enzyme is greatly reduced, and the tolerance to enzyme in vivo is better, expected to be slower in vivo metabolism, and the duration of drug efficacy is longer, which is beneficial to the development of long-acting preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to 17-benzimidazolyl-10α-methyl-steroid derivatives, their preparation, use and pharmaceutical compositions. BACKGROUND

[0002] Abiraterone acetate, chemical name 17-(3-pyridyl)-androst-5, 16-dien-3β-ol acetate (structural formula as follows), is a CYP17 inhibitor, which is clinically combined with prednisone to treat metastatic advanced prostate cancer that has occurred to traditional hormone therapy resistance, not only can reduce the level of prostate specific antigen, but also helps to shrink the tumor, and can prolong the life of patients with advanced prostate cancer.

[0003]

[0004] In structure, it is derived from the sterane skeleton structure as follows, the skeleton structure (hereinafter referred to as sterane ring) has four rings as shown below, and the carbon number (1-17) on each ring is as follows. Androsterone refers to the connection of one β-methyl at C-10 and C-13 positions, which can be recorded as 10β-methyl, 13β-methyl.

[0005] SUMMARY

[0006] The present application provides a kind of 17-benzimidazolyl-10α-methyl-steroid compound, which is a new class of compounds, and has inhibitory effect on prostate cancer, colon cancer, lung cancer and pancreatic cancer.

[0007] The present application provides a kind of 17-benzimidazolyl-10α-methyl-steroid compound, which has the following formula I structure or its pharmaceutically acceptable salt:

[0008]

[0009] R1 is selected from =O, -OH, halogen, -OC (O) R2, substituted or unsubstituted imidazolyl, triazolyl or benzimidazolyl, wherein R2 is selected from C1-C5 alkyl, C1-C5 haloalkyl, phenyl, halophenyl, imidazolyl, triazolyl or R2' O-, wherein R2' is selected from C1-C5 alkyl;

[0010] R3 is selected from halogen, C1-C5 alkyl, C1-C5 haloalkyl, C1-C5 alkoxy, hydroxyl or amino;

[0011] R4, R5, R6, R7 are the same or different, each independently selected from -OH, =0, halogen, amino, C1-C5 alkyl, C1-C5 haloalkyl, C1-C5 alkoxy, C2-C5 alkenyl, or C2-C5 ester;

[0012] i, j, k, m and n are each independently selected from 0, 1, 2, 3 or 4;

[0013] represents a single or double bond;

[0014] when a certain is a double bond, the adjacent is a single bond.

[0015] In embodiments of the present application, R1is selected from =0, -OH, -OAc, CH3OC(O)O-, imidazolyl, triazolyl, benzimidazolyl,

[0016] In embodiments of the present application, when said R1is selected from -OH, said -OH is a β-OH or an α-OH.

[0017] In embodiments of the present application, said R3is selected from C1-C5 alkyl or haloalkyl.

[0018] In embodiments of the present application, said k is 0.

[0019] In embodiments of the present application, said i, j, m and n are all 0, preferably said k is 0.

[0020] In embodiments of the present application, said halogen in R1to R7is selected from F, Cl, Br or I.

[0021] In embodiments of the present application, said compound is selected from the following structural formulae:

[0022]

[0023] In embodiments of the present application, said compound is selected from the following structural formulae:

[0024]

[0025] In embodiments of the present application, said compound is selected from the following structural formulae:

[0026]

[0027]

[0028]

[0029] The present application provides a method for preparing the compound of the present application, which comprises using an intermediate shown in formula V as raw material, and connecting a substituted or unsubstituted benzimidazolyl group at C-17 position;

[0030]

[0031] The substituted or unsubstituted benzimidazolyl group corresponds to the benzimidazolyl structure on the compound;

[0032] The R9corresponds to or corresponds to R4after reaction;

[0033] The R 10 corresponds to or corresponds to R5after reaction;

[0034] The R 11 corresponds to or corresponds to R6after reaction;

[0035] The R 12 corresponds to or corresponds to R7after reaction.

[0036] In an embodiment of the present application, i, j, m and n are all 0.

[0037] In an embodiment of the present application, the intermediate is selected from the following structural formula:

[0038]

[0039] In an embodiment of the present application, the intermediate is prepared by a method comprising the following steps: photochemical conversion of a compound of the following formula to flip the methyl group at C-10 position from β configuration to α configuration

[0040]

[0041] R8is selected from -OH or protected hydroxyl, preferably R8is selected from -OH or OAc;

[0042] R 13 is selected from =O or protected carbonyl, preferably R 13 is selected from =O or

[0043] In an embodiment of the present application, optionally, the photochemical conversion is a photocatalytic reaction of ultraviolet light, optionally, the photocatalytic reaction of ultraviolet light first opens the steroid ring in the wavelength range of 260-290 nm, and then closes the steroid ring in the wavelength range of 295-340 nm, and the reaction temperature is -10-50℃.

[0044] In a specific embodiment of the present application, the method for preparing the intermediate is as follows:

[0045]

[0046] Compound 1 is protected at the 3-hydroxyl group (e.g., using acetic anhydride) and the 17-ketone (e.g., using ethylene glycol) to give compound 3.

[0047] Compound 3 is oxidized at the allylic position (7) to the carbonyl group to give compound 4 using air oxidation with a catalyst such as N-hydroxyphthalimide and an initiator such as benzoyl peroxide.

[0048] Compound 4 is hydrazonated at the 7-carbonyl group and dehydrazonated to form the 5,7 double bond to give compound 6.

[0049] Compound 6 is photo-catalyzed using UV light to invert the 10-methyl group from the beta to the alpha configuration to give compound 7. The photo-catalysis first opens the steroid ring at a wavelength of 260-290 nm and then closes the ring at a wavelength of 295-340 nm. The reaction temperature is controlled between -10 and 50 °C.

[0050] In an embodiment of the application, the 17-benzimidazolyl-10a-methyl-steroid compounds are prepared by the following method.

[0051]

[0052] Compound 7 is hydrolyzed under base catalysis to give compound IN1F. Optionally, the reaction solvent can be ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, etc. The base can be sodium hydroxide, potassium hydroxide, potassium carbonate, etc. The reaction temperature is 0-60 °C.

[0053] Compound IN1F is hydrogenated under transition metal catalysis to give compound CK004-1A. Optionally, the reaction solvent is one or a mixture of several of ethyl acetate, ethanol, dioxane, tetrahydrofuran, dichloromethane. The catalyst is 5% palladium on carbon, 10% palladium on carbon, metallic platinum, platinum dioxide, platinum acetate, etc. The hydrogen pressure is 0.05-2.0 MPa and the reaction temperature is 40-70 °C.

[0054] Compound CK004-1A is hydrolyzed under acid catalysis to give compound CK004-1B. Optionally, the reaction solvent is ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, etc. The acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid, etc. The reaction temperature is 0-40 °C.

[0055] Compound CK004-1B is hydrazonated at the 17-position, iodinated, and coupled to give TM10.

[0056] Compound TM10 is reacted with carbon-based bimidazole in acetonitrile to give compound TM32. More preferably, the solvent can be tetrahydrofuran, toluene, dichloromethane, etc.

[0057] The compound TM10 is subjected to esterification reaction with acetic anhydride, acetyl chloride, etc. under the action of a catalyst and a base to obtain TM12, and more preferably, the catalyst is 4-dimethylaminopyridine, and the base can be triethylamine, pyridine, etc.

[0058] In an embodiment of the present application, the 17-benzimidazolyl-10α-methyl-steroidal compound is prepared by the following method.

[0059]

[0060] The compound 7 is hydrolyzed into compound IN1F under the catalysis of a base, and optionally, the reaction solvent can be ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, etc. The base can be sodium hydroxide, potassium hydroxide, potassium carbonate, etc., and the reaction temperature is 0-60°C.

[0061] The compound IN1F is hydrogenated under the catalysis of a transition metal to obtain compound CK004-1E, and optionally, the catalyst is 5% palladium-carbon, 10% palladium-carbon, metallic platinum, platinum dioxide, platinum acetate, etc., and the reaction temperature is 40-70°C.

[0062] The compound CK004-1E is hydrolyzed into compound CK004-1F under the catalysis of an acid, and optionally, the acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid, etc., and the reaction temperature is 0-40°C.

[0063] The compound CK004-1F is subjected to hydrazone formation, iodination, and coupling reaction at the 17-position to obtain TM16.

[0064] The compound TM16 is reacted with carbon-based bimidazole in acetonitrile to synthesize compound TM31, and more preferably, the solvent can be tetrahydrofuran, toluene, dichloromethane, etc.

[0065] The compound TM16 is subjected to esterification reaction with chloroformic acid methyl ester, etc. under the action of a base to obtain TM34.

[0066] The present application provides use of the compound of the present application in the preparation of a medicament for treating cancer.

[0067] In an embodiment of the present application, the cancer includes prostate cancer, colon cancer, lung cancer, or pancreatic cancer.

[0068] The present application provides a pharmaceutical composition comprising the 17-benzimidazolyl-10α-methyl-steroidal compound and a pharmaceutically acceptable excipient.

[0069] The dosage form of the pharmaceutical composition can be a common dosage form such as an oral preparation, an injection preparation, etc., and can be a solid preparation, a liquid preparation, etc., and the solid preparation is, for example, a tablet, a capsule, granules, etc., and the liquid preparation is, for example, a solution, a suspension, an emulsion, etc.

[0070] The pharmaceutically acceptable conventional excipients can be used in a conventional amount by a conventional preparation method. For example, the tablet excipients include fillers (diluents), binders, disintegrants, lubricants, glidants, etc. The fillers are selected from lactose, microcrystalline cellulose, mannitol, pregelatinized starch, etc. The binders are selected from hydroxypropyl methylcellulose, polyvinylpyrrolidone, methylcellulose, povidone, starch, etc. The disintegrants are selected from croscarmellose sodium, crospovidone, sodium carboxymethyl starch, corn starch, etc. The lubricants are selected from magnesium stearate, stearic acid, sodium stearyl fumarate, etc. The glidants are selected from talc, microfine silica, etc. The tablet preparation can adopt the wet or dry granulation compression method, or the direct powder compression method, or the blank granule compression method.

[0071] In comparison with the prior art, the present application at least obtains the following beneficial technical effects:

[0072] The compound of the present application, with the C-10 methyl group in the alpha configuration, can act on the Ano1 (Anoctamin 1) target and has a strong inhibitory effect on prostate cancer, colon cancer, lung cancer, and pancreatic cancer cells.

[0073] The IC50 of the compound of the present application against human prostate cancer cells is in the range of <150 μM, preferably <50 μM, more preferably <20 μM. The IC50 of the compound of the present application against human colon cancer cells is in the range of <100 μM, preferably <50 μM, more preferably <20 μM, more preferably <10 μM. The IC50 of the compound of the present application against human non-small cell lung cancer cells is preferably in the range of <150 μM, more preferably <50 μM, more preferably <25 μM. The IC50 of the compound of the present application against human pancreatic cancer cells is preferably in the range of <100 μM, more preferably <50 μM, more preferably <20 μM, more preferably <10 μM.

[0074] The C-10 methyl group of the compound of the present application is in the alpha configuration, which has lower or no reactivity with enzymes (such as 3α steroid dehydrogenase / 3α hydroxysteroid oxidoreductase, cholesterol oxidase, etc.), better molecular stability, is less likely to be degraded by enzymes, has better tolerance to enzymes in vivo, is expected to be metabolized more slowly in vivo, has a longer time to be excreted out of the body, has a longer duration of drug efficacy, and is beneficial for the development of long-acting formulations.

[0075] In addition, the compound of the present application has high bioavailability and fewer side effects. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Figure is the docking simulation result of TM16-3α steroid dehydrogenase;

[0077] Figure 2 Figure for docking simulation result of TM16'-3alpha steroid dehydrogenase;

[0078] Figure 3 Figure for docking simulation result of Abiraterone-3alpha steroid dehydrogenase;

[0079] Figure 4 Figure for docking simulation result of TM16-Ano1 target;

[0080] Figure 5 Figure for docking simulation result of TM31-Ano1 target;

[0081] Figure 6 Figure for docking simulation result of TM32-Ano1 target;

[0082] Figure 7 Figure for docking simulation result of TM34-Ano1 target. DETAILED DESCRIPTION

[0083] In order to make the objects, technical solutions and advantages of the present application clearer and more comprehensible, the present application will be further described in detail. However, it should be understood that the description herein is only used to explain the present application and is not intended to limit the scope of the present application.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the terms used herein should be interpreted only as the terms are commonly used by those in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. The reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be described herein.

[0085] In order to further understand the present application, the present application will be further described in detail below in combination with examples.

[0086] The specific compounds (compounds in examples, including intermediates) in the present application involving 10-position alpha methyl have the following default configuration

[0087]

[0088] If a certain group or H connected to the steroid ring on a certain compound is different from the above configuration, it is indicated separately, for example, if the group at position 14 is in beta configuration, it is indicated as 14beta.

[0089] For the general formula compounds (including at least two compounds), in addition to the configuration of the groups that have been indicated (for example, two angular methyl groups, i.e. methyl groups on C10 and C13), the other groups or H connected to the steroid ring can be in alpha or beta configuration.

[0090] Example 1

[0091]

[0092] In a 2000 mL three-necked flask, DHEA (3β-hydroxy-5-androsten-17-one) (i.e., Compound 1) 200 g, DCM (dichloromethane) 800 mL, triethylamine 140 g, nitrogen replacement 3 times, DMAP (4-dimethylaminopyridine) 4 g, nitrogen replacement 3 times. After stirring at room temperature to make it fully dissolved, acetic anhydride 140 g was added dropwise, and the dropping was completed in 2 h. After the dropping was completed, the stirring was continued for 15 min. TLC monitoring showed that the raw material was reacted completely, 40 mL of methanol was added, and the stirring was continued for 30 min. It was washed once with 200 mL of 5% hydrochloric acid and once with 200 mL of 5% sodium bicarbonate solution. The organic phase was rotary evaporated under reduced pressure in a water bath at 35°C, and DCM was replaced with methanol. Filtration was performed, the filter cake was washed once with a small amount of methanol, and drying was performed in a 50°C air-drying oven for 12 h to obtain 200 g of white solid (i.e., Compound 2) with a mass yield of 100%. In this context, the "mass yield" refers to the mass ratio of the obtained product to the raw material. Taking the above example as an example, the mass yield of 100% means that the mass ratio of the obtained white solid (i.e., Compound 2) to the added raw material (i.e., Compound 1) is 100%.

[0093]

[0094] In a 2000 mL three-necked flask, DHEA acetate (i.e., Compound 2 obtained as described above) 200 g, ethylene glycol 1200 mL, PTS (p-toluenesulfonic acid) 4 g, triethyl orthoformate 260 g. Stirring was performed at 50°C, and TLC monitoring showed that the raw material was reacted completely. Cooling to room temperature, adding triethylamine 8 mL, and continuing to stir for 30 min. Pouring into 1600 mL of water and stirring for 30 min. Filtration was performed, and the filter cake was washed twice with a small amount of water. The filter cake was dissolved in 800 mL of dichloromethane, 4 mL of triethylamine was added, and the stirring was continued for 15 min. The aqueous layer was separated, and the organic phase was rotary evaporated under reduced pressure in a water bath at 35°C, and DCM was replaced with methanol. Filtration was performed, the filter cake was washed once with a small amount of methanol, and drying was performed in a 50°C air-drying oven for 12 h to obtain 210 g of white solid 3 with a mass yield of 105%.

[0095]

[0096] In 2000 mL three-necked flask, add 100 g compound 3, cyclohexanone 800 mL, dry air, stirring at 50°C to make it fully dissolved, then add NOP (N-hydroxyphthalimide) 32 g, benzoyl peroxide 0.50 g. TLC monitoring of raw materials reaction complete. Cool to room temperature, water bath 55°C, rotary evaporation to dryness under reduced pressure. Add dichloromethane 200 mL, petroleum ether 500 mL, stirring at room temperature for 30 min. Filter, filter cake with a small amount of petroleum ether wash 1 times. Add triethylamine 60 g, acetic anhydride 60 g in ice water bath, continue to stir for 30 min. Stand 12 h. Water bath 55°C, rotary evaporation to dryness under reduced pressure. Add dichloromethane to fully dissolve, in water bath 35°C, rotary evaporation under reduced pressure, replaced with methanol dichloromethane. Filter, filter cake with a small amount of methanol wash 1 times, and in 50°C air drying oven drying 12 h, get 75 g white solid 4, mass yield 75%.

[0097]

[0098] In 2000 mL three-necked flask, add 100 g compound 4, TSH (p-toluenesulfonyl hydrazine) 66 g, toluene 400 mL, n-hexane 600 mL, stirring to reflux water. TLC monitoring of raw materials reaction complete. Cool to room temperature, water bath 55°C, rotary evaporation to dryness under reduced pressure. Add methanol 660 mL, n-hexane 130 mL, stirring at room temperature for 30 min. Filter, filter cake with a small amount of methanol wash 1 times, and in 50°C air drying oven drying 12 h, get 126 g white solid 5, mass yield 126%.

[0099]

[0100] In 1000 mL three-necked flask, add lithium amide 5.6 g, chlorobenzene 175 mL, remove ammonia under reduced pressure. After ammonia removal, add compound 5 chlorobenzene solution (35 g compound 5, chlorobenzene 350 mL), move the flask into 120°C oil bath, stirring for 1 h. TLC monitoring of raw materials reaction complete. Cool to room temperature, stirring in ice water bath, with 5% phosphoric acid to pH 6-8. Liquid-liquid, water layer with chlorobenzene 40 mL extraction 1 times, combined organic layer, and washed with water 1 times. Organic layer with anhydrous sodium sulfate drying 2 h. Filter, filter the liquid in water bath 55°C, rotary evaporation to dryness under reduced pressure. Add dichloromethane to fully dissolve, in water bath 35°C, rotary evaporation under reduced pressure, replaced with methanol dichloromethane. Filter, filter cake with a small amount of methanol wash 1 times, and in 50°C air drying oven drying 12 h, get 19 g white solid 6, mass yield 54.3%.

[0101]

[0102] Take 50 g of compound 6, 0.5 g of BHT (antioxidant), add 1.5 L of ethyl acetate to dissolve, pour into a photochemical reactor, start the internal cooling system, and irradiate with a 260-270 nm LED ultraviolet lamp (100 W) for 3 h, then irradiate with a 310-330 nm LED ultraviolet lamp (100 W) for 3 h, take a sample for HPLC monitoring, and after the reaction is completed, concentrate the reaction solution to an oil, add 150 ml of methanol, stir to precipitate compound 6, and filter under suction to recover compound 6 (20 g). After the mother liquor is concentrated to dryness, it is mixed with silica gel and passed through a chromatographic column to obtain 10.5 g of compound 7.

[0103] The 1H NMR of compound 7 was detected as follows: 1H NMR (400 MHz, CDCl3) δ 5.57 (dd, J = 5.5, 2.1 Hz, 1H), 5.41 (dt, J = 5.3, 2.5 Hz, 1H), 4.71 (tt, J = 11.4, 4.5 Hz, 1H), 4.03 - 3.80 (m, 4H), 2.51 (ddd, J = 14.2, 4.8, 2.2 Hz, 1H), 2.42 - 2.25 (m, 2H), 2.08 - 1.98 (m, 5H), 1.97 - 1.86 (m, 3H), 1.87 - 1.76 (m, 1H), 1.76 - 1.63 (m, 3H), 1.61 - 1.47 (m, 4H), 1.44 - 1.31 (m, 1H), 0.96 (s, 3H), 0.79 (s, 3H).

[0104] HRMS mass spectrum (EI) m / z: calculated 373.5: tested 372.9.

[0105] Example 2

[0106] Compound 7 prepared in Example 1 was used to synthesize 17-benzimidazolyl-10α-methyl-steroidal compounds, which are referred to as TM10, TM12 and TM32 in this synthesis.

[0107]

[0108] In a 250 mL three-necked flask, 5 g of compound 7, 30 g of acetone, and 1.26 g of sodium hydroxide were added, stirred at room temperature for 1 h, and the raw material was monitored by TLC to determine that the reaction was complete. The acetone was removed by rotary evaporation under reduced pressure, extracted with dichloromethane, and concentrated to obtain a light yellow oil IN1F4.3 g with a mass yield of 86.0%.

[0109]

[0110] Into a 250ml hydrogenation flask, 10g IN1F, 150ml anhydrous ethanol, 2g of 5% palladium on carbon, nitrogen replacement three times, hydrogen replacement three times, temperature to 55-60°C, hydrogen pressure 0.15-0.20MPa reaction 4 hours, control reaction completion, filter to remove palladium on carbon, concentrated, column chromatography (eluent petroleum ether: ethyl acetate = 10:1 ~ petroleum ether: ethyl acetate 5:1) to get solid 6.3g (i.e. compound CK004-1A), mass yield 63.0%.

[0111]

[0112] Into a 50ml reaction bottle, 1.5g CK004-1A, acetone 22ml, water 7.5ml, p-toluenesulfonic acid 0.3g, reaction complete, concentrated without fraction, 10ml dichloromethane, separated, 5ml of 5% sodium bicarbonate washing once, separated, organic layer concentrated dry to get CK004-1B 1.2g, mass yield 80%.

[0113]

[0114] Into a reaction bottle, 1.2g of CK004-1B, 0.86g hydrazine hydrate (85%), 5g anhydrous ethanol, 0.006g hydrazine sulfate, temperature to 30-35°C reaction overnight, reaction complete, slowly pour the reaction liquid into 50ml water, stirring for 1 hour, filter, water leaching, 50°C drying to get white solid 1.15g (i.e. compound CK004-1C), mass yield 95.8%.

[0115]

[0116] Into a reaction bottle, 1.05g CK004-1C, add 12ml tetrahydrofuran stirring to dissolve. Into another reaction bottle, 10ml tetrahydrofuran, temperature to -5 to 5°C, add 1.72g iodine, stirring for half an hour. At -5 to 5°C, dropwise add tetramethyl guanidine, dropwise complete, stirring for half an hour. At -5 to 5°C, dropwise add CK004-1C tetrahydrofuran solution, dropwise, at -5 to 5°C, reaction 1 hour, reaction complete, add 1.2g sodium thiosulfate, temperature to 40°C, stirring for half an hour, filter, filtrate concentrated to no fraction, add 10ml tetrahydrofuran, add 5ml 1M hydrochloric acid washing, again with 5ml 5% sodium bicarbonate solution washing, again with 5ml saturated sodium thiosulfate solution washing, separated, organic layer concentrated dry to get 1.21g, compound CK004-1D, mass yield 115.2%.

[0117]

[0118] To a reaction flask was added 720 mg of CK004-1D, 15 ml of dimethyl sulfoxide, 50 mg of cuprous iodide, 300 mg of benzimidazole, 80 mg of 8-hydroxyquinoline, 600 mg of potassium carbonate, and the temperature was raised to 180 °C for 16 hours. After the reaction was complete, the temperature was lowered, 50 ml of dichloromethane was added, and the mixture was washed with water 5 times, 20 ml each time. The organic phase was concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate = 10:1 to petroleum ether: ethyl acetate 2:1) was performed to obtain 420 mg of a solid (i.e., compound TM10) with a yield of 58.3%.

[0119] The 1H NMR of compound TM10 was detected as follows: 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.86-7.77 (m, 1H), 7.49 (s, 1H), 7.31-7.26 (m, 2H), 5.95 (s, 1H), 4.06 (s, 1H), 2.41 (s, 2H), 2.16-1.95 (m, 3H), 1.85 (d, J = 12.3 Hz, 3H), 1.79-1.40 (m, 12H), 1.27 (d, J = 12.4 Hz, 3H), 1.22-1.11 (m, 1H), 1.02 (s, 3H), 1.02 (s, 3H).

[0120] 13C NMR was as follows: 13C NMR (101 MHz, CDCl3) δ 147.66, 143.24, 141.47, 134.50, 123.40, 122.83, 122.49, 120.18, 111.27, 66.35, 48.82, 47.66, 45.30, 38.40, 37.88, 35.78, 34.30, 33.61, 30.96, 28.72, 28.14, 25.03, 20.77, 16.26, 14.85.

[0121] HRMS mass spectrum (EI) m / z: calculated 390.56: found 390.9.

[0122]

[0123] To a reaction flask was added 80 mg of TM10, 3 ml of dichloromethane, 200 mg of triethylamine, 0.5 mg of 4-dimethylaminopyridine, 200 mg of acetic anhydride was added at 10-20 °C, and the reaction was complete. After the reaction was complete, 0.5 ml of methanol was added, and the mixture was concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate = 5:1) was performed to obtain 75 mg of a white solid (TM12) with a mass yield of 93.8%.

[0124] The 1H NMR of compound TM12 was detected as follows: 1H NMR (400 MHz, CDC13) δ 7.98 (s, 1H), 7.86 - 7.77 (m, 1H), 7.53 - 7.47 (m, 1H), 7.34 - 7.27 (m, 2H), 5.96 (d, J = 2.9 Hz, 1H), 5.02 (s, 1H), 2.42 (s, 2H), 2.06 (s, 5H), 1.85 (d, J = 13.2 Hz, 2H), 1.82 - 1.56 (m, 9H), 1.52 (s, 3H), 1.19 (s, 4H), 1.03 (s, 6H).

[0125] 13C NMR was: 13C NMR (101 MHz, CDC13) δ 170.66, 147.65, 143.29, 141.47, 134.51, 123.40, 122.81, 122.49, 120.22, 111.25, 69.95, 48.80, 47.66, 45.22, 39.37, 37.57, 34.96, 33.58, 32.77, 30.95, 28.10, 25.77, 24.85, 21.58, 20.77, 16.25, 14.99.

[0126] HRMS mass spectrum (EI) m / z, calcd for 432.5: found 432.9.

[0127]

[0128] To the reaction bottle, 100 mg of TM10, 62 mg of carbon-based diimidazole, 8 ml of acetonitrile, were added, and the temperature was raised to 60°C for 5 hours. After the reaction was completed, it was concentrated to dryness, dissolved with 5 ml of dichloromethane, washed once with 2 ml of water, and the organic layer was concentrated and dried. Column chromatography (eluent petroleum ether: ethyl acetate = 5:1 ~ petroleum ether: ethyl acetate 1:1) was performed to obtain 83 mg of solid (TM32) with a yield of 83%.

[0129] The 1H NMR of compound TM32 was detected as follows: 1H NMR (400 MHz, CDC13) δ 8.16 (s, 1H), 7.98 (s, 1H), 7.86 - 7.78 (m, 1H), 7.50 (dd, J = 7.2, 1.8 Hz, 1H), 7.45 (s, 1H), 7.34 - 7.27 (m, 2H), 7.09 (s, 1H), 5.97 (d, J = 2.9 Hz, 1H), 5.27 (s, 1H), 2.51 - 2.36 (m, 2H), 2.20 - 2.05 (m, 2H), 1.94 - 1.48 (m, 13H), 1.44 - 1.16 (m, 4H), 1.09 (s, 3H), 1.04 (s, 3H).

[0130] 13C NMR: 13C NMR (101 MHz, CDC13) δ 148.07, 147.61, 143.29, 141.43, 137.07, 134.50, 130.59, 123.43, 122.83, 122.53, 120.24, 117.12, 111.23, 75.44, 48.79, 47.67, 45.31, 39.88, 37.61, 35.04, 33.53, 32.57, 30.93, 28.12, 25.74, 24.70, 20.80, 16.17, 14.99.

[0131] HRMS mass spectrum (EI) m / z: Calcd for 484.6: Found 484.9.

[0132] Example 3

[0133]

[0134] Into a reaction flask was added 3.0 g IN 1F, 0.6 g 5% Pd / C, 15 mL dioxane, water bath 60 °C, nitrogen replacement, hydrogen replacement, hydrogen balloon pressurization, reaction was completed, filtered, concentrated to dryness, column chromatography (petroleum ether: ethyl acetate = 15:1) to obtain 1.2 g of white solid CK004-1E, mass yield 40.0%.

[0135]

[0136] Into a reaction flask was added 2 g of CK004-1E, 15 ml of acetone, 0.8 g of p-toluenesulfonic acid, 5 ml of water, and the reaction was carried out at room temperature. After the reaction was completed, the acetone was removed by concentration, 10 ml of dichloromethane was added, washed once with 5 ml of 5% sodium bicarbonate solution, washed once with 5 ml of water, and the organic layer was concentrated to dryness to obtain 1.7 g of CK004-1F, mass yield 85.0%.

[0137]

[0138] Into a reaction flask was added 850 mg of CK004-1F, 3.5 ml of anhydrous ethanol, 612 mg of hydrazine hydrate, and 4 mg of hydrazine sulfate, and the reaction was carried out at 30-35 °C. After the reaction was completed, the reaction solution was slowly poured into 50 ml of ice water, stirred for 1 hour, filtered, washed with water, and dried at 50 °C to obtain 820 mg of white solid CK004-1G, mass yield 96.47%.

[0139]

[0140] Into a reaction flask was added 1.55 g of CK004-1G, 15 ml of tetrahydrofuran was added, and it was dissolved at 40°C. Into another reaction flask was added 15 ml of tetrahydrofuran, and it was cooled to -5 to 5°C, 2.66 g of iodine was added, and then 3 g of tetramethylguanidine was added dropwise. While controlling the temperature to be -5 to 5°C, the tetrahydrofuran solution of CK004-1G was added dropwise, after the dropwise addition was completed, it was reacted at -5 to 5°C for 1 hour, after the reaction was completed, 1.86 g of sodium thiosulfate was added, and it was stirred at 40°C for half an hour, it was filtered, the filtrate was concentrated to remove the distillate, 15 ml of tetrahydrofuran was added, 8 ml of 1M hydrochloric acid was added for washing, 8 ml of 5% sodium bicarbonate solution was added for washing, and 8 ml of saturated sodium thiosulfate solution was added for washing, the organic layer was concentrated to dryness to obtain 1.8 g of CK004-1H, and the mass yield was 116.1%.

[0141]

[0142] Into a reaction flask was added 800 mg of CK004-1H, 15 ml of dimethyl sulfoxide, 80 mg of cuprous iodide, 300 mg of benzimidazole, 80 mg of 8-hydroxyquinoline, and 600 mg of potassium carbonate, and it was reacted at 180°C for 16 hours, after the reaction was completed, it was cooled, 50 ml of dichloromethane was added, and it was washed with water 5 times, 20 ml each time, the organic phase was concentrated to dryness, and column chromatography (eluent: petroleum ether: ethyl acetate = 10:1 ~ petroleum ether: ethyl acetate 2:1) was performed to obtain 320 mg of a solid (i.e., compound TM16), and the yield was 40%.

[0143] It was confirmed that the 1H NMR of compound TM16 was as follows: 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.87-7.77 (m, 1H), 7.60-7.54 (m, 1H), 7.36-7.27 (m, 2H), 5.92 (d, J = 1.4 Hz, 1H), 5.37 (dd, J = 9.2, 6.4 Hz, 1H), 4.12-4.09 (m, 1H), 3.03-2.85 (m, 1H), 2.64-2.46 (m, 1H), 2.36 (dd, J = 10.5, 1.7 Hz, 2H), 2.03 (d, J = 12.4 Hz, 4H), 1.78-1.59 (m, 6H), 1.59-1.40 (m, 5H), 1.26 (s, 1H), 1.22 (s, 3H), 0.71 (s, 3H).

[0144] 13C NMR: 13C NMR (101 MHz, CDC13) δ 147.32, 143.42, 140.81, 136.92, 123.57, 122.70, 120.28, 118.67, 111.69, 66.20, 60.40, 50.68, 47.23, 45.62, 35.75, 35.20, 34.64, 31.52, 30.55, 29.12, 23.41, 21.06, 18.55, 14.21, 10.26.

[0145] HRMS mass spectrum (EI) m / z: Calcd for 388.25: Found, 388.9.

[0146]

[0147] To the reaction bottle was added 100 mg of TM16, 62 mg of carbon-based diimidazole, 8 ml of acetonitrile, and the temperature was raised to 60°C for 5 hours of reaction. After the reaction was completed, it was concentrated to dryness, dissolved with 5 ml of dichloromethane, washed once with 2 ml of water, and the layers were separated. The organic layer was concentrated and dried, and column chromatography (eluent petroleum ether: ethyl acetate = 5:1 ~ petroleum ether: ethyl acetate 1:1) was performed to obtain 63 mg of solid (i.e., compound TM31) at a yield of 63%.

[0148] Upon testing, the 1H NMR of compound TM31 was: 1H NMR (400 MHz, CDC13) δ 8.16 (s, 1H), 8.07 (s, 1H), 7.87 - 7.78 (m, 1H), 7.57 (dd, J = 6.0, 3.2 Hz, 1H), 7.45 (s, 1H), 7.37 - 7.27 (m, 2H), 7.08 (s, 1H), 5.93 (d, J = 1.1 Hz, 1H), 5.40 (s, 1H), 5.30 (s, 1H), 2.95 (d, J = 2.2 Hz, 1H), 2.56 (d, J = 3.2 Hz, 1H), 2.34 (ddd, J = 42.0, 25.5, 6.8 Hz, 2H), 2.18 - 1.37 (m, 15H), 1.25 (s, 3H), 0.78 (s, 3H).

[0149] 13C NMR was: 13C NMR (101 MHz, CDC13) δ 148.11, 147.25, 143.50, 140.80, 137.04, 134.01, 130.61, 123.59, 122.72, 120.36, 118.65, 118.41, 117.12, 111.64, 75.17, 50.65, 47.22, 45.59, 36.44, 34.43, 32.48, 31.32, 30.25, 29.06, 26.21, 23.53, 18.59, 10.43.

[0150] HRMS mass spectrum (EI) m / z: Calcd for 482.6: Found 482.9.

[0151]

[0152] To the reaction bottle was added 100 mg of TM16, 2 ml of pyridine, the solution was stirred to clear, 40 mg of methyl chloroformate was added, and the reaction was carried out for 16 hours. After the reaction was completed, 0.5 ml of methanol was added to quench, and concentrated to dryness, column chromatography (eluent petroleum ether: ethyl acetate = 20: 1 ~ petroleum ether: ethyl acetate 6: 1) to obtain a solid 56 mg (i.e., compound TM34), yield 56%.

[0153] It was detected that the 1H NMR (400 MHz, CDC13) of compound TM34 was δ 8.07 (s, 1H), 7.87-7.78 (m, 1H), 7.57 (d, J = 9.2 Hz, 1H), 7.35-7.28 (m, 2H), 5.92 (d, J = 1.3 Hz, 1H), 5.57-5.30 (m, 1H), 4.94 (s, 1H), 3.78 (s, 3H), 2.92 (d, J = 2.1 Hz, 1H), 2.55 (ddd, J = 15.4, 6.5, 3.3 Hz, 1H), 2.45-2.20 (m, 2H), 2.09-1.81 (m, 5H), 1.78-1.35 (m, 10H), 1.22 (s, 3H), 0.72 (s, 3H).

[0154] 13C NMR was: 13C NMR (101 MHz, CDC13) δ 155.32, 147.30, 143.41, 140.81, 136.92, 133.99, 123.58, 122.72, 120.29, 118.60, 111.67, 74.03, 54.49, 50.65, 46.98, 45.59, 35.84, 34.30, 32.69, 32.07, 31.42, 30.29, 29.06, 26.24, 23.38, 18.53, 10.38.

[0155] HRMS mass spectrum (EI) m / z: Calcd for 446.6: Found, 446.9.

[0156] In addition to the compounds prepared in the above examples, other example compounds can be prepared by the same / similar preparation process as the foregoing examples, and all of the example compounds are listed as follows:

[0157]

[0158]

[0159]

[0160] Example 4

[0161] I. Pharmacological experiment: inhibition of cancer cells

[0162] 1. Test method

[0163] 1.1 Experimental grouping and sample preparation

[0164] Each sample of the compounds of the examples was prepared into a 100 mM stock solution using dimethyl sulfoxide (DMSO) as the solvent, and diluted with the corresponding complete culture medium for each cell culture to prepare a working solution with a concentration of 100, 30, 10, 3, 1, 0.3 μM. A solvent control group, a positive control group with different concentrations, and a sample treatment group with different concentrations were set up.

[0165] 1.2 Cell culture

[0166] The culture medium for human prostate cancer cells (DU 145) was MEM medium containing 10% FBS (fetal bovine serum); the culture medium for human colon cancer cells (HCT-116) was McCoy's 5A containing 10% FBS; the culture medium for human non-small cell lung cancer cells (A549) was Ham's F-12K medium containing 10% FBS, and the culture conditions were all 37°C, 5% CO2. The culture medium for human pancreatic cancer cells (PANC-1) was DMEM medium containing 10% FBS. When the growth state was good, the cells were subcultured every 2 days at a ratio of 1:3. In a clean bench, the culture medium was discarded, and the cells were washed twice with 1x PBS, then 600 μL of 0.25% trypsin was added for digestion, and after about 1-3 min, the cells were detached, 3 mL of the corresponding culture medium containing 10% FBS was added to terminate the trypsin digestion, and the single cell suspension was blown into an EP tube and centrifuged at 1000 rpm for 5 min. The culture medium was discarded, and the cells were resuspended with fresh culture medium at a certain ratio (cell density was about 10 5 / mL) and inoculated into new culture bottles, which were placed in a 37°C, 5% CO2 incubator for culture.

[0167] 1.3 Cell Seeding

[0168] The well-grown cells were collected by routine digestion, and the cell density of DU 145 was adjusted to 2 x 10 4 cells / mL, the cell density of HCT-116 was adjusted to 2 x 10 4 cells / mL, the cell density of A549 was adjusted to 3 x 10 4 cells / mL, and the cell density of PANC-1 was adjusted to 4 x 10 4 cells / mL. Each cell suspension was inoculated into a 96-well culture plate at a density of 100 μL / well, and the cells were uniformly spread on the bottom of the well by cross-shaking for 10 times. The culture plate was placed in a CO2incubator for 24 h.

[0169] 1.4 Cell Treatment

[0170] The sample working solution of the compound prepared in step 1.1 was taken, and 100 μL / well was added to the corresponding well to make the final volume 200 μL (100 μL of cell culture medium and 100 μL of sample working solution) and the final concentration 50, 15, 5, 1.5, 0.5, 0.15 μM, respectively. A solvent control group was set up, and the positive control group was set up at a concentration of 50, 15, 5, 1.5, 0.5, 0.15 μM, respectively. The number of duplicate wells in each group was 3. The culture was carried out at 37 °C and 5% CO2for 72 h.

[0171] 1.5 Detection of Cell Proliferation OD Value

[0172] After 72 h of cell treatment, 20 μL of thiazolyl blue (MTT) was added to each well, and the culture was continued at 37 °C and 5% CO2for 4 h. The liquid in each well was carefully aspirated, and 150 μL / well of DMSO was added to the well, and shaken for 10 min.

[0173] The OD value of A1-H1 well (8 wells) was set as the zero value on the enzyme marker, and the OD value of each well was detected at 492 nm.

[0174] 1.6 Result Calculation

[0175] The OD value of the solvent control group was set as 100% cell viability, and the ratio of the OD value of the other groups to the OD value of the solvent control group was the relative cell viability. The activity of the sample on DU 145 or HCT-116 or A549 or PANC-1 cells was evaluated by cell proliferation rate. If the inhibition rate was > 100%, it was determined as a system error, and was calculated as 100%.

[0176] The inhibition rate calculation formula is: inhibition rate (%) = (1-OD 样品 / OD 溶媒 ) x 100%

[0177] The half maximal inhibitory concentration (IC50) was calculated using SPSS software. 50 ).

[0178] 2. Experimental results

[0179] Table 1 IC50values of compounds of some examples and comparative examples against each cancer cell

[0180]

[0181] Note: DU145 is human prostate cancer cell, HCT-116 is human colon cancer cell, A549 is human non-small cell lung cancer cell, PANC-1 is human pancreatic cancer cell.

[0182] The compounds of the present application, whose methyl group at C-10 position is in a configuration of a, have inhibitory effects on prostate cancer, colon cancer, lung cancer, and pancreatic cancer cells. The IC50of the compounds of the present application against human prostate cancer cells is in the range of < 150 μM, preferably in the range of < 50 μM, more preferably in the range of < 20 μM. The IC50of the compounds of the present application against human colon cancer cells is in the range of < 100 μM, preferably in the range of < 50 μM, more preferably in the range of < 20 μM, more preferably in the range of < 10 μM. The IC50of each compound of the present application against human non-small cell lung cancer cells is in the range of < 150 μM, except for individual > 150 μM, more preferably in the range of < 50 μM, more preferably in the range of < 25 μM. The IC50of each compound of the examples of the present application against human pancreatic cancer cells is preferably in the range of < 100 μM, more preferably in the range of < 50 μM, more preferably in the range of < 20 μM, more preferably in the range of < 10 μM.

[0183] In the present application, it is considered that IC50in the range of 50-150 μM indicates that the compound has a certain degree of inhibitory effect on cancer cells, and IC50< 50 μM indicates that the compound has a good inhibitory effect on cancer cells.

[0184] In addition, the applicant points out that in the prior art, abiraterone acetate (or abiraterone) is clinically used in combination with prednisone to treat prostate cancer, however, the present application surprisingly found and proved by experiments (as shown in Table 1) that abiraterone acetate not only has an anti-prostate cancer effect, but also shows an unexpected inhibitory effect on colon cancer cells or lung cancer cells, in particular, the IC50of abiraterone acetate against human colon cancer cells is in the range of < 50 μM, the IC50of abiraterone acetate against human colon cancer cells is in the range of < 10 μM; the IC50of abiraterone acetate against human non-small cell lung cancer cells is in the range of < 50 μM.

[0185] Further, from the results of Table 1, it can be seen that the compound TM10 of the present application shows good inhibition for both human prostate cancer cells with IC50 in the range of <20 μM, human colon cancer cells with IC50 in the range of <10 μM, human non-small cell lung cancer cells with IC50 in the range of <25 μM, and human pancreatic cancer cells with IC50 in the range of <20 μM, indicating that the compound TM10 of the present application even has better inhibitory effect than abiraterone acetate for these two cancers (i.e., human non-small cell lung cancer cells and human pancreatic cancer cells). This fully indicates that the compound TM10 of the present application shows good inhibition for human prostate cancer cells, human colon cancer cells, human non-small cell lung cancer cells, and human pancreatic cancer cells. The compound TM12 of the present application shows good inhibition for human prostate cancer cells with IC50 in the range of <15 μM, human colon cancer cells with IC50 in the range of <10 μM, and human pancreatic cancer cells with IC50 in the range of <50 μM, and has certain inhibition for human non-small cell lung cancer cells. The compound TM16 of the present application shows good inhibition for both human prostate cancer cells with IC50 in the range of <20 μM and human colon cancer cells with IC50 in the range of <10 μM, and has good inhibition for human non-small cell lung cancer cells with IC50 in the range of <20 μM and human pancreatic cancer cells with IC50 in the range of <10 μM, indicating that the compound TM16 of the present application even has better inhibitory effect than abiraterone acetate for these two cancers (i.e., human non-small cell lung cancer cells and human pancreatic cancer cells). This fully indicates that the compound TM16 of the present application shows good inhibition for human prostate cancer cells, human colon cancer cells, human non-small cell lung cancer cells, and human pancreatic cancer cells.

[0186] II. Docking of compounds with enzyme molecules

[0187] Docking scoring was performed on the following compounds using the docking software MOE (Molecular Operating Environment, a comprehensive software system for pharmaceutical and life sciences developed by Chemical Computing Group ULC, Canada), and the specific results are shown in Table 2.

[0188] Among them, the docking simulation results of TM16-3α steroid dehydrogenase (HSD) are shown in Figure 1

[0189] The docking simulation results of compound 16'-3α steroid dehydrogenase are shown in Figure 2 ​As shown in the figure, compound 16' has the following structural formula, which is different from TM16 in that the methyl group at C10 is in the beta configuration without flipping;

[0190]

[0191]

[0192] The docking simulation results of abiraterone-3alpha steroid dehydrogenase are as shown in the figure Figure 3 As shown in the figure, abiraterone has the following structural formula

[0193]

[0194] Table 2: Docking score of each compound with 3alpha steroid dehydrogenase

[0195] Compound Docking score TM16-3 alpha steroid dehydrogenase -4.48 Compound 16'-3 alpha steroid dehydrogenase -4.72 Abiraterone-3 alpha steroid dehydrogenase -6.98

[0196] The scoring of the MOE software is calculated according to the parameters of electrostatic parameters between molecules and enzymes, hydrogen bond, molecular attraction, molecular orbit, etc. The more negative the score, the lower the free energy, the more stable the conformation, and the better the binding.

[0197] The above results show that: the compound TM16 of the present application, after flipping the methyl group at C10 (alpha configuration), is not as good as the non-flipped compound in docking with the above enzyme, indicating that the flipped compound becomes insensitive to the above enzyme, or the enzyme has lower activity to the flipped compound. It can be known that the compound has better tolerance to the enzyme in vivo, is expected to be metabolized more slowly in vivo, the time of excretion out of the body will be prolonged, the action time will also be prolonged, and the drug efficacy duration will be longer, which is beneficial to the development of long-acting preparations. In addition, even compared with abiraterone, the compound TM16 of the present application is not as good as abiraterone in docking with the above enzyme, indicating that the compound TM16 after flipping the methyl group at C10 becomes insensitive to the above enzyme, or the enzyme has lower activity to the flipped compound. It can be known that the compound has better tolerance to the enzyme in vivo, is expected to be metabolized more slowly in vivo, the time of excretion out of the body will be prolonged, the action time will also be prolonged, and the drug efficacy duration will be longer, which is beneficial to the development of long-acting preparations.

[0198] III. Docking of compounds with Ano1 target

[0199] Further, the present application also simulates and scores the docking of the following compounds TM16, TM31, TM32, TM34 with Ano1 target by using the docking software MOE, and the scoring results are shown in Table 3 as shown in the figure Figure 4 、 Figure 5 、 Figure 6 and Figure 7 as shown in the figure.

[0200] Table 3: docking score of each compound with Ano1 (Anoctamin 1) target

[0201] Compound Docking score with Anol (Anoctamin 1) target TM16 -5.79 TM31 -6.39 TM32 -6.40 TM34 -6.59

[0202] In combination with Table 3 and Figure 4 It can be seen that the compound TM16 of the present application can be well combined with the Ano1 (Anoctamin 1) target, and the docking score thereof is -5.79, so it is expected to have a good inhibitory effect on colon cancer, lung cancer and pancreatic cancer, which shows good correspondence and consistency with the aforementioned IC50 value. As for other compounds TM31, TM32 and TM34, the docking score thereof with the Ano1 (Anoctamin 1) target is even lower than that of TM16, so it is even better combined with the Ano1 (Anoctamin 1) target, and therefore it is also expected to have a good inhibitory effect on colon cancer, lung cancer and pancreatic cancer.

[0203] Four, enzyme-linked immunosorbent assay to detect the effect of the compound of the present application on the Ano1 expression of cancer cells

[0204] The effect of the compound of the present application on the Ano1 expression of cancer cells is studied by enzyme-linked immunosorbent assay as follows.

[0205] Calcium-activated chloride channel protein 1 (Ano1) is located in the 11q13 region of human chromosome, and the expression of Ano1 in human prostate cancer cell lines is significantly higher than that in human normal prostate epithelial cell line RWPE-1. Blocking the Ano1 channel can significantly inhibit the proliferation, migration and invasion ability of prostate cancer cells. After blocking the Ano1 channel, cancer cells will express more calcium-activated chloride channel protein 1 (Ano1) in order to survive, thereby causing the detected Ano1 to be up-regulated. In other words, if the Ano1 channel is blocked, the compensatory expression of the protein will be increased. The compound of the present application can block the Ano1 channel, thereby causing the compensatory expression of the protein to increase, which will be described in detail below.

[0206] 4.1 Test materials

[0207] 4.1.1 Sample

[0208] TM16 (working concentration 10 μM, 5 μM), D4A (abiraterone oxide, structure as follows, working concentration 10 μM, 5 μM), with abiraterone acetate (working concentration 10 μM, 5 μM) as a positive control.

[0209]

[0210] 4.1.2 Cell strain

[0211] Human prostate cancer cells (DU 145), epithelioid adherent cells, numbered CL-0075, culture conditions: MEM medium containing 10% fetal bovine serum (FBS), 37°C, 5% CO2; human colon cancer cells (HCT-116), epithelioid adherent cells, numbered CL-0096, McCoy's 5A medium containing 10% fetal bovine serum (FBS), 37°C, 5% CO2. Both strains of cells were purchased from Wuhan Punsai Life Science Co., Ltd.

[0212] 4.1.3 Main reagents

[0213] Human Ano1 ELISA kit, size 48T, batch number 202201, Shanghai Zoben Biological Technology Co., Ltd.; BCA kit, size 500 times, product of Biyun Tian Biological Technology Co., Ltd.

[0214] 4.2 Test method

[0215] 4.2.1 Cell treatment

[0216] According to the growth conditions of DU 145 or HCT-116 cells, the cells were cultured, and 1×10 5 cells / well were inoculated in a 6-well plate. After the cells adhered, TM16, D4A, and abiraterone acetate were added at a working concentration of 10 μM and 5 μM, respectively. The 6-well plate with the added samples was placed in a CO2 incubator and cultured for 72 h.

[0217] 4.2.2 Protein harvesting and protein concentration determination

[0218] After 72 h of incubation in the incubator, the cells in each group were lysed with cell lysis buffer, and the supernatant was obtained by low-temperature centrifugation. The cell protein was harvested, and the protein concentration of each sample was detected using a BCA kit.

[0219] BCA kit detection method:

[0220] Protein standard preparation: Take 0.8 mL of protein standard preparation solution and add it to the protein standard (20 mg BSA, bovine serum albumin). Dissolve thoroughly to prepare a 25 mg / mL protein standard solution. Take 20 μL of 25 mg / mL protein standard solution and add 980 μL of diluent to prepare a 0.5 mg / mL protein standard solution.

[0221] 2. Preparation of BCA working solution: According to the sample quantity, add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix well. In this test, 12.24 mL of BCA working solution was prepared (12 mL of reagent A: 0.24 mL of reagent B).

[0222] ③ Add 0, 1, 2, 4, 8, 12, 16, 20 μL of standard to the standard wells of the 96-well plate, and add standard diluent to make up to 20 μL, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL, respectively.

[0223] ④ Take 20 μL of the sample to be tested into the sample wells of the 96-well plate.

[0224] ⑤ Add 200 μL of BCA working solution to each well, and place at 37°C for 30 min.

[0225] ⑥ Measure the absorbance at 492 nm using an enzyme marker.

[0226] ⑦ Calculate the protein concentration of the sample according to the standard curve and the volume of the sample used.

[0227] 4.2.3 Ano1 content result detection

[0228] According to the Ano1 ELISA kit detection instruction, the Ano1 expression was determined, and each protein sample was used.

[0229] ELISA kit detection operation steps:

[0230] ① Standard sample addition: set standard sample wells and sample wells, and add different concentrations of standard sample 50 μL to each standard sample well.

[0231] ② Sample addition: set zero adjustment wells (add sample diluent without sample and enzyme marker reagent, and the rest of the steps are the same), and set sample wells to be tested. Add sample diluent 40 μL to the sample wells to be tested on the enzyme marker coating plate, and then add sample to be tested 10 μL (sample dilution is 5 times). Add sample to the bottom of the enzyme marker plate well, and mix gently.

[0232] ③ Add enzyme marker solution: add enzyme marker reagent 100 μL to each well except the blank well.

[0233] ④ Incubation: after sealing the plate with a sealing film, incubate at 37°C for 60 min.

[0234] ⑤ Washing: remove the sealing film, discard the liquid, and shake dry. Add enough washing liquid to each well, stand for 30 s, then discard, and repeat the washing 5 times, and pat dry.

[0235] ⑥ Color development: add color developer A and color developer B 50 μL each to each well, mix gently, and incubate at 37°C for 15 min.

[0236] ⑦ Termination: add termination solution 50 μL to each well to terminate the reaction (at this time, the blue color immediately turns yellow).

[0237] 8. Detection: Within 15 min after adding the stop solution, adjust the zero hole to zero, and detect the absorbance value (OD value) of each hole at 450 nm wavelength.

[0238] 9. Calculate the Anol concentration of the sample according to the standard curve and the sample volume used.

[0239] 4.3 Test results

[0240] 4.3.1 Protein concentration determination results

[0241] According to the OD value measured by the BCA kit, according to the standard curve, the protein concentration of each sample is calculated in mg. The standard curve equation is: y = 0.2611x + 0.1308, R 2 = 0.9964.

[0242] Table 4: BSA standard detection results (n = 2)

[0243] Standard concentration (mg / mL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5 OD value 1 0.129 0.136 0.144 0.159 0.185 0.211 0.233 0.253 OD value 2 0.126 0.137 0.142 0.157 0.189 0.214 0.239 0.261 Average OD value 0.1275 0.1365 0.1430 0.1580 0.1870 0.2125 0.2360 0.2570

[0244] Table 5: Total protein concentration results of each cell lysate

[0245]

[0246] 4.3.2 Anol content determination results

[0247] After detection, calculate the average value of the duplicate holes, and adjust the average value of the detection results to zero with the average value of the zero hole. Draw a standard curve graph to obtain the standard curve equation. Calculate the Anol results of each sample according to the standard curve equation, and calculate the relative Anol results according to the total protein concentration. The Anol protein expression is expressed as the Anol protein concentration (ng) contained in each mg of total protein.

[0248] The standard curve equation is: y = 0.284x + 0.1113, R 2 = 0.9863.

[0249] Table 6: Anol protein standard detection results

[0250] Standard concentration (mg / mL) 0 0.625 1.25 2.5 5 10 Zeroing well OD value 1 0.045 0.236 0.487 0.947 1.681 2.874 0.034 OD value 2 0.040 0.256 0.508 0.980 1.699 2.861 0.034 Average OD value after zeroing 0.008 0.212 0.464 0.930 1.724 2.834 0.034

[0251] Table 7: Anol expression results of each sample of DU 145 cells (n = 2)

[0252]

[0253] Table 8: Anol expression results of each sample of HCT-116 cells (n = 2)

[0254]

[0255] From the above, for DU 145 cells, the expression of Ano1 in the solvent control group was 0.9621 at 72 h. Compared with the blank control group, the expression of Ano1 in the DU145 cells treated by the samples TM16, D4A and abiraterone acetate at various concentrations was increased, and the expression results showed that low concentration expression was low and high concentration expression was high. This shows that the compound of the present application can increase the compensatory expression of Ano1, which is due to the blocking of the Ano1 channel by the compound of the present application. In the case of blocking the Ano1 channel, the compound of the present application can inhibit the proliferation, migration and invasion of prostate cancer cells. The results are in good agreement with the results of the aforementioned molecular simulation and docking and the determination of IC50 values. For HCT-116 cells, the compound of the present application also shows the result of blocking the Ano1 channel and increasing the compensatory expression of the protein channel.

[0256] The above only describes the embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A 17-benzimidazolyl-10α-methyl-steroidal compound, characterized in that, It has the following structural formula:

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