CN116621902A

CN116621902A - 17-benzimidazolyl-10 alpha-methyl-steroid derivatives, preparation method, application and pharmaceutical composition thereof

Info

Publication number: CN116621902A
Application number: CN202210541892.0A
Authority: CN
Inventors: 蒋红平; 刘喜荣; 唐杰; 何群; 罗桂芳
Original assignee: Hunan Jiujian Pharmaceutical Technology Co ltd; Shanghai Chunjian Industrial Development Co ltd
Current assignee: Hunan Keyixin Biomedical Co ltd
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2023-08-22

Abstract

The present application relates to a compound, a process for its preparation, its use, and pharmaceutical compositions thereof. The C-10 methyl of the steroid derivative is in a reversed alpha configuration, is a brand new compound, and has a strong inhibition effect on prostate cancer, colon cancer and lung cancer cells. In order to prepare the steroid derivative, the application also synthesizes a series of intermediates corresponding to the steroid derivative. The steroid derivatives of the present application can be used as medicaments for effectively treating cancers, particularly cancers including prostate cancer, colon cancer or lung cancer. The C-10 methyl of the steroid derivative is alpha configuration, compared with beta configuration, the activity of the steroid derivative with enzyme is greatly reduced, the steroid derivative has better tolerance to in vivo enzyme, is expected to be metabolized slowly in vivo, has longer duration of drug effect, and is favorable for developing long-acting preparations.

Description

17-benzimidazolyl-10 alpha-methyl-steroid derivatives, preparation method, application and pharmaceutical composition thereof

Technical Field

The application relates to the technical field of medicines, in particular to a 17-benzimidazolyl-10 alpha-methyl-steroid derivative, a preparation method, application and a pharmaceutical composition thereof.

Background

Abiraterone acetate (Abiraterone acetate), known as 17- (3-pyridyl) -androsta-5, 16-dien-3 beta-ol acetate (structural formula shown below), is a CYP17 inhibitor, which clinically treats metastatic advanced prostate cancer resistant to traditional hormone therapy in combination with prednisone, can reduce the level of prostate specific antigen, helps to reduce tumors, and can prolong the life of advanced prostate patients.

Structurally, it is derived from a stane skeleton structure having four rings as shown below (hereinafter, steroid rings), and the carbon numbers (1 to 17) on each ring are as follows. Androstane refers to a beta-methyl group, which may be designated as 10beta-methyl or 13beta-methyl, attached to each of the C-10 and C-13 positions.

Disclosure of Invention

The invention provides a 17-benzimidazolyl-10 alpha-methyl-steroid compound, which is a novel compound and has an inhibiting effect on prostate cancer, colon cancer, lung cancer and pancreatic cancer.

The present invention provides a 17-benzimidazolyl-10α -methyl-steroid compound having the structure of formula I or a pharmaceutically acceptable salt thereof:

R ₁ selected from =o, -OH, halogen, -OC (O) R ₂ Substituted or unsubstituted imidazolyl, triazolyl or benzimidazolyl, wherein R ₂ Selected from C1-C5 alkyl, C1-C5 haloalkyl, phenyl, halophenyl, imidazolyl, triazolyl or R ₂ ' O-, wherein R ₂ ' is selected from C1-C5 alkyl;

R ₃ selected from halogen, C1-C5 alkyl, C1-C5 haloalkyl, C1-C5 alkoxy, hydroxy or amino;

R ₄ 、R ₅ 、R ₆ 、R ₇ identical or different, each independently selected from-OH, =o, halogen, amino, C1-C5 alkyl, C1-C5 haloalkyl, C1-C5 alkoxy, C2-C5 alkenyl or C2-C5 ester group;

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

represents a single bond or a double bond;

when a certain isIn the case of double bonds, adjacent thereto +.>Is a single bond.

In an embodiment of the invention, R ₁ Selected from =o, -OH, -OAc, CH ₃ OC (O) O-, imidazolyl-, triazolyl-, benzimidazolyl,

In the embodiment of the inventionIn the scheme, the R ₁ When selected from the group consisting of-OH, the-OH is β -OH or α -OH.

In an embodiment of the invention, said R ₃ Selected from C1-C5 alkyl or haloalkyl.

In an embodiment of the invention, k is 0.

In an embodiment of the invention, the i, j, m and n are all 0, preferably the k is 0.

In an embodiment of the invention, said R ₁ To R ₇ Wherein the halogen is selected from F, cl, br or I.

In an embodiment of the invention, the compound is selected from the following structural formulae:

the invention provides a preparation method of a compound, which comprises the steps of taking an intermediate shown in a formula V as a raw material, and connecting a substituted or unsubstituted benzimidazolyl at a C-17 position;

the substituted or unsubstituted benzimidazolyl is correspondingly identical in structure to the benzimidazolyl on the compound;

the R is ₉ And R is R ₄ Corresponding to the same or after reaction;

the R is ₁₀ And R is R ₅ Corresponding to the same or after reaction;

the R is ₁₁ And R is R ₆ Corresponding to the same or after reaction;

the R is ₁₂ And R is R ₇ The corresponding is the same or the corresponding is the same after the reaction.

In an embodiment of the invention, the i, j, m and n are all 0.

In an embodiment of the invention, the intermediate is selected from the following formulae:

in an embodiment of the invention, the intermediate is prepared by a process comprising the steps of: the C-10 methyl group is converted from beta configuration to alpha configuration by photochemistry conversion

R ₈ Selected from-OH or protected hydroxy groups, preferably R ₈ Selected from-OH or OAc;

R ₁₃ selected from =o or protected carbonyl, preferably R ₁₃ Selected from =o or

In an embodiment of the invention, optionally, the photochemical conversion is an ultraviolet photocatalytic reaction, optionally, the ultraviolet photocatalytic reaction opens the steroid ring first in the wavelength range of 260-290nm, and then closes the steroid ring in the wavelength range of 295-340nm, the reaction temperature being-10-50 ℃.

In one embodiment of the invention, the process for the preparation of the intermediate is as follows:

compound 1 is protected with hydroxyl at the 3-position (e.g., using a reagent such as acetic anhydride) and carbonyl at the 17-position (e.g., using a reagent such as ethylene glycol) to afford compound 3.

The compound 3 is oxidized to carbonyl group at allylic position (7 position) to obtain compound 4, and air oxidation is carried out by using a catalyst N-hydroxyphthalimide and an initiator benzoyl peroxide.

And (3) hydrazone formation and 5,7 double bond formation of the compound 4 are carried out through 7-carbonyl hydrazone removal to obtain a compound 6.

The compound 6 undergoes ultraviolet photocatalytic reaction, and the 10-position methyl is converted into alpha configuration from beta configuration to obtain the compound 7. The photocatalytic reaction opens the steroid ring in the wavelength range of 260-290nm and then closes the ring in the wavelength range of 295-340 nm. The reaction temperature is controlled between-10 ℃ and 50 ℃.

In an embodiment of the present invention, the 17-benzimidazolyl-10α -methyl-steroid compound is prepared by the following method.

Compound 7 is hydrolyzed to compound IN1F under base catalysis, alternatively, the reaction solvent may be ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone, or the like. The alkali can be sodium hydroxide, potassium carbonate and the like, and the reaction temperature is 0-60 ℃.

The compound IN1F is hydrogenated to synthesize the compound CK004-1A under the catalysis of transition metal, and optionally, the reaction solvent is one or a mixture of more of ethyl acetate, ethanol, dioxane, tetrahydrofuran and dichloromethane. The catalyst is 5% palladium carbon, 10% palladium carbon, metal platinum, platinum dioxide, platinum acetate, etc. The hydrogen pressure is 0.05-2.0MPa, and the reaction temperature is 40-70 ℃.

The compound CK004-1A is hydrolyzed into the compound CK004-1B under the catalysis of acid, and optionally, the reaction solvent is ethyl acetate, tetrahydrofuran, dichloromethane, acetonitrile, acetone and the like. The acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid, etc., and the reaction temperature is 0-40 ℃.

The compound CK004-1B is subjected to 17-position hydrazone formation, iodination and coupling reaction to obtain TM10.

Compound TM10 is reacted with carbodiimidazole in acetonitrile to synthesize compound TM32, and more preferably the solvent may be tetrahydrofuran, toluene, methylene chloride, etc.

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

The compound IN1F is hydrogenated under the catalysis of transition metal to prepare the compound CK004-1E, and optionally, the catalyst is 5% palladium carbon, 10% palladium carbon, metal platinum, platinum dioxide, platinum acetate and the like, and the reaction temperature is 40-70 ℃.

The compound CK004-1E is hydrolyzed into the compound CK004-1F under the catalysis of acid, and optionally, the acid is p-toluenesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, acetic acid and the like, and the reaction temperature is 0-40 ℃.

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

Compound TM16 is reacted with carbodiimidazole in acetonitrile to synthesize compound TM31, and more preferably the solvent may be tetrahydrofuran, toluene, methylene chloride, etc.

The compound TM16 and methyl chloroformate are subjected to esterification reaction under the action of alkali to obtain TM34.

The invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of cancer.

In embodiments of the invention, the cancer comprises prostate cancer, colon cancer, lung cancer or pancreatic cancer.

The invention provides a pharmaceutical composition, which comprises 17-benzimidazolyl-10 alpha-methyl-steroid compounds and pharmaceutically acceptable auxiliary materials.

The dosage form of the pharmaceutical composition can be common dosage forms such as oral preparations, injection preparations and the like, and can be solid preparations such as tablets, capsules, granules and the like, liquid preparations such as solutions, suspensions, emulsions and the like.

Pharmaceutically acceptable common auxiliary materials can be selected, and the preparation method is adopted according to the conventional dosage and the conventional preparation method. For example, tablet excipients include fillers (diluents), binders, disintegrants, lubricants, glidants, and the like. The filler is selected from lactose, microcrystalline cellulose, mannitol, pregelatinized starch, etc. The binder is selected from hydroxypropyl methylcellulose, polyvinylpyrrolidone, methylcellulose, povidone, starch, etc. The disintegrating agent is selected from croscarmellose sodium, crospovidone, sodium carboxymethyl starch, corn starch, etc. The lubricant is selected from magnesium stearate, stearic acid, sodium stearyl fumarate, etc. The glidant is selected from talcum powder, micro powder silica gel, etc. The preparation of the tablet can adopt a wet or dry granulating and tabletting method or a direct powder tabletting method or a blank granule tabletting method.

In contrast to the prior art, the present invention achieves at least the following advantageous technical effects:

the C-10 methyl of the compound is alpha configuration, can act on an Ano1 (Anoctamin 1) target spot, and has strong inhibition effect on prostate cancer, colon cancer, lung cancer and pancreatic cancer cells.

The compounds of the invention have an IC50 for human prostate cancer cells in the range < 150. Mu.M, preferably < 50. Mu.M, more preferably < 20. Mu.M. The compounds of the invention have an IC50 for human colon carcinoma cells in the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 20. Mu.M, more preferably in the range < 10. Mu.M. The compounds of the invention preferably have an IC50 in the range < 150. Mu.M, more preferably in the range < 50. Mu.M, and even more preferably in the range < 25. Mu.M against human non-small cell lung cancer cells. The compounds of the invention preferably have an IC50 for human pancreatic cancer cells in the range < 100. Mu.M, more preferably in the range < 50. Mu.M, more preferably in the range < 20. Mu.M, more preferably in the range < 10. Mu.M.

The C-10 methyl of the compound is in an alpha configuration, compared with a beta configuration, the compound has reduced or no reactivity with enzymes (such as 3 alpha steroid dehydrogenase/3 alpha hydroxyl steroid oxidoreductase, cholesterol oxidase and the like), has better molecular stability, is not easy to degrade under the action of the enzymes, has better tolerance to enzymes in vivo, is expected to be metabolized in vivo slowly, can be discharged out of the body for a prolonged period of time, has longer duration of drug effect, and is favorable for developing long-acting preparations.

In addition, the compound of the invention has high bioavailability and less other side effects.

Drawings

FIG. 1 is a graph showing the results of a docking simulation of a TM16-3 alpha steroid dehydrogenase;

FIG. 2 is a graph showing the results of a docking simulation of the TM16' -3. Alpha. Steroid dehydrogenase;

FIG. 3 is a graph of docking simulation results for abiraterone-3 alpha steroid dehydrogenase;

FIG. 4 is a graph of docking simulation results for the TM16-Ano1 target;

FIG. 5 is a graph of the results of a docking simulation of the TM31-Ano1 target;

FIG. 6 is a graph of docking simulation results for the TM32-Ano1 target;

FIG. 7 is a graph of the results of a docking simulation of the TM34-Ano1 target.

Detailed Description

The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.

Unless defined otherwise, 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 invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.

The present invention will be described in further detail with reference to examples.

Specific compounds (compounds in the examples, including intermediates) referred to in the present invention having an alpha methyl group at the 10-position have the following default configuration

If a group attached to the steroid ring or H on a compound is different from the above configuration, it is designated separately, e.g., 14 beta if the group at position 14 is in beta configuration.

For compounds of the general formula (including at least two compounds), other groups or H attached to the steroid ring may be in the alpha or beta configuration, in addition to the configuration of the groups already indicated (e.g. two angular methyl groups, i.e. methyl groups on C10, C13).

Example 1

In a 2000mL three-necked flask, 200g of DHEA (3β -hydroxy-5-androsten-17-one) (i.e., compound 1), 800mL of DCM (dichloromethane), 140g of triethylamine, 3 times of nitrogen substitution, 4g of DMAP (4-dimethylaminopyridine), and 3 times of nitrogen substitution were added. After stirring at room temperature to dissolve it sufficiently, 140g of acetic anhydride was added dropwise over 2 hours. After the completion of the dropwise addition, stirring was continued for 15min. TLC monitored complete reaction of starting material, 40mL of methanol was added and stirring was continued for 30min. Washed 1 time with 200mL of 5% hydrochloric acid and 1 time with 200mL of 5% sodium bicarbonate solution. The organic phase was distilled off under reduced pressure at 35℃in a water bath, and DCM was replaced by methanol. The mixture was filtered, and the filter cake was washed 1 time with a small amount of methanol and dried in a forced air drying oven at 50℃for 12 hours to give 200g of a white solid (i.e., compound 2) in a mass yield of 100%. Herein, "mass yield" refers to the ratio of the mass of the resulting product to the mass of the starting material. Taking the above examples as an example, the mass yield of 100% means: the ratio of the obtained white solid (i.e., compound 2) to the mass of the added raw material (i.e., compound 1) was 100%.

Into a 2000mL three-necked flask, 200g of DHEA acetate (i.e., the compound 2 obtained above), 1200mL of ethylene glycol, 4g of PTS (p-toluenesulfonic acid), and 260g of triethyl orthoformate were placed. Stirring was carried out at 50℃and TLC monitored for complete reaction of starting materials. Cooled to room temperature, added with triethylamine 8mL and stirred for 30min. Pouring into 1600mL of water, and stirring for 30min. The mixture was filtered and the filter cake was washed 2 times with a small amount of water. The filter cake was dissolved in 800mL of dichloromethane, and 4mL of triethylamine was added thereto and stirred for 15min. The aqueous layer was separated and the organic phase was distilled off under reduced pressure in a water bath at 35℃and the dichloromethane was replaced by methanol. Filtering, washing the filter cake with a small amount of methanol for 1 time, and drying in a blast drying oven at 50 ℃ for 12 hours to obtain 210g of white solid 3 with a mass yield of 105%.

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Into a 2000mL three-necked flask, 100g of Compound 3 and 800mL of cyclohexanone were charged, and after stirring at 50℃until they were sufficiently dissolved, 32g of NOP (N-hydroxyphthalimide) and 0.50g of benzoyl peroxide were added. TLC monitored complete reaction of starting material. Cooled to room temperature, dried by rotary evaporation under reduced pressure at 55 ℃ in water bath. 200mL of methylene chloride and 500mL of petroleum ether are added, and the mixture is stirred at room temperature for 30min. Filtering, washing the filter cake with a small amount of petroleum ether for 1 time. 60g of triethylamine is added into the filtrate, 60g of acetic anhydride is added into the ice water bath dropwise, and stirring is continued for 30min. Standing for 12h. Steaming under reduced pressure at 55deg.C in water bath until dry. After the dichloromethane was sufficiently dissolved, the mixture was distilled under reduced pressure in a water bath at 35℃to replace the dichloromethane with methanol. The mixture was filtered, and the filter cake was washed 1 time with a small amount of methanol and dried in a forced air drying oven at 50℃for 12 hours to give 75g of white solid 4 in 75% mass yield.

In a 2000mL three-necked flask, 100g of Compound 4, 66g of TSH (p-toluenesulfonyl hydrazide), 400mL of toluene, and 600mL of n-hexane were added, and the mixture was heated to reflux for water separation under stirring. TLC monitored complete reaction of starting material. Cooled to room temperature, dried by rotary evaporation under reduced pressure at 55 ℃ in water bath. 660mL of methanol, 130mL of n-hexane and stirring at room temperature for 30min were added. The mixture was filtered, and the filter cake was washed 1 time with a small amount of methanol and dried in a forced air drying oven at 50℃for 12 hours to give 126g of a white solid 5 in a mass yield of 126%.

Into a 1000mL three-necked flask, 5.6g of lithium amide and 175mL of chlorobenzene were charged, and ammonia gas was removed under reduced pressure. After the ammonia gas was removed, a chlorobenzene solution of compound 5 (35 g of compound 5, chlorobenzene 350 mL) was added, and the flask was transferred to an oil bath at 120℃and reacted under stirring for 1 hour. TLC monitored complete reaction of starting material. Cooling to room temperature, and regulating pH to 6-8 with 5% phosphoric acid under stirring in ice water bath. The mixture was separated, the aqueous layer was extracted 1 time with chlorobenzene 40mL, the organic layers were combined, and washed 1 time with water. The organic layer was dried over anhydrous sodium sulfate for 2h. Filtering, and steaming the filtrate under reduced pressure at 55deg.C in water bath until it is dry. After the dichloromethane was sufficiently dissolved, the mixture was distilled under reduced pressure in a water bath at 35℃to replace the dichloromethane with methanol. The mixture was filtered, and the filter cake was washed 1 time with a small amount of methanol and dried in a forced air drying oven at 50℃for 12 hours to give 19g of an off-white solid 6 in a mass yield of 54.3%.

50g of compound 6,0.5g of BHT (antioxidant) is weighed, 1.5L of ethyl acetate is added for dissolution, the mixture is poured into an photochemical reactor, an internal cooling system is started, an LED ultraviolet lamp (100W) with the wavelength of 260-270nm is used for illumination for 3 hours, then an LED ultraviolet lamp (100W) with the wavelength of 310-330nm is used for illumination for 3 hours, sampling HPLC is used for monitoring the reaction, after the reaction is completed, the reaction solution is concentrated to be oily, 150ml of methanol is added, compound 6 is stirred and separated, suction filtration is carried out, compound 6 (20 g) is recovered, after mother solution is concentrated to be dry, silica gel is stirred, and a chromatographic column is used for obtaining 10.5g of compound 7.

The 1H NMR of compound 7 was detected as: 1H NMR (400 MHz, CDCl 3) delta 5.57 (dd, J=5.5, 2.1Hz, 1H), 5.41 (dt, J=5.3, 2.5Hz, 1H), 4.71 (tt, J=11.4, 4.5Hz, 1H), 4.03-3.80 (m, 4H), 2.51 (ddd, J=14.2, 4.8,2.2Hz, 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).

HRMS mass spectrum (EI) m/z: theoretical calculation 373.5: test value: 372.9.

example 2

The compound 7 prepared in example 1 was used to synthesize 17-benzimidazolyl-10α -methyl-steroid compounds, herein designated as TM10, TM12 and TM32.

In a 250mL three-necked flask, 5g of Compound 7, 30g of acetone and 1.26g of sodium hydroxide were added, and the mixture was stirred at room temperature for 1 hour, followed by TLC monitoring the completion of the reaction of the starting materials. Acetone is removed by rotary evaporation under reduced pressure, dichloromethane is added for extraction, and the mixture is concentrated to dryness to obtain light yellow oily matter IN1F 4.3g, and the mass yield is 86.0%.

10g of IN1F,150ml of absolute ethyl alcohol, 2g of 5% palladium carbon, nitrogen substitution three times and hydrogen substitution three times are added into a 250ml hydrogenation kettle, the temperature is raised to 55-60 ℃, the hydrogen pressure is 0.15-0.20MPa, the reaction is carried out for 4 hours, the center control reaction is completed, palladium carbon is removed by filtration, the concentration is carried out, and column chromatography (eluent petroleum ether: ethyl acetate=10:1-petroleum ether: ethyl acetate 5:1) is carried out to obtain 6.3g of solid (namely, compound CK 004-1A) with the mass yield of 63.0%.

1.5g of CK004-1A, 22ml of acetone, 7.5ml of water and 0.3g of p-toluenesulfonic acid are added into a 50ml reaction bottle, after the reaction is finished, no fraction is concentrated, 10ml of dichloromethane is added, the mixture is separated, 5ml of 5% sodium bicarbonate is washed once, the mixture is separated, and the organic layer is concentrated and dried to obtain CK004-1B1.2g, and the mass yield is 80%.

1.2g of CK004-1B,0.86g of hydrazine hydrate (85%), 5g of absolute ethyl alcohol and 0.006g of hydrazine sulfate are added into a reaction bottle, the temperature is raised to 30-35 ℃ for reaction overnight, the reaction liquid is slowly poured into 50ml of water after the reaction is completed, the mixture is stirred for 1 hour, filtered, rinsed by water and dried at 50 ℃ to obtain 1.15g of white solid (namely, compound CK 004-1C), and the mass yield is 95.8%.

1.05g of CK004-1C was added to the reaction flask, and 12ml of tetrahydrofuran was added thereto and the mixture was dissolved by stirring. 10ml of tetrahydrofuran was added to the other flask, cooled to-5 to 5℃and 1.72g of iodine was added thereto and stirred for half an hour. Tetramethyl guanidine is added dropwise at-5 to 5 ℃ and stirred for half an hour after the addition. Adding dropwise a CK004-1C tetrahydrofuran solution at the temperature of-5 to 5 ℃, reacting for 1 hour at the temperature of-5 to 5 ℃, adding 1.2g of sodium thiosulfate after the reaction is finished, heating to 40 ℃, stirring for half an hour, filtering, concentrating the filtrate until no fraction exists, adding 10ml of tetrahydrofuran, adding 5ml of 1M hydrochloric acid for washing, then washing with 5ml of 5% sodium bicarbonate solution, then washing with 5ml of saturated sodium thiosulfate solution for layering, concentrating an organic layer to obtain 1.21g of compound CK004-1D, and obtaining the mass yield of 115.2%.

720mgCK004-1D, dimethyl sulfoxide 15ml, cuprous iodide 50mg, benzimidazole 300mg, 8-hydroxyquinoline 80mg, potassium carbonate 600mg are added into a reaction bottle to react for 16 hours at 180 ℃, after the reaction is completed, the temperature is reduced, 50ml of dichloromethane is added, the reaction is washed 5 times with water, 20ml of dichloromethane are added each time, the organic phase is concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate=10:1-petroleum ether: ethyl acetate 2:1) is carried out to obtain 420mg of solid (namely, compound TM 10) with the yield of 58.3 percent.

The 1H NMR of compound TM10 was detected as: 1H NMR (400 MHz, CDCl 3) delta 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).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 390.56: test value: 390.9.

80mg of TM10,3ml of dichloromethane, 200mg of triethylamine, 0.5mg of 4-dimethylaminopyridine and 200mg of acetic anhydride were added to a reaction flask at 10-20℃and 0.5ml of methanol were added after the completion of the reaction, and the mixture was concentrated to dryness to give 75mg of a white solid (TM 12) in a mass yield of 93.8% by column chromatography (petroleum ether: ethyl acetate=5:1).

The 1H NMR of Compound TM12 was found to be: 1H NMR (400 MHz, CDCl 3) delta 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).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 432.5: test value: 432.9.

100mg of TM10, 62mg of carbodiimidazole and 8ml of acetonitrile are added into a reaction bottle, the temperature is raised to 60 ℃ for reaction for 5 hours, after the reaction is finished, the reaction is concentrated to dryness, 5ml of dichloromethane is added for dissolution, 2ml of water is used for washing once, the layers are separated, the organic layer is concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate=5:1-petroleum ether: ethyl acetate 1:1) is carried out to obtain 83mg of solid (TM 32) with the yield of 83%.

The 1H NMR of Compound TM32 was found to be: 1H NMR (400 MHz, CDCl 3) delta 8.16 (s, 1H), 7.98 (s, 1H), 7.86-7.78 (m, 1H), 7.50 (dd, J=7.2, 1.8Hz, 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).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 484.6: test value: 484.9.

example 3

3.0g of IN1F,0.6g of 5% Pd/C,15mL of dioxane, water bath at 60 ℃, nitrogen substitution, hydrogen balloon pressurization, reaction completion, filtration, concentration to dryness, column chromatography (petroleum ether: ethyl acetate=15:1) to obtain 1.2g of white solid CK004-1E with a mass yield of 40.0%.

2g of CK004-1E,15ml of acetone, 0.8g of p-toluenesulfonic acid and 5ml of water are added into a reaction flask for reaction at room temperature, after the reaction is finished, the acetone is concentrated and removed, 10ml of dichloromethane is added, 5ml of 5% sodium bicarbonate solution is added for washing once, 5ml of water is used for washing once, layering is carried out, and 1.7g of CK004-1F is obtained after the organic layer is concentrated to dryness, and the mass yield is 85.0%.

850mg of CK004-1F, 3.5ml of absolute ethyl alcohol, 612mg of hydrazine hydrate, 4mg of hydrazine sulfate and 30-35 ℃ are added into a reaction bottle for reaction. After the reaction, the reaction solution was slowly poured into 50ml ice water, stirred for 1 hour, filtered, washed with water and dried at 50℃to obtain 820mg of white solid CK004-1G with a mass yield of 96.47%.

1.55G of CK004-1G was added to the reaction flask, 15ml of tetrahydrofuran was added thereto, and the mixture was dissolved at 40 ℃. 15ml of tetrahydrofuran was added to the other flask, the temperature was lowered to-5 to 5℃and 2.66g of iodine was added thereto, followed by dropwise addition of 3g of tetramethylguanidine. Adding dropwise CK004-1G tetrahydrofuran solution at the temperature of-5 to 5 ℃, reacting for 1 hour at the temperature of-5 to 5 ℃ after the dropwise addition is finished, adding 1.86G sodium thiosulfate after the reaction is finished, heating to 40 ℃, stirring for half an hour, filtering, concentrating the filtrate until no fraction is generated, adding 15ml tetrahydrofuran, adding 8ml 1M hydrochloric acid for washing, washing with 8ml 5% sodium bicarbonate solution, washing with 8ml saturated sodium thiosulfate solution for layering, concentrating the organic layer to obtain 1.8G CK004-1H, and obtaining the mass yield of 116.1%.

800mg of CK004-1H, 15ml of dimethyl sulfoxide, 80mg of cuprous iodide, 300mg of benzimidazole, 80mg of 8-hydroxyquinoline and 600mg of potassium carbonate are added into a reaction bottle to react for 16 hours at the temperature of 180 ℃, after the reaction is finished, the temperature is reduced, 50ml of dichloromethane is added, the reaction is washed 5 times with water, 20ml of dichloromethane are added each time, the organic phase is concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate=10:1-petroleum ether: ethyl acetate 2:1) is carried out to obtain 320mg of solid (namely, compound TM 16) with the yield of 40%.

The 1H NMR of Compound TM16 was found to be: 1H NMR (400 MHz, CDCl 3) delta 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, 6.4Hz, 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, 1.7Hz, 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).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 388.25: test value: 388.9.

100mg of TM16, 62mg of carbodiimidazole and 8ml of acetonitrile are added into a reaction bottle, the temperature is raised to 60 ℃ for reaction for 5 hours, after the reaction is finished, the reaction is concentrated to dryness, 5ml of dichloromethane is added for dissolution, 2ml of water is used for washing once, the layers are separated, the organic layer is concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate=5:1-petroleum ether: ethyl acetate 1:1) is carried out to obtain 63mg of solid (namely, compound TM 31) with the yield of 63%.

The 1H NMR of compound TM31 was detected as: 1H NMR (400 MHz, CDCl 3) delta 8.16 (s, 1H), 8.07 (s, 1H), 7.87-7.78 (m, 1H), 7.57 (dd, J=6.0, 3.2Hz, 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, 25.5,6.8Hz, 2H), 2.18-1.37 (m, 15H), 1.25 (s, 3H), 0.78 (s, 3H).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 482.6: test value: 482.9.

100mg of TM16,2ml of pyridine, and 40mg of methyl chloroformate were added to the reaction flask and reacted for 16 hours. After the reaction, 0.5ml of methanol was added to quench, and concentrated to dryness, and column chromatography (eluent petroleum ether: ethyl acetate=20:1 to petroleum ether: ethyl acetate 6:1) gave 56mg of solid (i.e., compound TM 34) in 56% yield.

1H NMR (400 MHz, CDCl 3) delta 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, 6.5,3.3Hz, 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) of compound TM34 was detected.

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 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.

HRMS mass spectrum (EI) m/z: theoretical calculation 446.6: test value: 446.9.

in addition to the compounds prepared in the above examples, the present invention can also prepare other examples by the same/similar preparation process as the previous examples, and all the examples are now listed as follows:

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Example 4

1. Pharmacological experiments: inhibition of cancer cells

1. Test method

1.1 Experimental grouping and sample preparation

The compound samples of each example were prepared as 100mM stock solutions using the vehicle dimethyl sulfoxide (DMSO), and diluted to working solutions at concentrations of 100, 30, 10, 3, 1, 0.3. Mu.M with the corresponding complete medium for each cell culture. A vehicle control group, a positive control group with different concentrations and a sample treatment group with different concentrations are arranged.

1.2 cell culture

Human prostate cancer cells (DU 145) medium was MEM medium containing 10% fbs (fetal bovine serum); human colon cancer cell (HCT-116) medium is McCoy's 5A with 10% FBS; human non-small cell lung cancer cells (A549) are Ham's F-12K medium containing 10% FBS, and the culture conditions are 37deg.C and 5% CO ₂ . Human pancreatic cancer cells (PANC-1) were in DMEM medium containing 10% FBS. When the growth state is good, the culture medium is passaged every 2 days, and the passaging ratio is 1:3. the medium was discarded in a clean bench, washed 2 times with 1 XPBS, then added with 600. Mu.L of 0.25% trypsin for digestion, after about 1-3 min, after cell shedding, 3mL of medium corresponding to each cell containing 10% FBS was added to terminate the digestion of pancreatin, blown into single cell suspensions, transferred into EP tubes, and centrifuged at 1000rpm for 5min. Discarding the culture The medium was resuspended in fresh medium at a ratio (cell density of about 10 ⁵ /mL) was inoculated into a new flask, and placed at 37℃with 5% CO ₂ Culturing in an incubator.

1.3 cell seeding

Taking cells with good growth state, conventionally digesting and collecting the cells, and regulating the cell density of DU 145 to 2×10 ⁴ Modulation of HCT-116 cells to 2X 10 cells per mL ⁴ Modulating A549 cells to 3X 10 per mL ⁴ PANC-1 cell density was adjusted to 4X 10 per mL ⁴ Each cell suspension was inoculated into 96-well plates at a density of 100. Mu.L/well, each cell suspension was shaken in a crisscross manner 10 times to spread the cells uniformly at the bottom of the wells, and the plates were placed in CO ₂ Culturing in an incubator for 24 hours.

1.4 cell treatment

The working solution of the compound sample of the example prepared in the step 1.1 is taken, and the concentrations of the working solution are respectively added into the corresponding wells at 100 mu L/well, so that the final volume of each well is 200 mu L (100 mu L of cell culture medium, 100 mu L of sample working solution), the final concentrations are respectively 50, 15, 5, 1.5, 0.5 and 0.15 mu M, meanwhile, a solvent control group is arranged, and the concentrations of the positive control groups are respectively 50, 15, 5, 1.5, 0.5 and 0.15 mu M, and the number of each compound well is 3. At 37℃with 5% CO ₂ Culturing for 72h under the condition.

1.5 detection of OD value of cell proliferation

After 72h of cell treatment, 20. Mu.L of thiazole blue (MTT) was added to each well at 37℃with 5% CO ₂ Culturing was continued for 4 hours under the condition, the liquid in each well was carefully aspirated, 150. Mu.L/well DMSO was added to the well, and shaking was performed for 10 minutes.

The average value of OD values of the A1-H1 wells (8 wells) is set to be zero on an enzyme-labeled instrument, and the OD values of all the wells are detected at 492 nm.

1.6 calculation of results

The OD value of the solvent control group is set as 100% of the cell activity, and the ratio of the OD value of each of the other groups to the OD value of the solvent control group is the relative cell activity. The activity of the samples on DU145, HCT-116, A549 or PANC-1 cells was evaluated by the cell proliferation rate, and if the proliferation inhibition rate was > 100%, the samples were judged to be systematic errors, based on 100%.

The inhibition rate calculation formula is: inhibition (%) = (1-OD _{Sample of} /OD _Solvent(s) )×100％

Calculation of half-maximal Inhibition (IC) using SPSS software ₅₀ )。

2. Experimental results

Table 1 part of the IC50 values of the compounds of examples and comparative examples for each cancer cell

Note that: DU145 is a human prostate cancer cell, HCT-116 is a human colon cancer cell, A549 is a human non-small cell lung cancer cell, and PANC-1 is a human pancreatic cancer cell.

The C-10 methyl of the compound is alpha configuration, and has inhibition effect on prostate cancer, colon cancer, lung cancer and pancreatic cancer cells. The compounds of the invention have an IC50 for human prostate cancer cells in the range < 150. Mu.M, preferably < 50. Mu.M, more preferably < 20. Mu.M. The compounds of the invention have an IC50 for human colon carcinoma cells in the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 20. Mu.M, more preferably in the range < 10. Mu.M. Each compound of the invention is in the range < 150 μm, more preferably in the range < 50 μm, more preferably in the range < 25 μm, for human non-small cell lung cancer cell IC50, except for individual >150 μm. The compounds of the various embodiments of the present invention preferably have an IC50 for human pancreatic cancer cells in the range of < 100. Mu.M, more preferably in the range of < 50. Mu.M, more preferably in the range of < 20. Mu.M, more preferably in the range of < 10. Mu.M.

In the application, the IC50 is considered to be in the range of 50-150 mu M, which indicates that the compound has a certain degree of inhibition on cancer cells, and the IC50 is less than 50 mu M, which indicates that the compound has a good inhibition on cancer cells.

Furthermore, the applicant has pointed out that abiraterone acetate (or abiraterone) was clinically used in combination with prednisone in the prior art for the treatment of prostate cancer, however, the present application unexpectedly found and experimentally demonstrated (as shown in the results of table 1), that abiraterone acetate not only has an anti-prostate cancer effect, but also exhibits an unexpected inhibitory effect against colon cancer cells or lung cancer cells, in particular, abiraterone acetate has an IC50 in the range < 50 μm against human colon cancer cells and abiraterone acetate has an IC50 in the range < 10 μm against human colon cancer cells; abiraterone acetate has an IC50 in the range < 50. Mu.M against human non-small cell lung cancer cells.

Further, as can be seen from the results of Table 1, the compound TM10 of the present application has an IC50 in the range of < 20. Mu.M against human prostate cancer cells and an IC50 in the range of < 10. Mu.M against human colon cancer cells, showing good inhibitory effect against both cancers; IC50 for human non-small cell lung cancer cells is in the range of < 25 mu M, and IC50 for human pancreatic cancer cells is in the range of < 20 mu M, which shows that the compound TM10 of the application has better inhibition effect than abiraterone acetate for the two cancers (namely, human non-small cell lung cancer cells and human pancreatic cancer cells). This is a full indication that the compound TM10 of the present application showed good inhibition against human prostate cancer cells, human colon cancer cells, human non-small cell lung cancer cells and human pancreatic cancer cells. The compound TM12 provided by the application has IC50 in the range of < 15 mu M for human prostate cancer cells, IC50 in the range of < 10 mu M for human colon cancer cells and IC50 in the range of < 50 mu M for human pancreatic cancer cells. This fully shows that the compound TM10 of the application has good inhibition effect on human prostate cancer cells, human colon cancer cells and human pancreatic cancer cells, and has certain inhibition effect on human non-small cell lung cancer cells. The compound TM16 provided by the application has good inhibition effect on human prostatic cancer cells, the IC50 of the compound is less than 20 mu M, and the IC50 of the compound is less than 10 mu M; IC50 for human non-small cell lung cancer cells is in the range of < 20 mu M, and IC50 for human pancreatic cancer cells is in the range of < 10 mu M, which shows that the compound TM16 of the application has better inhibition effect on the two cancers (namely, human non-small cell lung cancer cells and human pancreatic cancer cells) than abiraterone acetate. This is a full indication that the compound TM16 of the present application showed good inhibition against human prostate cancer cells, human colon cancer cells, human non-small cell lung cancer cells and human pancreatic cancer cells.

2. Docking of compounds to enzyme molecules

Docking software MOE (Molecular Operating Environment, integrated software system for pharmaceutical and life sciences developed by canadian chemical computing group Chemical Computing Group ULC) was used to score the following compounds in a docking manner, with the specific results shown in table 2.

Among them, the results of the docking simulation of TM16-3 alpha steroid dehydrogenase (HSD) are shown in FIG. 1;

the results of the docking simulation for the compound 16'-3α steroid dehydrogenase are shown in FIG. 2, wherein compound 16' has the following structural formula, which differs from TM16 in that the methyl group at the C10 position is in the non-inverted β configuration;

the results of the docking simulation of abiraterone-3 alpha steroid dehydrogenase are shown in FIG. 3, wherein abiraterone has the following structural formula

Table 2: butt scoring values for each compound and 3 alpha steroid dehydrogenase

Compounds of formula (I)	Butt joint scoring value
		TM16-3 alpha steroid dehydrogenase	-4.48
Compound 16' -3 alpha steroid dehydrogenase	-4.72
		Abiraterone-3 alpha steroid dehydrogenase	-6.98

Scoring by the MOE software is the result of a calculation based on parameters of electrostatic parameters, hydrogen bonding, molecular attraction, molecular orbitals, etc. between molecules and enzymes. The more negative the score, the lower the free energy, the more stable the conformation and the better the binding.

The above results indicate that: the compound TM16 disclosed by the invention has no non-inverted compound in butt joint with the enzyme after the C10 methyl is inverted (alpha configuration), so that the inverted compound is insensitive to the enzyme or has lower activity on the inverted compound, the compound is better in-vivo enzyme tolerance, the metabolism in the body is expected to be slower, the in-vitro discharge time is prolonged, the acting time is prolonged, the duration of the drug effect is longer, and the long-acting preparation is favorable for development. In addition, even though the docking of the compound TM16 of the present invention with the above enzyme is not as good as that of abiraterone, indicating that the compound TM16 turned over by the methyl group at the C10 position becomes insensitive to the above enzyme or that the enzyme has lower activity against the turned-over compound, the compound is expected to have better tolerance to in vivo enzymes, to be metabolized in vivo more slowly, to be excreted in vitro for longer duration of action, and to have longer duration of efficacy, which is advantageous for the development of a long-acting preparation.

3. Docking of Compounds with Ano1 target

Further, the invention also uses the docking software MOE to simulate and score the docking of the following compounds TM16, TM31, TM32 and TM34 with Ano1 targets, the scoring results are shown in the following table 3, and the docking simulation results are shown in FIG. 4, FIG. 5, FIG. 6 and FIG. 7.

Table 3: butt scoring values of each compound against Ano1 (Anostamin 1) target

Compounds of formula (I)	Docking scoring value with Ano1 (Anostamin 1) target
		TM16	-5.79
TM31	-6.39
		TM32	-6.40
TM34	-6.59

As can be seen from the combination of table 3 and fig. 4, the compound TM16 of the present invention can well bind to the Ano1 (Anoctamin 1) target, and the docking score thereof is-5.79, so that 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 values. As for the other compounds TM31, TM32 and TM34, the docking ratio with the Ano1 (Anoctamin 1) target was even lower than TM16, and thus binding with the Ano1 (Anoctamin 1) target was even better, so that good inhibition of colon cancer, lung cancer, pancreatic cancer was also expected.

4. ELISA test for detecting influence of the Compounds of the present invention on Ano1 expression of cancer cells

The effect of the compounds of the invention on the expression of Ano1 by cancer cells was investigated by enzyme-linked immunosorbent assay as follows.

Calcium activated chloride channel protein 1 (Ano 1) is positioned in a 11q13 region of a human chromosome, so that compared with a RWPE-1 normal prostate epithelial cell line of a human, the expression of Ano1 in the prostate cancer cell line of the human is obviously increased, and proliferation, migration and invasion capacity of prostate cancer cells can be obviously inhibited by blocking an Ano1 channel; after blocking the Ano1 channel, cancer cells express more calcium activated chloride channel protein 1 (Ano 1) for survival, thereby up-regulating Ano1 detected. In other words, if the Ano1 channel is blocked, the compensatory expression of the protein will be increased. The compounds of the present invention are capable of blocking the Ano1 channel, which in turn leads to an increase in compensatory expression of the protein, as will be described in detail below.

4.1 test materials

4.1.1 sample

TM16 (working concentration 10. Mu.M, 5. Mu.M), D4A (Abiraterone oxide, structure as follows, working concentration 10. Mu.M, 5. Mu.M) and Abiraterone acetate (working concentration 10. Mu.M, 5. Mu.M) were used as positive control.

4.1.2 cell lines

Human prostate cancer cells (DU 145), epithelial-like adherent cells, numbered CL-0075, were cultured under the following conditions: MEM medium containing 10% Fetal Bovine Serum (FBS), 37℃and 5% CO ₂ The method comprises the steps of carrying out a first treatment on the surface of the Human colon cancer cells (HCT-116), epithelial-like adherent cells, accession number CL-0096, mcCoy's 5A medium containing 10% Fetal Bovine Serum (FBS), 37 ℃, 5% CO ₂ . Both cells were purchased from marsupenario life technologies limited.

4.1.3 major reagents

Human Ano1 ELISA kit, specification 48T, lot number 202201, shanghai enzyme-linked biosciences Co., ltd; BCA kit, 500 times in specification, product of the bio-technology company, bi yun tian.

4.2 test methods

4.2.1 cell treatment

According to DU 145 or HCT-cellsGrowing conditions culture cells according to 1X 10 ⁵ The cells/holes are inoculated in a 6-hole plate, and after the cells are attached, TM16, D4A and abiraterone acetate are respectively added, and the working concentration is 10 mu M and 5 mu M. Placing the 6-well plate with the added sample in CO ₂ The incubators were incubated for 72h, respectively.

4.2.2 protein harvesting and protein concentration determination

Culturing in an incubator for 72h, respectively lysing each group of cells by using a cell lysate, centrifuging at low temperature to obtain supernatant, harvesting cell proteins, and detecting the protein concentration of each sample by using a BCA kit.

BCA kit detection method:

protein standard preparation: 0.8mL of protein standard preparation solution is taken and added into protein standard substance (20 mg BSA, bovine serum albumin) to be fully dissolved to prepare 25mg/mL of protein standard solution, 20 mu L of 25mg/mL of protein standard solution is taken and 980 mu L of diluent is added to prepare 0.5mg/mL of protein standard solution.

(2) Preparing BCA working solution: according to the number of samples, 1 volume BCA reagent B (50:1) was added to 50 volumes of BCA reagent A, and the mixture was repeatedly mixed, and 12.24mL of BCA working fluid (12 mL of reagent A:0.24mL of reagent B) was prepared in this test.

(3) Standard substances are added into standard substance holes of a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 mu L, and standard substance diluent is added to be 20 mu L, which is equivalent to the concentration of the standard substances of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL respectively.

(4) 20. Mu.L of the sample to be tested was taken into the sample wells of a 96-well plate.

(5) 200. Mu.L BCA working solution was added to each well, and the wells were left at 37℃for 30min.

(6) The absorbance at a wavelength of 492nm was measured with a microplate reader.

(7) The protein concentration of the sample was calculated from the standard curve and the sample volume used.

4.2.3Ano1 content results detection

Ano1 expression was determined according to the Ano1 ELISA kit detection instructions, and was used for each protein sample.

ELISA kit detection operation steps:

(1) sample addition of standard substance: standard wells and sample wells were set, and 50 μl of standard of different concentrations was added to each standard well.

(2) Sample adding: zero-setting holes (sample diluent is added, sample and enzyme-labeled reagent are not added, and other steps are the same) and sample holes to be measured are respectively arranged. Sample Kong Xianjia to be tested was diluted 40. Mu.L in the enzyme-labeled coated plate, and 10. Mu.L (5-fold sample dilution) of the sample to be tested was added. Adding the sample to the bottom of the ELISA plate hole, and gently shaking and mixing.

(3) Adding an enzyme-labeled liquid: except for blank wells, 100 μl of enzyme-labeled reagent was added to each well.

(4) Incubation: sealing the plates by using sealing plates, and then placing the plates at 37 ℃ for incubation for 60min.

(5) Washing: uncovering the sealing plate membrane, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 30s, discarding, repeatedly washing for 5 times, and beating to dry.

(6) Color development: 50 mu L of each color developing agent A and 50 mu L of each color developing agent B are added into each hole, mixed by gentle shaking, and incubated at 37 ℃ in dark for 15min.

(7) And (3) terminating: the reaction was stopped by adding 50. Mu.L of stop solution to each well (at which time the blue color immediately turned yellow).

(8) And (3) detection: the absorbance (OD) of each well was measured at a wavelength of Kong Diaoling, 450nm, which was zeroed within 15min after the addition of the stop solution.

(9) The Ano1 concentration of the sample was calculated from the standard curve and the sample volume used.

4.3 test results

4.3.1 results of protein concentration determination

After the OD value measured according to the BCA kit, the protein concentration of each sample was calculated in mg according to a standard curve. The standard curve equation is: y=0.2611x+0.1308, r ² ＝0.9964。

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

Standard substance 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

Table 5: results of total protein concentration of cell lysates

4.3.2Ano1 content determination results

After the detection is finished, calculating the average value of the multiple wells, zeroing the average value of the detection result by using the average value of the zeroing wells, drawing a standard curve graph to obtain a standard curve equation, calculating the Ano1 result of each sample according to the standard curve equation, calculating the relative Ano1 result according to the total protein concentration, and expressing the Ano1 protein expression condition by using the Ano1 protein concentration (ng) contained in each mg of total protein.

The standard curve equation is: y=0.284 x+0.1113, r ² ＝0.9863。

Table 6: ano1 protein standard detection result

Standard substance concentration (mg/mL)	0	0.625	1.25	2.5	5	10	Zero setting hole
								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

Table 7: results of Ano1 expression of each sample of DU145 cells (n=2)

Table 8: results of Ano1 expression in each sample of HCT-116 cells (n=2)

From the above, the expression of the vehicle control group Ano1 was 0.9621 at 72h for DU145 cells. Compared with the blank control group, samples TM16, D4A and abiraterone acetate have increased Ano1 expression levels of DU145 cells under the treatment of various concentrations, and the expression results show the conditions of low-concentration expression and high-concentration expression. This demonstrates that the compounds of the present invention are capable of increasing the compensatory expression of Ano1, since the compounds of the present invention block the Ano1 channel, and in the case of blocking the Ano1 channel, the compounds of the present invention inhibit proliferation, migration and invasion of prostate cancer cells, which have good agreement with the results of the aforementioned molecular simulation and docking and the results of the determination of IC50 values. For HCT-116 cells, the compounds of the invention also show the result of blocking the Ano1 channel and thus increasing compensatory expression of this protein channel.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as various modifications, equivalent arrangements, or improvements within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Claims

1. A 17-benzimidazolyl-10α -methyl-steroid compound having the structure of formula I:

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

represents a single bond or a double bond;

2. 17-benzimidazolyl-10α -methyl-steroid according to claim 1, wherein R is ₁ Selected from =o, -OH, -OAc, CH ₃ OC (O) O-, imidazolyl-, triazolyl-, benzimidazolyl,

3. 17-benzimidazolyl-10α -methyl-steroid according to claim 2, wherein R is ₁ When selected from the group consisting of-OH, the-OH is β -OH or α -OH.

4. 17-benzimidazolyl-10α -methyl-steroid according to claim 1, wherein R is ₃ Selected from C1-C5 alkyl or haloalkyl.

5. The 17-benzimidazolyl-10α -methyl-steroid according to claim 4, wherein k is 0.

6. 17-benzimidazolyl-10α -methyl-steroid according to claim 1, characterized in that i, j, m and n are all 0, preferably that k is 0.

7. 17-benzimidazolyl-10α -methyl-steroid according to any one of claims 1 to 6, characterized in that R ₁ To R ₇ Wherein the halogen is selected from F, cl, br or I.

8. 17-benzimidazolyl-10α -methyl-steroid compound according to any one of claims 1 to 6, characterized in that the compound is selected from the following structural formulae:

9. 17-benzimidazolyl-10α -methyl-steroid compound according to claim 8, characterized in that the compound is selected from the following structural formulae:

10. 17-benzimidazolyl-10α -methyl-steroid compound according to claim 8, characterized in that the compound is selected from the following structural formulae: