CN116917306A

CN116917306A - 17-pyridyl-10α-methyl-steroid derivatives and intermediate compounds, their preparation methods, their uses, and their pharmaceutical compositions

Info

Publication number: CN116917306A
Application number: CN202380008382.XA
Authority: CN
Inventors: 刘喜荣; 唐杰; 蒋红平; 何群; 罗桂芳; 张沐; 李龙; 吴四清
Original assignee: Shanghai Chunjian Pharmaceutical Technology Co ltd
Current assignee: Hunan Keyixin Biomedical Co ltd
Priority date: 2022-02-18
Filing date: 2023-02-16
Publication date: 2023-10-20
Also published as: WO2023155846A1

Abstract

The present invention relates to a 17-pyridyl-10α-methyl-steroid derivative and intermediate compound, its preparation method, its use, and its pharmaceutical composition. The methyl group at position C-10 of the steroid derivative of the present invention has an inverted α configuration, and is a brand-new compound that has a strong inhibitory effect on prostate cancer, breast cancer, colon cancer, and lung cancer cells. In order to prepare the above-mentioned steroid derivatives, the present invention also synthesizes a series of corresponding intermediates of the steroid derivatives. The steroid derivative of the present invention can be used as a drug to effectively treat cancer, especially cancer including prostate cancer, breast cancer, colon cancer or lung cancer. The methyl group at position C-10 of the steroid derivative of the present invention has an α configuration. Compared with the β configuration, its reactivity with enzymes is greatly reduced, and it has better tolerance to enzymes in the body. It is expected to be metabolized slowly in the body, and the drug The effect lasts longer, which is beneficial to the development of long-acting preparations.

Description

17-pyridyl-10 alpha-methyl-steroid derivatives and intermediate compounds, preparation method, use thereof, and pharmaceutical compositions thereof

Technical Field

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

Background

Abiraterone acetate (Abiraterone acetate) with chemical name 17- (3-pyridyl) -androstane-5, 16-diene-3 beta-Alcohol acetate (structural formula shown below) is a CYP17 inhibitor, which clinically treats metastatic advanced prostate cancer resistant to traditional hormone treatment by combining with prednisone, can reduce the prostate specific antigen level, is helpful for reducing 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 methyl group attached at each of the C-10 and C-13 positions. For abiraterone acetate, the methyl groups connected at the C-10 and C-13 positions are beta-forms and can be marked as 10 beta-methyl and 13 beta-methyl.

Disclosure of Invention

The invention provides a 17-pyridyl-10 alpha-methyl-steroid derivative and an intermediate compound, a preparation method and application thereof, and a pharmaceutical composition thereof. The invention synthesizes 17-pyridyl-10 alpha-methyl-steroid derivative with C-10 methyl as alpha configuration, which is a new compound with inhibiting effect on prostate cancer, breast cancer, colon cancer and lung cancer.

The present invention provides a 17-pyridinyl-10α -methyl-steroid derivative having the structure of formula I:

R ₁ selected from =o, -OH, halogen or-OC (O) R ₂ ；

R ₂ Selected from C1-C5 alkyl, C1-C5 haloalkyl, phenyl or halophenyl;

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, C1-C5 alkenyl or C1-C5 ester group;

represents a single bond or a double bond;

when a certain isIn the case of double bonds, adjacent theretoIs a single bond;

i. j, k, m and n are each independently selected from 0, 1, 2, 3, 4 or 5.

In an embodiment of the invention, the steroid derivative has the structural formula of formula ii:

in an embodiment of the invention, said R ₁ Selected from = O, -OH or OAc.

In an embodiment of the invention, said 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 steroid derivative is selected from the following structural formulas:

the invention provides a preparation method of a 17-pyridyl-10 alpha-methyl-steroid derivative, which comprises the steps of taking an intermediate shown in a formula V as a raw material, and connecting a substituted or unsubstituted pyridyl on a C-17 position;

the substituted or unsubstituted pyridyl group corresponds to the same pyridyl structure on the 17-pyridyl-10α -methyl-steroid derivative;

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.

The present invention provides an intermediate compound for preparing 17-pyridyl-10 alpha-methyl-steroid derivatives, said intermediate compound being selected from the following structural formulas:

R ₈ selected from-OH or protected hydroxy;

R ₁₃ selected from = O or protected carbonyl.

In an embodiment of the invention, said R ₈ Selected from-OH or OAc; and/or;

the R is ₁₃ Selected from =o or

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

the invention provides a preparation method of an intermediate compound, which comprises the following steps: the C-10 methyl group is converted from beta configuration to alpha configuration by photochemistry conversion

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

in an embodiment of the present invention, the preparation method of the intermediate compound of the 17-pyridyl-10α -methyl-steroid derivative is as follows:

in particular, in embodiments of the present invention,

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 into carbonyl through allylic position (7 position) to obtain a compound 4, and air oxidation is carried out by adopting 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-methyl is converted from beta configuration to alpha configuration to obtain a compound 7 (namely an intermediate compound). 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 ℃.

The compound 7 is subjected to two-step catalytic hydrolysis by acid (such as p-toluenesulfonic acid) and alkali (such as sodium hydroxide and the like) to remove the protecting groups at the 3 position and the 17 position, so as to obtain a compound 8.

In the present invention, a part of the compounds in the 17-pyridyl-10α -methyl-steroid derivatives are prepared by the following method:

the compound 8 is subjected to 17-position hydrazone formation, iodination and coupling reaction to obtain TM1, wherein the coupling reaction reagent is diethyl- (3-pyridine) -borane and bis (triphenylphosphine) palladium dichloride as a catalyst.

The TM1 is acetylated at 3 position under the action of catalyst and alkali to obtain TM2, and the acetylating agent can be acetic anhydride, acetyl chloride, etc.

Hydrogenation of TM2 under transition metal catalysis, reduction of 5,7,16 double bond to obtain TM4, optionally Pd/CaCO ₃ The catalyst has the reaction temperature of 40-70 ℃, is reduced in the hydrogen atmosphere, and has high selectivity and yield.

TM1 is protected by a D-a-like reaction (diels-alder reaction) using PTAD (4-phenyl-1, 2, 4-triazolin-3, 5-dione), hydroxy oxidation at the 3-position, deprotection to give TM8, optionally with oxidation system Tempo (2, 6-tetramethylpiperidine oxide) -NaClO-NaBr. In one embodiment, the deprotection is carried out at a temperature of 50 to 65℃because the target compound is not available at normal temperature.

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 TM5, wherein the coupling reaction reagent is diethyl- (3-pyridine) -borane, and bis (triphenylphosphine) palladium dichloride is adopted for catalysis.

The synthesis scheme of the TM7 from the compound TM5 is that the compound TM5 is subjected to esterification reaction with acetic anhydride, acetyl chloride and the like under the action of a catalyst and alkali to obtain the TM7, and the reaction solvent can be dichloromethane, chloroform, tetrahydrofuran, ethyl acetate and the like. The catalyst is 4-dimethylaminopyridine, the alkali can be triethylamine, pyridine and the like, and the reaction temperature is 0-40 ℃.

The synthesis scheme of the compound TM5 to synthesize the TM6 is that the compound TM5 is oxidized to synthesize the compound TM6 under the action of an oxidant, and the oxidant can be Jones reagent, TEMPO/sodium hypochlorite, european brain oxide 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 TM23.

The compound TM23 is oxidized to synthesize the compound TM22 by an oxidizing agent, which may alternatively be Jones reagent, TEMPO/sodium hypochlorite, european brain oxide, or the like.

The compound TM23 is subjected to esterification reaction with acetic anhydride, acetyl chloride and the like under the action of a catalyst and alkali to obtain TM9, wherein the catalyst is optionally 4-dimethylaminopyridine, and the alkali can be triethylamine, pyridine and the like.

The invention provides an application of 17-pyridyl-10 alpha-methyl-steroid derivatives in preparing medicines for treating cancers.

In an embodiment of the invention, the cancer comprises prostate cancer, breast cancer, colon cancer or lung cancer.

The invention provides a pharmaceutical composition, which comprises 17-pyridyl-10 alpha-methyl-steroid derivatives 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.

The invention provides application of steroid compounds with the following structures in preparation of medicines for treating colon cancer or lung cancer

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

the 17-pyridyl-10 alpha-methyl-steroid derivative is a brand new compound, wherein the C-10 methyl is alpha-configuration, has a strong inhibition effect on prostate cancer, breast cancer, colon cancer and lung cancer cells, can be used as a CYP17 inhibitor, has a strong inhibition effect on prostate cancer and breast cancer, can act on Ano1 (Anoctamin 1) targets, and has a strong inhibition effect on colon cancer and lung cancer cells.

The compounds of the invention are directed to human prostate cancer cell IC ₅₀ In the range < 140. Mu.M, preferably in the range < 80. Mu.M, more preferably in the range < 40. Mu.M, more preferably in the range < 20. Mu.M. The compounds of the invention are directed to human colon cancer cell IC ₅₀ In the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 30. Mu.M, more preferably in the range < 15. Mu.M. The compound of the invention aims at human non-small cell lung cancer cell IC ₅₀ In the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 30. Mu.M, more preferably in the range < 15. Mu.M.

The C-10 methyl of the 17-pyridyl-10 alpha-methyl-steroid derivative is alpha configuration, compared with beta configuration, the C-10 methyl of the 17-pyridyl-10 alpha-methyl-steroid derivative has greatly reduced reactivity with enzyme, better molecular stability, difficult degradation under the action of the enzyme, better tolerance to in vivo enzyme, slower metabolism in vivo, longer duration of drug effect and contribution to development of long-acting preparations.

In addition, the 17-pyridyl-10 alpha-methyl-steroid derivative has high bioavailability and less other side effects.

Drawings

FIG. 1 is a single crystal structure diagram of Compound I;

FIG. 2 is a graph of TLC results of compound D4A in an enzyme tolerance test;

wherein a is the TLC result of reaction monitoring 1 (0.5 hour); b is the TLC result of reaction monitoring 2 (2 hours); c is the TLC result of reaction monitoring 3 (5 hours);

FIG. 3 is a graph of TLC results of Compound TM22 in an enzyme tolerance test;

wherein d is the TLC result of reaction monitoring 1 (0.5 hour); e is the TLC result of reaction monitoring 2 (2 hours); f is the TLC result of reaction monitoring 3 (5 hours);

the left point in TLC is the raw material, the middle point is the mixing point of the raw material and the enzyme reduction reaction solution, and the right point is the enzyme reduction reaction solution.

FIG. 4 is a graph of docking simulation results for abiraterone-3 alpha steroid dehydrogenase;

FIG. 5 is a graph of scoring data for abiraterone-3 alpha steroid dehydrogenase;

FIG. 6 is a graph showing the results of a docking simulation of TM23-3 alpha steroid dehydrogenase;

FIG. 7 is a graph of scoring data for TM23-3 alpha steroid dehydrogenase;

FIG. 8 is a graph showing the results of a simulation of the docking of D4A-cholesterol oxidase;

FIG. 9 is a graph of scoring data for D4A-cholesterol oxidase;

FIG. 10 is a graph showing the results of a simulation of the docking of TM 22-cholesterol oxidase;

FIG. 11 is a graph of scoring data for TM 22-cholesterol oxidase.

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 of a different configuration than that described above, it is designated separately, e.g. 14β.

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

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. Organic phase inThe DCM was replaced with methanol by rotary evaporation under reduced pressure in a water bath at 35 ℃. 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%.

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. Filtering, filtering The 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.

Upon detection, compound 7 ¹ H NMR is: 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.

into a 250mL three-necked flask, 4.2g of Compound 7, 42g of acetone, 0.84g of PTS, and 21g of water were charged. Stirring at room temperature for 12h. TLC monitored complete reaction of starting material, 1.26g of sodium hydroxide was added, and stirring was performed under reflux for 1h, TLC monitored complete reaction of starting material. Acetone was removed by rotary evaporation under reduced pressure at 40℃in a water bath. Extraction with dichloromethane 20ml×3, combining the organic layers, and washing with water 1 time, spin-evaporating to dryness under reduced pressure at 45 ℃ in a water bath, gave compound 8 as a pale yellow oil, 2.9g, mass yield 69.0%.

Example 2

The compound 8 prepared in example 1 was used for the synthesis of 17-pyridinyl-10α -methyl-steroid derivatives, the compound synthesized here being denoted TM1, the synthesis procedure of TM1 being as follows:

in a 250mL three-necked flask, 3.6g of Compound 8, 14.4g of ethanol, 2.60g of hydrazine hydrate and 0.015g of hydrazine sulfate were charged. Stirred at 35℃for 12h. TLC monitors the completion of the reaction of the starting material, adding 115.2g of ice water, stirring for 30min, filtering, washing the filter cake with a small amount of water for 1 time, and drying in a blast drying oven at 50℃for 12 hours to obtain 2.5g of an off-white solid (i.e., compound 9) in a mass yield of 70%.

Into a 250mL three-necked flask, 30g of THF (tetrahydrofuran) was added, and the flask was stirred in an ice-water bath, followed by adding 5.16g of iodine thereto, and stirring was performed for 15 minutes with a constant temperature. 5.88g of tetramethyl guanidine is added dropwise after 10min, and the mixture is stirred for 15min under heat preservation. A THF solution of compound 9 (compound 9 3g, 36g of THF) was added dropwise over 120 min. The ice water bath is kept warm and stirred for 60min. TLC monitored complete reaction of starting material, sodium thiosulfate 4.20g was added and stirred at 50℃for 30min. The mixture was filtered, the filter cake was washed 2 times with a small amount of THF, and the filtrate was distilled to dryness under reduced pressure in a water bath at 50 ℃. 30g of THF was added thereto, and after complete dissolution, 20mL of 5% hydrochloric acid was slowly added thereto, and the mixture was stirred at 50℃for 30 minutes. The mixture was separated, the aqueous layer was extracted with THF 20 mL. Times.2, the organic layers were combined, and saturated sodium bicarbonate solution 20mL was added and stirred at room temperature for 30min. The mixture was separated, and the organic layer was washed 1 time with 20mL of saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate for 2h. The mixture was filtered and the filter cake was washed 2 times with a small amount of THF. The filtrate was distilled to dryness under reduced pressure in a water bath at 50℃to give 3.6g of a brown oil (i.e., compound 10) in a mass yield of 120%.

Into a 100mL three-necked flask, 3g of compound 10, 1.3g of diethyl-3-pyridylborane, 0.015g of diphenylphosphine palladium dichloride, 36g of DMF (N, N-dimethylformamide), 1.8g of potassium carbonate, 4.5g of water and 3 times of nitrogen substitution were added, and the temperature was raised to 80℃and the mixture was kept at the temperature and stirred for 12 hours. TLC monitored complete reaction of starting materials, cooling to room temperature, adding 20mL of water, extracting with 20 mL. Times.3 of dichloromethane, combining organic layers, washing the organic layers with 20 mL. Times.3 of water, and spin-evaporating to dryness under reduced pressure at 50℃in a water bath to give 2.5g of brown oil (TM 1) in a mass yield of 83.3%.

Detected, compound TM1 ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.69 (d, J=1.8 Hz, 1H), 8.44 (dd, J=4.7, 1.3Hz, 1H), 7.70 (s, 1H), 7.23 (dd, J=8.0, 4.2Hz, 1H), 6.14 (d, J=3.0 Hz, 1H), 5.71 (dd, J=5.2, 2.4Hz, 1H), 5.65-5.59 (m, 1H), 4.14-4.11 (m, 1H), 3.06-2.94 (m, 1H), 2.56-2.44 (m, 2H), 2.40-2.26 (m, 3H), 2.15-2.06 (m, 1H), 2.04 (s, 1H), 1.92-1.76 (m, 5H), 1.75-1.63 (m, 6H), 1.27 (J, 12.5,5 =25).0Hz,9H),1.06(s,3H),0.78(s,3H).

HRMS mass spectrum (EI) m/z: theoretical calculated value: 348.5, test value: 348.1.

example 3

TM1 prepared in example 2 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM2, the synthesis procedure of TM2 being as follows:

In a 50mL three-necked flask, 500mg of Compound (TM 1), 10mL of methylene chloride, 0.015g of DMAP, 0.36g of triethylamine and 3 times of nitrogen substitution were placed. After stirring at room temperature to dissolve sufficiently, 0.36g of acetic anhydride was added dropwise over 30 minutes. Stirring was continued for 30min after the completion of the dripping. TLC monitors that the raw materials are reacted completely, the raw materials are distilled to dryness under reduced pressure at 50 ℃ in water bath, 1g of ethanol is added for dissolution, 30g of water is added dropwise under stirring at room temperature, and stirring is continued for 30min after the dripping. The mixture was filtered, and the cake was washed with a small amount of water 1 time and dried in a forced air drying oven at 50℃for 12 hours to give 400mg of an off-white solid (TM 2) in 80% mass yield.

Detected, compound TM2 ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.70 (s, 1H), 8.44 (s, 1H), 7.71 (d, J=7.9 Hz, 1H), 7.23 (dd, J=7.7, 4.7Hz, 1H), 6.14 (s, 1H), 5.63 (s, 2H), 5.08 (s, 1H), 3.01 (s, 1H), 2.45 (s, 6H), 2.07 (s, 5H), 1.96-1.47 (m, 13H), 1.27 (d, J=10.9 Hz, 7H), 1.09 (s, 3H), 0.79 (s, 3H).

HRMS mass spectrum (EI) m/z: theoretical calculated value: 390.5 test value: 390.1.

example 4

TM2 prepared in example 3 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM4, the synthesis procedure of TM4 being as follows:

in a 50mL three-necked flask, 300mg of Compound (TM 2), 10mL of ethanol, 0.1g of palladium calcium carbonate and 3 times of hydrogen substitution were placed. Stirring was carried out at room temperature under hydrogen balloon pressure for 24h. TLC monitored complete reaction of the starting material, filtered and the filter cake was washed 1 time with a small amount of ethanol. The filtrate is distilled to dryness under reduced pressure at 50 ℃ in water bath, 1g of ethanol is added for dissolution, 30g of water is added dropwise under stirring at room temperature, and stirring is continued for 30min after the dripping. The mixture was filtered, and the cake was washed with a small amount of water 1 time and dried in a forced air drying oven at 50℃for 12 hours to give 200mg of an off-white solid (TM 4) in 67% mass yield.

Example 5

TM1 prepared in example 2 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM8, the synthesis procedure of TM8 being as follows:

in a 50mL three-necked flask, 500mg of Compound (TM 1), 170mg of PTAD (4-phenyl-1, 2, 4-triazolin-3, 5-dione) and 10mL of chloroform were placed. Stirring for 1h at room temperature. TLC monitored complete reaction of starting material. And (3) performing rotary evaporation under reduced pressure at the water bath temperature of 30 ℃, and replacing chloroform by petroleum ether. Filtering, washing the filter cake with a small amount of petroleum ether for 1 time, and drying in a blast drying oven at 50 ℃ for 12 hours to obtain 486mg of off-white solid 12 with a mass yield of 97.2%.(note: wherein "protection" means a protecting group)

In a 50mL three-necked flask, 500mg of Compound 12, 50mg of Tempo (2, 6-tetramethylpiperidine oxide), 50mg of sodium bromide, 10mL of chloroform and 0.5mL of saturated sodium bicarbonate solution were added. After stirring at room temperature to dissolve sufficiently, 1.1g of sodium hypochlorite was added dropwise. Stirring was continued for 30min after the completion of the dropping. TLC monitored complete reaction of starting material. And (3) performing rotary evaporation under reduced pressure at the water bath temperature of 30 ℃, and replacing chloroform by petroleum ether. Filtering, washing the filter cake with a small amount of petroleum ether for 1 time, and drying in a blast drying oven at 50 ℃ for 12 hours to obtain 500mg of yellowish solid with mass yield of 100%.

In a 50mL three-necked flask, 500mg of Compound 13, 10mL of methanol and 2mL of an ethanol solution of hydrogen chloride were added. Stirred at 60℃for 1h. TLC monitored complete reaction of starting material. Steaming under reduced pressure to dry at 55deg.C in water bath. 10mL of methanol was added for dissolution, 1mL of water was added, 0.2g of sodium hydroxide was added, and the mixture was stirred at room temperature for 1h. Steaming under reduced pressure to dry at 55deg.C in water bath. 50mL of water, 30mL of methylene chloride was added, the mixture was dissolved and separated, and the aqueous layer was extracted 1 time with 20mL of methylene chloride. The organic layers were combined, distilled under reduced pressure in a water bath at 55℃and the dichloromethane was replaced by petroleum ether. The mixture was filtered, and the filter cake was washed 1 time with a small amount of petroleum ether and dried in a forced air drying oven at 50℃for 12 hours to give 100g of pale yellow solid (TM 8) in 20% mass yield.

Example 6

The compound 7 prepared in example 1 was used for the synthesis of 17- (3-pyridyl) -10α -methyl-steroid derivatives, the synthesized compound was designated TM5, and the synthesis procedure of TM5 was as follows:

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 substance IN1F4.3g with the mass yield of 86.0 percent.

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 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 with the yield of 63.0%.

Adding 1.5g of CK004-1A, 22ml of acetone, 7.5ml of water and 0.3g of p-toluenesulfonic acid into a 50ml reaction bottle, concentrating until the reaction is finished, adding 10ml of dichloromethane, layering, washing once with 5ml of 5% sodium bicarbonate, layering, and organicThe layer was concentrated to dryness to give CK004-1B1.2g, with a yield of 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 finished, the mixture is stirred for 1 hour, filtered, leached by water and dried at 50 ℃ to obtain 1.15g of white solid with the mass yield of 95.8%.

1.05g of CK004-1C was added to the reaction flask, and 12ml of tetrahydrofuran was added thereto, followed by stirring to dissolve the solution. 10ml of tetrahydrofuran is added into another reaction flask to be cooled to-5 to 5 ℃, 1.72g of iodine is added into the reaction flask, and the reaction flask is stirred for half an hour. And dropwise adding tetramethyl guanidine at the temperature of-5 to 5 ℃ and stirring for half an hour after the dropwise adding is finished. Adding CK004-1C tetrahydrofuran solution dropwise at-5 ℃, reacting for 1 hour at-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 is generated, adding 10ml of tetrahydrofuran, adding 5ml of 1M hydrochloric acid for washing, then using 5ml of 5% sodium bicarbonate solution for washing, and then using 5ml of saturated sodium thiosulfate solution for washing and layering, concentrating an organic layer to obtain 1.21g, and obtaining 115.2% mass yield.

800mg of CK004-1D,340mg of diethyl- (3-pyridine) -borane, 4mg of bis (triphenylphosphine) palladium dichloride, 10.0ml of N, N-dimethylformamide, 480mg of anhydrous potassium carbonate and 1.2g of water are added into a reaction flask, and the temperature is raised to 85-90 ℃ for reaction. After the reaction, the mixture was filtered, 10ml of methylene chloride was added to the filtrate, the mixture was washed 3 times with saturated sodium chloride and concentrated to dryness, and column chromatography (eluent: petroleum ether: ethyl acetate=13:1 to petroleum ether: ethyl acetate=6:1) was performed to obtain 590g of a white solid (TM 5) in a mass yield of 73.8%.

Compound TM5 was detected ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) δ8.64 (d, J=1.7Hz, 1H), 8.44 (d, J=4.7Hz, 1H), 7.66(d,J＝7.9Hz,1H),7.20(dd,J＝7.9,4.8Hz,1H),6.02(d,J＝2.9Hz,1H),4.04(s,1H),2.35–2.15(m,2H),2.15–2.01(m,1H),1.94(dd,J＝13.9,11.0Hz,1H),1.89–1.53(m,13H),1.50(dd,J＝12.8,2.7Hz,1H),1.47–1.35(m,2H),1.36–1.11(m,4H),1.06(s,3H),0.94(s,3H).

¹³ C NMR was: 13C NMR (101 MHz, CDCl 3) delta 152.10,147.75,133.49,132.77,66.40,50.31,47.40,45.14,37.88,37.59,35.87,34.45,34.01,33.54,32.50,28.73,28.26,25.20,20.90,17.14,14.70.

HRMS mass spectrum (EI) m/z: theoretical calculated value: 352.2 test value: 352.0.

the applicant has synthesized compound I below using CK004-1B and determined the configuration of CK004-1B by determining the single crystal structure of compound I.

Under the protection of nitrogen, 540mg of CK004-1B and 12ml of pyridine are added into a reaction bottle, 760mg of 4-chlorobenzoyl chloride is added, stirring reaction is carried out at room temperature for 2 hours, the reaction completion of raw materials is detected, 100ml of dichloromethane is added, the pH is regulated to 5-6 by 5% of diluted hydrochloric acid, liquid separation is carried out, an organic layer is washed by 5% of sodium bicarbonate, water washing, layering and concentrating are carried out, and column chromatography (petroleum ether: ethyl acetate=20:1-10:1) is carried out, thus 510mg of compound I is obtained.

Compound I ¹ H NMR is: ¹ H NMR(400MHz,CDCl ₃ )δ7.98(d,J＝8.7Hz,2H),7.42(d,J＝8.7Hz,2H),5.26(s,1H),2.45(dd,J＝19.2,9.0Hz,1H),2.16–1.91(m,4H),1.88–1.73(m,5H),1.67–1.56(m,8H),1.42(s,1H),1.34–1.23(m,3H),1.15(d,J＝13.4Hz,1H),0.97(s,3H),0.91(s,3H).

the single crystal structure of compound I is shown in FIG. 1, demonstrating that the configuration of compound I corresponds to the above formula.

Example 7

TM5 prepared in example 6 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM7, the synthesis procedure of TM7 being as follows:

130mg of TM5,5ml of dichloromethane, 75.4mg of triethylamine, 0.6mg of 4-dimethylaminopyridine and 75.4mg of acetic anhydride are added to a reaction flask at 10-20 ℃, 0.5ml of methanol is added after the completion of the reaction, and the mixture is concentrated to dryness and subjected to column chromatography (petroleum ether: ethyl acetate=5:1) to obtain 131mg of a white solid (TM 7) with a mass yield of 100.7%.

Compound TM7 was detected ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.64 (d, J=1.8 Hz, 1H), 8.45 (dd, J=4.8, 1.5Hz, 1H), 7.67 (dt, J=7.9, 1.9Hz, 1H), 7.24-7.18 (m, 1H), 6.11-5.93 (m, 1H), 5.01 (d, J=2.4 Hz, 1H), 2.35-2.17 (m, 2H), 2.14-2.04 (m, 4H), 2.00-1.89 (m, 1H), 1.86-1.61 (m, 11H), 1.60-1.45 (m, 5H), 1.34-1.13 (m, 4H), 1.07 (s, 3H), 0.96 (d, J=4.7 Hz, 3H).

¹³ C NMR was: 13C NMR (101 MHz, CDCl 3) delta 170.69,152.10,147.81,133.47,132.72,128.49,123.02,70.05,50.29,47.41,45.07,38.86,37.28,35.11,33.99,33.52,32.84,32.49,28.23,25.80,25.01,21.58,20.91,17.14,14.84.

HRMS mass spectrum (EI) m/z: theoretical calculated value: 394.5 test value: 394.2.

example 8

TM5 prepared in example 6 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM6, the synthesis procedure for TM6 being as follows:

250mg of TM5,1.5ml of dichloromethane, 0.25ml of 5% sodium bicarbonate solution, 0.01g of sodium bromide and 10% of sodium hypochlorite are added dropwise at the temperature of 0-5 ℃ in a reaction bottle, after the reaction is finished, the layers are separated, the aqueous layer is back extracted with dichloromethane, the organic phases are combined, the organic phases are washed by adding saturated sodium thiosulfate solution and then water, the organic phases are concentrated to dryness, and column chromatography (petroleum ether: ethyl acetate=5:1) yields 120mg of white solid TM6 with a mass yield of 48.0%.

Example 9

The compound 7 prepared in example 1 was used for the synthesis of 17- (3-pyridyl) -10α -methyl-steroid derivatives, the syntheses hereinThe compound was designated TM23, and the synthesis procedure for TM23 was as follows:

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%.

To the reaction flask was added 850mg of CK004-1F, 3.5ml of absolute ethanol, 612mg of hydrazine hydrate, 4mg of hydrazine sulfate, and the mixture was reacted at 30-35 ℃. 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-5-5℃and 2.66g of iodine was added thereto, followed by dropwise addition of 3g of tetramethylguanidine. Dropwise adding CK004-1G tetrahydrofuran solution at-5-5 ℃ under controlled temperature, reacting for 1 hour at-5-5 ℃, adding 1.86G sodium thiosulfate after the completion of the reaction, heating to 40 ℃ and stirring for half an hour, filtering, concentrating the filtrate until no fraction exists, adding 15ml tetrahydrofuran, adding 8ml 1M hydrochloric acid for washing, then adding 8ml 5% sodium bicarbonate solution for washing, and then The layer was washed with 8ml of saturated sodium thiosulfate solution, and the organic layer was concentrated to dryness to give 1.8g of CK004-1H with a mass yield of 116.1%.

1.0mg of CK004-1H,425mg of diethyl- (3-pyridine) -borane, 5mg of bis (triphenylphosphine) palladium dichloride, 12ml of N, N-dimethylformamide, 600mg of anhydrous potassium carbonate and 1.52g of water are added into a reaction flask, and the temperature is raised to 85-90 ℃ for reaction. After the reaction, the mixture was filtered, 15ml of methylene chloride was added to the filtrate, the mixture was washed 3 times with saturated sodium chloride and concentrated to dryness, and column chromatography (eluent: petroleum ether: ethyl acetate=13:1 to petroleum ether: ethyl acetate=6:1) was carried out to give compound TM23 as a white solid of 410mg in a mass yield of 41.0%.

Compound TM23 was detected ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.69 (s, 1H), 8.43 (d, J=3.8 Hz, 1H), 7.78-7.67 (m, 1H), 7.26-7.18 (m, 1H), 6.12 (s, 1H), 5.32 (s, 1H), 4.31 (s, 1H), 4.13-4.07 (m, 1H), 2.76 (d, J=2.4 Hz, 1H), 2.38 (dd, J=6.6, 3.3Hz, 1H), 2.35-2.13 (m, 3H), 2.12-1.90 (m, 4H), 1.90-1.59 (m, 9H), 1.60-1.35 (m, 7H), 1.26 (s, 2H), 1.17 (s, 3H), 0.96 (t, J=7.4 Hz, 1H), 0.69 (s, 3H).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 167.71,151.40,147.61,137.95,133.06,130.91,128.85,127.17,123.16,118.23,66.32,65.57,51.44,47.37,45.68,35.83,35.23,34.59,32.64,31.61,30.64,29.18,23.67,19.10,13.73,10.24.

HRMS mass spectrum (EI) m/z: theoretical calculated value: 350.3, test value: 349.9.

example 10

TM23 prepared in example 9 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM9, the synthesis procedure for TM9 being as follows:

160mg of TM23,5ml of dichloromethane, 92.8mg of triethylamine and 0.8mg of 4-dimethylaminopyridine are added to a reaction flask, 92.8mg of acetic anhydride is added at 10-20 ℃,0.8 ml of methanol is added after the reaction, and the mixture is concentrated to dryness, and column chromatography (petroleum ether: ethyl acetate=5:1) gives 150mg of a white solid (TM 9) with a mass yield of 93.8%.

Detected, compound TM9 ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.70 (d, J=1.5 Hz, 1H), 8.44 (d, J=3.7 Hz, 1H), 7.81-7.67 (m, 1H), 7.22 (dd, J=7.9, 4.8Hz, 1H), 6.13 (dd, J=3.0, 2.0Hz, 1H), 5.45-5.24 (m, 1H), 5.16-4.88 (m, 1H), 2.87-2.71 (m, 1H), 2.40 (d, J=3.3 Hz, 1H), 2.24 (dd, J=10.7, 1.6Hz, 2H), 2.07 (s, 3H), 2.02 (d, J=4.5 Hz, 2H), 1.91 (s, 2H), 1.85-1.74 (m, 2H), 1.73-1.52 (m, 1H), 2.87-2.71 (m, 1H), 2.40 (d, J=10.7, 1.6Hz, 2H), 2.07 (s, 3H), 2.02 (d, 3.3H), 1.7.7 Hz, 1.3H (s, 3H).

13C NMR was: 13C NMR (101 MHz, CDCl 3) delta 170.73,151.36,147.66,138.00,133.05,131.74,127.17,123.17,118.13,69.81,51.43,47.29,45.68,36.12,34.32,32.79,32.58,32.37,30.54,26.29,23.74,21.53,19.03,10.38.

HRMS mass spectrum (EI) m/z: theoretical calculated value: 392.5 test value: 391.9.

example 11

TM23 prepared in example 9 was used to synthesize 17- (3-pyridyl) -10α -methyl-steroid derivatives, the compound synthesized here being denoted TM22, the synthesis procedure of TM22 being as follows:

140mg of TM23,5ml of dichloromethane, 0.5ml of 5% sodium bicarbonate solution, 5mg of sodium bromide, 1.5ml of 10% sodium hypochlorite are added dropwise at a temperature of 0-5 ℃ in a reaction bottle, after the reaction is finished, the layers are separated, a water layer is back extracted by dichloromethane, organic phases are combined, the organic phases are added for washing by saturated sodium thiosulfate solution, the organic phases are concentrated to dryness by water washing, and column chromatography (petroleum ether: ethyl acetate=5:1) is carried out to obtain 48mg of white solid (TM 22) with a mass yield of 34.28%.

Compound TM22 was detected ¹ H NMR is: 1H NMR (400 MHz, CDCl 3) delta 8.70 (s, 1H), 8.45 (s, 1H), 7.74-7.66 (m, 1H), 7.23 (dd, J=7.8, 4.8Hz, 1H), 6.13 (dd, J=3.2, 2.0Hz, 1H), 5.44-5.30 (m, 1H), 2.79 (dd, J=6.3, 2.3Hz, 1H), 2.54-2.37 (m, 2H), 2.37-2.28 (m, 1H), 2.29-2.18 (m, 4H), 2.16 (d, J=1.7 Hz, 1H), 2.15-1.96 (m, 4H), 1.91-1.38 (m, 9H), 1.28 (dd, J=1.28 (m, 1H)3.6,10.4Hz,2H),1.16(s,3H),0.94(d,J＝3.8Hz,3H).

HRMS mass spectrum (EI) m/z: theoretical calculated value: 348.3 test value: 347.9.

in addition to the compounds prepared in the examples above, the present invention also prepares some other example compounds by the same/similar preparation process as the previous examples, and all the example compounds are listed below:

Example 12

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 . 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. The medium was discarded, resuspended in fresh medium, and the medium was added 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 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 sample on DU145, HCT-116 or A549 cells was evaluated by the cell proliferation rate, and if the proliferation inhibition rate was > 100%, the sample was judged to be a systematic error, 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 sectionIC of the compounds of the examples and comparative examples on individual cancer cells ₅₀ Value of Note that: DU145 is a human prostate cancer cell, HCT-116 is a human colon cancer cell, and A549 is a human non-small cell lung cancer cell.

The 17- (pyridyl) -10 alpha-methyl-steroid derivative is a brand new compound, and the C-10 methyl of the derivative is alpha-configuration, and has inhibition effect on prostate cancer, colon cancer and lung cancer cells. Compounds of the various embodiments of the invention are directed to human prostate cancer cell IC ₅₀ In the range < 140. Mu.M, preferably in the range < 80. Mu.M, more preferably in the range < 40. Mu.M, more preferably in the range < 20. Mu.M. Compounds of the various embodiments of the invention are directed to human colon cancer cell IC ₅₀ In the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 30. Mu.M, more preferably in the range < 15. Mu.M. Compounds of various embodiments of the application are directed to human non-small cell lung cancer cell IC ₅₀ In the range < 100. Mu.M, preferably in the range < 50. Mu.M, more preferably in the range < 30. Mu.M, more preferably in the range < 15. Mu.M.

In the present application, IC is considered ₅₀ Within the range of 50-140 mu M, the compound has a certain inhibition effect on cancer cells and IC ₅₀ < 50. Mu.M indicates that the compounds have good inhibitory effect on cancer cells.

Furthermore, the applicant has shown that abiraterone acetate (or abiraterone) is clinically used in combination with prednisone in the prior art for the treatment of prostate cancer, however, the present application has unexpectedly found and proved by experiments (as shown in the results of table 1), that abiraterone, abiraterone acetate and abiraterone oxide have not only an anti-prostate cancer effect, but also unexpectedly significant inhibitory effect against colon cancer cells or lung cancer cells, in particular abiraterone against human colon cancer cells IC ₅₀ Abiraterone acetate is directed against humans in the range < 50. Mu.MColon cancer cell IC ₅₀ Abiraterone oxide was directed against human colon cancer cell IC in the range < 10. Mu.M ₅₀ In the range < 10. Mu.M; abiraterone acetate against human non-small cell lung cancer cell IC ₅₀ Abiraterone oxide is directed against human non-small cell lung cancer cell IC in the range < 50. Mu.M ₅₀ In the range < 20. Mu.M. Because of the different pathogenesis of cancer and the different mechanism of drug action, prior to the present application, it was not expected by those skilled in the art that abiraterone, abiraterone acetate and abiraterone oxide could have such good inhibitory effect on specific cancer species, i.e. colon cancer or human non-small cell lung cancer.

Further, as can be seen from the results of Table 1, compound TM1 of the present application was directed to IC of human prostate cancer cells ₅₀ Shows that the composition has good inhibition effect on the prostate cancer cells, and the inhibition effect on the prostate cancer cells is even better than that of Abiraterone; furthermore, in contrast to abiraterone, compound TM5 of the application is directed against IC of human prostate cancer cells ₅₀ Shows that the composition has a certain inhibition effect on prostate cancer cells and simultaneously aims at human colon cancer cell IC ₅₀ 24.100, even better than Abiraterone against human colon cancer cell IC ₅₀ According to 41.910, it is shown that the compound TM5 of the present application shows a good inhibitory effect against human colon cancer cells. In contrast to Abiraterone, compound TM23 of the present application is directed against human prostate cancer cell IC ₅₀ Within the range of less than 80 mu M, the preparation shows that the preparation has a certain inhibition effect on prostate cancer cells, and simultaneously aims at human colon cancer cell IC ₅₀ 25.242, even better than Abiraterone against human colon cancer cell IC ₅₀ Is 41.910 of the present invention, compound TM23 is directed to human non-small cell lung cancer cell IC ₅₀ 41.485, even better than Abiturin for human non-small cell lung cancer cell IC ₅₀ The method comprises the steps of carrying out a first treatment on the surface of the In other words, the compound TM23 of the present invention shows a good inhibitory effect against both human colon cancer cells and human non-small cell lung cancer cells, which was not previously expected by the person skilled in the art.

Further, the applicant points out that the compound TM4 of the present invention is directed against human prostate compared to abiraterone acetateCancer cell IC ₅₀ In the range < 40. Mu.M, and at the same time, IC against human colon cancer cells ₅₀ IC for human non-small cell lung cancer cells in the range of < 20. Mu.M ₅₀ In the range < 30. Mu.M; the compound TM4 of the invention shows good inhibition effect on human prostate cancer cells, human colon cancer cells and human non-small cell lung cancer cells. Compared with abiraterone acetate, the compound TM7 of the invention aims at human prostatic cancer cell IC ₅₀ In the range < 20. Mu.M, and at the same time, IC against human colon cancer cells ₅₀ IC for human non-small cell lung cancer cells in the range of < 20. Mu.M ₅₀ In the range < 80. Mu.M. In other words, the compound TM7 of the invention has good inhibition effect on human prostate cancer cells and human colon cancer cells, and has certain inhibition effect on human non-small cell lung cancer cells. Compared with abiraterone acetate, the compound TM9 of the invention aims at human prostatic cancer cell IC ₅₀ 11.389 even better than abiraterone acetate against human prostate cancer cell IC ₅₀ 12.717 against human colon carcinoma cell IC ₅₀ IC for human non-small cell lung cancer cells in the range of < 10. Mu.M ₅₀ 24.850, even better than abiraterone acetate against human non-small cell lung cancer cell IC ₅₀ 30.607 of (2); the compound TM9 of the invention shows good inhibition effect on human prostate cancer cells, human colon cancer cells and human non-small cell lung cancer cells.

Still further, the applicants have noted that compound TM6 of the present invention is directed against human prostate cancer cells and human non-small cell lung cancer cells IC, as compared to Abiraterone oxide ₅₀ Within the range of less than 70 mu M, the composition shows that the composition has a certain inhibition effect on prostate cancer cells and human non-small cell lung cancer cells, and simultaneously aims at human colon cancer cells IC ₅₀ Within the range of < 30 mu M, the compound TM6 of the invention shows good inhibition effect on human colon cancer cells; compared with Abiraterone oxide, the compound TM23 of the invention has the IC50 in the range of < 140 mu M for human prostatic cancer cells, the IC50 in the range of < 100 mu M for human colon cancer cells and the IC50 in the range of < 100 mu M for human non-small cell lung cancer cellsA certain inhibition effect.

2. Tolerance test to enzyme

9ml of water and 1.5ml of absolute ethanol were added to a clean reaction flask, and after the compound D4A had been completely dissolved, 0.5g of compound D4A (purity: 99.1%) and 1.5ml of absolute ethanol were added with stirring, the pH was adjusted to 7 to 8 with a 1N hydrogen chloride solution, the reaction temperature was adjusted to 30 to 35℃and 12g of glucose, 0.1g of 3. Alpha. Reductase, 0.22g of glucose dehydrogenase, 0.015g of coenzyme I and 0.015g of coenzyme II were added. After stirring uniformly, pH=7-8 is adjusted by 1N sodium hydroxide solution for reaction for 0.5-3 hours, TLC is used for monitoring the reaction (developing agent is petroleum ether, dichloromethane and ethyl acetate=5:2:1, color developing agent is phosphomolybdic acid), the specific result is shown in figure 2, and TLC results show that the compound D4A is completely reacted for 0.5 hours.

The 3 alpha reductase is an abbreviation for 3 alpha hydroxy steroid oxidoreductase, available from Kyoto Lai Biotechnology Co., ltd. Nap, which belongs to the Comamonas testosteroni source. Glucose dehydrogenase, available from Shanghai Meilin Biochemical technologies Co. Coenzyme one (NAD): nicotinamide adenine dinucleotide is purchased from Shanghai Biotechnology Co. Coenzyme two (NADP), nicotinamide adenine dinucleotide phosphate, available from Hangzhou Weitai biopharmaceutical Co.

The enzyme reduction of compound TM22 was carried out as described above, and the specific results are shown in FIG. 3, and TLC results show that compound TM22 was converted to a small fraction in 0.5 to 5 hours.

The results show that compared with D4A, the reaction activity of TM22 and reductase is greatly reduced, the molecular stability is better, the degradation is not easy to occur under the action of the enzyme, the tolerance to in vivo metabolic enzyme is better, the in vivo metabolism is expected to be slower, the in vitro discharging time can be prolonged, the action time can be prolonged, the duration of the drug effect is longer, and the long-acting preparation can be developed.

Similar to TM22, the remaining example compounds of the invention also only converted a small fraction, if not all, in 0.5 to 5 hours, again indicating: the compound has greatly reduced or no reactivity with reductase, better molecular stability, difficult degradation under the action of enzyme, better tolerance to in vivo metabolic enzyme, slower metabolism in vivo, prolonged in vitro discharge time, prolonged action time and longer duration of drug effect, and is beneficial to developing long-acting preparations.

3. Docking with enzyme molecules (3 alpha steroid dehydrogenase/cholesterol oxidase)

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.

Wherein, the docking simulation result of the abiraterone-3 alpha steroid dehydrogenase is shown in figure 4, and the scoring data is shown in figure 5;

the results of the docking simulation for the TM23-3 alpha steroid dehydrogenase are shown in FIG. 6, and the scoring data are shown in FIG. 7;

the results of the docking simulation of D4A-cholesterol oxidase are shown in FIG. 8, and the scoring data are shown in FIG. 9;

the results of the docking simulation for the TM 22-cholesterol oxidase are shown in FIG. 10, and the scoring data are shown in FIG. 11.

TABLE 2

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 show that: in the 17- (3-pyridyl) -10 alpha-methyl-steroid derivative prepared by the invention, after C10 methyl is overturned, the compound which is not overturned is not well butted with enzyme, which indicates that the overturned compound is insensitive to the above 2 enzymes or the enzyme is inactive to the overturned compound, so that the 17- (3-pyridyl) -10 alpha-methyl-steroid derivative has better tolerance to in-vivo enzyme, is expected to have slower metabolism in vivo, prolonged in-vitro discharge time, prolonged action time and longer duration of drug effect, and is beneficial to developing a long-acting preparation.

Similar to TM22 and TM23, the remaining example compounds of the invention, after inversion of the methyl group at the C10 position, have no non-inverted compound in the docking with the enzyme, indicating that the inverted compound is insensitive to the above 2 enzymes, or the enzyme is inactive to the inverted compound, and the 17- (3-pyridyl) -10α -methyl-steroid derivatives of the invention are well tolerated by enzymes in vivo, are expected to be metabolized relatively slowly in vivo, are prolonged in time to be expelled from the body, are prolonged in duration of action, and are useful for developing long-acting formulations.

4. Solubility test

Weighing about 20mg of each test sample (TM 1, abiraterone and Abiraterone acetate), placing into different 10mL measuring flasks, adding buffer mediums with different pH values (hydrochloric acid solution with pH value of 1.0, acetic acid-sodium acetate buffer solution with pH value of 4.5 and potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH value of 6.8), diluting to scale, shaking uniformly, oscillating for 24 hours, keeping solid undissolved state, filtering with a filter membrane with 0.45 μm, taking 1.0mL of each supernatant, mixing with 0.5mL of corresponding buffer mediums with different pH values uniformly, and carrying out quantitative analysis on solubility (mass/mug of TM1, abiraterone acetate contained in each mL of solution) by HPLC. HPLC conditions were as follows:

Chromatographic column: waters (Waters SunFire C18 (150 mm. Times.4.6 mm, 3.5 μm)

Mobile phase: acetonitrile-water (75/25; V/V)

Flow rate: 1.0 ml/min

A detector: UV detector (205 nm)

Sample injection amount: 20 μl of

The results are shown in Table 3:

TABLE 3 Table 3

From this, the solubility of TM1 in water under acidic conditions is far higher than Yu Abi tex, which is favorable for the dissolution of the drug in the gastrointestinal tract, and is expected to have higher bioavailability.

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

A 17-pyridinyl-10 a-methyl-steroid derivative, characterized in that said steroid derivative has the structure of formula I:

R ₁ selected from =o, -OH, halogen or-OC (O) R ₂ ；

R ₂ Selected from C1-C5 alkyl, C1-C5 haloalkyl, phenyl or halophenyl;

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, C1-C5 alkenyl or C1-C5 ester group;

Represents a single bond or a double bond;

when a certain isIn the case of double bonds, adjacent theretoIs a single bond;

i. j, k, m and n are each independently selected from 0, 1, 2, 3, 4 or 5.
17-pyridinyl-10α -methyl-steroid derivatives according to claim 1, characterized in that said steroid derivatives have the structural formula ii:
17-pyridinyl-10α -methyl-steroid derivatives according to claim 2, characterized in that,

the R is ₁ Selected from = O, -OH or OAc.
A17-pyridinyl-10α -methyl-steroid derivative according to claim 3, wherein,

the R is ₁ When selected from the group consisting of-OH, the-OH is β -OH or α -OH.
17-pyridinyl-10α -methyl-steroid derivatives according to claim 2, characterized in that,

the R is ₃ Selected from C1-C5 alkyl or haloalkyl.
17-pyridinyl-10α -methyl-steroid derivatives according to claim 5, characterized in that,

and k is 0.
17-pyridinyl-10α -methyl-steroid derivatives according to claim 2, characterized in that,

the i, j, m and n are all 0, preferably the k is 0.
17-pyridinyl-10α -methyl-steroid derivatives according to any of the claims 1-7, characterized in that,

The R is ₁ To R ₇ Wherein the halogen is selected from F, cl, br or I.
17-pyridinyl-10 a-methyl-steroid derivative according to any of the claims 1-7, characterized in that said steroid derivative is selected from the following structural formulas:
17-pyridinyl-10α -methyl-steroid derivatives according to claim 9, characterized in that said steroid derivatives are selected from the following structural formulas:
a process for the preparation of a 17-pyridinyl-10α -methyl-steroid derivative according to any of claims 1-10, characterized in that the process comprises starting from an intermediate of formula V, connecting a substituted or unsubstituted pyridinyl group in the C-17 position;

the substituted or unsubstituted pyridyl group corresponds to the same pyridyl structure on the 17-pyridyl-10α -methyl-steroid derivative;

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.
An intermediate compound for the preparation of a 17-pyridinyl-10 α -methyl-steroid derivative, characterized in that said intermediate compound is selected from the following structural formulas:

R ₈ Selected from-OH or protected hydroxy;

R ₁₃ selected from = O or protected carbonyl.
An intermediate compound according to claim 12, characterized in that,

the R is ₈ Selected from-OH or OAc; and/or;

the R is ₁₃ Selected from =o or
An intermediate compound according to claim 12, wherein the intermediate compound is selected from the following structural formulae:
a process for the preparation of an intermediate compound according to any one of claims 12 to 14, comprising the steps of: the C-10 methyl group is converted from beta configuration to alpha configuration by photochemistry conversion
Use of a 17-pyridinyl-10 a-methyl-steroid derivative according to any of claims 1-10 for the preparation of a medicament for the treatment of cancer.
The use according to claim 16, wherein the cancer comprises prostate cancer, breast cancer, colon cancer or lung cancer.
A pharmaceutical composition comprising the 17-pyridinyl-10 a-methyl-steroid derivative according to any of the claims 1-10 and a pharmaceutically acceptable adjuvant.
Application of steroid compound with following structure in preparation of medicine for treating colon cancer or lung cancer