An anti-cancer steroid compound, its preparation method, uses, and pharmaceutical composition

Abstract

The present invention relates to an anti-cancer steroid compound, a preparation method, uses thereof, and a pharmaceutical composition. The methyl group at the C-10 position of the steroid compound of the present invention has an inverted α configuration, and it is a class of completely new steroid compounds, which have strong inhibitory effects on prostate cancer, colon cancer, and lung cancer cells. In order to prepare the above steroid compound, the present invention has also synthesized a series of corresponding intermediates. The steroid compound of the present invention can be used as a drug to effectively treat cancer, especially cancers including prostate cancer, colon cancer, or lung cancer, etc.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to an anti-cancer steroid compound, a preparation method, a use and a pharmaceutical composition thereof. Background Art

[0002] Abiraterone acetate, chemically named 17-(3-pyridyl)-androst-5,16-dien-3β-ol acetate (structural formula is as follows), is a CYP17 inhibitor. Clinically, it is used in combination with prednisone to treat metastatic advanced prostate cancer that has become resistant to traditional hormonal therapy. It can not only reduce the prostate specific antigen level, but also help shrink tumors and extend the life of advanced prostate cancer patients.

[0003]

[0004] Structurally, it is derived from the following steroid skeleton structure, and this skeleton structure (hereinafter referred to as the steroid ring) has the following four rings, and the carbon numbers (1-17) on each ring are as follows. Androstane means that each of the C-10 and C-13 positions is connected to a β-methyl group, which can be denoted as 10β-methyl and 13β-methyl.

[0005] Summary of the Invention

[0006] The present invention provides a steroid compound, which is a new type of compound and has inhibitory effects on prostate cancer, colon cancer, lung cancer and pancreatic cancer. The steroid compound has the structure of formula I or its pharmaceutically acceptable salt:

[0007]

[0008] R1 and R2 are the same or different and are each independently selected from an unsubstituted or halogen- or C 1-5 alkyl-substituted imidazolyl, triazolyl, benzimidazolyl, benzotriazolyl, quinolinyl, pyrrolidinyl, piperidinyl or piperazinyl;

[0009] R3, R4, R5 and R6 are the same or different and are each independently selected from -OH, =O, halogen, amino, C 1-5 alkyl, C 1-5 haloalkyl, C 1-5 alkoxy, C 2-5 alkenyl, C 2-5 alkynyl or C 2-5 ester group;

[0010] i, j, m and n are each independently selected from 0, 1, 2, 3 or 4;

[0011] Represents a single bond or a double bond.

[0012] In an embodiment of the present invention, R1 and R2 are selected from imidazolyl groups.

[0013] In an embodiment of the present invention, i, j, m, and n are all 0.

[0014] In an embodiment of the present invention, the halogen in R1 to R6 is selected from F, Cl, Br, or I.

[0015] In an embodiment of the present invention, the steroid compound is selected from the following structural formulas:

[0016]

[0017] In an embodiment of the present invention, the steroid compound is selected from the following structural formulas:

[0018]

[0019] The present invention provides a method for preparing a steroid compound, the method comprising using the intermediate shown in formula II as a raw material, and connecting a substituted or unsubstituted imidazole ester group at the C-3 position and the C-17 position;

[0020]

[0021] The substituted or unsubstituted imidazole ester group has the same structure as the imidazole ester group on the steroid compound;

[0022] R7 is the same as R3 or becomes the same after reaction;

[0023] R8 is the same as R4 or becomes the same after reaction;

[0024] R9 is the same as R5 or becomes the same after reaction;

[0025] The R 10 is the same as R6 or becomes the same after reaction;

[0026] i, j, m, and n are each independently selected from 0, 1, 2, 3, or 4;

[0027] Represents a single bond or a double bond.

[0028] In an embodiment of the present invention, the intermediate is selected from the following structural formulas:

[0029]

[0030] In an embodiment of the present invention, the intermediate is prepared by a method comprising the following steps: subjecting a compound of the following formula to photochemical conversion to invert the methyl group at the C-10 position from the β-configuration to the α-configuration:

[0031]

[0032] R 11 is selected from -OH or a protected hydroxyl group, preferably R 11 is selected from -OH or OAc;

[0033] R 12 is selected from ═O or a protected carbonyl group, preferably R 12 is selected from ═O or

[0034] In an embodiment of the present invention, optionally, the photochemical conversion is an ultraviolet photocatalytic reaction. Optionally, the ultraviolet photocatalytic reaction first opens the steroid ring in the wavelength range of 260 - 290 nm, and then closes the steroid ring in the wavelength range of 295 - 340 nm, and the reaction temperature is -10 to 50 °C.

[0035] In a specific embodiment of the present invention, the preparation method of intermediate 9 is as follows:

[0036]

[0037] Compound 1 is subjected to protection of the 3-position hydroxyl group (for example, using reagents such as acetic anhydride) and protection of the 17-position carbonyl group (for example, using ethylene glycol reagent) to obtain compound 3.

[0038] Compound 3 is oxidized to a carbonyl group at the allylic position (7-position) to obtain compound 4, for example, using catalyst N-hydroxyphthalimide and initiator benzoyl peroxide for air oxidation.

[0039] Compound 4 is subjected to hydrazonation of the 7-position carbonyl group and dehrazonation to form a 5,7 double bond to obtain compound 6.

[0040] Compound 6 undergoes an ultraviolet photocatalytic reaction, and the methyl group at the 10-position is inverted from the β-configuration to the α-configuration to obtain compound 7. The photocatalytic reaction first opens the steroid ring in the wavelength range of 260 - 290 nm, and then closes the ring in the wavelength range of 295 - 340 nm. The reaction temperature is controlled at -10 to 50 °C.

[0041] Compound 7 is catalytically hydrolyzed with an acid (such as p-toluenesulfonic acid, etc.) to remove the protecting group at the 3-position to obtain compound 8.

[0042] Compound 8 is successively subjected to Oppenauer oxidation of the 3-position hydroxyl group and hydrolysis of the 17-position ketal to obtain compound 9.

[0043] In a specific embodiment of the present invention, the steroidal compound of the present invention is prepared by the following method.

[0044]

[0045] Compound 9 is hydrogenated under Ni catalysis to form Compound 10. Optionally, the reaction solvent can be dioxane, dichloromethane, acetonitrile, etc. The amount of the catalyst used is 0.01 to 0.10 times the mass of Compound 9, and the reaction temperature is 0 to 30 °C.

[0046] Compound 10 undergoes an esterification reaction under the catalysis of p-toluenesulfonic acid, etc. The esterifying reagent can be isopropenyl acetate or acetic anhydride, and the reaction temperature is 20 °C to 60 °C.

[0047] Compound 11 is reduced to form Compound 12. The reaction solvent is a mixed solvent of dichloromethane, tetrahydrofuran, ethyl acetate, etc. and methanol, ethanol, etc. The reducing agent is sodium borohydride, potassium borohydride, etc., and the catalyst is calcium chloride, cerium chloride, etc. The reaction temperature is 0 - 20 °C.

[0048] Compound 12 reacts with carbonyldiimidazole to obtain TM35. The reaction solvent can be acetonitrile, dichloromethane, tetrahydrofuran, etc., and the reaction temperature is 40 - 80 °C.

[0049] In a specific embodiment of the present invention, the compound of the present invention is prepared by the following method.

[0050]

[0051] Compound 9 is hydrogenated under transition metal catalysis to obtain Compound 13. Optionally, the catalyst is 5% palladium on carbon, 10% palladium on carbon, etc., and the reaction temperature is 10 - 30 °C.

[0052] Compound 13 undergoes a reduction reaction to form Compound 14 and Compound 15. The reaction solvent is a mixed solvent of dichloromethane, tetrahydrofuran, ethyl acetate, etc. and methanol, ethanol, etc. The reducing agent is sodium borohydride, potassium borohydride, etc., and the reaction temperature is 0 - 20 °C.

[0053] Compound 14 and Compound 15 react with carbonyldiimidazole to obtain TM36 and Compound 37. The reaction solvent can be acetonitrile, dichloromethane, tetrahydrofuran, etc., and the reaction temperature is 40 - 80 °C.

[0054] The present invention provides the use of a steroidal compound in the preparation of a drug for treating cancer.

[0055] In the embodiments of the present invention, the cancer includes prostate cancer, colon cancer, lung cancer or pancreatic cancer.

[0056] The present invention provides a pharmaceutical composition comprising the steroidal compound of the present invention and a pharmaceutically acceptable excipient.

[0057] In an embodiment of the present invention, the dosage form of the pharmaceutical composition can be tablets, capsules, granules, powders, suspensions, solutions, emulsions, injections, etc. According to the characteristics of their respective dosage forms, the administration routes include oral, sublingual, injection, etc.

[0058] In an embodiment of the present invention, the pharmaceutical composition provided by the present invention is an oral solid preparation, preferably tablets. In addition to the active ingredient, the steroidal compound of the present invention, this oral solid preparation also contains pharmaceutical excipients. The said pharmaceutical excipients are all conventional pharmaceutical excipients in the art, including fillers (also known as diluents), disintegrants, binders or wetting agents, lubricants (including glidants), etc. The dosage of the pharmaceutical excipients can be according to the conventional dosage.

[0059] The said fillers generally include lactose, microcrystalline cellulose, mannitol, pregelatinized starch, starch, sucrose, dextrin, sorbitol, calcium carbonate, calcium hydrogen carbonate, hydroxypropyl methylcellulose and ethylcellulose, etc. They can be used alone or in combination.

[0060] The said disintegrants generally include starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethyl starch, cross-linked sodium carboxymethylcellulose, cross-linked povidone and low-substituted hydroxypropyl cellulose, etc. They can be used alone or in combination.

[0061] The said binders or wetting agents generally include povidone (polyvinylpyrrolidone), hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethylcellulose, polyethylene glycol, starch paste, water and ethanol solutions of various concentrations, etc. They can be used alone or in combination.

[0062] The said lubricants generally include zinc stearate, magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, sucrose fatty acid ester, colloidal silicon dioxide, stearic acid and solid polyethylene glycol, etc. They can be used alone or in combination.

[0063] If necessary, other excipients can also be added to the above composition, such as sweeteners (such as aspartame, steviol glycosides, etc.), colorants (such as yellow iron oxide, red iron oxide, etc.), stabilizers (such as citric acid, lactic acid, malic acid, etc.), pH regulators (such as sodium bicarbonate, fumaric acid, citric acid, etc.).

[0064] If necessary, the above composition can also contain other suitable active ingredients.

[0065] The preparation of the above oral solid preparations can be carried out according to the conventional methods for preparing oral solid preparations in the technical field, such as: tablets can be prepared by wet granulation tableting, dry granulation tableting, fluidized bed granulation tableting, direct powder compression tableting, etc. When the oral solid preparation is a tablet, it can be further coated as needed to form film-coated tablets or sugar-coated tablets. The coating materials include cellulose-based, acrylic resin-based, and sugar-based materials, such as hydroxypropyl methylcellulose and sucrose, etc., and plasticizers, anti-adhesives, and light-blocking agents can also be added thereto.

[0066] The dosage of the above composition is adjusted according to the nature and severity of the patient's condition, the administration route, and the patient's age, weight, etc.

[0067] "Treatment" of any disease or disorder means improving the disease or disorder (i.e., preventing (inhibiting) the disease or reducing the manifestation, degree, or severity of at least one of its clinical symptoms). In another embodiment, "treatment" means improving at least one physical parameter, which may not be distinguishable to the subject. In yet another embodiment, "treatment" means physically (e.g., stabilization of distinguishable symptoms), physiologically (e.g., stabilization of physical parameters), or regulating the disease or disorder in both ways. In additional embodiments, "treatment" involves slowing the progression of the disease.

[0068] Compared with the prior art, the present invention at least obtains the following beneficial technical effects, for example:

[0069] The steroidal compound of the present invention has an α configuration at the C-10 position methyl, and both the C-3 position and the C-17 position are imidazole or triazole ester groups, and has a strong inhibitory effect on prostate cancer cells, colon cancer cells, lung cancer cells, and pancreatic cancer cells.

[0070] The IC50 of the steroidal compound of the present invention against human prostate cancer cells is within the range of <100 μM, preferably within the range of <50 μM, and more preferably within the range of <20 μM. The IC50 of the steroidal compound of the present invention against human colon cancer cells is within the range of <100 μM, preferably within the range of <50 μM, and more preferably within the range of <20 μM. The IC50 of the steroidal compound of the present invention against human non-small cell lung cancer cells is within the range of <100 μM, more preferably within the range of <50 μM, and more preferably within the range of <15 μM. The IC50 of the steroidal compound of the present invention against human pancreatic cancer cells is preferably within the range of <100 μM, more preferably within the range of <50 μM, and more preferably within the range of <25 μM.

[0071] Compared with the C-10 methyl group being in the β configuration, the C-10 methyl group of the steroidal compounds of the present invention is in the α configuration. Moreover, since both the C-3 and C-17 positions are imidazole or triazole ester groups, the reactivity with in vivo enzymes (such as 3α-steroid dehydrogenase / 3α-hydroxysteroid oxidoreductase, cholesterol oxidase, etc.) is reduced or there is no reactivity, the molecular stability is better, it is not easily degraded by the action of enzymes, and it is expected to have a slower metabolism in vivo and a longer duration of drug effect.

[0072] In addition, the C-10 methyl group of the steroidal compounds of the present invention is in the α configuration. Compared with the C-10 methyl group being in the β configuration, the steroidal compounds of the present invention have high bioavailability and fewer other side effects. Detailed implementation manners

[0073] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art and will not be elaborated herein.

[0075] In the present invention, the term "alkyl" refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon group. In some embodiments, the alkyl has 1-8 carbon atoms (denoted as C 1-8 alkyl). In some embodiments, the alkyl has 1-6 carbon atoms (C 1-6 alkyl). In some embodiments, the alkyl has 1-3 carbon atoms (C 1-3 alkyl). C 1-6 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, and various branched isomers thereof. The alkyl can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available connection point, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, carbocyclic group, alkoxy, halogen, hydroxyl, oxo group, amino, amine group, acyl group, acyloxy group or ester group.

[0076] The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond. In some embodiments, the alkenyl is C 2-8 alkenyl. In some embodiments, the alkenyl is C 2-6 alkenyl. In some embodiments, the alkenyl is C 2-4 alkenyl. The carbon-carbon double bond can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). C 2–6 Non-limiting examples of alkenyl include vinyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl, etc. The alkenyl can be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxy, oxo, amino, amine, acyl, acyloxy or ester.

[0077] The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond. In some embodiments, the alkynyl is C 2-8 alkynyl. In some embodiments, the alkynyl is C 2-6 alkynyl. In some embodiments, the alkynyl is C 2-4 alkynyl. Non-limiting examples include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, etc. The alkynyl can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxy, oxo, amino, amine, acyl, acyloxy or ester.

[0078] "Alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl and carbocyclic are defined as above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopentyloxy, cyclohexyloxy, etc.

[0079] "Ester group" refers to the group -COOR c , where R c is alkyl, alkenyl, alkynyl, carbocyclic or aryl as defined herein.

[0080] "Haloalkyl" refers to an alkyl group substituted by one or more halogens, where alkyl is defined as above.

[0081] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0082] When listing a range of numerical values, it is intended to include each numerical value and sub-range within the said range. For example, "C 1-6 alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl group.

[0083] "Plural" means more than two, such as 2 - 5, 2 - 3, 2, 3, 4, or 5, etc.

[0084] Different expressions such as "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", "X is A, B and C", etc. all express the same meaning, that is, X can be any one or more of A, B, and C.

[0085] "Optional" or "optionally", "selectable" or "selectably" means that the subsequent described event or circumstance can but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur. For example, "aryl optionally substituted by alkyl" means that the alkyl may or may not be present, and this description includes the case where the aryl is substituted by alkyl and the case where the aryl is not substituted by alkyl.

[0086] "Substituted (substitution)" means that one or more hydrogen atoms in the group are independently replaced by the corresponding number of substituents. It goes without saying that the substituents are only at their possible chemical positions, and those skilled in the art can determine (by experiment or theory) the possible or impossible substitutions without excessive effort.

[0087] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Specifically, such salts are non - toxic and can be organic or inorganic acid addition salts and base addition salts.

[0088] The protection or de - protection reaction of functional groups is carried out according to known methods, such as the methods described in the following literature: Protective Groups in Organic Synthesis, 4th Edition (Theodora W. Greene, Peter G. M. Wuts), Wiley - Interscience (2007).

[0089] Examples of alcohol hydroxyl protecting groups include: ether-type protecting groups such as methoxymethyl ether (-OMOM), trimethylsilyl ether (-OTMS), tetrahydropyranyl ether, etc.; carboxylic acid ester-type protecting groups such as acetate (-OAc), etc.; sulfonic acid ester-type protecting groups such as p-toluenesulfonate (-OTs), etc.

[0090] Examples of ketone carbonyl protecting groups include: ketal-type protecting groups such as dimethyl ketal, etc.; cyclic ketal-type protecting groups such as 1,3-dioxolane, 1,3-dioxane, etc.; oxime-type protecting groups such as O-methyl oxime, etc.; hydrazone-type protecting groups such as N,N-dimethyl hydrazone, etc.

[0091] To further understand the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0092] Example 1

[0093]

[0094] In a 2000 mL three-necked flask, add 200 g of DHEA (3β-hydroxy-5-androsten-17-one) (i.e., Compound 1), 800 mL of DCM (dichloromethane), 140 g of triethylamine, displace with nitrogen 3 times, add 4 g of DMAP (4-dimethylaminopyridine), and displace with nitrogen 3 times. After stirring at room temperature to dissolve it completely, add 140 g of acetic anhydride dropwise, and finish dropping in 2 h. After dropping, continue to stir for 15 min. Monitor the reaction of the raw materials by TLC. After the reaction is complete, add 40 mL of methanol and continue to stir for 30 min. Wash once with 200 mL of 5% hydrochloric acid and once with 200 mL of 5% sodium bicarbonate solution. The organic phase is rotary evaporated under reduced pressure at a water bath temperature of 35 °C, and DCM is replaced with methanol. Filter, wash the filter cake once with a small amount of methanol, and dry it in a blast drying oven at 50 °C for 12 h to obtain 200 g of a white solid (i.e., Compound 2), with a mass yield of 100%. In this article, "mass yield" refers to the ratio of the mass of the obtained product to the mass of the raw material. Taking the above example as an example, a mass yield of 100% means that the ratio of the mass of the obtained white solid (i.e., Compound 2) to the mass of the added raw material (i.e., Compound 1) is 100%.

[0095]

[0096] In a 2000 mL three-necked flask, add 200 g of DHEA acetate (i.e., the aforementioned obtained compound 2), 1200 mL of ethylene glycol, 4 g of PTS (p-toluenesulfonic acid), and 260 g of triethyl orthoformate. Stir at 50 °C and monitor the reaction of the raw materials by TLC until completion. Cool to room temperature, add 8 mL of triethylamine, and continue stirring for 30 min. Pour into 1600 mL of water and stir for 30 min. Filter, and wash the filter cake with a small amount of water twice. Dissolve the filter cake in 800 mL of dichloromethane, add 4 mL of triethylamine, and stir for 15 min. Separate the aqueous layer, and under reduced pressure, rotary evaporate the organic phase at a water bath temperature of 35 °C, and displace dichloromethane with methanol. Filter, wash the filter cake with a small amount of methanol once, and dry in a forced-air drying oven at 50 °C for 12 h to obtain 210 g of white solid 3 with a mass yield of 105%.

[0097]

[0098] In a 2000 mL three-necked flask, add 100 g of compound 3, 800 mL of cyclohexanone, introduce dry air, stir at 50 °C until fully dissolved, then add 32 g of NOP (N-hydroxyphthalimide) and 0.50 g of benzoyl peroxide. Monitor the reaction of the raw materials by TLC until completion. Cool to room temperature, and under reduced pressure, rotary evaporate to dryness at a water bath temperature of 55 °C. Add 200 mL of dichloromethane and 500 mL of petroleum ether, and stir at room temperature for 30 min. Filter, and wash the filter cake with a small amount of petroleum ether once. Add 60 g of triethylamine to the filtrate, dropwise add 60 g of acetic anhydride in an ice-water bath, and continue stirring for 30 min. Let stand for 12 h. Under reduced pressure, rotary evaporate to dryness at a water bath temperature of 55 °C. After fully dissolving with dichloromethane, under reduced pressure, rotary evaporate at a water bath temperature of 35 °C, and displace dichloromethane with methanol. Filter, wash the filter cake with a small amount of methanol once, and dry in a forced-air drying oven at 50 °C for 12 h to obtain 75 g of white solid 4 with a mass yield of 75%.

[0099]

[0100] In a 2000 mL three-necked flask, add 100 g of compound 4, 66 g of TSH (p-toluenesulfonyl hydrazide), 400 mL of toluene, and 600 mL of n-hexane, heat to reflux with water separation under stirring. Monitor the reaction of the raw materials by TLC until completion. Cool to room temperature, and under reduced pressure, rotary evaporate to dryness at a water bath temperature of 55 °C. Add 660 mL of methanol and 130 mL of n-hexane, and stir at room temperature for 30 min. Filter, wash the filter cake with a small amount of methanol once, and dry in a forced-air drying oven at 50 °C for 12 h to obtain 126 g of white solid 5 with a mass yield of 126%.

[0101]

[0102] In a 1000 mL three-necked flask, 5.6 g of lithium amide and 175 mL of chlorobenzene were added, and ammonia was removed under reduced pressure. After the ammonia was removed completely, a chlorobenzene solution of Compound 5 (35 g of Compound 5 and 350 mL of chlorobenzene) was added. The flask was transferred into an oil bath at 120 °C and stirred for heat preservation for 1 h. The reaction of the raw materials was monitored by TLC. After cooling to room temperature, under stirring in an ice-water bath, the pH was adjusted to 6 - 8 with 5% phosphoric acid. Liquid separation was carried out. The aqueous layer was extracted once with 40 mL of chlorobenzene, and the organic layers were combined and washed once with water. The organic layer was dried with anhydrous sodium sulfate for 2 h. After filtration, the filtrate was rotary evaporated to dryness under reduced pressure in a water bath at 55 °C. After being fully dissolved with dichloromethane, it was rotary evaporated under reduced pressure in a water bath at 35 °C, and dichloromethane was replaced with methanol. After filtration, the filter cake was washed once with a small amount of methanol and dried in a forced-air drying oven at 50 °C for 12 h to obtain 19 g of off-white solid 6 with a mass yield of 54.3%.

[0103]

[0104] Weigh 50 g of Compound 6 and 0.5 g of BHT (antioxidant), add them to 1.5 L of ethyl acetate for dissolution, pour it into a photoreactor, turn on the internal cooling system, irradiate with a 260 - 270 nm LED ultraviolet lamp (100 W) for 3 h, then irradiate with a 310 - 330 nm LED ultraviolet lamp (100 W) for 3 h. Take a sample and monitor the reaction by HPLC. After the reaction was completed, the reaction solution was concentrated to an oily state, 150 ml of methanol was added, and Compound 6 was precipitated by stirring. It was filtered by suction to recover Compound 6 (20 g). After the mother liquor was concentrated to dryness, it was mixed with silica gel and passed through a chromatography column to obtain 10.5 g of Compound 7.

[0105] After detection, the 1 1H NMR of Compound 7 was: 1H NMR (400 MHz, CDCl3) δ 5.57 (dd, J = 5.5, 2.1 Hz, 1H), 5.41 (dt, J = 5.3, 2.5 Hz, 1H), 4.71 (tt, J = 11.4, 4.5 Hz, 1H), 4.03–3.80 (m, 4H), 2.51 (ddd, J = 14.2, 4.8, 2.2 Hz, 1H), 2.42–2.25 (m, 2H), 2.08–1.98 (m, 5H), 1.97–1.86 (m, 3H), 1.87–1.76 (m, 1H), 1.76–1.63 (m, 3H), 1.61–1.47 (m, 4H), 1.44–1.31 (m, 1H), 0.96 (s, 3H), 0.79 (s, 3H).

[0106] HRMS mass spectrum (EI) m / z: The theoretically calculated value was 373.5, and the measured value was 372.9.

[0107]

[0108] In a 250 mL three-necked flask, 4.2 g of compound 7, 42 g of acetone, 0.84 g of PTS, and 21 g of water were added. Stir at room temperature for 12 h. Monitor the reaction of the raw materials by TLC. Remove acetone by rotary evaporation under reduced pressure. Extract with 20 mL of dichloromethane three times, combine the organic layers, wash once with water, and rotary evaporate to dryness at 45 °C in a water bath to obtain 2.9 g of compound 8 as a pale yellow oil, with a mass yield of 69.0%.

[0109]

[0110] Add 5 g of compound 8, 75 mL of toluene, and 10 mL of cyclohexanone to the reaction flask. Heat to 110 °C and reflux for 1 hour to separate water. Cool to 50 - 60 °C under nitrogen protection, add 1.35 g of aluminum isopropoxide, and continue to heat to 90 - 100 °C for 1 hour until the reaction is complete. Cool down, add 40 mL of 3.5% hydrochloric acid, separate the layers, wash the organic layer once with water, dry, and filter. Add 10 mL of trimethyl orthoacetate and 0.25 g of p-toluenesulfonic acid to the filtrate. After the reaction is complete, add a small amount of triethylamine to adjust the pH to 7 - 8, concentrate to obtain an oil. Add 20 mL of methanol, stir to precipitate a solid, and filter. Dissolve the filter cake in 30 mL of dichloromethane, add 30 mL of 40% hydrogen chloride / ethanol solution, react completely, transfer to 50 mL of ice water, separate the layers, extract the aqueous phase with dichloromethane, combine the organic layers, concentrate, and replace with methanol to obtain 2.6 g of compound 9, with a mass yield of 62%.

[0111] After detection, the 1 1H NMR of compound 9 was: 1H NMR (400 MHz, CDCl3) δ 6.40–6.06 (m, 2H), 5.70 (s, 1H), 2.71–2.39 (m, 4H), 2.29 (ddd, J = 13.2, 5.3, 2.1 Hz, 1H), 2.23–2.01 (m, 3H), 1.94 (tt, J = 5.1, 4.2 Hz, 2H), 1.90–1.60 (m, 5H), 1.60–1.41 (m, 1H), 1.26 (s, 3H), 0.98 (s, 3H).

[0112] HRMS mass spectrum (EI) m / z: Theoretical calculated value 284.1: Measured value: 284.9.

[0113] Example 2

[0114]

[0115] In a three-necked flask, 1 g of Compound 9, 20 ml of dioxane, and 0.1 g of Raney nickel were added. Hydrogenation reaction was carried out under normal pressure at 10 - 20 °C by passing hydrogen for 16 hours. After the reaction was complete, filtration was performed. The filtrate was concentrated to dryness, 2 ml of methanol was added for pulping, filtration was carried out, and drying gave 0.73 g of Compound 10 with a mass yield of 73%.

[0116] Upon detection, the 1 1H NMR of Compound 10 was: 1H NMR (400 MHz, CDCl3) δ 5.75 (s, 1H), 2.60–2.28 (m, 5H), 2.24–2.13 (m, 3H), 2.05 (d, J = 6.2 Hz, 4H), 1.92–1.82 (m, 1H), 1.77 (d, J = 3.6 Hz, 2H), 1.73–1.59 (m, 5H), 1.51 (ddt, J = 14.2, 9.7, 6.0 Hz, 2H), 1.30 (s, 3H), 0.94 (s, 3H).

[0117] HRMS mass spectrum (EI) m / z: Theoretical calculated value 286.1: Measured value: 287.2.

[0118]

[0119] 500 mg of Compound 10, 1 ml of acetic anhydride, and 10 mg of p-toluenesulfonic acid were added to the reaction flask. The reaction was carried out at 40 °C under nitrogen protection. After the reaction was completed, 1 ml of methanol was added and stirred, concentrated, dichloromethane and water were added for liquid separation, and the organic layer was concentrated. Column chromatography (petroleum ether:ethyl acetate = 8:1 - 5:1) gave 230 mg of Compound 11 with a yield of 46%.

[0120] Upon detection, the 1 1H NMR of Compound 11 was: 1H NMR (400 MHz, CDCl3) δ 5.74 (d, J = 2.1 Hz, 1H), 5.63–5.53 (m, 1H), 2.14 (s, 3H), 2.04–1.87 (m, 5H), 1.86–1.58 (m, 8H), 1.58–1.49 (m, 3H), 1.49–1.36 (m, 2H), 1.36–1.23 (m, 1H), 1.02 (s, 3H), 0.95 (s, 3H).

[0121] HRMS mass spectrum (EI) m / z: Theoretical calculated value 328.4: Measured value: 328.9.

[0122]

[0123] Add 60 mg of calcium chloride to a reaction flask, dissolve it in 3 ml of absolute ethanol, cool down to -5 to 5 °C, add sodium borohydride, stir for 0.5 hour, add a 6 ml dichloromethane solution of 200 mg of compound 11, after the reaction is complete, add 5% dilute hydrochloric acid until the reaction solution becomes clear, separate the layers, extract the aqueous layer with dichloromethane, and perform column chromatography (petroleum ether:ethyl acetate = 8:1 to 3:1) to obtain 125 mg of compound 12.

[0124] After detection, the 1 1H NMR of compound 12 is: 1H NMR (400 MHz, CDCl3) δ 5.33 (dd, J = 11.7, 9.8 Hz, 1H), 3.68 (t, J = 8.6 Hz, 1H), 3.55 (dtd, J = 21.5, 10.7, 5.3 Hz, 1H), 2.41–2.10 (m, 3H), 2.09–1.97 (m, 2H), 1.90 (ddd, J = 16.8, 9.0, 4.2 Hz, 3H), 1.86–1.72 (m, 4H), 1.57 (dddd, J = 18.3, 15.6, 11.0, 5.0 Hz, 12H), 1.45–1.31 (m, 3H), 1.31–1.22 (m, 2H), 1.18 (s, 3H), 1.09–0.93 (m, 2H), 0.80 (s, 3H).

[0125] HRMS mass spectrum (EI) m / z: Theoretical calculated value 290.4: Measured value: 290.9.

[0126]

[0127] Add 100 mg of compound 12, 341 mg of carbonyldiimidazole, and 5 ml of acetonitrile to a reaction flask, heat up to 60 °C and react for 8 hours. After the reaction is complete, concentrate, add ethyl acetate and water to separate the layers, evaporate the organic layer to dryness, and perform column chromatography (petroleum ether:ethyl acetate = 3:1 to 2:1) to obtain 84 mg of TM35.

[0128] After detection, the 1 1H NMR of compound TM35 is: 1H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 4.2 Hz, 2H), 7.42 (d, J = 4.4 Hz, 2H), 7.07 (s, 2H), 5.47 (s, 1H), 4.88 (dd, J = 21.5, 12.6 Hz, 2H), 2.74–2.41 (m, 2H), 2.40–2.17 (m, 3H), 1.99 (dd, J = 23.3, 9.6 Hz, 5H), 1.74 (qdd, J = 21.6, 16.8, 5.8 Hz, 12H), 1.26 (d, J = 4.3 Hz, 4H), 0.98 (s, 3H).

[0129] For compound TM35 13 13C NMR is as follows: 13C NMR (101 MHz, CDCl3) δ 148.60, 148.11, 137.07 (d, J =, 136.73, 130.56, 122.12, 117.10, 87.55, 78.69, 43.91, 42.70, 42.24, 39.23, 37.71, 36.66, 34.86, 32.77, 27.21, 26.95, 26.22, 24.09, 22.43, 11.83.

[0130] HRMS mass spectrum (EI) m / z: Theoretical calculated value 478.5: Measured value: 478.9.

[0131] Example 3

[0132]

[0133] In a three-necked flask, 1 g of compound 9, 5 ml of tetrahydrofuran, and 0.1 g of 10% palladium on carbon were added. Hydrogenation reaction was carried out at normal pressure with hydrogen at 10 - 20 °C for 16 hours. After the reaction was complete, filtration was carried out. The filtrate was concentrated to dryness, 2 ml of methanol was added for pulping, filtration was carried out, and drying gave 0.86 g with a mass yield of 86%.

[0134] After detection, for compound 13 1 1H NMR is as follows: 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 1H), 2.33 (dd, J = 5.6, 3.1 Hz, 4H), 2.25–1.97 (m, 5H), 1.93 (ddd, J = 11.1, 5.8, 2.8 Hz, 1H), 1.90–1.66 (m, 7H), 1.66–1.47 (m, 4H), 1.42 (dd, J = 13.6, 6.8 Hz, 2H), 1.31–1.21 (m, 1H), 1.18 (s, 3H), 0.98 (s, 3H).

[0135] For compound 13 13 13C NMR is as follows: 13C NMR (101 MHz, CDCl3) δ 212.83, 47.22, 46.48, 43.18, 41.65, 37.36, 36.21, 35.77, 35.20, 33.40, 30.48, 26.57, 25.28, 22.75, 19.73, 16.24.

[0136] HRMS mass spectrum (EI) m / z: Theoretical calculated value 288.4: Measured value: 288.9.

[0137]

[0138] 1 g of compound 13 was added to a reaction flask and dissolved in 10 ml of dichloromethane and 10 ml of ethanol. 0.6 g of sodium borohydride was added in batches and the reaction was completed for 0.5 hour. 5% dilute hydrochloric acid was added dropwise until the reaction solution became clear. The layers were separated and the aqueous layer was extracted three times with 5 ml of dichloromethane. The organic layers were combined, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 10:1 to 3:1) to obtain 0.43 g of compound 14 and 0.25 g of compound 15.

[0139]

[0140] To a reaction flask were added 200 mg of compound 14, 641 mg of carbonyldiimidazole, and 10 ml of acetonitrile. The temperature was raised to 60°C and the reaction was carried out for 8 hours. After the reaction was completed, the mixture was concentrated, and ethyl acetate and water were added to separate the layers. The organic layer was concentrated to dryness and purified by column chromatography (petroleum ether:ethyl acetate = 3:1 to 2:1) to obtain 153 mg of TM36.

[0141] After testing, the compound TM36 1 H NMR is: 1H NMR (400MHz, CDCl3) δ8.05(s,2H),7.34(s,2H),7.00(dd,J=3.0,0.7Hz,2H),4.83(d,J=8.2Hz,2H),2.46–2.14(m,2H),2.08–1.87(m,5H),1 .86–1.76(m,3H),1.77–1.60(m,6H),1.62–1.50(m,4H),1.44(ddd,J=31.5,17.8,8.5Hz,3H),1.33–1.10(m,3H),1.05(s,3H),0.86(s,3H).

[0142] Compound TM36 13 C NMR is: 13C NMR (101 MHz, CDCl3) δ 148.61, 148.19, 137.05, 130.55, 117.08, 87.48, 79.26, 43.23, 42.62, 41.94, 36.64, 35.48, 34.22, 32.12, 31.30, 29.40, 27.24, 26.75, 24.38, 22.97, 21.35, 20.94, 11.47.

[0143] HRMS mass spectrum (EI) m / z: theoretical calculated value 480.6: measured value: 480.9.

[0144]

[0145] Add 100 mg of compound 15, 321 mg of carbonyldiimidazole, and 5 ml of acetonitrile to a reaction flask. Heat the mixture to 60 °C and react for 8 hours. After the reaction is completed, concentrate the mixture, add ethyl acetate and water for liquid separation. Evaporate the organic layer to dryness, and perform column chromatography (petroleum ether:ethyl acetate = 3:1 - 2:1) to obtain 79 mg of TM37.

[0146] Upon detection, the 1 1H NMR of compound TM37 is as follows: 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 2H), 7.35 (s, 2H), 7.00 (d, J = 4.3 Hz, 2H), 4.83 (d, J = 8.4 Hz, 2H), 2.35–2.10 (m, 1H), 2.02–1.81 (m, 4H), 1.79–1.40 (m, 13H), 1.38 (dd, J = 18.8, 12.6 Hz, 2H), 1.35–1.06 (m, 8H), 0.98 (s, 4H), 0.86 (s, 3H).

[0147] The 13 13C NMR of compound TM37 is as follows: 13C NMR (101 MHz, CDCl3) δ 148.63, 148.23, 137.09, 130.59, 117.11, 87.51, 78.42, 45.28, 44.69, 43.27, 42.62, 38.37, 37.32, 35.19, 33.25, 31.43, 30.20, 29.69, 28.34, 26.75, 24.93, 24.34, 21.27, 16.27, 14.12, 11.61.

[0148] HRMS mass spectrum (EI) m / z: theoretical calculated value 480.6; measured value: 480.9.

[0149] In addition to the compounds prepared in the above examples, through the same / similar preparation processes as those in the foregoing examples, the present invention also prepares some other example compounds. All the example compounds are listed as follows:

[0150]

[0151] Example 4

[0152] I. Pharmacological experiment: inhibitory effect on cancer cells

[0153] 1. Test method

[0154] 1.1 Experimental grouping and sample preparation

[0155] For each compound sample of the embodiments, a stock solution of 100 mM was prepared using dimethyl sulfoxide (DMSO) as the solvent, and then diluted with the corresponding complete medium for each cell culture to obtain a working solution with concentrations of 100, 30, 10, 3, 1, and 0.3 μM. A solvent control group, positive control groups with different concentrations, and sample treatment groups with different concentrations were set up.

[0156] 1.2 Cell culture

[0157] The culture medium for human prostate cancer cells (DU 145) was MEM medium containing 10% fetal bovine serum (FBS); the culture medium for human colon cancer cells (HCT-116) was McCoy's 5A containing 10% FBS; the culture medium for human non-small cell lung cancer cells (A549) was Ham's F-12K containing 10% FBS, and the culture conditions were all 37 °C and 5% CO2; the culture medium for human pancreatic cancer cells (PANC-1) was DMEM medium containing 10% FBS. When the growth state was good, subculture was performed every 2 days at a subculture ratio of 1:3. In the laminar flow hood, the culture medium was discarded, and the cells were washed twice with 1×PBS, then 600 μL of 0.25% trypsin was added for digestion. After about 1 - 3 minutes, when the cells detached, 3 mL of the corresponding medium containing 10% FBS for each cell was added to terminate the digestion of trypsin. The cells were blown into a single-cell suspension, transferred into an EP tube, and centrifuged at 1000 rpm for 5 minutes. The culture medium was discarded, and the cells were resuspended with fresh medium and inoculated into a new culture flask at a certain ratio (cell density was about 10 5 / mL), and then placed in an incubator at 37 °C and 5% CO2 for culture.

[0158] 1.3 Cell seeding

[0159] Cells in good growth state were taken, digested and collected routinely. The density of DU 145 cells was adjusted to 2×10 4 cells / mL, the density of HCT-116 cells was adjusted to 2×10 4 cells / mL, the density of A549 cells was adjusted to 3×10 4 cells / mL, and the density of PANC-1 cells was adjusted to 4×10 4 cells / mL. Each cell suspension was seeded into a 96-well culture plate at a density of 100 μL / well, and shaken 10 times crosswise to evenly spread the cells on the bottom of the wells. The culture plate was placed in a CO2 incubator for 24 h.

[0160] 1.4 Cell treatment

[0161] Take the working solution of the example compound sample prepared in step 1.1, and add 100 μL / well to the corresponding wells at different concentrations, so that the final volume in each well is 200 μL (100 μL cell culture medium, 100 μL sample working solution), and the final concentrations are 50, 15, 5, 1.5, 0.5, 0.15 μM respectively. At the same time, set a solvent control group and a positive control group with concentrations of 50, 15, 5, 1.5, 0.5, 0.15 μM respectively. The number of replicate wells in each group is 3. Incubate at 37 °C and 5% CO2 for 72 h.

[0162] 1.5 Detection of OD value of cell proliferation

[0163] After 72 h of cell treatment, add 20 μL of thiazolyl blue (MTT) to each well, and continue to culture at 37 °C and 5% CO2 for 4 h. Carefully aspirate the liquid in each well, and add 150 μL / well of DMSO to the wells, and shake for 10 min.

[0164] Set the average OD value of wells A1-H1 (8 wells) on the microplate reader as the zero adjustment value, and detect the OD value of each well at 492 nm.

[0165] 1.6 Result calculation

[0166] Set the OD value of the solvent control group as 100% cell viability, and the ratio of the OD value of the remaining groups to the OD value of the solvent control group is the relative cell viability. Evaluate the activity of the sample on DU 145 or HCT-116 or A549 or PANC-1 cells by the cell proliferation rate. If the proliferation inhibition rate > 100% appears, it is determined as a systematic error and calculated as 100%.

[0167] The formula for calculating the inhibition rate is: Inhibition rate (%) = (1 - ODsample / ODsolvent) × 100%

[0168] Use SPSS software to calculate the half-maximal inhibitory concentration (IC50).

[0169] 2. Experimental results

[0170] Table 1 IC50 values of compounds in some examples and comparative examples against various cancer cells

[0171]

[0172] Note: 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.

[0173] The compounds of the present invention, with the C-10 methyl group in the α configuration, have inhibitory effects on prostate cancer cells, colon cancer cells, lung cancer cells, and pancreatic cancer cells. For the compounds of each example of the present invention, the IC50 against human prostate cancer cells is in the range of <100 μM, preferably in the range of <50 μM, and more preferably in the range of <20 μM. For each example of the present invention, the IC50 against human colon cancer cells is in the range of <100 μM, preferably in the range of <50 μM, and more preferably in the range of <20 μM. For each example of the present invention, the IC50 against human non-small cell lung cancer cells is in the range of <100 μM, more preferably in the range of <50 μM, and more preferably in the range of <15 μM. For the compounds of each example of the present invention, the IC50 against human pancreatic cancer cells is preferably in the range of <100 μM, more preferably in the range of <50 μM, and more preferably in the range of <25 μM.

[0174] In addition, the applicant points out that in the prior art, abiraterone acetate (or abiraterone) is clinically used in combination with prednisone to treat prostate cancer. However, the present application unexpectedly discovers and confirms through experiments (as shown in the results of Table 1) that abiraterone acetate not only has an anti-prostate cancer effect, but also shows unexpected inhibitory effects on colon cancer cells, lung cancer cells, and human pancreatic cancer cells. In particular, the IC50 of abiraterone acetate against human colon cancer cells is in the range of <10 μM, the IC50 of abiraterone acetate against human non-small cell lung cancer cells is in the range of <50 μM; the IC50 of abiraterone acetate against human pancreatic cancer cells is in the range of <50 μM.

[0175] Furthermore, from the results of Table 1, it can be seen that for the compound TM35 of the present invention, the IC50 against human prostate cancer cells is in the range of <20 μM, and the IC50 against human colon cancer cells is in the range of <20 μM, showing good inhibitory effects on these two types of cancers; the IC50 against human non-small cell lung cancer cells is in the range of <15 μM, and the IC50 against human pancreatic cancer cells is in the range of <25 μM, indicating that the compound TM35 of the present invention has an even better inhibitory effect on these two types of cancers (i.e., human non-small cell lung cancer cells and human pancreatic cancer cells) than abiraterone acetate. This fully shows that the compound TM35 of the present invention shows good inhibitory effects on human prostate cancer cells, human colon cancer cells, human non-small cell lung cancer cells, and human pancreatic cancer cells.

[0176] In addition, due to the C-10 methyl group of the compounds of the present invention being the inverted α configuration, and both the C-3 and C-17 positions being imidazole or triazole ester groups, the reaction activity with in vivo enzymes (such as 3α-steroid dehydrogenase / 3α-hydroxysteroid oxidoreductase, cholesterol oxidase, etc.) is reduced or there is no reaction activity, the molecular stability is better, it is not easily degraded by the action of enzymes, and it is expected to have a slower metabolism in vivo and a longer duration of drug effect.

[0177] The above are only embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A steroid compound, characterized in that, The steroid compound has a structure of the following formula or a pharmaceutically acceptable salt thereof: Among them, R1 and R2 are imidazolyl groups; represents a single bond or a double bond.

2. The steroid compound according to claim 1, wherein The steroid compound has the following structural formula:

3. A method for preparing a steroid compound according to any one of claims 1 to 2, characterized in that, The method comprises using an intermediate represented by the following formula as a raw material and connecting an imidazole ester group at the C-3 position and the C-17 position; 4. The preparation method according to claim 3, wherein The intermediate is prepared by a method comprising the following steps: subjecting a compound of the following formula to photochemical conversion to invert the methyl group at the C-10 position from the β configuration to the α configuration: wherein, R 11 is selected from -OH or OAc; R 12 is selected from =O or 5. Use of the steroid compound according to any one of claims 1 to 2 in the preparation of a medicament for treating cancer.

6. The use according to claim 5, wherein The cancer includes prostate cancer, colon cancer, lung cancer or pancreatic cancer.

7. A pharmaceutical composition, characterized in that, Comprising the steroid compound according to any one of claims 1 to 2 and a pharmaceutically acceptable excipient.

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