4-azasteroid compound, preparation method, use and pharmaceutical composition thereof

Abstract

The application discloses a 4-azasteroid compound, wherein the C-10 position methyl is in alpha configuration, and the 4-azasteroid compound has strong inhibiting effect on prostate cancer and colon cancer cells in addition to the drug activity of finasteride / dutasteride. The preparation method of the 4-azasteroid compound is simple in operation, high in efficiency, low in cost, environment-friendly, high in product purity, free of toxic by-products, and high in yield, and is suitable for large-scale industrial production. The application further discloses the use of the 4-azasteroid compound in the preparation of a drug for inhibiting cancer cells, and a pharmaceutical composition composed of the 4-azasteroid compound, which is verified by pharmacological experiments to have strong inhibiting effect on prostate cancer and colon cancer cells, and has a good application prospect in the preparation of the drug for inhibiting cancer cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a 4-azasteroid compound, a preparation method and use thereof, and a pharmaceutical composition. BACKGROUND

[0002] Finasteride, with a chemical name of N-tert-butyl-3-oxo-4-aza-5alpha-androst-1-ene-17beta-carboxamide, has a chemical formula of C 23 H 36 N2O2, and a structural formula of: Finasteride is a specific inhibitor of intracellular enzyme type II 5alpha-reductase in the process of testosterone metabolism into dihydrotestosterone. 5alpha-reductase promotes the conversion of testosterone into dihydrotestosterone (DHT) in the prostate, causing prostate hypertrophy. Finasteride reduces the serum dihydrotestosterone level by inhibiting the enzyme, thereby reducing the prostate volume and increasing the urine flow, and is clinically used for treating benign prostatic hypertrophy. On the other hand, DHT is the main cause of male hair loss (androgenic alopecia), and finasteride is successfully used for treating male hair loss by blocking the conversion of testosterone into DHT.

[0003] Dutrsteride, with a chemical name of N-{2,5-bis(trifluoromethyl)phenyl}-3-oxo-4-azasteroid-5alpha-androst-1-ene-17beta-carboxamide, has a chemical structure of: is a drug developed by GSK for preventing and treating benign prostatic hyperplasia. Dutrsteride belongs to 5alpha-reductase inhibitors, and is also a drug capable of simultaneously inhibiting two types of 5alpha-reductase. Dutrsteride has a more rapid action and more significant effect than finasteride, and has the advantage of dual inhibition.

[0004] At present, finasteride and dutrsteride and related compounds thereof have not been found to be applied to inhibiting cancer cells. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a 4-azasteroid compound and a preparation method thereof, and to provide the use of the 4-azasteroid compound in preparing a drug for inhibiting cancer cells, and a pharmaceutical composition composed of the 4-azasteroid compound.

[0006] To solve the above technical problem, the technical solution provided by the present application is as follows:

[0007] A 4-azasteroid compound has the following structure (as shown in formula I) or a pharmaceutically acceptable salt thereof: Figure 1

[0008] ​

[0009] wherein R is selected from C 1-5 alkyl, C 1-5 haloalkyl, phenyl, C 1-3 alkyl-substituted phenyl or C 1-3 haloalkyl-substituted phenyl;

[0010] R1, R2, R3 are the same or different, 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;

[0011] m, n and i are each independently selected from 0, 1 or 2.

[0012] The 4-azasteroid compound is preferably, wherein R is selected from C The halogen is selected from F, Cl, Br or I.

[0013] Preferably, m, n and i are all 0.

[0014] More preferably, the 4-azasteroid compound has the following structure of Formula II or Formula III:

[0015]

[0016] Based on the overall inventive concept, the present application also provides a method for preparing a 4-azasteroid compound, comprising the following steps:

[0017] amide reaction of a compound of Formula IV with NH2-R to obtain a compound having the structure of Formula I; or

[0018] amide reaction of a compound of Formula V with NH2-R followed by 1,2-dehydrogenation, or 1,2-dehydrogenation followed by amide reaction of NH2-R to obtain a compound having the structure of Formula I;

[0019] The structural formula of Formula IV and Formula V is as follows:

[0020]

[0021] wherein R is selected from C 1-5 alkyl, C 1-5 haloalkyl, C 1-3 alkyl-substituted phenyl or C 1-3 haloalkyl-substituted phenyl;

[0022] R1, R2, R3 are the same or different, each independently selected from -OH, =O, halogen, amino, C1-5 Alkyl, C 1-5 Haloalkyl, C 1-5 Alkoxy, C 2-5 alkenyl, C 2-5 alkynyl or C 2-5 Ester group;

[0023] m, n, and i are each independently selected from 0, 1, or 2.

[0024] Preferably, in the above preparation method, the compound of formula V is compound 5, and the preparation method includes the following steps:

[0025] Compound 5 undergoes an amide reaction with tert-butylamine in the presence of thionyl chloride and a base to give compound 6, which is then dehydrogenated in the presence of DDQ and BSTFA to give compound II; or

[0026] Compound 5 was dehydrogenated in the presence of DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and BSTFA (bis(trimethylsilyl)trifluoroacetamide) to give compound 8. Then, compound 8 was amided with 3,5-ditrifluoromethylaniline in the presence of thionyl chloride and a base to give compound III.

[0027] The structural formulas of compounds 5, 6 and 8 are as follows:

[0028]

[0029] More preferably, the preparation method includes the following steps (the structures of compounds 1-9 are as follows: Figures 1-2 As shown):

[0030] (1) Dissolve compound 1 and base in a mixed solvent, slowly add aqueous solution of oxidant to carry out the reaction, filter out the salt after the reaction is complete, add sodium bisulfite aqueous solution to quench excess oxidant, remove organic solvent by vacuum distillation, cool to crystallize, filter, rinse with water, and dry with forced air to obtain compound 2.

[0031] (2) Compound 2 and amine reagent were added to the solvent to react. After the reaction was completed, drinking water was added, the organic solvent was removed by vacuum distillation, the crystals were cooled and crystallized, filtered and washed with water, and dried by blowing air to obtain compound 3.

[0032] (3) Dissolve compound 3 in a solvent, add a catalyst, replace with hydrogen, and carry out the reaction. After the reaction is complete, add an appropriate amount of drinking water, remove the organic solvent by vacuum distillation, cool to crystallize, filter, rinse with water, and dry with a forced air to obtain compound 4.

[0033] (4) Compound 4 is dissolved in 1,4-dioxane, an aqueous solution of a base is added, halogenated reagent is added slowly in batches, reaction is carried out, after reaction is completed, an aqueous solution of sodium bisulfite is added to quench the reaction, hydrochloric acid is used to adjust pH to acidity, 1,4-dioxane is recovered by normal pressure distillation, filtration is carried out, and air blowing drying is performed to obtain compound 5;

[0034] Steps (5) and (6) are optionally selected from the following two schemes:

[0035] Scheme 1:

[0036] (5) Compound 5 is dissolved in a solvent with a base, dichlorosulfoxide is added dropwise, after reaction under heat preservation, tert-butylamine is added dropwise, and the reaction is continued under heat preservation, after the reaction is completed, an aqueous solution of sodium hydroxide is added to quench the reaction, the organic phase is separated after adding water, and distilled under reduced pressure, and compound 6 is obtained by crystallization under cooling, filtration, and air blowing drying;

[0037] (6) Compound 6 is dissolved in a solvent, DDQ and BSTFA are added, and reaction is carried out, after reaction is completed, an aqueous solution of sodium bisulfite is added to quench the reaction, filtration is carried out, the organic phase is separated after water washing with an aqueous solution of sodium carbonate and an aqueous solution of sodium bisulfite, distilled under reduced pressure, replaced with acetone, crystallized under cooling, filtered, and air blowing dried to obtain compound 7, i.e., the 4-azasteroid compound of formula II;

[0038] Scheme 2:

[0039] (5) Compound 5 is dissolved in a solvent, DDQ and BSTFA are added, and reaction is carried out, after reaction is completed, an aqueous solution of potassium carbonate is added to extract the product, acetic acid is added for acidification, filtration is carried out, and air blowing drying is performed to obtain compound 8;

[0040] (6) Compound 8 is dissolved in a solvent with a base, dichlorosulfoxide is added dropwise, after reaction under heat preservation, 3,5-ditrifluoromethylaniline is added dropwise, and the reaction is continued under heat preservation, after the reaction is completed, an aqueous solution of sodium hydroxide is added to quench the reaction, the organic phase is separated after adding an appropriate amount of water, distilled under reduced pressure, crystallized under cooling, filtered, and air blowing dried to obtain compound 9, i.e., the 4-azasteroid compound of formula III.

[0041] Further preferably, in step (1), the oxidizing agent is potassium permanganate or a mixture of potassium permanganate and sodium periodate, the base is any one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate, the solvent of the base solution is a mixed solvent of tert-butyl alcohol or isopropyl alcohol and water, and the reaction temperature is 40-70°C;

[0042] In step (2), the amine reagent is ammonium formate or ammonium acetate, the solvent is toluene or xylene, and the reaction temperature is 100-140°C;

[0043] In step (3), the catalyst is palladium-carbon or active nickel, the solvent is formic acid or acetic acid, the reaction temperature is 20-40 DEG C, and the reaction pressure is 0.2-0.8 MPa;

[0044] In step (4), the halogenating agent is any one or more of bromine, dibromohydantoin, dichlorohydantoin, NBS and NCS, the base is sodium hydroxide or potassium hydroxide, the solvent is 1,4-dioxane, and the reaction temperature is 0-20 DEG C.

[0045] In steps (5) and (6) of the scheme one and the scheme two, the base is any one or more of triethylamine, pyridine and DMF, the solvent is any one or more of dichloromethane, chloroform and tetrahydrofuran, the temperature of the incubation reaction is 0-20 DEG C, and the time is 1.5-2.5 h; the solvent is toluene or xylene, and the reaction temperature is 100-140 DEG C.

[0046] Based on the overall inventive concept, the application further provides a use of the 4-azasteroid compound in the preparation of a drug for inhibiting cancer cells.

[0047] Preferably, the cancer cells include prostate cancer cells or colon cancer cells.

[0048] Based on the overall inventive concept, the application further provides a pharmaceutical composition comprising the 4-azasteroid compound and a pharmaceutically acceptable excipient.

[0049] Compared with the prior art, the application has the following beneficial effects:

[0050] 1. The 4-azasteroid compound has an alpha configuration at the C-10 position (and a beta H at the C-9 position), and the overall conformation is changed, so that, in addition to the drug activity of finasteride and dutasteride (which have a beta configuration at the C-10 position), the 4-azasteroid compound has a stronger inhibiting effect on prostate cancer and colon cancer cells, and has a good application prospect in the preparation of a drug for treating cancer.

[0051] 2. The 4-azasteroid compound has an IC 50 In the range of <50 μM, preferably in the range of <20 μM, and more preferably in the range of <12 μM. 50 In the range of <40 μM, preferably in the range of <15 μM, and more preferably in the range of <10 μM.

[0052] 3. Because the overall conformation is changed, the 4-azasteroid compound has a high solubility and a higher bioavailability.

[0053] 4. The preparation method of the present invention is simple to operate, highly efficient, low in cost, environmentally friendly, and produces products with high purity and high yield. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 A schematic diagram illustrating the synthesis principle of 4-azasteroid compounds with the structure of formula II;

[0056] Figure 2 A schematic diagram illustrating the synthesis principle of 4-azasteroid compounds with the structure of formula III;

[0057] Figure 3 This is a schematic diagram of the preparation process of the 4-azasteroid compound in Example 1 when it is of formula II;

[0058] Figure 4 This is a schematic diagram of the preparation process of the 4-azasteroid compound in Example 2 when it is of formula III. Detailed Implementation

[0059] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0060] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0061] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0062] In this invention, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1-8 carbon atoms (denoted as C). 1-8 Alkyl group). In some embodiments, the alkyl group has 1-6 carbon atoms (C60-C60). 1-6 Alkyl group). In some embodiments, the alkyl group has 1-3 carbon atoms (C1-C2). 1-3 Alkyl). C 1-6Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, s-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 the like. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available attachment point, and are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, carbocyclyl, alkoxy, halo, hydroxyl, oxo, amino, amido, acyl, acyloxy, or ester.

[0063] 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 (as in 2-butenyl) or terminal (as in 1-butenyl). In some embodiments, the alkenyl is C 2–6 Non-limiting examples of alkenyl groups include ethenyl, 1 -propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like. Alkenyl groups can be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, alkoxy, halo, hydroxyl, oxo, amino, amido, acyl, acyloxy, or ester.

[0064] 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, and the like. Alkynyl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, alkoxy, halo, hydroxyl, oxo, amino, amido, acyl, acyloxy, or ester.

[0065] "Alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl and carbocyclyl are as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propyloxy, butyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

[0066] "Ester" or "ester group" refers to the radical -COOR c , where Rc It is an alkyl, alkenyl, ynyl, carbocyclic or aryl group as defined herein.

[0067] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0068] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0069] When listing a range of values, the intention is to include every value within that range and its subranges. For example, "C" 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 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.

[0070] "Multiple" refers to two or more, such as 2-5, 2-3, 2, 3, 4 or 5, etc.

[0071] The different terms "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", and "X is A, B and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0072] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted aryl" means that the alkyl group may but does not have to be present, and the description includes cases where the aryl group is substituted with an alkyl group and cases where the aryl group is not substituted with an alkyl group.

[0073] "Substitution" means that one or more hydrogen atoms in a group are independently replaced by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0074] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Specifically, such salt is non-toxic and may be an organic or inorganic acid addition salt and a base addition salt.

[0075] The label of the carbon atom of the steroid compound steroid ring is as follows, for example, the C atom of label 10 is "C-10", the C-1 and C-2 are "1,2".

[0076]

[0077] 1. In the first embodiment of the present application, when the 4-azasteroid compound is formula II (i.e. compound 7), as shown in the following synthesis principle: Figure 1

[0078] (1) Compound 1 (known compound, can be synthesized by the method reported in patent document CN101360832A) undergoes oxidative ring-opening reaction in the presence of an oxidizing agent and a base, the oxidizing agent can be potassium permanganate or a mixture of potassium permanganate and sodium periodate, the base can be sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, the solvent can be t-butyl alcohol or a mixed solvent of isopropyl alcohol and water, to obtain compound 2;

[0079] (2) Compound 2 undergoes ring-closing reaction in the presence of an amine reagent, the amine reagent can be ammonium formate or ammonium acetate, the solvent can be toluene or xylene to obtain compound 3;

[0080] (3) Compound 3 undergoes reduction reaction in the presence of a metal catalyst and hydrogen, the catalyst can be palladium on carbon or active nickel, the solvent can be formic acid or acetic acid to obtain compound 4;

[0081] (4) Compound 4 undergoes halogenation reaction in the presence of a halogenating reagent and a base, the halogenating reagent can be bromine, dibromohydrazine, dichlorohydrazine, NBS, NCS, the base can be sodium hydroxide or potassium hydroxide, the solvent can be 1,4-dioxane, the reaction temperature is 0-20℃, to obtain compound 5;

[0082] (5) Compound 5 undergoes amide reaction with t-butylamine in the presence of dichlorosulfoxide and a base, the base can be triethylamine, pyridine, DMF, the solvent can be dichloromethane, trichloromethane, tetrahydrofuran, to obtain compound 6;

[0083] (6) Compound 6 undergoes dehydrogenation reaction in the presence of DDQ and BSTFA, the solvent can be toluene or xylene, to obtain compound 7.

[0084] When the 4-azasteroid compound is formula II (i.e. compound 7 of Figure 1 The specific preparation method of the compound 7 includes the following steps:

[0085] ​(1) Compound 1 is dissolved in a mixed solvent with a base, an aqueous solution of an oxidant is slowly added dropwise, and the reaction is carried out at a temperature of 40-70°C, after the reaction is completed, the salt is filtered off, an appropriate amount of an aqueous solution of sodium bisulfite is added to quench the excess oxidant, the organic solvent is removed by distillation under reduced pressure, and the compound 2 is obtained by crystallization after cooling, filtration, water elution and air drying;

[0086] (2) Compound 2 is added to a solvent with an amine reagent, and the reaction is carried out at a temperature of 100-140°C, after the reaction is completed, an appropriate amount of drinking water is added, the organic solvent is removed by distillation under reduced pressure, and the compound 3 is obtained by crystallization after cooling, filtration, water elution and air drying;

[0087] (3) Compound 3 is dissolved in a solvent, a catalyst is added, and the reaction is carried out under hydrogen replacement at a temperature of 20-40°C and a pressure of 0.2-0.8 MPa, after the reaction is completed, an appropriate amount of drinking water is added, the organic solvent is removed by distillation under reduced pressure, and the compound 4 is obtained by crystallization after cooling, filtration, water elution and air drying;

[0088] (4) Compound 4 is dissolved in 1,4-dioxane, an appropriate amount of an aqueous solution of a base is added, and a halogenated reagent is slowly added in batches at a temperature of 0-20°C, after the reaction is completed, an aqueous solution of sodium bisulfite is added to quench the reaction, hydrochloric acid is added to adjust the pH to be acidic, 1,4-dioxane is recovered by distillation under normal pressure, and the compound 5 is obtained by filtration and air drying;

[0089] (5) Compound 5 is dissolved in a solvent with a base, dichlorosulfoxide is added dropwise, and the reaction is carried out at 0-20°C for 1.5-2.5 h, then tert-butylamine is added dropwise, the reaction temperature is maintained, after the reaction is completed, an aqueous solution of sodium hydroxide is added to quench the reaction, the organic phase is separated, and the compound 6 is obtained by crystallization after cooling, filtration and air drying;

[0090] (6) Compound 6 is dissolved in a solvent, DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and BSTFA (bis (trimethylsilyl) trifluoroacetamide) are added, and the reaction temperature is maintained at 100-140°C, after the reaction is completed, an aqueous solution of sodium bisulfite is added to quench the reaction, the mixture is filtered, separated, the organic phase is washed with an aqueous solution of sodium carbonate and an aqueous solution of sodium bisulfite, distilled under reduced pressure, replaced with acetone, crystallized after cooling, filtered, and air dried to obtain the compound 7.

[0091] 2. In the second specific embodiment of the present application, when the structure of the 4-azasteroid compound is formula III (i.e. compound 9), as shown in the following formula: Figure 2 the synthesis principle is as follows:

[0092] (1)-(4) Compound 5 is synthesized from compound 1, and the method is the same as that in the first specific embodiment;

[0093] (5) Compound 5 is dehydrogenated under the action of DDQ and BSTFA, the solvent can be toluene or xylene, to obtain compound 8;

[0094] (6) Compound 8 is subjected to amide reaction with 3, 5-ditrifluoromethylaniline under the action of dichlorosulfoxide and a base, the base can be triethylamine, pyridine, DMF, the solvent can be dichloromethane, chloroform, tetrahydrofuran, to obtain compound 9.

[0095] When the structure of the 4-azasteroid compound is formula III (i.e. Figure 2 The specific preparation method of compound 9 of formula (III) comprises the following steps:

[0096] (1)-(4) Compound 5 is synthesized from compound 1, and the method is the same as that in the first specific embodiment;

[0097] (5) Compound 5 is dissolved in a solvent, DDQ and BSTFA are added, the reaction temperature is kept at 100-140℃, after the reaction is completed, potassium carbonate aqueous solution is added to extract the product, glacial acetic acid is added for acidification, filtration is carried out, and air blowing drying is performed to obtain compound 8;

[0098] (6) Compound 8 is dissolved in a solvent with a base, dichlorosulfoxide is added dropwise, the reaction is carried out at 0-20℃ for 1.5-2.5h, 3, 5-ditrifluoromethylaniline is added dropwise, the reaction temperature is kept, after the reaction is completed, sodium hydroxide aqueous solution is added to quench the reaction, liquid separation is carried out, after the organic phase is added with water, vacuum distillation is carried out, cooling is carried out for crystallization, filtration is carried out, and air blowing drying is performed to obtain compound 9.

[0099] 3. In the third specific embodiment of the present application, a pharmaceutical composition comprising a 4-azasteroid compound and a pharmaceutically acceptable excipient is provided.

[0100] The dosage forms of the pharmaceutical composition of the present application include tablets, capsules, granules, powders, suspensions, solutions, emulsions, injections and the like. They are administered according to the characteristics of the respective dosage forms, and the administration routes include oral, sublingual, injection and the like.

[0101] In a preferred embodiment, the pharmaceutical composition provided by the present application is an oral solid preparation, preferably a tablet. The oral solid preparation contains, in addition to the active ingredient 4-azasteroid compound, a pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, including fillers (also known as diluents), disintegrants, binders or wetting agents, lubricants (including glidants) and the like. The amount of the pharmaceutical excipient can be used according to the conventional amount.

[0102] The fillers generally include lactose, microcrystalline cellulose, mannitol, pregelatinized starch, starch, sucrose, dextrin, sorbitol, calcium carbonate, calcium bicarbonate, hydroxypropyl methylcellulose and ethyl cellulose and the like. They can be used alone or in combination.

[0103] The disintegrants include starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, and low-substituted hydroxypropyl cellulose, etc. They can be used alone or in combination.

[0104] The binders or wetting agents include povidone (polyvinylpyrrolidone), hydroxypropyl methyl cellulose, hydroxypropyl cellulose, ethyl cellulose, polyethylene glycol, starch paste, water, and various concentrations of ethanol solution, etc. They can be used alone or in combination.

[0105] The lubricants include zinc stearate, magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, sucrose fatty acid ester, microfine silica, stearic acid, and solid polyethylene glycol, etc. They can be used alone or in combination.

[0106] If necessary, other excipients such as sweeteners (e.g., aspartame, steviol, etc.), colorants (e.g., yellow iron oxide, red iron oxide, etc.), stabilizers (e.g., citric acid, lactic acid, malic acid, etc.), and pH adjustors (e.g., sodium bicarbonate, fumaric acid, citric acid, etc.) can be added to the above composition.

[0107] If necessary, other suitable active ingredients can be included in the above composition.

[0108] The above oral solid preparation can be prepared according to the conventional methods for preparing oral solid preparations in the art, e.g., tablets can be prepared by wet granulation, dry granulation, fluidized bed granulation, and direct compression of powder mixture, etc. When the oral solid preparation is a tablet, it can be further coated to form a film-coated tablet or sugar-coated tablet, if necessary. The coating materials include cellulose-based, acrylic resin-based, and sugar-based materials, e.g., hydroxypropyl methyl cellulose and sucrose, etc., to which plasticizers, anti-adherents, and opacifiers, etc. can be added.

[0109] The amount of the above composition to be administered is adjusted according to the nature and severity of the patient's condition, the route of administration, and the patient's age and body weight, etc.

[0110] 4. In the fourth embodiment of the present application, the use of 4-azasteroid compounds for the manufacture of a medicament for the treatment of cancer, specifically prostate cancer or colon cancer, is provided.

[0111] "Treating" any disease or disorder means ameliorating the disease or disorder (i.e., arresting / inhibiting the disease or reducing the manifestation, extent, or severity of at least one of its clinical symptoms). In another embodiment, "treating" means improving at least one physical parameter that can not be discernible by the subject. In yet another embodiment, "treating" means modulating a disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In further embodiments, "treating" involves slowing the progression of a disease.

[0112] Example 1:

[0113] A 4-azasteroid compound, as shown below, Figure 3 The preparation method comprises the following steps:

[0114] Dissolve 30 g of compound 1 in 600 ml of t-butyl alcohol and 60 ml of water, and warm to 80°C. Add a mixed aqueous solution of potassium permanganate and sodium periodate (0.6 g of potassium permanganate and 105 g of sodium periodate dissolved in 600 ml of water) dropwise while maintaining the temperature at 80-90°C. After the dropwise addition is completed, maintain the temperature at 80-90°C for 2 hours. After the reaction is completed, cool to room temperature, and filter. Add an aqueous solution of sodium bisulfite (60 g of sodium bisulfite dissolved in 120 ml of water) to the filtrate, distill off the t-butyl alcohol under reduced pressure, cool, filter, and wash the filter cake with water until neutral. Dry the filter cake at 50°C under a blast of air to obtain compound 2, with a yield of 96.7%.

[0115] Dissolve 30 g of compound 2 and 28 g of ammonium acetate in 60 ml of toluene, and warm to 100-110°C for 8 hours. After the reaction is completed, add 300 ml of water, distill off the toluene under reduced pressure, cool, filter, and wash the filter cake with water until neutral. Dry the filter cake at 50°C under a blast of air to obtain compound 3, with a yield of 97.2%.

[0116] Dissolve 25 g of compound 3 in 500 ml of glacial acetic acid, and add 2.5 g of palladium on carbon. Replace the atmosphere with hydrogen three times, and add hydrogen gas until the pressure is P H2 = 0.4-0.5 MPa. Maintain the temperature at 20-30°C for 16 hours. After the reaction is completed, add 500 ml of water, distill off the glacial acetic acid under reduced pressure, cool, filter, and wash the filter cake with water until neutral. Dry the filter cake at 50°C under a blast of air to obtain compound 4, with a yield of 96.9%.

[0117] 22g of compound 4 was dissolved in 250ml of 1,4-dioxane, and 100ml of 40% sodium hydroxide aqueous solution was added. 45g of dibromohydantoin was slowly added in three portions. After stirring at room temperature for 2 hours, 20ml of 15% sodium bisulfite was added to quench the reaction. The pH was adjusted to 2 with concentrated hydrochloric acid. 1,4-dioxane was recovered by atmospheric distillation. 500ml of water was added, stirred, filtered, and the filter cake was washed with water until neutral. The mixture was dried at 50℃ to obtain compound 5, with a yield of 98.8%.

[0118] 20g of compound 5 was dissolved in 200ml of chloroform, cooled to 0℃, and 16g of thionyl chloride was added dropwise while maintaining the temperature at 0-10℃. After the addition was complete, the reaction was maintained at 0-10℃ for 2 hours. Then, 40g of tert-butylamine was added dropwise while maintaining the temperature at 0-10℃. After the addition was complete, the reaction was maintained at 0-10℃ for 2 hours. After the reaction was complete, 200ml of water was added to quench the reaction. The mixture was separated, and 200ml of water was added to the organic phase. The mixture was then distilled under reduced pressure until dichloromethane was completely removed. The mixture was cooled, filtered, and the filter cake was washed with water until neutral. The mixture was then dried at 50℃ to obtain compound 6, with a yield of 98.2%.

[0119] 20g of compound 6 was dissolved in 400ml of toluene, 16g of DDQ and 70g of BSTFA were added, the mixture was heated to 100-110℃ and reacted for 5 hours, then cooled to room temperature, and an aqueous solution of sodium bisulfite (30g of sodium bisulfite dissolved in 400ml of water) was added. After stirring for 1 hour, the mixture was filtered, and the filtrate was separated. The organic phase was washed once with 15% sodium carbonate water and once with 15% sodium bisulfite water, respectively. The mixture was distilled under reduced pressure until nearly dry, and acetone was added once until a white solid precipitated. A small amount of acetone was added and stirred, the mixture was filtered, washed with a small amount of acetone, and dried at 50℃ to obtain compound 7 (formula II), with a yield of 89.8%.

[0120] Compound 7 was detected, 1 H-NMR (500MHz, CDCl3) δ (ppm): 0.57 (s, 3H), 0.72 (s, 3H), 1.01~1.08 (m, 3H), 1.27~1.52 (m, 5H), 1.32 (s, 9H) ), 1.61~1.81 (m, 7H), 2.14 (dd, 1H), 3.35~3.45 (m, lH), 5.21 (s, lH), 5.56 (s, 1H), 5.82 (d, 1H), 6.80 (d, lH). MS-EI m / z:373[M + H] + .

[0121] Example 2:

[0122] A 4-azasteroid compound, such as Figure 4 As shown, its preparation method includes the following steps:

[0123] 30g of compound 1 was dissolved in 600ml of tert-butanol and 60ml of water. The mixture was heated to 80℃ and kept at 80-90℃. A mixed aqueous solution of potassium permanganate and sodium periodate (0.6g of potassium permanganate and 110g of sodium periodate dissolved in 600ml of water) was added dropwise. After the addition was complete, the mixture was kept at 80-90℃ for 2 hours. After the reaction was complete, the mixture was cooled to room temperature and filtered. An aqueous solution of sodium bisulfite (60g of sodium bisulfite dissolved in 120ml of water) was added to the filtrate. The tert-butanol was removed by vacuum distillation. The mixture was cooled, filtered, and the filter cake was washed with water until neutral. The mixture was then dried at 50℃ to obtain compound 2, with a yield of 97.2%.

[0124] 30g of compound 2 and 30g of ammonium acetate were added to 70ml of toluene, and the mixture was heated to 100-110℃ and reacted for 8 hours. After the reaction was completed, 300ml of water was added, and the mixture was distilled under reduced pressure until all toluene was removed. The mixture was then cooled, filtered, and the filter cake was washed with water until neutral. The mixture was then dried in a forced-air dryer at 50℃ to obtain compound 3, with a yield of 97.2%.

[0125] Dissolve 25g of compound 3 in 600ml of glacial acetic acid, add 2.5g of palladium on carbon, purge three times with hydrogen, and add hydrogen until the solution reaches pH P. H2 =0.4~0.5Mpa; keep the reaction at 20~30℃ for 18 hours. After the reaction is complete, add 500ml of water, distill under reduced pressure until glacial acetic acid is completely removed, cool down, filter, wash the filter cake with water until neutral, and dry it at 50℃ to obtain compound 4, with a yield of 96.9%.

[0126] 22g of compound 4 was dissolved in 240ml of 1,4-dioxane, and 100ml of 40% sodium hydroxide aqueous solution was added. 40g of dichlorohydantoin was slowly added in three portions. After stirring at room temperature for 2 hours, 20ml of 15% sodium bisulfite was added to quench the reaction. The pH was adjusted to 2 with concentrated hydrochloric acid. 1,4-dioxane was recovered by atmospheric distillation. 500ml of water was added, stirred, filtered, and the filter cake was washed with water until neutral. The mixture was dried at 50℃ to obtain compound 5, with a yield of 98.8%.

[0127] 20g of compound 5 was dissolved in 800ml of toluene, 16g of DDQ and 70g of BSTFA were added, the mixture was heated to 100-110℃ and reacted for 6 hours, then cooled to room temperature, 150ml of 10% potassium carbonate aqueous solution was added, the mixture was stirred for 1 hour, the mixture was separated, 80g of glacial acetic acid was added dropwise to the aqueous phase, a large amount of white solid precipitated out, the mixture was filtered, washed with water until neutral, and dried at 50℃ to obtain compound 8, with a yield of 90.2%.

[0128] Dissolve 15 g of compound 8 in 300 ml of chloroform, cool to 0°C, and drop 16 g of dichloro sulfoxide at a temperature of 0-10°C. After the drop is completed, keep the temperature at 0-10°C for 2 hours. Drop 30 g of 3,5-ditrifluoromethyl aniline at a temperature of 0-10°C. After the drop is completed, keep the temperature at 0-10°C for 2 hours. After the reaction is completed, quench the reaction by adding 200 ml of water, separate the liquid, distill the organic phase under reduced pressure until it is nearly dry, replace it with ethyl acetate, and then add a small amount of ethyl acetate, stir for 2 hours, filter, rinse the filter cake with a small amount of ethyl acetate, and dry it at 50°C to obtain compound 9 (formula III) with a yield of 98.2%.

[0129] Compound 9 is detected, 1 H-NMR (500 MHz, CDCl3) δ (ppm): 0.62 (s, 3H), 0.96 (s, 3H), 1.01-1.18 (m, 2H), 1.32-1.97 (m, 8H), 2.13-2.30 (m, 2H), 2.33-2.45 (m, 6H), 3.32-3.42 (m, 1H), 5.76 (s, 1H), 7.45 (d, 1H), 7.50 (s, 1H), 7.74 (d, 1H), 8.78 (s, 1H). MS-EI m / z: 529 [M + H] + .

[0130] Example 3:

[0131] Use of the 4-azasteroid compound of Example 1 or Example 2 in the preparation of a medicament for treating prostate cancer or colon cancer. In order to verify the inhibitory effect of the 4-azasteroid compound on prostate cancer and colon cancer, and to prove the application effect of the 4-azasteroid compound in the preparation of a medicament for treating prostate cancer or colon cancer, the present application provides the following pharmacological experiments.

[0132] 1. Test method

[0133] 1.1 Experimental grouping and sample preparation

[0134] The sample of the compound of each example is prepared into a 100 mM mother liquor using the solvent dimethyl sulfoxide (DMSO), and is diluted into a working solution using the corresponding complete culture medium for cell culture, with a concentration of 100, 30, 10, 3, 1, 0.3 μM. A solvent control group, different concentrations of a positive control group, and different concentrations of a sample treatment group are set.

[0135] 1.2 Cell culture

[0136] The medium for human prostate cancer cells (DU 145) is MEM medium containing 10% FBS (fetal bovine serum); the medium for human colon cancer cells (HCT-116) is McCoy's 5A containing 10% FBS. When the cells are in good growth condition, they are subcultured every 2 days at a ratio of 1:3. In a clean bench, the culture medium is discarded, the cells are washed twice with 1x PBS, then 600 μL of 0.25% trypsin is added for about 1-3 min until the cells are detached, 3 mL of the corresponding medium containing 10% FBS for each type of cell is added to terminate the trypsin digestion, and the cells are blown into a single cell suspension and transferred into an EP tube, which is centrifuged at 1000 rpm for 5 min. The culture medium is discarded, the cells are resuspended in fresh culture medium, and inoculated into new culture bottles at a certain ratio (the cell density is about 10 5 / mL) and cultured in a 37°C, 5% CO2 incubator.

[0137] 1.3 Cell inoculation

[0138] The cells in good growth condition are routinely digested and collected, the density of DU 145 cells is adjusted to 2x10 4 / mL, and the density of HCT-116 cells is adjusted to 2x10 4 / mL, and each type of cell suspension is inoculated into a 96-well culture plate at a density of 100 μL / well, the plate is shaken crosswise for 10 times to make the cells evenly spread on the bottom of the wells, and the plate is placed in a CO2 incubator for 24 h.

[0139] 1.4 Cell treatment

[0140] The sample working solution of the compound of the example prepared in step 1.1 is added to the corresponding wells at a concentration of 100 μL / well, so that the final volume of each well is 200 μL (100 μL of cell culture medium and 100 μL of sample working solution), and the final concentration is 50, 15, 5, 1.5, 0.5, 0.15 μM, respectively. At the same time, a solvent control group is set up, and the positive control group has a concentration of 50, 15, 5, 1.5, 0.5, 0.15 μM, respectively. Each group has 3 replicates. The plate is incubated at 37°C, 5% CO2 for 72 h.

[0141] 1.5 Detection of cell proliferation OD value

[0142] After 72 h of cell treatment, 20 μL of thiazolyl blue (MTT) is added to each well, and the plate is incubated at 37°C, 5% CO2 for 4 h. The liquid in each well is carefully aspirated, 150 μL / well of DMSO is added to each well, and the plate is shaken for 10 min.

[0143] The OD value of A1-H1 wells (8 wells) is set as the zero value on the enzyme label instrument, and the OD value of each well is detected at 492 nm.

[0144] 1.6 Result calculation

[0145] The OD value of the solvent control group was set as 100% cell viability, and the ratio of the OD value of each group to the OD value of the solvent control group was the relative cell viability. The activity of the sample on DU 145 or HCT-116 cells was evaluated by the cell proliferation rate, and if the inhibition rate was > 100%, it was determined to be a system error, and was calculated as 100%.

[0146] The inhibition rate calculation formula was: inhibition rate (%) = (1-OD 样品 / OD 溶媒 ) x 100%

[0147] The half-inhibition rate (IC 50 ) was calculated using SPSS software.

[0148] 2. Experimental results

[0149] Table 1 IC 50 values of compounds of some examples and comparative examples on each cancer cell

[0150]

[0151] Note: DU145 is a human prostate cancer cell, and HCT-116 is a human colon cancer cell.

[0152] In summary, compound 7 and compound 9 have strong inhibitory effects on human prostate cancer cells and human colon cancer cells with IC50<10 μM, while the inhibitory effects of finasteride and dutasteride are not obvious.

[0153] The above only describes the embodiments of the present application and does not limit the present application. Any modification, equivalent replacement or improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A 4-azasteroid compound characterized by, The 4-azasteroid compound has the following formula II or formula III structure: (Formula II), (Formula III).

2. A process for the preparation of a 4-azasteroid compound as claimed in claim 1, characterized in that, The method comprises the following steps: Compound 5 is subjected to amide reaction with tert-butylamine in the presence of dichloro sulfoxide and a base to obtain compound 6, and then compound 6 is subjected to dehydrogenation reaction in the presence of DDQ and BSTFA to obtain the compound of formula II; or Compound 5 is subjected to dehydrogenation reaction in the presence of DDQ and BSTFA to obtain compound 8, and then compound 8 is subjected to amide reaction with 2,5-ditrifluoromethyl aniline in the presence of dichloro sulfoxide and a base to obtain the compound of formula III; The structural formulae of the compound 5, the compound 6 and the compound 8 are as follows: ， ， 。 3. Use of a 4-azasteroid compound as claimed in claim 1 or a 4-azasteroid compound prepared by the process as claimed in claim 2 for the manufacture of a medicament for inhibiting cancer cells, characterized in that, The cancer cells include prostate cancer cells or colon cancer cells.

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