Steroid phosphate compound and application thereof

Abstract

The invention discloses a steroid phosphate ester compound, a pharmaceutically acceptable salt, a tautomer, a preparation method of the steroid phosphate ester compound, the pharmaceutically acceptable salt, the tautomer, the preparation method of the steroid phosphate ester compound, the pharmaceutical composition and application of the steroid phosphate ester compound, and the steroid phosphate ester compound is a compound almost free of hepatotoxicity and can be used for preventing and treating androgen receptor related diseases such as prostatic cancer. Specifically, the invention relates to a steroid phosphate compound represented by a formula (I) or a pharmaceutically acceptable salt thereof, wherein the definition of each substituent is shown in the specification. # imgabs0 #

Description

[0001] This application is a divisional application of the application with the application date of June 30, 2022, application number 202210759806.3, and invention title "A Steroidal Phosphate Ester Compound and Its Application". Technical Field

[0002] The present invention relates to the field of pharmaceutical technology, and particularly to a class of steroidal phosphate ester derivatives with almost no liver toxicity, a preparation method thereof as an androgen antibody inhibitor, and their pharmaceutical uses, especially the pharmaceutical uses in preventing and treating androgen receptor-related diseases such as prostate cancer. Background Art

[0003] The androgen receptor (AR) belongs to the nuclear receptor family and contains four main regions: the N-terminal active transcriptional control region (NTD), the DNA binding region (DBD), the hinge region, and the ligand binding region (LBD). AR is mainly expressed in androgen target tissues such as the prostate, skeletal muscle, liver, and central nervous system. The highest expression levels are observed in the prostate, adrenal gland, and epididymis. AR can be activated by the binding of endogenous androgens including testosterone and 5α-dihydrotestosterone (5α-DHT).

[0004] Prostate cancer (PCa) is a malignant tumor that occurs in the prostate tissue of men and is the result of abnormal and disordered growth of prostatic acinar cells.

[0005] The occurrence and development of PCa are closely related to androgens. Endocrine therapy is currently the main treatment method for advanced prostate cancer. Androgen receptor antagonist (AR antagonist) drugs such as nilutamide, flutamide, and bicalutamide (trade name Casodex) were originally designed to avoid the side effects of hormone therapy and eliminate drug resistance in prostate cancer patients. Although these androgen receptor antagonists have good effects on patients with advanced prostate cancer who receive treatment for the first time when used in combination with hormone therapy, their efficacy against drug-resistant prostate cancer is very limited whether used alone or in combination therapy. In recent years, studies have shown that bicalutamide has relatively weak specificity as an androgen receptor (AR) antagonist and has a certain receptor activation effect (agonism) (a similar problem has also been found for flutamide). The latest research shows that the reactivation of the androgen receptor signaling pathway may be the fundamental cause of resistance to hormone therapy, which limits the efficacy of currently commonly used androgen receptor antagonist drugs such as bicalutamide in treating prostate cancer, especially for advanced resistant prostate cancer. In addition, the efficacy of these androgen receptor antagonist drugs is generally limited by their significant side effects, such as toxicity to the liver or central nervous system. Therefore, there is an urgent need in the clinical treatment of prostate cancer for a new generation of androgen receptor antagonists with high activity, low toxicity, and no residual activation property (agonism).

[0006] Many corticosteroids have been used as anti-inflammatory, antirheumatic, anti-allergic, and anti-shock agents. In particular, 11-deoxy-cortisol esters and their derivatives have been widely used as anti-inflammatory agents. 17- or 21-monoesters of carboxylic acids containing no more than six carbon atoms are also known.

[0007] For example, the preparation of 17α,21-diacetoxypregn-4-ene-3,20-dione is disclosed in US3,530,038, which also mentions the use of propionyl derivatives and a series of aliphatic acyl derivatives with chains containing up to six carbon atoms.

[0008] US3,152,154 discloses the preparation of 21-hydroxy-pregn-4,9-diene-3,20-dione-17α-butyrate, which is used as an intermediate in the preparation of 3,21-diacyloxy-17α-butyryloxypregn-3,5,9-triene-20-one, where the 3- and 21-acyl groups are the same and are acetyl, propionyl, butyryl, and isobutyryl. All the examples cited in these documents relate to compounds in which the 17α- and 21-positions are esterified with the same acyl group.

[0009] As the sole example of a mixed ester, the preparation of 21 - acetoxypregn - 4 - ene - 3,20 - dione - 17α dimethylpropionate is described in Liebigs Ann. Chem. 1983, 705 - 71: The authors state that the preparation of the mixed ester is only possible when the substituent in the 21 - position is an acetyl group.

[0010] US3,530,038 discloses a method for preparing 11β - 17α - 21 - trihydroxysteroids, which comprises oxidizing 11 - deoxy - 17α - OR - 21 - OR'steroids, where R is a carboxylic acid residue of 1 - 18 carbon atoms and R' is hydrogen or an acyl group of 1 - 18 carbon atoms, with a microorganism of the genus Curvularia to obtain the corresponding 11β - hydroxysteroids.

[0011] According to the same patent, compounds of the pregnane, androstane or estrane series are mentioned as possible starting steroids, but no transformation of 11 - deoxy - 17α - OR - 21 - OR'steroids where R is an acyl group of 1 - 18 carbon atoms and R' is hydrogen is mentioned.

[0012] The preparation of these products is described by R.B. Turner with reference to the preparation of pregn - 4 - ene - 3,20 - dione - 17α,21 - diacetate (J.Am.Chem.Soc. 75 (1953) 3489) and by R. Gardi et al. (Gazz.Chim.It. 93 (1963) 431 - 450).

[0013] US3,780,177 discloses the preparation of 21 - hydroxy - pregn - 4,9 - diene - 3,20 - dione - 17α - butyrate by means of a butyrate and its use as an intermediate in the preparation of 6α,9α - difluoroprednisolone - 17 - butyrate - 21 - ester derivatives.

[0014] Finally, CN1246328C discloses the preparation and use of mixed esters of 17α,21 - dihydroxypregnene, and describes 17 - and 21 - acyl derivatives with the same aliphatic chain containing no more than four carbon atoms.

[0015] In summary, no steroidal phosphate derivatives are known in the disclosed content, and in particular, no mixed esters of 17α,21 - dihydroxypregnene involving phosphate derivatives are known.

[0016] In the present invention, after chemically substituting the 21 - hydroxyl group with a phosphate derivative, specificity can be improved and the side effects of the androgen receptor antagonist drugs currently used clinically (such as toxicity to the liver or central nervous system) can be reduced. These compounds can bind to androgen receptors, inhibit the movement of the receptor into the cell nucleus and reduce its transcriptional activity, and are expected to become candidate drugs for androgen receptor antagonists in the treatment of advanced prostate cancer. SUMMARY OF THE INVENTION

[0017] The inventor of the present invention has developed a steroid phosphate compound represented by formula (I), its pharmaceutically acceptable salts, and its tautomers,

[0018]

[0019] In formula (I),

[0020] R0 is selected from the following groups or atoms: H, -OCH2OCH3, -OCH3, -OAc, -OH, =NOH, =NH, or =O;

[0021] The dotted line indicates the presence or absence of a double bond;

[0022] Y is selected from O or S;

[0023] R1 and R2 are each independently selected from a cation, hydrogen, or the following groups which are unsubstituted or substituted by one or more groups A: C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 alkylamino, 3-8 membered cycloalkyl, 3-8 membered heterocycle, C5-C20 aryl;

[0024] Alternatively, R1 and R2 are linked together and together with P and the O atoms respectively linked to R1 and R2 form a 5-7 membered ring;

[0025] R3 is selected from the following groups which are unsubstituted or substituted by one or more groups A: C1-C12 alkyl, C1-C12 alkanoyl, C5-C20 aryl, or aroyl.

[0026] Group A is selected from: halogen, trifluoromethyl, trifluoromethoxy, nitro, nitrile, hydroxyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylamino, C1-C8 alkylcarbonyl, C1-C8 alkoxycarbonyl, C1-C8 alkylcarbonyloxy, 3-8 membered cycloalkyl.

[0027] In the solution of the present invention, the dotted line indicates the presence or absence of a double bond, that is, the connected atoms can be a single bond or a double bond.

[0028] In some solutions of the present invention, R0 is selected from: H, -OCH2OCH3, -OCH3, -OAc, -OH, and in this case, R0 forms a single bond with the connected carbon atom;

[0029] In some solutions of the present invention, R0 is selected from: =NOH, =NH, or =O, and in this case, R0 forms a double bond with the connected carbon atom.

[0030] In some solutions of the present invention, the above-mentioned Y is selected from O or S; preferably, Y is O.

[0031] In some embodiments of the present invention, the pharmaceutically acceptable salts thereof and their tautomers, the steroid phosphate compound is represented by the structural formula (II),

[0032]

[0033] wherein, the definitions of the substituents in formula (II) are as defined in formula (I).

[0034] In some embodiments of the present invention, both R1 and R2 are hydrogen;

[0035] In some embodiments of the present invention, R1 and R2 are selected from cations;

[0036] In some specific embodiments of the present invention, R1 and R2 are each independently selected from metal cations; preferably, R1 and R2 are each independently selected from: Li + 、Na + 、K + 、Zn 2+ 、Mg 2+ 、Ca 2+ 、Ag + ;

[0037] In some specific embodiments of the present invention, R 1 and R 2 are each independently selected from quaternary ammonium salts or ammonium ions; preferably, R1 and R2 are each independently selected from N(C1-C4-alkyl or substituted alkyl)4 + ;

[0038] In some specific embodiments of the present invention, R1 and R2 are each independently selected from the following groups which are unsubstituted or substituted by one or more Gene A: C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 alkylamino, 3-8 membered cycloalkyl, 3-8 membered heterocycle, C5-C20 aryl;

[0039] In some specific embodiments of the present invention, R1 and R2 are each independently selected from: C1-C4 alkyl, C1-C4 alkylamino, C1-C4 alkylphenyl, C1-C4 alkylheterocycle, C1-C4 alkylaryl,

[0040] In some embodiments of the present invention, R1 and R2 are connected, and together with P and the O atoms respectively connected to R1 and R2, form a 5-7 membered ring.

[0041] In some embodiments of the present invention, R3 is selected from the following groups which may or may not be substituted by one or more gene A: C1-C12 alkyl, C1-C12 alkanoyl, C5-C20 aryl, or arylcarbonyl; preferably, R3 is selected from C1-C8 acyl; more preferably, R3 is propionyl.

[0042] In the embodiments of the present invention, group A is selected from: halogen, trifluoromethyl, trifluoromethoxy, nitro, cyano, hydroxyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylamino, C1-C8 alkylcarbonyl, C1-C8 alkoxycarbonyl, C1-C8 alkylcarbonyloxy, 3-8 membered cycloalkyl.

[0043] The steroidal phosphate compounds of the present invention are selected from:

[0044]

[0045]

[0046] Certain steroidal phosphate compounds of the present invention may exist in non-solvated form or solvated form, including hydrate form. Generally, the solvated form and the non-solvated form are equivalent and both are included in the scope of the present invention.

[0047] Certain steroidal phosphate compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, enantiomers, diastereomers, stereoisomers and individual isomers are all included in the scope of the present invention.

[0048] Definitions and explanations:

[0049] The following are the term definitions used in this specification. The initial definitions provided for the groups or terms herein apply to the groups or terms throughout this specification, either alone or as part of other groups, unless otherwise indicated.

[0050] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0051] The terms "alkyl" and "alkane" refer to saturated hydrocarbons containing 1 - 12 carbon atoms, including straight-chain, branched-chain or cycloalkanes. Exemplary "alkyls" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, n-hexyl, heptyl, octyl, cyclopropyl, etc.

[0052] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group containing 2 - 12 carbon atoms and at least one carbon-carbon double bond. Exemplary groups of this kind include vinyl or allyl.

[0053] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group containing 2 - 12 carbon atoms and at least one carbon-carbon triple bond. Exemplary groups of this kind include ethynyl.

[0054] The term "aryl" refers to a cyclic aromatic hydrocarbon group containing 1 - 5 aromatic rings, especially monocyclic or bicyclic groups, and containing 5 - 20 carbon atoms. Exemplary groups of this kind include phenyl, biphenyl, naphthyl, etc. If containing two or more aromatic rings (bicyclic, etc.), the aromatic rings of the aryl can be connected at a single point (e.g., biphenyl) or fused (e.g., naphthyl, phenanthryl, etc.).

[0055] The term "heterocycle" refers to a fully saturated, partially unsaturated or fully unsaturated, including aromatic (i.e., "heteroaromatic") 3 - 8 membered cyclic group having at least one heteroatom in at least one carbon-containing ring. Each ring of the heterocyclic group containing heteroatoms can have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and / or sulfur atoms, where nitrogen and sulfur heteroatoms can be optionally oxidized and sulfur heteroatoms can be optionally quaternized. Exemplary monocyclic heterocyclic groups include, but are not limited to, azetidinyl, pyrrolidinyl, pyrrolyl, oxetanyl, pyrroline, imidazolyl, imidazoline, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazoline, isoxazolyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, etc.

[0056] The term "alkoxy" refers to a group in which an alkyl is linked to an oxygen atom;

[0057] The term "alkylamino" refers to a group in which an alkyl is linked to a nitrogen atom.

[0058] The term "pharmaceutically acceptable salt" refers to salts of the steroid phosphate compounds of the present invention, prepared from the compounds with specific substituents discovered in the present invention and relatively non-toxic acids or bases. When the steroid phosphate compounds of the present invention contain relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the steroid phosphate compounds of the present invention contain relatively basic functional groups, the acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific steroid phosphate compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0059] Preferably, the salt is contacted with a base or an acid in a conventional manner, and then the parent steroid phosphate compound is separated, thereby regenerating the neutral form of the steroid phosphate compound. The parent form of the steroid phosphate compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0060] As used herein, "pharmaceutically acceptable salts" are derivatives of the steroid phosphate compounds of the present invention, wherein the parent steroid phosphate compounds are modified by salt formation with acids or with bases. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid radicals such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent steroid phosphate compounds, such as salts formed from non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxynaphthalene, hydroxyethanesulfonic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, and p-toluenesulfonic acid.

[0061] The term "chiral" refers to a molecule having the property of non-superimposability with its mirror image counterpart, while the term "achiral" refers to a molecule that can be superimposed on its mirror image counterpart.

[0062] The term "stereoisomer" refers to a compound having the same chemical constitution but different arrangements of atoms or groups in space.

[0063] The term "diastereomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. A mixture of diastereomers can be separated by high-resolution analytical methods, such as electrophoresis and chromatography.

[0064] The term "treatment", insofar as it relates to a disease or condition to some extent, includes preventing the disease or condition from occurring, inhibiting the disease or condition, eliminating the disease or condition, and / or relieving one or more symptoms of the disease or condition.

[0065] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent steroid phosphate compounds containing acid radicals or bases by conventional chemical methods. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of both, these steroid phosphate compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid. Generally, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0066] The novel steroid phosphate compounds disclosed in the present invention, in addition to having a higher inhibitory effect on prostate cancer cells, through hepatotoxicity tests, surprisingly, it was found that this series of compounds can significantly reduce the commonly occurring hepatotoxic side effects of the androgen receptor antagonist drugs currently used clinically. These compounds are expected to become modified candidate drugs for androgen receptor antagonists in the treatment of advanced prostate cancer. Brief Description of the Drawings

[0067] Figure 1 Shows the results of the hepatotoxicity test of the present invention: A. Blank group; B. High-dose abiraterone group; C. High-dose compound AR01-1 group; D. High-dose compound AR01-3 group; E. High-dose compound AR01-9 group. Detailed Embodiments

[0068] Here, for example, many exemplary preparation methods of the steroid phosphate compounds of the present invention are provided in the following embodiments. The present invention will be described in detail through examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and its specific embodiments have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Some of the present invention can be used as intermediates for preparing other compounds of the present invention.

[0069] The steroid phosphate compounds of the present invention were synthesized using commercially available compounds AR01-1-1, magnesium chloride, zinc chloride, ammonium bicarbonate, sodium bicarbonate, pyrophosphoryl chloride, diethyl chlorophosphate, dibenzyl chlorophosphate, and diphenyl chlorophosphate as starting materials.

[0070] Example 1: (8R,9S,10R,13S,14S,17R)-10,13-Dimethyl-3-oxo-17-(2-(phosphoryl)acetyl)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl propionate (Compound AR01-1)

[0071] Synthesis route:

[0072]

[0073] Preparation method:

[0074] Under nitrogen protection, tetrahydrofuran (100 mL) and compound (AR01-1-1) (8.05 g, 20.0 mmol) (AR01-1-1) were added to a reaction flask, and the temperature was lowered to -40 °C to -30 °C. Phosphoryl chloride (6.04 g, 24.0 mmol) was added to tetrahydrofuran (10 mL), and it was slowly dropped into the reaction flask. During the dropping process, the temperature of the reaction solution should be maintained at -40 °C to -30 °C. After heat preservation and stirring reaction, and after monitoring the reaction to completion by TLC, water (20 mL) was slowly added to the system, and the temperature was raised to 0 °C to 5 °C. After the hydrolysis was completed, sodium bicarbonate was added to adjust the pH value to 7 - 8. Toluene (50 mL) was added, and after layering, the aqueous phase was adjusted to pH = 3 - 4 with hydrochloric acid, filtered, and dried to obtain the crude product of compound AR01-1. The crude product was added to methyl tert-butyl ether / 1,2-methoxybutane (1:2), the temperature was raised to 45 °C, and after heat preservation and stirring reaction, it was slowly cooled to room temperature after the reaction ended, filtered, and dried to obtain the refined product of AR01-1 (8.27 g), with a yield of 85.7%. ESI-MS(+): m / z = 482.9. 1 1H NMR(CDCl3): δ 5.77(s, 1H), 4.60 - 4.87(m, 2H), 3.44 - 3.56(m, 1H), 2.85(s, 1H), 0.70 - 2.43(m, 30H).

[0075] Example 2: Preparation of disodium 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propanoyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl phosphate (Compound AR01-2)

[0076] Synthetic route:

[0077]

[0078] Preparation method:

[0079] The compound AR01-1 (9.66 g, 0.02 mol) prepared in Example 1 above was added to 30 mL of ethanol, cooled to 0 - 10 °C, and 10 mL of an aqueous solution containing 3.4 g of sodium bicarbonate (0.04 mol) was dropped. The temperature was maintained during the reaction. After the reaction was completed, ethanol was distilled off under reduced pressure at 20 - 30 °C. 20 mL of acetone was added, crystallization occurred, and after filtration and drying, AR01-2 (9.24 g) was obtained, with a yield of 87.8%. ESI-MS(+): m / z = 549.18.

[0080] Example 3: Preparation of Zinc Salt of 2-((8R,9S,10R,13S,14S,17R)-10,13-Dimethyl-3-oxo-17-(propanoyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl Phosphate (Compound AR01-3)

[0081] Synthesis Route:

[0082]

[0083] Preparation Method:

[0084] Take the compound AR01-2 (10.52 g, 0.02 mol) prepared in the above Example 2 and add it to 20 ml of water. At 20 - 30 °C, slowly add 10 ml of an aqueous solution containing 1.36 g of zinc chloride (0.01 mol). Keep the temperature and react. Solids will precipitate during the reaction. After the reaction is complete, filter and dry to obtain AR01-3 (9.96 g), with a yield of 91.2%. ESI-MS(+): m / z = 567.10.

[0085] Example 4: Preparation of Magnesium Salt of 2-((8R,9S,10R,13S,14S,17R)-10,13-Dimethyl-3-oxo-17-(propanoyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl Phosphate (Compound AR01-4)

[0086] Synthesis Route:

[0087]

[0088] Preparation Method:

[0089] Refer to the synthesis process of Example 3, replace zinc chloride with magnesium chloride, and synthesize compound AR01-4 (3.21 g), with a yield of 88.2%. ESI-MS(+): m / z = 527.20.

[0090] Example 5: Preparation of Diammonium Salt of 2-((8R,9S,10R,13S,14S,17R)-10,13-Dimethyl-3-oxo-17-(propanoyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl Phosphate (Compound AR01-5)

[0091] Synthesis Route:

[0092]

[0093] Preparation method:

[0094] Referring to Example 2, ammonium bicarbonate was used to replace sodium bicarbonate to synthesize Compound AR01-5 (2.11 g), with a yield of 86.3%. ESI-MS(+): m / z = 539.28

[0095] Example 6: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(diethoxyphosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl propionate (Compound AR01-6)

[0096] Synthesis route:

[0097]

[0098] Preparation method:

[0099] Compound AR01-1-1 (4.03 g, 10 mmol), DMAP (0.12 g, 1 mmol) and triethylamine (1.1 g, 11 mmol) were dissolved in THF (100 mL). Diethyl chlorophosphate (1.9 g, 11 mmol) was added via syringe over 30 minutes. The mixture was allowed to react overnight at room temperature. The mixture was then quenched with saturated aqueous NH4Cl and extracted with methyl tert-butyl ether (75 mL x 3). The combined organic phases were dried over anhydrous Na2SO4 and then concentrated to give a crude product, which was purified by column chromatography (eluting with petroleum ether:ethyl acetate = 5:1 - 2:1) to give Product AR01-6 (3.65 g), with a yield of 67.7%. ESI-MS(+): m / z = 539.29.

[0100] Example 7: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(diphenoxyphosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl propionate (Compound AR01-7)

[0101] Synthesis route:

[0102]

[0103] Preparation method:

[0104] Replace diethyl chlorophosphate with diphenyl chlorophosphate, and refer to the synthesis procedure of Example 6 to prepare Compound AR01-7 (1.23 g) with a yield of 63.9%. ESI-MS(+): m / z = 635.28.

[0105] Example 8: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(bis(benzyloxy)phosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl acetate (Compound AR01-8)

[0106] Synthesis route:

[0107]

[0108] Preparation method:

[0109] Replace diethyl chlorophosphate with dibenzyl chlorophosphate, and refer to the synthesis procedure of Example 6 to prepare Compound AR01-8 (1.43 g) with a yield of 66.2%. ESI-MS(+): m / z = 663.32.

[0110] Example 9: Preparation of disilver salt of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propanoyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl phosphate (Compound AR01-9)

[0111] Synthesis route:

[0112]

[0113] Preparation method:

[0114] Refer to the synthesis procedure of Example 3, replace zinc chloride with silver nitrate, and synthesize Compound AR01-9 (0.94 g) with a yield of 85.7%. ESI-MS(+): m / z = 695.02.

[0115] Example 10: Preparation of (8R,9S,10R,13S,14S,17R)-10,13-dimethyl-17-(2-(2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)acetyl)-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl propionate (Compound AR01-10)

[0116]

[0117] Preparation of Compound AR01-10-SM:

[0118] Compound AR01-10-SM was synthesized exactly according to the method described in the literature Tetrahedron: Asymmetry 11(2000)125–138.

[0119] Preparation of Compound AR01-10:

[0120] Using diethyl chlorophosphate replaced by compound AR01-10-SM, referring to the synthesis process of Example 6, compound AR01-10 (1.33 g) was prepared with a yield of 62.1%. ESI-MS(+): m / z = 523.25.

[0121] In order to clarify that the compounds of the present invention show improved activity compared to first-line anti-androgen drugs (such as abiraterone) known in clinical applications and reduce the side effects of androgen receptor antagonist drugs used clinically (such as toxicity to the liver), a series of in vitro studies were designed.

[0122] Example 11: Inhibitory Activity Test on Prostate Cancer Cells

[0123] Prostate cancer 22Rv1 cells were added to a flat-bottom 96-well cell culture plate at a density of 5000 cells / well with a volume of 100 μl. After adherent growth for 12 h, the test compound was added to a final volume of 200 μl; the test compound started from the highest concentration of 100 μM and was diluted in a 2-fold concentration gradient, with a total of 6 concentrations; after culturing for 48 h, 10 μl of Cell Counting Kit-8 cell counting reagent (CCK-8) was added. After incubation for 4 h, the absorbance at 450 nm was detected using a microplate reader. The IC50 values of the compounds were calculated using Graphpad Prism 5.0, and the results are shown in Table 1:

[0124] Table 1 Data Sheet of Test Results of Different Compounds in Inhibiting Prostate Cancer Cells

[0125] Compound Name IC50 (μM) Abiraterone ＞50 AR01-1 5.5 AR01-2 14.2 AR01-3 7.2 AR01-4 10.2 AR01-5 7.9 AR01-6 21.5 AR01-7 14.2 AR01-8 5.3 AR01-9 4.8 AR01-10 11.8

[0126] As can be seen from Table 1, the series of compounds of the present invention all have significant inhibitory activity against prostate cancer cells compared to abiraterone.

[0127] Example 12: Mouse Prostate Cancer Model Experiment

[0128] SPF-grade Kunming mice (KM mice), male, 6 - 7 weeks old, weighing 15 - 20 g. The mice were divided into a tumor-bearing control group and a drug administration group. RM-1 cells in the logarithmic growth phase were collected, and the cell seeding density was 5×106 cells / mL, wipe the right front axilla of the mouse with 75% alcohol, and inject 0.2 mL for each mouse. After tumor formation, the tumor-bearing control group was intraorally injected with 200 μL of normal saline every day, and the drug administration groups were intraorally injected with abiraterone, AR01-1, AR01-2, AR01-3, AR01-4, AR01-5, AR01-6, AR01-7, AR01-8, AR01-9, AR01-10 at 0.04 mg / g respectively. After 30 days of drug administration, the mice were sacrificed by cervical dislocation, and the subcutaneous tumors were isolated and weighed. The tumor weights of the group of rats treated with the drug were compared with those of the tumor-bearing control group, and the percentage of tumor inhibitory effect was calculated. The results are shown in Table 2.

[0129] Tumor inhibition rate % = (tumor weight of tumor-bearing control group - tumor weight of compound sample) / tumor weight of tumor-bearing control group * 100%

[0130] Table 2 Inhibitory effect on mouse prostate cancer cells

[0131] Compound Name Tumor Weight (g) Tumor Inhibition Rate % Tumor-Bearing Control Group 5.02 / Abiraterone 3.51 30.1 AR01-1 0.73 85.5 AR01-2 2.47 50.8 AR01-3 0.80 84.1 AR01-4 1.93 61.6 AR01-5 2.12 57.8 AR01-6 2.02 59.8 AR01-7 1.35 73.1 AR01-8 1.66 66.9 AR01-9 0.54 89.2 AR01-10 2.44 51.4

[0132] The data of the prostate cancer inhibition test of mice in Table 2 show that, compared with the 30.1% tumor inhibition rate of abiraterone, the tumor inhibition rates of the compounds AR01-1, AR01-2, AR01-3, AR01-4, AR01-5, AR01-6, AR01-7, AR01-8, AR01-9, AR01-10 of the present invention for inhibiting prostate tumors are all greater than 50%. Among them, the tumor inhibition rates of the compounds AR01-1, AR01-3, AR01-9 for inhibiting prostate tumors are greater than 80%, showing a significant effect on inhibiting prostate cancer.

[0133] Example 13: Detection of hepatotoxicity test:

[0134] Take 130 SPF-grade SD rats (120 - 140 g), with 65 males and 65 females. Breeding environment: temperature 20 - 25 °C, humidity 40% - 70%, separated by gender, 5 rats per cage. Divide them into 1 blank control group (10 rats), 1 abiraterone group, and 3 experimental groups according to the random allocation method, with 10 rats in each group. The abiraterone group and the experimental groups are divided into 3 dose groups of low, medium, and high. Based on the clinically common dose of abiraterone, design according to 4, 2, and 1 times the clinical equivalent dose of the recommended daily prescription dosage (1.0 g) for adults (60 kg). Set the high, medium, and low doses to be 66.8 mg / kg, 33.4 mg / kg, and 16.7 mg / kg respectively; the blank control group (equal volume of distilled water), the abiraterone group and the experimental groups are given gastric gavage once a day (1 mL / 100 g of rat body weight), weigh the body weight once a week, adjust the drug dosage according to the body weight, and continuously administer the drug for 60 d.

[0135] Test method:

[0136] (1) General condition observation: The body weight was measured once a week, and the mental state, hair color, food intake, excreta, activities, etc. of the rats after medication were observed every day.

[0137] (2) Blood routine test: The blood of the rats was collected on the 20th, 40th, and 60th days after medication respectively, and the blood routine was detected.

[0138] (3) Liver function test: The rats were fasted for 12 hours before the last administration, with normal drinking water. In the blank control group, an equal volume of distilled water was given. The rats in other dose groups were administered by gavage. After 2 hours, the orbital venous blood of the rats was taken, allowed to stand for 30 minutes and then centrifuged. The upper serum was taken and detected according to the requirements of the relevant kit instructions to determine the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0139] (4) Histopathological examination:

[0140] After the rats were bled, they were immediately dissected, and the livers of the rats were taken. After removing the surrounding connective tissues, they were rinsed in physiological saline, and then the surface liquid was blotted dry with filter paper, weighed, and the liver index was calculated (liver coefficient = liver mass / body weight) × 100%. Another right lobe of the liver was taken, fixed with 10% formalin solution, embedded in paraffin, stained with conventional HE, and the histological morphological changes were observed under an optical microscope. On the 20th, 40th, and 60th days after administration, some rats were sacrificed, the livers were removed, and their morphology, color, and texture were observed, and histopathological examinations of the liver tissues were performed.

[0141] Data processing:

[0142] Statistical analysis was performed using SPSS 13.0 statistical software. The t-test and analysis of variance were used for comparison between groups, and the measurement data were expressed as P < 0.05 was considered statistically significant.

[0143] Test results:

[0144] (1) During the medication period, the food intake, excreta, and activities of the rats were relatively normal, the hair color had no obvious abnormal changes, and there were no cases of death in each group.

[0145] (2) The body weights of the rats in the abiraterone group and the experimental group were within the normal range on the 20th, 40th, and 60th days after administration. There was no statistically significant difference between the groups (P ≥ 0.05). The results are shown in Table 3.

[0146] Table 3 Changes in body weight of rats in each group after 60 days of administration (g, n = 10)

[0147]

[0148] (3) Blood routine test

[0149] At 20 and 40 days after drug administration, all blood routine indexes of rats were within the normal range. There was no significant difference between each dose group of the abiraterone group and the experimental group and the blank group (P>0.05). At 60 days after drug administration, there was a significant difference between the abiraterone group and the blank group, while all blood routine indexes of each dose group in the experimental group were within the normal range, and there was no significant difference between them and the blank group (P>0.05). The results are shown in Table 4.

[0150] Table 4 Comparison of blood routine test results of rats at 60 days after drug administration ( n = 10)

[0151]

[0152] (4) Liver function test

[0153] Compared with the blank group, the serum AST and ALT values of rats in the abiraterone group were in a relatively high range, while the levels of serum AST and ALT in each dose group of different compounds were relatively normal, and there was no significant difference between them and the blank group (P>0.05). However, it was found that as the dose increased, the AST and ALT values gradually increased, showing a certain dose-dependent relationship. The results are shown in Table 5.

[0154] Table 5 Comparison of liver function test results of rats at 60 days after drug administration ( n = 10)

[0155]

[0156]

[0157] (5) Liver / body ratio

[0158] Compared with the blank group, each dose group in the abiraterone group led to an increase in the liver / body ratio, while there was no significant difference in each dose group of the experimental group (P>0.05). The results are shown in Table 6.

[0159] Table 6 Comparison of liver organ index results of rats at 60 days after drug administration ( n = 10)

[0160]

[0161]

[0162] (6) Histopathological examination of liver tissue

[0163] Gross observation: The livers of rats in the blank group and each dose group of the experimental group were normal in shape, soft in texture, rosy and shiny, and no nodules, masses, cysts, etc. were seen.

[0164] Observation under light microscope after HE staining: In the livers of rats in each dose group of the abiraterone group, varying degrees of damage were observed, mainly manifested as hepatocyte swelling, and a small number of cases showed focal or single-cell necrosis. The necrotic cells were diffusely present in the central part of the hepatic lobule, accompanied by inflammatory cell infiltration (see Figure B for the high dose of abiraterone); while in the blank group (see Figure A for the blank group) and each dose group of the experimental group, the hepatic lobular structure was normal, hepatocytes were not swollen or necrotic, the central vein was not dilated, there was no hyperplasia in the portal area, and no inflammatory cell infiltration was seen. Some hepatocytes showed microvesicular steatosis. At 40 days, some hepatocytes showed mild cloudy swelling, but there was no significant difference among groups (P>0.05); at 60 days, the hepatic lobular structure was intact, the boundaries of hepatocytes were clear, the central vein was not dilated, there was no hyperplasia in the portal area, and no inflammatory cell infiltration was seen. Some hepatocytes showed macrovesicular and microvesicular steatosis, and some hepatocytes showed mild to moderate hydropic degeneration, but there was no significant difference among groups (P>0.05) (see Figures C, D, and E for the high doses of compound AR01-1, compound AR01-3, and compound AR01-9 respectively). See Figure 1 。

Claims

1. A steroid phosphate compound represented by formula (I), a pharmaceutically acceptable salt thereof, or a tautomer thereof, In formula (I), R0 is selected from the following groups or atoms: H, -OCH2OCH3, -OCH3, -OAc, -OH, =NOH, =NH, or =O; The dotted line indicates the presence or absence of a double bond; Y is selected from O or S; R1 and R2 are each independently selected from a cation, hydrogen, or the following groups which are substituted or unsubstituted by one or more groups A: C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 alkylamino, 3-8 membered cycloalkyl, 3-8 membered heterocycle, C5-C20 aryl; Alternatively, R1 and R2 are linked together and together with P and the O atoms respectively linked to R1 and R2 form a 5-7 membered ring; R3 is selected from the following groups which are substituted or unsubstituted by one or more genes A: C1-C12 alkyl, C1-C12 alkanoyl, C5-C20 aryl, or aroyl; Group A is selected from: halogen, trifluoromethyl, trifluoromethoxy, nitro, nitrile, hydroxyl, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylamino, C1-C8 alkylcarbonyl, C1-C8 alkoxycarbonyl, C1-C8 alkylcarbonyloxy, 3-8 membered cycloalkyl.

2. The steroid phosphate compound, pharmaceutically acceptable salt or tautomer thereof according to claim 1, wherein, R0 is selected from H, -OCH3, -OH, =NOH or =O, preferably H or =O.

3. The steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claim 1 or 2, wherein the structural formula of the steroid phosphate compound is formula (II), The definitions of the substituents in formula (II) are as defined in formula (I) of claim 1.

4. The steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to any one of claims 1-3, wherein R3 is selected from C1-C12 alkanoyl, and preferably, R3 is selected from C1-C8 acyl.

5. The steroid phosphate compound, pharmaceutically acceptable salt or tautomer thereof according to any one of claims 1-4, wherein R1 and R2 are each independently selected from: alkali metal cations, alkaline earth metal cations, quaternary ammonium salts, ammonium ions, zinc ions, silver ions; preferably Li + , Na + , K + , Zn + , Mg 2+ , Ca 2+ , Ag + , ammonium ion, or N(C1-C4-alkyl)4 + .

6. The partial steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to any one of claims 1-5, which is selected from one of the following compounds:

7. A pharmaceutical composition comprising the steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to any one of claims 1-6.

8. Use of the steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claims 1-6, and the pharmaceutical composition of claim 7 in the manufacture of a medicament for the treatment of conditions related to androgen receptor activity.

9. Use of the steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claims 1-6, and the pharmaceutical composition of claim 7 in the manufacture of a medicament for the treatment of prostate cancer in patients sensitive and / or insensitive to hormone therapy.

10. Use of the steroid phosphate compound, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claims 1-6, and the pharmaceutical composition of claim 7 in the manufacture of a medicament for the treatment of hair loss or hair regrowth.

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