A steroidal phosphate compound and its application

Abstract

The present invention discloses a steroidal phosphate compound, a pharmaceutically acceptable salt, a tautomer, and a preparation method, a pharmaceutical composition and use thereof. The steroidal phosphate compound is a compound with almost no hepatotoxicity and can be used for preventing and treating diseases related to androgen receptors, such as prostate cancer. Specifically, it relates to a steroidal phosphate compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the definition of each substituent is detailed in the specification.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a class of steroidal phosphate derivatives with almost no hepatotoxicity, a preparation method thereof as androgen antibody inhibitors and their medical uses, in particular, their use as drugs for preventing and treating androgen receptor-related diseases, such as prostate cancer. Background Art

[0002] Androgen receptor (AR) belongs to the nuclear receptor family and contains four main regions: N-terminal active transcriptional control region (NTD), DNA binding region (DBD), hinge region and ligand binding region (LBD). AR is mainly expressed in androgen target tissues such as prostate skeletal muscle, liver and central nervous system. The maximum expression level is 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).

[0003] Prostate cancer (PCa) is a malignant tumor that occurs in male prostate tissue. It is the result of abnormal and disordered growth of prostate acinar cells. The incidence of prostate cancer has obvious geographical and racial differences. In developed countries and regions such as Europe and the United States, it is the most common malignant tumor in men, and its mortality rate ranks second among all cancers. In Asia, its incidence is lower than that in Western countries, but it has been rising rapidly in recent years. At present, the incidence of prostate cancer in my country is increasing year by year, and most of the cases are found in the middle and late stages.

[0004] The occurrence and development of PCa are closely related to androgens, and endocrine therapy is currently the main treatment 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 the drug resistance of prostate cancer patients. Although these androgen receptor antagonists are effective for first-time advanced prostate cancer patients when used in combination with hormone therapy, their efficacy in drug-resistant prostate cancer is very limited whether used alone or in combination therapy. Studies in recent years have shown that bicalutamide has weak specificity as an androgen receptor (AR) antagonist and has a certain receptor activation effect (agonism) (flutamide has also been found to have similar problems). The latest research shows that the reactivation of the androgen receptor signaling pathway may be the root cause of resistance to hormone therapy, which limits the efficacy of androgen receptor antagonists such as bicalutamide, which are currently commonly used in the market, in the treatment of prostate cancer, especially for late-stage resistant prostate cancer. In addition, the efficacy of these androgen receptor antagonists is also generally limited by their significant side effects, such as toxicity to the liver or central nervous system. Therefore, the clinical treatment of prostate cancer urgently needs a new generation of androgen receptor antagonists with high activity, low toxicity and no residual activation properties (agonism).

[0005] Many corticosteroids have been used as anti-inflammatory, anti-rheumatic, anti-allergic and anti-shock agents. In particular, 11-deoxy-cortisol esters and their derivatives have been widely used as anti-inflammatory agents. Carboxylic acid 17 or 21 monoesters containing no more than six carbon atoms are also known.

[0006] For example, the preparation of 17α,21-diacetoxypregn-4-ene-3,20-dione is disclosed in US Pat. No. 3,530,038, which also mentions the use of propionyl derivatives and a series of aliphatic acyl derivatives containing chains of up to six carbon atoms.

[0007] US 3,152,154 discloses the preparation of 21-hydroxypregn-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-trien-20-one, wherein the 3 and 21 acyl groups are the same and are acetyl, propionyl, butyryl and isobutyryl. All examples cited in these documents relate to compounds in which the 17α and 21 positions are esterified by the same acyl group.

[0008] As the only 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 mixed esters is possible only when the substituent in position 21 is acetyl.

[0009] US3,530,038 discloses a method for preparing 11β-17α-21-trihydroxysteroids, which comprises oxidizing 11-deoxy-17α-OR-21-OR' steroids, wherein R is a carboxylic acid residue of 1 to 18 carbon atoms and R' is hydrogen or an acyl group of 1 to 18 carbon atoms, using a microorganism of the genus Curvularia to obtain the corresponding 11β-hydroxysteroid.

[0010] According to the same patent, compounds of the pregnane, androstane or estrane series are mentioned as possible starting steroids, but there is no mention of any conversion of 11 deoxy-17α-OR-21-OR′ steroids in which R is an acyl group of 1 to 18 carbon atoms and R′ is hydrogen.

[0011] The preparation of these products is described by RB 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).

[0012] US Pat. No. 3,780,177 discloses the preparation of 21-hydroxy-pregna-4,9-diene 3,20-dione-17a-butyrate by means of orthobutyrate and its use as an intermediate for the preparation of 6α,9α-difluoroprednisolone-17-butyrate-21 ester derivatives.

[0013] Finally, CN 1246328C discloses the preparation and use of mixed esters of 17α,21-dihydroxypregnene and describes 17 and 21 acyl derivatives having the same aliphatic chain containing not more than four carbon atoms.

[0014] In summary, no steroidal phosphate derivatives are known from the disclosed contents, and in particular, no mixed esters of 17α,21-dihydroxypregnene involving phosphate derivatives are known.

[0015] In the present invention, substitution in the chemical structure, such as replacement of the 21-hydroxyl group with a phosphate derivative, can improve specificity and reduce the side effects of androgen receptor antagonist drugs currently used in clinical practice (such as toxicity to the liver or central nervous system). These compounds can bind to androgen receptors, inhibit the movement of receptors into the cell nucleus and reduce their transcriptional activity, and are expected to become candidate androgen receptor antagonist drugs for the treatment of advanced prostate cancer. Summary of the invention

[0016] The present inventors have developed a steroidal phosphate compound represented by formula (I), a pharmaceutically acceptable salt thereof and a tautomer thereof.

[0017]

[0018] In formula (I),

[0019] R0 is selected from the following groups or atoms: H, -OCH2OCH3, -OCH3, -OAc, -OH, =NOH, =NH, or =O;

[0020] The dashed lines indicate the presence or absence of double bonds;

[0021] Y is selected from O or S;

[0022] R1 and R2 are independently selected from cations, 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;

[0023] Alternatively, R1 and R2 are connected to form a 5-7 membered ring together with P and O connected to R1 and R2 respectively;

[0024] R3 is selected from the following groups which may be substituted or unsubstituted by one or more radicals A: C1-C12 alkyl, C1-C12 alkanoyl, C5-C20 aryl, or aroyl.

[0025] 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, and 3-8 membered cycloalkyl.

[0026] In the scheme of the present invention, the dotted line indicates the presence or absence of a double bond, that is, the connected atoms can be single bonds or double bonds.

[0027] In some embodiments of the present invention, R0 is selected from: H, -OCH2OCH3, -OCH3, -OAc, -OH, in which case R0 forms a single bond with the connected carbon atom;

[0028] In some embodiments of the present invention, R0 is selected from: =NOH, =NH, or =O, in which case R0 forms a double bond with the carbon atom to which it is connected.

[0029] In some embodiments of the present invention, the above Y is selected from O or S; preferably, Y is O.

[0030] In some embodiments of the present invention, the pharmaceutically acceptable salts and tautomers thereof, the steroidal phosphate compound is as shown in the structural formula (II),

[0031]

[0032] Wherein, the substituents in formula (II) are as defined in formula (I).

[0033] In some embodiments of the present invention, R1 and R2 are both hydrogen;

[0034] In some embodiments of the present invention, R1 and R2 are selected from cations;

[0035] In some specific embodiments of the present invention, the above R1 and R2 are independently selected from metal cations; preferably, R1 and R2 are independently selected from: Li + 、Na + , K + 、Zn 2+ Mg 2+ , Ca 2+ 、Ag + ;

[0036] In some specific embodiments of the present invention, the above R 1 and R 2 Preferably, R1 and R2 are independently selected from N(C1-C4-alkyl or substituted alkyl)4 + ;

[0037] In some specific embodiments of the present invention, the above R1 and R2 are independently selected from the following groups which are substituted or unsubstituted 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 heterocyclic, C5-C20 aryl;

[0038] In some specific embodiments of the present invention, the above R1 and R2 are independently selected from: C1-C4 alkyl, C1-C4 alkylamino, C1-C4 alkylphenyl, C1-C4 alkyl heterocycle, C1-C4 alkylaryl,

[0039] In some embodiments of the present invention, R1 and R2 are connected to form a 5-7 membered ring together with P and O connected to R1 and R2 respectively.

[0040] In some embodiments of the present invention, the above R3 is selected from the following groups which are substituted or unsubstituted by one or more gene A: C1-C12 alkyl, C1-C12 alkanoyl, C5-C20 aryl, or aromatic acyl; preferably, R3 is selected from C1-C8 acyl; more preferably, R3 is selected from propionyl.

[0041] In the scheme 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, and 3-8 membered cycloalkyl.

[0042] The steroidal phosphate compound of the present invention is selected from:

[0043]

[0044]

[0045] Certain steroidal phosphate compounds of the present invention may exist in unsolvated or solvated forms, including hydrate forms. Generally speaking, the solvated forms are equivalent to the unsolvated forms and are all included in the scope of the present invention.

[0046] 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 within the scope of the present invention.

[0047] Definition and Explanation:

[0048] The following are definitions of terms used in this specification. The initial definitions provided for a group or term herein apply to the group or term throughout this specification, either alone or as part of another group, unless otherwise indicated.

[0049] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood in accordance with the ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commodity or its active ingredient. The term "pharmaceutically acceptable" used here refers to those compounds, materials, compositions and / or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0050] The terms "alkyl" and "alkane" refer to saturated hydrocarbons containing 1 to 12 carbon atoms, including straight chain, branched chain or cycloalkanes. Exemplary "alkyl" 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.

[0051] The term "alkenyl" refers to a straight or branched hydrocarbon group containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include vinyl or allyl.

[0052] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Exemplary such groups include ethynyl.

[0053] The term "aryl" refers to a cyclic aromatic hydrocarbon group containing 1 to 5 aromatic rings, especially a monocyclic or bicyclic group, and contains 5 to 20 carbon atoms. Exemplary such groups include phenyl, biphenyl or naphthyl, etc. If two or more aromatic rings are contained (bicyclic, etc.), the aromatic rings of the aryl group can be connected at a single point (e.g., biphenyl) or fused (e.g., naphthyl, phenanthrenyl, etc.).

[0054] The term "heterocycle" refers to a 3-8 ring group having at least one heteroatom in at least one carbon-containing ring, which is fully saturated or partially unsaturated or fully unsaturated, including aromatics (i.e., "heteroaromatics"). 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, wherein nitrogen and sulfur heteroatoms can be optionally oxidized and heteroatoms can be optionally quaternized. Exemplary monocyclic heterocyclic groups include, but are not limited to, azetidinyl, pyrrolidinyl, pyrrolyl, oxetanyl. Pyrrolinyl, imidazolyl. Imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuranyl, thienyl, etc.

[0055] The term "alkoxy" refers to a group in which an alkyl group is attached to an oxygen atom;

[0056] The term "alkylamino" refers to a group in which an alkyl group is attached to a nitrogen atom.

[0057] The term "pharmaceutically acceptable salt" refers to a salt of the steroidal phosphate compound of the present invention, prepared from a compound having a specific substituent discovered by the present invention and a relatively non-toxic acid or base. When the steroidal phosphate compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound 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 amino or magnesium salts or similar salts. When the steroidal phosphate compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such compound 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, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as 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 and methanesulfonic acid, etc.; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific steroidal phosphate compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

[0058] Preferably, the neutral form of the steroidal phosphate compound is regenerated by contacting the salt with a base or acid in a conventional manner and then isolating the parent steroidal phosphate compound. The parent form of the steroidal phosphate compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0059] As used herein, "pharmaceutically acceptable salts" are derivatives of the steroidal phosphate compounds of the present invention, wherein the parent steroidal phosphate compound is modified by salification with an acid or alkali. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali 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 steroidal phosphate compound, such as salts formed with 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, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxy, hydroxynaphthalene, isethionic 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, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid.

[0060] The term "chiral" refers to molecules that have the property of being non-superimposable on their mirror image partner, whereas the term "achiral" refers to molecules that are superimposable on their mirror image partner.

[0061] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0062] 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 points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated under high-resolution analytical methods, such as electrophoresis and chromatography.

[0063] The term "treating" refers to the extent it relates to a disease or condition, including 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.

[0064] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent steroidal phosphate compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these steroidal phosphate compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0065] The novel steroidal phosphate compounds disclosed in the present invention not only have a higher inhibitory effect on prostate cancer cells, but also surprisingly found through liver toxicity tests that this series of compounds can significantly reduce the common liver toxicity side effects of androgen receptor antagonist drugs currently used in clinical practice. These compounds are expected to become improved androgen receptor antagonist candidate drugs for the treatment of advanced prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The figures show the results of the hepatotoxicity test of the present invention: A. blank group B. abiraterone high-dose group C. compound AR01-1 high-dose group D. compound AR01-3 high-dose group E. compound AR01-9 high-dose group. DETAILED DESCRIPTION

[0067] Here, for example, many exemplary preparation methods of steroidal phosphate compounds of the present invention are provided in the following examples. The present invention is described in detail below by way of example, but is not intended to be any adverse restriction to the present invention. The present invention has been described in detail herein, and its specific embodiment modes are also disclosed, and it will be apparent to those skilled in the art that various changes and modifications will 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.

[0068] The steroidal phosphate compound of the present invention is synthesized using commercially available compound AR01-1-1, magnesium chloride, zinc chloride, ammonium bicarbonate, sodium bicarbonate, pyrophosphoryl chloride, diethyl chlorophosphate, dibenzyl chlorophosphonate, and diphenyl chlorophosphonate as starting materials.

[0069] Example 1: (8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(2-(phosphono)acetyl)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentanoic acid phenanthren-17-yl propionate (Compound AR01-1)

[0070] Synthesis route:

[0071]

[0072] Preparation method:

[0073] Under nitrogen protection, tetrahydrofuran (100 mL) and compound (AR01-1-1) (8.05 g, 20.0 mmol) (AR01-1-1) were added to the reaction bottle and the temperature was lowered to -40°C to -30°C. Pyrophosphoryl chloride (6.04 g, 24.0 mmol) was added to tetrahydrofuran (10 ml) and slowly dripped into the reaction bottle. During the dripping process, the temperature of the reaction liquid should be maintained at -40°C to -30°C, and the reaction was stirred and kept warm. After the reaction was completed by TLC monitoring, 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, and toluene (50 ml) was added. The layers were separated, and the aqueous phase was adjusted to pH = 3-4 with hydrochloric acid, filtered, and dried to obtain the crude compound AR01-1. The crude product was added to methyl tert-butyl ether / 1,2-methoxybutane (1:2), and the temperature was raised to 45°C. The reaction was stirred and kept warm. After the reaction was completed, the temperature was slowly lowered to room temperature, 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 HNMR(CDCl3): δ5.77(s,1H), 4.60~4.87(m,2H), 3.44~3.56(m,1H), 2.85(s,1H), 0.70~2.43(m,30H).

[0074] Example 2: Preparation of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propionyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl phosphate disodium salt (Compound AR01-2)

[0075] Synthesis route:

[0076]

[0077] Preparation method:

[0078] The compound AR01-1 (9.66 g, 0.02 mol) prepared in Example 1 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 added dropwise. The reaction was maintained at this temperature. After the reaction was completed, the ethanol was evaporated under reduced pressure at 20-30°C, 20 ml of acetone was added, crystallized, filtered, and dried to obtain AR01-2 (9.24 g), with a yield of 87.8%. ESI-MS (+): m / z=549.18.

[0079] Example 3: Preparation of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propionyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl phosphate zinc salt (Compound AR01-3)

[0080] Synthesis route:

[0081]

[0082] Preparation method:

[0083] The compound AR01-2 (10.52 g, 0.02 mol) prepared in Example 2 was added to 20 ml of water, and 10 ml of an aqueous solution containing 1.36 g of zinc chloride (0.01 mol) was added dropwise at 20-30°C. The reaction was maintained at this temperature, and solids were precipitated during the reaction. After the reaction was completed, the solids were filtered and dried to obtain AR01-3 (9.96 g), with a yield of 91.2%. ESI-MS (+): m / z = 567.10.

[0084] Example 4: Preparation of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propionyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl phosphate magnesium salt (Compound AR01-4)

[0085] Synthesis route:

[0086]

[0087] Preparation method:

[0088] Referring to the synthesis process of Example 3, magnesium chloride was used instead of zinc chloride to synthesize compound AR01-4 (3.21 g) with a yield of 88.2%. ESI-MS (+): m / z = 527.20.

[0089] Example 5: Preparation of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propionyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl phosphate diamine salt (Compound AR01-5)

[0090] Synthesis route:

[0091]

[0092] Preparation method:

[0093] Referring to Example 2, ammonium bicarbonate was used instead of sodium bicarbonate to synthesize compound AR01-5 (2.11 g) with a yield of 86.3%. ESI-MS (+): m / z = 539.28

[0094] Example 6: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(diethoxyphosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxy-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentanoic acid phenanthren-17-yl propionate (Compound AR01-6)

[0095] Synthesis route:

[0096]

[0097] Preparation method:

[0098] 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 a syringe within 30 minutes. The mixture was reacted overnight at room temperature. The mixture was then quenched with saturated NH4Cl aqueous solution 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 (eluted with petroleum ether:ethyl acetate = 5:1-2:1) to give product AR01-6 (3.65 g) in a yield of 67.7%. ESI-MS (+): m / z = 539.29.

[0099] Example 7: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(diphenyloxyphosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxy-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentanoic acid phenanthren-17-yl propionate (Compound AR01-7)

[0100] Synthesis route:

[0101]

[0102] Preparation method:

[0103] Substituting diethyl chlorophosphate with diphenyl chlorophosphonate, the compound AR01-7 (1.23 g) was prepared with a yield of 63.9%. ESI-MS (+): m / z = 635.28.

[0104] Example 8: Preparation of (8R,9S,10R,13S,14S,17R)-17-(2-(di(benzyloxy)phosphoryl)oxy)acetyl)-10,13-dimethyl-3-oxy-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentanoic acid phenanthrate (Compound AR01-8)

[0105] Synthesis route:

[0106]

[0107] Preparation method:

[0108] Substituting diethyl chlorophosphate with dibenzyl chlorophosphonate, the compound AR01-8 (1.43 g) was prepared with a yield of 66.2%. ESI-MS (+): m / z = 663.32.

[0109] Example 9: Preparation of 2-((8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-17-(propionyloxy)-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl phosphate disilver salt (Compound AR01-9)

[0110] Synthesis route:

[0111]

[0112] Preparation method:

[0113] Referring to the synthesis process of Example 3, silver nitrate was used instead of zinc chloride to synthesize compound AR01-9 (0.94 g) with a yield of 85.7%. ESI-MS (+): m / z = 695.02.

[0114] Example 10: Preparation of (8R,9S,10R,13S,14S,17R)-10,13-dimethyl-17-(2-(2-oxido-1,3,2-dioxophosphinol-2-yl)oxy)acetyl)-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentanoic acid phenanthren-17-yl propionate (Compound AR01-10)

[0115]

[0116] Preparation of compound AR01-10-SM:

[0117] Compound AR01-10-SM was synthesized in full accordance with the method described in Tetrahedron: Asymmetry 11 (2000) 125-138.

[0118] Preparation of compound AR01-10:

[0119] Compound AR01-10-SM was used to replace diethyl chlorophosphate, and the synthetic procedure of Example 6 was referred to to prepare compound AR01-10 (1.33 g) with a yield of 62.1%. ESI-MS (+): m / z=523.25.

[0120] In order to demonstrate that the compounds of the present invention exhibit improved activity over first-line anti-androgen drugs known to be in clinical use (e.g., abiraterone) and reduced side effects (e.g., toxicity to the liver) of clinically used androgen receptor antagonists, a series of in vitro studies were designed.

[0121] Example 11: Test of inhibitory activity on prostate cancer cells

[0122] Prostate cancer 22Rv1 cells were added to a flat-bottom 96-well cell culture plate at 5000 cells / well in a volume of 100μl. After 12 hours of adherent growth, the test compound was added in a final volume of 200μl; the test compound was diluted 2-fold from the highest concentration of 100uM, for a total of 6 concentrations; after 48 hours of culture, 10μl Cell Counting Kit-8 cell counting reagent (CCK-8) was added, and after incubation for 4 hours, the 450nm absorption value was detected using an enzyme reader. The IC50 value of the compound was calculated using GraphpadPrism5.0, and the results are shown in Table 1:

[0123] Table 1 Data table of the test results of different compounds inhibiting prostate cancer cells

[0124] 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

[0125] As can be seen from Table 1, the series of compounds of the present invention have significant prostate cancer cell inhibition activity relative to abiraterone.

[0126] Example 12: Mouse prostate cancer model test

[0127] SPF 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-treated group. RM-1 cells in the logarithmic growth phase were collected and inoculated at a cell density of 5×106 cells / mL, wipe the axilla of the right forelimb of the mouse with 75% alcohol, and inject 0.2mL into each mouse. After the tumor was formed, the tumor-bearing control group was orally injected with 200μL of normal saline every day, and the drug-treated group was orally injected with 0.04mg / g of abiraterone, AR01-1, AR01-2, AR01-3, AR01-4, AR01-5, AR01-6, AR01-7, AR01-8, AR01-9, and AR01-10, respectively. After 30 days of drug administration, the mice were killed by dislocation of the neck, and the subcutaneous tumors were separated and weighed. The tumor weight of the group of rats treated with the drug was compared with that of the tumor-bearing control group, and the percentage of tumor inhibition was calculated. The results are shown in Table 2.

[0128] Tumor inhibition rate % = (tumor weight of tumor-bearing control group - tumor weight of compound sample) / tumor weight of tumor-bearing control group * 100%

[0129] Table 2 Inhibitory effect on mouse prostate cancer cells

[0130] 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

[0131] The data from the prostate cancer inhibition test on mice in Table 2 show that, relative to 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, and AR01-10 of the present invention are all greater than 50%, among which the tumor inhibition rates of the compounds AR01-1, AR01-3, and AR01-9 are greater than 80%, showing a significant effect in inhibiting prostate cancer.

[0132] Example 13: Hepatotoxicity test detection:

[0133] 130 SD rats (120-140g) of specific pathogen-free (SPF) grade were selected, half of which were male and half were female. The feeding environment was: temperature 20-25℃, humidity 40%-70%, male and female were separated, and 5 rats were placed in each cage. According to the random allocation method, they were divided into 1 blank control group (10 rats), 1 abiraterone group, and 3 experimental groups, each with 10 rats. The abiraterone group and the experimental group were divided into 3 dose groups: low, medium, and high. Based on the commonly used clinical dose of abiraterone, the high, medium, and low doses were set to 66.8mg / kg, 33.4mg / kg, and 16.7mg / kg, respectively, according to the clinical equivalent of the recommended daily dose (1.0g) of the adult (60kg) prescription, 4, 2, and 1 times. The blank control group (equal volume of distilled water), the abiraterone group, and the experimental group were gavaged once a day (1mL / 100g rat body weight), and the body weight was weighed once a week. The dosage was adjusted according to the body weight, and the drug was administered continuously for 60 days.

[0134] Test method:

[0135] (1) General condition observation: The rats were weighed once a week, and their mental state, coat color, eating, excretion, activities, etc. were observed every day after medication.

[0136] (2) Routine blood test: Blood was collected from rats at 20, 40, and 60 days after medication for routine blood test.

[0137] (3) Liver function test: Rats were fasted for 12 h before the last administration and were allowed to drink normal water. The blank control group was given an equal volume of distilled water. Rats in other dose groups were given the drug by gavage. Ocular venous blood was collected 2 h later and centrifuged after standing for 30 min. The upper serum was collected and tested according to the instructions of the relevant kit to determine the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0138] (4) Histopathological examination:

[0139] After blood was drawn from the rats, the rats were immediately dissected and the livers were taken. The surrounding connective tissues were removed and rinsed in physiological saline. The surface liquid was then wiped dry with filter paper, weighed and the liver index (liver coefficient = liver weight / body weight) × 100% was calculated. The right lobe of the liver was taken, fixed with 10% formalin solution, embedded in paraffin, stained with conventional HE, and its tissue morphological changes were observed under an optical microscope. Some rats were killed at 20, 40, and 60 days after administration, and the livers were removed to observe their morphology, color, and texture, and liver tissue pathology was performed.

[0140] Data processing:

[0141] Statistical analysis was performed using SPSs13.0 statistical software. The t-test and variance analysis were used for comparison between groups. P<0.05 indicated statistically significant differences.

[0142] Test results:

[0143] (1) During the medication period, the rats' eating, excretion, and activities were relatively normal, and there was no obvious abnormal change in coat color. There was no mortality in any group.

[0144] (2) The body weights of rats in the abiraterone group and the experimental group were within the normal range at 20, 40, and 60 days after administration, and there was no significant difference between the groups (P ≥ 0.05). The results are shown in Table 3.

[0145] Table 3 Changes in body weight of rats in each group after 60 days of drug administration (g, n=10)

[0146]

[0147] (3) Routine blood test

[0148] At 20 and 40 days of drug administration, all blood routine indexes of rats were within the normal range, and there was no statistically significant difference between the abiraterone group and the experimental group and the blank group (P>0.05); at 60 days of drug administration, there was a significant difference between the abiraterone group and the blank group, while all blood routine indexes of the experimental group were within the normal range, and there was no statistically significant difference between the experimental group and the blank group (P>0.05). The results are shown in Table 4.

[0149] Table 4 Comparison of blood routine test results of rats after 60 days of drug administration ( n=10)

[0150]

[0151] (4) Liver function test

[0152] Compared with the blank group, the serum AST and ALT values ​​of rats in the abiraterone group were in a higher range, while the serum AST and ALT levels of the different compound dose groups were relatively normal, and there was no statistically significant difference compared with the blank group (P>0.05). However, it can be found that with the increase of the dose, the AST and ALT values ​​gradually increased, forming a certain dose-dependent relationship. The results are shown in Table 5.

[0153] Table 5 Comparison of liver function test results in rats after 60 days of drug administration ( n=10)

[0154]

[0155]

[0156] (5) Liver / body ratio

[0157] Compared with the blank group, each dose group of abiraterone resulted in an increase in liver / body ratio, while each dose group of the experimental group showed no significant difference (P>0.05). The results are shown in Table 6.

[0158] Table 6 Comparison of liver organ index results of rats after 60 days of drug administration ( n=10)

[0159]

[0160]

[0161] (6) Liver histopathological examination

[0162] Macroscopic observation: The livers of rats in the blank group and the experimental group at each dose were normal in morphology, soft in texture, ruddy and shiny, without nodules, masses, cysts, etc.

[0163] After HE staining, the livers of rats in each dose group of abiraterone were observed under a light microscope to different degrees of damage, mainly manifested as swelling of hepatocytes, and a small number of small focal or single cell necrosis was observed. Necrotic cells were diffusely present in the center of the liver lobule, accompanied by inflammatory cell infiltration (high dose of abiraterone was seen in Figure 1 B in the figure); while the blank group (blank group see Figure 1 The hepatic lobule structure of rats in the experimental group was normal, with no swelling or necrosis of hepatocytes, no dilatation of central veins, no hyperplasia of portal areas, no inflammatory cell infiltration, microvesicular fatty degeneration of some hepatocytes, and mild turbid swelling of some hepatocytes at 40 days, but there was no significant difference among the groups (P>0.05); at 60 days, the hepatic lobule structure was intact, with clear boundaries of hepatocytes, no dilatation of central veins, no hyperplasia of portal areas, no inflammatory cell infiltration, macrovesicular and microvesicular fatty degeneration of some hepatocytes, and mild and moderate watery degeneration of some hepatocytes, but there was no significant difference among the groups (P>0.05) (high doses of compound AR01-1, compound AR01-3 and compound AR01-9 were respectively Figure 1 C, D and E in ). See Figure 1 .

Claims

1. A steroidal phosphate compound represented by formula (I), a pharmaceutically acceptable salt thereof or a tautomer thereof, In formula (I), R0 is selected from =0; There is a single bond between carbons at positions 1 and 2; there is a double bond between carbons at positions 4 and 5; Y is selected from O; R1 and R2 are independently selected from Na + 、Zn 2+ Mg 2+ 、Ag + NH4 + , hydrogen, ethyl, phenyl, benzyl, or R1 and R2 together form propylene; R3 is selected from propionyl.

2. The steroidal phosphate compound according to claim 1, its pharmaceutically acceptable salt or its tautomer, selected from one of the following compounds:

3. A pharmaceutical composition comprising the steroidal phosphate compound, a pharmaceutically acceptable salt thereof or a tautomer thereof as claimed in any one of claims 1 to 2.

4. Use of the steroidal phosphate compound according to claims 1-2, its pharmaceutically acceptable salt or tautomer, and the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating a disease associated with androgen receptor activity.

5. Use of the steroidal phosphate compound according to claims 1-2, its pharmaceutically acceptable salt or tautomer, and the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating prostate cancer in patients who are sensitive and / or insensitive to hormone therapy.

6. Use of the steroidal phosphate compound according to claims 1-2, its pharmaceutically acceptable salt or tautomer, and the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating hair loss or regenerating hair.

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