WO2024088395A1 - Bridged-ring compound and pharmaceutical use thereof - Google Patents

Abstract

Disclosed in the present invention are a series of bridged-ring compounds and a pharmaceutical use thereof, and in particular, disclosed are a compound as shown in formula (I) and a pharmaceutically acceptable salt thereof.

Description

Bridged ring compounds and their pharmaceutical applications

This application claims the following priority:

Application number: CN202211338895.0, application date: October 28, 2022;

Application number: CN202211407218.X, application date: November 10, 2022.

Technical Field

The present invention relates to a series of bridged ring compounds and their pharmaceutical applications, and in particular to compounds represented by formula (I) and pharmaceutically acceptable salts thereof.

Background technique

The androgen receptor (AR) is a 110 kDa steroidal nuclear receptor that mediates the induction of a variety of biological effects through interaction with endogenous androgens, one of its key functions being androgen-activated gene transcription. Endogenous androgens include steroids such as testosterone and dihydrotestosterone. Testosterone is converted to dihydrotestosterone in many tissues by the enzyme 5a reductase. The androgen receptor plays an important role in many androgen-related diseases, such as prostate cancer, androgenic alopecia, and acne. Compounds that weaken the action of androgens on the androgen receptor can be used to treat diseases or conditions in which the androgen receptor plays a role.

Summary of the invention

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in,

T is selected from CH and N;

each R ₁ is independently selected from F, Cl, Br, I, C _1-3 alkyl and C _1-3 alkoxy, and the C _1-3 alkyl and C _1-3 alkoxy are each independently optionally substituted by 1, 2 or 3 R _a ;

R ₂ is selected from H, C _1-4 alkyl and C _1-3 alkyl-S(═O) ₂ , wherein the C _1-4 alkyl and C _1-3 alkyl-S(═O) ₂ are each independently optionally substituted by 1, 2 or 3 R _b ;

Ring A is selected from 5-8 membered bridged cycloalkyl groups, wherein the 5-8 membered bridged cycloalkyl groups are optionally substituted by 1, 2 or 3 R groups;

each Ra _is independently selected from F, Cl, Br, I, OH, _NH2 and CN;

each R _b and R is independently selected from F, Cl, Br, I, OH, CN, NH ₂ , C _1-3 alkyl and C _1-3 alkoxy;

X is selected from O and NH;

n is selected from 0, 1, 2 or 3.

In some embodiments of the present invention, each _Ra is selected from F, and other variables are as defined in the present invention.

In some embodiments of the present invention, each R ₁ is independently selected from F, OCH ₃ and CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, each R ₁ is selected from CF ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, each R _b is independently selected from F, Cl, Br and OH, and other variables are as defined in the present invention.

In some embodiments of the present invention, each R _b is selected from OH, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from C _1-4 alkyl and C _1-3 alkyl-S(=O) ₂ , and the C _1-4 alkyl and C _1-3 alkyl-S(=O) ₂ are each independently optionally substituted by 1, 2 or 3 R _b , and other variables are as defined in the present invention.

In some embodiments of the present invention, _R2 is selected from _CH3 , _CH2CH3 , CH( _CH3 ) ₂ , _{CH2CH2CH3 , CH2OH, CH2CH2OH , CH2CH2CH2OH ,} CH2CH ₍ OH) _CH2 (OH), _{CH2CH ( OH ) CH3} and _{CH3 -} S(=O) ₂ , and other variables are as defined _in the present invention.

In some embodiments of the present invention, R ₂ is selected from CH ₂ OH, CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ OH, CH ₂ CH(OH)CH ₂ (OH) and CH ₂ CH(OH)CH ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from CH ₂ CH ₂ OH, and other variables are as defined in the present invention.

In some embodiments of the present invention, each R is independently selected from F, OH, CN, CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ and OCH ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from Other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from Other variables are as defined in the present invention.

In some embodiments of the present invention, the above compound has a structure shown in formula (I-1),

wherein T, X, R ₁ , R ₂ and n are as defined in the present invention.

Some other solutions of the present invention are obtained by arbitrarily combining the above variables.

The present invention also provides the following compounds or pharmaceutically acceptable salts thereof:

The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in preparing a drug for treating diseases associated with androgen receptor antagonists.

In some embodiments of the present invention, the above-mentioned application is characterized in that the disease associated with androgen receptor antagonists is androgenic alopecia.

Technical Effects

The compound of the present invention exhibits good AR antagonistic activity at the cellular level and inhibitory activity on LNCaP cell proliferation. The compound of the present invention has a significant therapeutic improvement effect on the pathological changes of C57BL/6 androgenic alopecia model mice. This study provides a good scientific basis for the regulation of hair follicles and hair growth and development and its production and utilization.

Definition and Description

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 to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are within the scope of medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, 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 compounds of the present invention contain relatively basic functional groups, 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. Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into either base or acid addition salts.

Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Denotes a solid wedge bond or dotted wedge key Or use a wavy line Represents a straight solid bond and straight dashed key

Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds to become a group with a corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group represents the nitrogen atom in the The two ends of the group are connected to other groups; The wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group.

The compounds of the present invention may exist in specific forms. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, different functional group isomers are in dynamic equilibrium and can quickly convert to each other. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomerism can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valence tautomers) include interconversions by the reorganization of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).

The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

The term "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes the The circumstances under which the described event or condition occurs and the circumstances under which the described event or condition does not occur.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituent can be arbitrary on the basis of chemical achievable.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

When the number of a linking group is 0, such as -(CRR) ₀ -, it means that the linking group is a single bond.

When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituent does not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0043] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

Unless otherwise specified, the term "C _1-3 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-3 alkyl _groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

Unless otherwise specified, the term "C _1-4 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. The C _1-4 alkyl group includes C _1-2 , C _1-3 and C _2-3 alkyl groups, etc.; they can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-4 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), etc.

Unless otherwise specified, the term "C _1-3 alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms connected to the rest of the molecule through an oxygen atom. The C _1-3 alkoxy includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy, etc. Examples of C _1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.

Unless otherwise specified, the number of atoms in a ring is generally defined as the ring member number, for example, "5-7 membered ring" refers to a "ring" having 5-7 atoms arranged around it.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any specific situation of n to n+m carbon atoms, for example, _C1-12 includes _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , C9, _C10 , _C11 , and _C12 , and also includes any range from n to n+m, for example, _C1-12 includes _C1-3 , _C1-6 , _C1-9 , _C3-6 , _C3-9 , _C3-12 , _C6-9 , _C6-12 , and _C9-12 , etc.; similarly, n- _membered to n+m-membered means that the number of atoms in the ring is n to n+m _. For example, a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any range from n to n+m, for example, a 3-12 membered ring includes a 3-6 membered ring, a 3-9 membered ring, a 5-6 membered ring, a 5-7 membered ring, a 6-7 membered ring, a 6-8 membered ring, and a 6-10 membered ring, etc.

Unless otherwise specified, the term "5-8 membered bridged cycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 5 to 8 ring atoms. It belongs to a bicyclic ring system. The 5-8 membered bridged cycloalkyl includes 5-6 membered, 5-7 membered, 5-8 membered, 5 membered, 6 membered, 7 membered and 8 membered bridged cycloalkyl, etc. Examples of 5-8 membered bridged cycloalkyl include, but are not limited to wait.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8venture diffractometer, the light source is CuKα radiation, and the scanning mode is φ/ω scanning. After collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure, and the absolute configuration can be confirmed.

The present invention uses the following abbreviations: DMF stands for dimethylformamide; DIPEA stands for N,N-diisopropylethylamine; HATU stands for 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

Detailed ways

The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed therein. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

Reference Example 1

synthetic route:

Step 1: Synthesis of Compound I-B

Compound Ⅰ-A (5 g, 28.15 mmol) was dissolved in acetonitrile (100 mL), potassium carbonate (19.45 g, 140.74 mmol) was added, and the mixture was stirred at 20°C for 10 minutes, and 2-bromoisobutyric acid (9.40 g, 56.30 mmol) was added, and the mixture was stirred at 20°C for 16 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was extracted with ethyl acetate (100 mL) and water (100 mL), and the organic phase was discarded. The aqueous phase was adjusted to pH 4-5 with dilute hydrochloric acid (4 M), and the mixture was directly concentrated under reduced pressure to obtain compound Ⅰ-B.

MS-ESI m/z: 227.9 [M+H] ⁺ .

Step 2: Synthesis of Compound Ⅰ-C

Compound Ⅰ-B (6.40g, 28.15mmol) was dissolved in toluene (20mL) and dichloromethane (20mL), triethylamine (2.85g, 28.15mmol) was added, and the mixture was stirred at 20°C for 10 minutes. Then 4-cyano-3-trifluoromethylphenyl isothiocyanate (9.64g, 42.23mmol) was dissolved in toluene (20mL) and added dropwise to the reaction solution, and the mixture was stirred at 20°C for 4 hours. The reaction solution was concentrated under reduced pressure to remove the solvent. The residue was diluted with ethyl acetate (100mL), the organic phase was washed with water (100mL), and the liquid was washed with saturated brine (100mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The crude product was purified by pulping (petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain compound Ⅰ-C.

MS-ESI m/z: 437.9 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J=8.3 Hz, 1H), 7.83 (d, J=1.8 Hz, 1H), 7.70 (dd, J=1.9, 8.2 Hz, 1H), 3.73 (s, 3H), 2.83 (s, 6H), 1.68 (s, 6H).

Step 3: Synthesis of Compound Ⅰ

Compound Ⅰ-C (1.00 g, 2.29 mmol) was dissolved in methanol (15 mL), and then sodium hydroxide (0.46 g, 11.43 mmol) was dissolved in water (5 mL) and added to the reaction solution. The temperature was raised to 40°C and stirred for reaction for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent. The residue was extracted with dichloromethane (20 mL) and water (20 mL). The organic phase was discarded, the aqueous phase was adjusted to pH 2-3 with dilute hydrochloric acid (4 M), extracted with dichloromethane (20 mL*2), the organic phases were combined, and the separated liquids were washed with saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain compound Ⅰ.

MS-ESI m/z: 424.0 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 12.66-12.19 (br, 1H), 8.34 (d, J=8.3 Hz, 1H), 8.23 (d, J=1.3 Hz, 1H), 7.99 (dd, J=1.4, 8.2 Hz, 1H), 2.72 (s, 6H), 1.60 (s, 6H).

Intermediate II

Triethylamine (11.75 g, 116.14 mmol) was added to a solution of compound Ⅰ-A (10 g, 38.71 mmol) in dioxane (40 mL), stirred at 20 ° C for 15 min, acetone (44.97 g, 774.30 mmol) and trimethylsilyl cyanide (19.20 g, 193.57 mmol) were added, and stirred at 80 ° C for 11.75 hours. After the reaction was complete, the reaction solution was evaporated under reduced pressure to remove excess solvent, ethyl acetate (50 mL) was added, and the reaction solution was washed with saturated sodium bicarbonate solution (50 mL*3), water (50 mL), and brine (10 mL), dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to remove excess solvent. The crude product was slurried with petroleum ether (50 mL) at 20 ° C (room temperature) for 30 minutes, filtered, and the filter cake was collected to obtain compound Ⅱ.

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 3.69 (s, 3H), 2.23 (s, 6H), 1.50 (s, 6H).

Example 1

synthetic route:

Step 1: Synthesis of compound 1

Compound Ⅰ-C (0.50 g, 1.18 mmol) was dissolved in dimethylformamide (10 mL), and N,N-diisopropylethylamine (0.46 g, 3.54 mmol) and HATU (0.67 g, 1.77 mmol) were added to the reaction solution in sequence, and finally ethanolamine (0.22 g, 3.54 mmol) was added, and the mixture was stirred at 20°C for 12 hours. The reaction solution was purified by high performance liquid chromatography (preparative column model: Xtimate C18, length × inner diameter: 150 mm × 40 mm, 5 μm; mobile phase system: acetonitrile/water (containing 0.05% HCl); gradient elution method: acetonitrile was gradiently eluted from 25% to 55%, and the elution time was 10 minutes) to obtain compound 1.

MS-ESI m/z: 467.2 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.70 (dd, J = 1.8, 8.3 Hz, 1H), 6.11(t, J＝5.0Hz, 1H), 3.76(t, J＝5.0Hz, 2H), 3.46(q, J＝5.5Hz, 2H), 2.81(s, 6H), 1.68(s, 6H).

Example 2

synthetic route:

Step 1: Synthesis of compound 2

Compound I-C (0.40 g, 0.94 mmol) was dissolved in dimethylformamide (10 mL), and N,N-diisopropylethylamine (0.37 g, 2.83 mmol) and HATU (0.54 g, 1.42 mmol) were added to the reaction solution in sequence, and finally (R)-3-amino-1,2-propanediol (0.17 g, 1.89 mmol) was added, and the mixture was stirred at 20°C for 12 hours. The reaction solution was purified by high performance liquid chromatography (preparative column model: Xtimate C18, length × inner diameter: 150 mm × 40 mm, 5 μm; mobile phase system: acetonitrile/water (containing 0.05% HCl); gradient elution method: acetonitrile gradient elution from 23% to 53%, elution time 10 minutes) to obtain compound 2.

MS-ESI m/z: 497.3 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J=8.3 Hz, 1H), 7.83 (s, 1H), 7.70 (d, J=8.0 Hz, 1H), 6.23 (t, J=5.1 Hz, 1H), 3.90-3.74 (m, 1H), 3.68-3.32 (m, 4H), 2.82 (s, 6H), 1.68 (s, 6H).

Example 3

synthetic route:

Step 1: Synthesis of compound 3

Compound I-C (0.50 g, 1.18 mmol) was dissolved in dimethylformamide (10 mL), and N,N-diisopropylethylamine (0.46 g, 3.54 mmol) and HATU (0.67 g, 1.77 mmol) were added to the reaction solution in sequence, and finally (S)-3-amino-1,2-propanediol (0.22 g, 2.36 mmol) was added, and the mixture was stirred at 20°C for 12 hours. The reaction solution was purified by high performance liquid chromatography (preparative column model: Xtimate C18, length × inner diameter: 150 mm × 40 mm, 5 μm; mobile phase system: acetonitrile/water (containing 0.05% HCl); gradient elution method: acetonitrile gradient elution from 25% to 55%, elution time 10 minutes) to obtain compound 3.

MS-ESI m/z: 497.3 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J=8.3 Hz, 1H), 7.83 (d, J=1.8 Hz, 1H), 7.70 (dd, J=2.0, 8.3 Hz, 1H), 6.21 (t, J=5.8 Hz, 1H), 3.89-3.75 (m, 1H), 3.68-3.32 (m, 4H), 2.82 (s, 6H), 1.68 (s, 6H).

Example 4

synthetic route:

Step 1: Synthesis of compound 4

Compound I-C (0.45 g, 1.06 mmol) was dissolved in dimethylformamide (10 mL), and N,N-diisopropylethylamine (0.41 g, 3.19 mmol) and HATU (0.61 g, 1.59 mmol) were added to the reaction solution in sequence, and finally (R)-(-)-1-amino-2-propanol (0.16 g, 2.13 mmol) was added, and the mixture was stirred at 20°C for 12 hours. The reaction solution was subjected to high performance liquid chromatography (preparative column model: Xtimate C18, length × inner diameter: 150 mm × 40 mm, 5 μm; mobile phase system: acetonitrile/water (containing 0.05% HCl); gradient elution method: acetonitrile gradient elution from 25% to 55%, elution time 10 minutes) to obtain compound 4.

MS-ESI m/z: 481.2 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J=8.0 Hz, 1H), 7.83 (d, J=1.3 Hz, 1H), 7.70 (dd, J=1.5, 8.3 Hz, 1H), 6.11 (t, J=5.0 Hz, 1H), 4.03-3.86 (m, 1H), 3.57-3.39 (m, 1H), 3.19-3.04 (m, 1H), 2.81 (s, 6H), 1.91 (s, 2H), 1.98-1.84 (m, 1H), 1.68 (s, 6H).

Example 5

synthetic route:

Step 1: Synthesis of compound 5

Compound Ⅰ-C (0.45 g, 1.06 mmol) was dissolved in dimethylformamide (10 mL), and N,N-diisopropylethylamine (0.41 g, 3.19 mmol) and HATU (0.61 g, 1.59 mmol) were added to the reaction solution in sequence, and finally (S)-(+)-1-amino-2-propanol (0.16 g, 2.13 mmol) was added, and the mixture was stirred at 20°C for 12 hours. The reaction solution was purified by high performance liquid chromatography (preparative column model: Xtimate C18, length × inner diameter: 150 mm × 40 mm, 5 μm; mobile phase system: acetonitrile/water (containing 0.05% HCl); gradient elution method: acetonitrile was gradiently eluted from 22% to 52%, and the elution time was 10 minutes) to obtain compound 5.

MS-ESI m/z: 481.2 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.96 (d, J=8.3 Hz, 1H), 7.83 (d, J=2.0 Hz, 1H), 7.70 (dd, J=1.9, 8.2 Hz, 1H), 6.06 (t, J=4.9 Hz, 1H), 4.02-3.88 (m, 1H), 3.55-3.44 (m, 1H), 3.19-3.06 (m, 1H), 2.81 (s, 6H), 1.77 (s, 3H), 1.68 (s, 6H).

Example 6

synthetic route:

Step 1: Synthesis of compound 6-B

Compound 6-A (10 g, 64.89 mmol) was added to methanol (100 mL), and sodium methoxide (5.26 g, 97.33 mmol) was added, and the mixture was refluxed at 80 ° C for 16 hours. The reaction solution was poured into water (300 mL), extracted with ethyl acetate (200 mL × 3), the organic phases were combined, washed with saturated brine (200 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was separated and purified by column chromatography (silica gel mesh: 100-200 mesh, eluent: petroleum ether/ethyl acetate = 1/0–10/1, volume ratio) to obtain compound 6-B.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 7.17 (dd, J=1.3, 8.5 Hz, 1H), 6.50 (t, J=8.3 Hz, 1H), 6.29 (s, 2H), 3.97 (d, J=2.3 Hz, 3H).

Step 2: Synthesis of compound 6-D

Compound 6-B (2.2 g, 13.24 mmol) was added to ethyl acetate (30 mL), and then compound 6-C (4.61 g, 19.86 mmol) was added, and the mixture was heated at 50°C for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and then separated and purified by column chromatography (silica gel mesh: 100-200 mesh, eluent: petroleum ether/ethyl acetate = 1/0–25/1, volume ratio) to obtain the target compound 6-D.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 7.65 (dd, J=1.9, 8.4 Hz, 1H), 7.33-7.20 (m, 1H), 4.11 (d, J=3.3 Hz, 3H).

Step 3: Synthesis of compound 6-E

Compound 6-D (4 g, 19.21 mmol) and intermediate II (4.00 g, 19.21 mmol) were added to tetrahydrofuran (80 mL), and triethylamine (5.83 g, 57.63 mmol) was added, and the mixture was stirred at 20°C for 16 hours. 4M hydrochloric acid (10 mL) and methanol (10 mL) were added, and the mixture was stirred at 20°C for 2 hours. After the reaction was completed, the reaction solution was added to water (300 mL), and extracted with ethyl acetate (200 mL × 2). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude product was separated and purified by column chromatography (silica gel mesh: 100-200 mesh, eluent: petroleum ether/ethyl acetate = 1/0–3/1, volume ratio) to obtain compound 6-E.

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.45 (dd, J=1.9, 8.4 Hz, 1H), 7.05 (dd, J=6.1, 8.4 Hz, 1H), 4.17 (d, J=3.0 Hz, 3H), 3.73 (s, 3H), 2.82 (s, 6H), 1.66 (d, J=1.5 Hz, 6H).

Step 4: Synthesis of compound 6

Compound 6-E (2.1 g, 5.03 mmol) was added to acetonitrile (20 mL), and then 2-aminoethanol (1.54 g, 25.15 mmol) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (210.07 mg, 1.51 mmol) were added, and the mixture was stirred at 20 °C for 16 hours. After the reaction was completed, the solvent was removed under reduced pressure, ethyl acetate (100 mL) was added to dissolve, and then washed with 0.5 M hydrochloric acid (100 mL) and saturated brine (100 mL) in turn, and the organic phase was spin-dried. Drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate to dryness under reduced pressure. The crude product was separated and purified by column chromatography (silica gel mesh: 100-200 mesh, eluent: dichloromethane/methanol = 1/0–25/1, volume ratio) to obtain compound 6.

MS-ESI m/z: 447.2 [M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.45 (dd, J=1.8, 8.5 Hz, 1H), 7.05 (dd, J=6.0, 8.3 Hz, 1H), 6.14 (t, J=5.1 Hz, 1H), 4.17 (d, J=3.3 Hz, 3H), 3.75 (t, J=4.9 Hz, 2H), 3.45 (q, J=5.4 Hz, 2H), 2.80 (s, 6H), 1.66 (s, 6H).

Example 7

synthetic route:

Step 1: Synthesis of compound 7-B

Compound 7-A (5 g, 29.38 mmol) was dissolved in toluene (150 mL), triethylamine (8.92 g, 88.15 mmol) and diphenylphosphoryl azide (16.17 g, 58.77 mmol) were added, stirred at 20 ° C for 30 minutes, and heated to 120 ° C for 3 hours. After the reaction solution was cooled to 20 ° C, benzyl alcohol (9.53 g, 88.15 mmol) was added, stirred at 20 ° C for 30 minutes, and heated to 120 ° C for 4 hours. The solvent was removed by concentration under reduced pressure. The residue was dissolved in ethyl acetate (150 mL), and the organic phase was washed with water (50 mL × 3) and saturated brine (50 mL × 3) in turn, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1:0-4:1). Compound 7-B was obtained.

MS-ESI m/z:276.2[M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.09 (s, 1H), 7.41-7.27 (m, 5H), 5.00 (s, 2H), 3.60 (s, 3H), 2.17 (s, 6H).

Step 2: Synthesis of compound 7-C

Compound 7-B (3.6 g, 13.08 mmol) was dissolved in tetrahydrofuran (36 mL) and water (18 mL), sodium hydroxide (1.05 g, 26.15 mmol) was added, and the mixture was stirred at 15°C for 1 hour and 30 minutes. The reaction solution was adjusted to pH 4 by adding 2M dilute hydrochloric acid, diluted with water (100 mL), and the aqueous phase was extracted with ethyl acetate (80 mL × 3), and the organic phases were combined and washed with saturated brine (80 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Compound 7-C was obtained.

MS-ESI m/z:262.2[M+H] ⁺ .

Step 3: Synthesis of compound 7-D

Compound 7-C (3.25 g, 12.44 mmol) was dissolved in dichloromethane (100 mL), and 2-chloro-1-methylpyridine (salt) iodide (3.81 g, 14.93 mmol), 4-dimethylaminopyridine (75.98 mg, 621.96 μmol) and methanesulfonamide (2.37 g, 24.88 mmol) were added. After stirring at 15°C for 10 minutes, triethylamine (3.78 g, 37.32 mmol, 5.19 mL) was added and stirred at 15°C for 1 hour. The reaction solution was dissolved in ethyl acetate (200 mL), and aqueous ammonium chloride solution (150 mL) was added for separation. The aqueous phase was extracted with ethyl acetate (100 mL × 2), and the organic phases were combined and washed with 1M dilute hydrochloric acid (50 mL × 2) and saturated brine (100 mL × 2) in turn. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1:0-0:1) to obtain compound 7-D.

MS-ESI m/z:339.1[M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 11.75 (s, 1H), 8.06 (s, 1H), 7.41-7.28 (m, 5H), 5.00 (s, 2H), 3.23 (s, 3H), 2.21 (s, 6H).

Step 4: Synthesis of compound 7-E

Under argon protection, wet palladium/carbon (130 mg, 10% palladium content) was placed in a hydrogenation bottle, methanol (100 mL) was added to wet it, and then compound 7-D (1.3 g, 3.84 mmol) was added, and the mixture was stirred at 15 psi and 15 ° C for 4 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and methanol (50 mL × 3) was added for elution. The filtrate was collected and concentrated under reduced pressure to remove the solvent. Compound 7-E was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 3.17 (s, 1H), 2.82 (s, 3H), 1.98 (s, 6H).

Step 5: Synthesis of compound 7-F

Compound 7-E (650 mg, 3.18 mmol) was dissolved in acetonitrile (13 mL), cooled to 0 °C, acetone (31.82 mmol, 2.34 mL) and zinc chloride (0.7 M, 909.27 μL) were added, and the mixture was stirred at 0 °C for 1 hour, and trimethylsilyl cyanide (1.26 g, 12.73 mmol) was added, and the mixture was heated to 80 °C and stirred for 12 hours. The reaction solution was concentrated under reduced pressure to remove the solvent. The residue was dissolved in ethyl acetate (30 mL), filtered, and rinsed with ethyl acetate (5 mL × 2). The filtrate was concentrated under reduced pressure to remove the solvent. Compound 7-F was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) δ: 11.62 (s, 1H), 3.82 (s, 1H), 3.22 (s, 3H), 2.17 (s, 6H), 1.37 (s, 6H).

Step 6: Synthesis of compound 7

Compound 7-F (450 mg, 1.66 mmol) was dissolved in N, N-dimethylformamide (9 mL), and compound 4-cyano-3-trifluoromethylphenyl isothiocyanate (454.14 mg, 1.99 mmol) was added, and the temperature was raised to 60 ° C and stirred for 12 hours. Then methanol (9 mL), water (9 mL) and hydrochloric acid (2M, 9.00 mL) were added, and the mixture was heated to 60 ° C and stirred for 2 hours. The solvent was removed by concentration under reduced pressure. The residue was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3), and the organic phases were combined and washed with saturated brine (20 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was subjected to high performance liquid chromatography (preparative column model: Phenomenex Luna C18, length * inner diameter: 250 mm * 50 mm, 10 μm). Mobile phase system: acetonitrile / water (containing 0.04% hydrochloric acid). Gradient elution method: acetonitrile was eluted from 30% to 70% in a gradient manner, and the elution time was 10 minutes to obtain compound 7.

MS-ESI m/z:500.9[M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 11.91 (s, 1H), 8.35 (d, J=8.0 Hz, 1H), 8.23 (s, 1H), 7.99 (dd, J=1.2, 8.0 Hz, 1H), 3.26 (s, 3H), 2.80 (s, 6H), 1.60 (s, 6H).

Example 8

synthetic route:

Step 1: Synthesis of compound 8-B

Dissolve compound 8-A (0.95 g, 3.94 mmol) in tetrahydrofuran (11 mL), methanol (3.3 mL) and water (3.3 mL), add lithium hydroxide monohydrate (330.45 mg, 7.87 mmol). Stir the reaction solution at 15 ° C for 16 hours. After the reaction is completed, dilute the reaction solution with water (30 mL) and adjust the pH to 3-4 with hydrochloric acid (1 M). Extract with ethyl acetate (30 mL*3), combine the organic phases, wash with saturated brine (50 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain compound 8-B.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 12.36 (s, 1H), 7.58 (s, 1H), 2.08 (s, 6H), 1.37 (s, 9H).

Step 2: Synthesis of compound 8-C

Compound 8-B (79 mg, 1.17 mmol) was dissolved in DMF (5 mL). Triethylamine (355.19 mg, 3.51 mmol), compound methylamine hydrochloride (398.86 mg, 1.76 mmol) and HATU (667.34 mg, 1.76 mmol) were added at 0-5°C. The reaction solution was stirred at 20°C for 2 hours. After the reaction was completed, the reaction solution was diluted with DCM (50 mL), washed with water (30 mL*2), the organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 8-C.

¹ H NMR (400 MHz, CDCl ₃ ) δ: 5.56 (s, 1H), 2.82 (d, J=4.8 Hz, 1H), 2.25 (s, 6H), 1.45 (s, 9H).

Step 3: Synthesis of compound 8-D

Compound 8-C (120 mg, 499.38 μmol) was dissolved in ethyl acetate hydrochloride (4 M, 2 mL), and the reaction solution was stirred at 15° C. for 3 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure to obtain the hydrochloride of compound 8-D.

Step 4: Synthesis of compound 8-E

Compound 8-D (0.045 g, 254.75 μmol, hydrochloride) was dissolved in acetonitrile (0.5 mL), and potassium carbonate (176.04 mg, 1.27 mmol) was added. A solution of 2-bromo-2-methylpropionic acid (85.09 mg, 509.5 μmol) in acetonitrile (0.5 mL) was added. The reaction solution was stirred at 20°C under nitrogen protection for 16 hours. After the reaction was completed, the reaction solution was adjusted to pH 1 with hydrochloric acid (2 M). The solution was concentrated under reduced pressure to obtain compound 8-E.

MS-ESI m/z:226.9[M+H] ⁺ .

Step 5: Synthesis of compound 8

Compound 8-E (0.04 g, 176.78 μmol) was suspended in toluene (0.5 mL) and dichloromethane (0.5 mL), triethylamine (17.89 mg, 176.78 μmol) was added, and the suspension was stirred at 20°C for 10 minutes. A toluene solution (0.5 mL) of compound 4-cyano-3-trifluoromethylphenylisothiocyanate (60.51 mg, 265.17 μmol) was added, and the reaction solution was stirred at 60°C for 4 hours. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure, and compound 8 was obtained by separation using a thick preparative chromatography plate and high performance liquid chromatography (ammonium bicarbonate method).

MS-ESI m/z:437.1[M+H] ⁺ .

¹ H NMR (400 MHz, CDCl ₃ -d) δ: 7.96 (d, J=8.0 Hz, 1H), 7.83 (d, J=1.6 Hz, 1H), 7.71-7.69 (m, 1H), 5.56 (s, 1H), 2.85 (d, J=4.8 Hz, 3H), 2.80 (s, 6H), 1.68 (s, 6H).

Biological Testing

Experimental Example 1: In vitro functional activity of compounds in androgen receptor reporter gene assay

Cells: HEK-293 cells

Reagents and consumables:

equipment:

Experimental steps:

1. Cell Culture

1) HEK-293 cell lines were cultured in DMEM medium containing 10% fetal bovine serum; the cells were cultured in an incubator at 37°C and 5% CO ₂ .

2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of TrypLE ^TM Express solution and incubate at 37°C for about 2 minutes. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

3) To maintain the physiological activity of cells, the cell confluence was controlled at about 80%.

4) Cell passaging, thawing and cryopreservation were performed according to conventional methods.

2. Experimental Procedure

1) Day 1: Plate and inoculate HEK-293 cells into 10 cm dishes;

2) The next day: transfection. Transfect pBIND-AR/pGL4.35/AR ORF plasmid into HEK-293 cells;

3) Day 3: Collect transfected cells, resuspend in phenol red-free DMEM medium containing 5% carbon-adsorbed serum, count and inoculate into 96-well plates, add test compounds, incubate at 37°C for 30 min, then add agonists, and then incubate in a 37°C incubator with 5% _CO2 ;

4) Day 4: 20-24 hours after drug addition, add Bright-Glo Reagent to the experimental wells according to the instructions of the kit Bright-Lite Luciferase Assay System, shake and lyse, and read the luminescence signal value (Luminescence) using a Biotek multi-function microplate reader.

3. Results Analysis

The mean and standard deviation of 0% inhibition (DMSO wells, MAX) and 100% inhibition (day0, DMSO) of each group were calculated;
Inhibition rate (%) = (1-(sample value-average value of 100% inhibition)/(average value of 0% inhibition-average value of 100% inhibition))*100;

The curves were fitted by GraphPad software and the _IC50 values were calculated.

The experimental results are shown in Table 1.

Table 1 Results of in vitro functional inhibition activity of the compounds of the present invention in in vitro detection of androgen receptor reporter gene assay

Conclusion: The compounds of the present invention exhibited good AR antagonistic activity.

Experimental Example 2: Study on the inhibitory effect of compounds on LNCaP cell proliferation in vitro

Cells: Human prostate cancer epithelial cells LNCaP

Reagents and consumables:

Phenol red-free 1640 medium, Invitrogen (Cat. No. 11835-030)

Fetal bovine serum, ExCell Bio (Cat. No.: FSP500)

Detection reagent CellTiter-Glo, Promega (Cat. No. G7568)

Trypsin, Gibco (Cat. No. 25200-072)

Penicillin-Streptomycin Gibco (Cat. No.: 15140-122)

96-well cell plate, Greiner (Cat. No.: 655090)

equipment:

Microplate reader, Perkin Elemer (model: Envision)

Cell counter, BECKMAN (model: Vi-CELL XR)

CO2 incubator, Thermo (model: 311)

Biological safety cabinet, AIRTECH (Model: BSC-1304IIA2)

Experimental steps:

1) Compound stock solution

The test compound was a 10 mM DMSO solution and stored in a 4°C refrigerator.

2) Preparation of cell culture medium

Cell culture medium configuration: 89% phenol red-free 1640 medium, 10% fetal bovine serum and 1% penicillin-streptomycin.

3) Cell plating

a) Take out the cultured cells from the incubator, remove the culture medium, wash twice with phosphate buffered saline, add 3 mL of trypsin to digest the cells, and incubate at 37°C for 3 to 4 minutes.

b) Terminate the digestion with 9 mL of assay medium, disperse the cells by blowing, resuspend by centrifugation, and count.

c) Dilute the cells to 0.2×10 ⁵ cells/mL with culture medium and seed 4000 cells per well in a 96-well cell plate with 200 μL.

d) Place in a 5% CO ₂ , 37°C incubator and incubate overnight.

4) Compound preparation and incubation

The compound was first diluted with culture medium to a final concentration of 100X, and the negative control group was DMSO diluted at the same ratio. Finally, the dilution was added to the cell supernatant at a ratio of 1:100 (0.1% DMSO, 2 μL of each concentration was added to the cell culture medium). The final maximum treatment concentration of 10 μM was diluted 3 times with culture medium to 9 concentrations. After the cell culture plate was incubated with the compound, it was returned to a 5% CO ₂ , 37°C incubator for 6 days.

5) Compound activity detection

a) Discard 50 μL of supernatant from each well of the cell plate.

b) Add 50 μL CTG detection reagent, shake for 10 min, and incubate at room temperature for 5 min. Read using Envision multi-label analyzer, program: US LUM 96 (CPS).

6) Result analysis
a) %Inhibition = ((RFU Cpd - AVER (RFU Neg.Ctrl)) / ((AVER (PBS) - AVER (RFU Neg.Ctrl)) × 100%

b) Using GraphPad Prism 8 data analysis software, select Dose-response-Inhibition-log [inhibitor] vs. response --

The variable slope (four parameters) model was used for fitting analysis to obtain the IC ₅₀ value of each test sample.

Table 3 Test results of the inhibitory effect of compounds on LNCaP cell proliferation

Conclusion: The compounds of the present invention exhibited good activity in inhibiting the proliferation of LNCaP cells.

Experimental Example 3: Pharmacodynamic evaluation study of compounds in AGA mouse model

1. Purpose of the experiment

The androgenic seborrheic alopecia (AGA) model was established by subcutaneous injection of testosterone propionate, and the hair growth effect of the test compound on androgenic seborrheic alopecia mice was preliminarily observed to provide a reference for the preclinical pharmacodynamic study of the test compound.

2. Main Reagents

2.1 Rosin, properties: yellow irregular crystals, specification: 100g, CAS number: 8050-09-7, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., storage method: room temperature.

2.2 Hualing brand high-efficiency slicing paraffin wax, properties: white irregular crystals, specification: 500g, manufacturer: Shanghai Peiou Biotechnology Co., Ltd., storage method: room temperature.

2.3 Testosterone propionate injection, properties: light yellow clear oily liquid, specification: 1mL: 25mg, batch number: veterinary drug 110201054, manufacturer: Hangzhou Animal Drug Factory, storage method: light-shielded, sealed and stored; olive oil (pharmaceutical grade), specification: 500mL/bottle, density: 0.9135g/mL, CAS number: 8001-25-0, manufacturer: Shanghai McLean Biochemical Technology Co., Ltd. Preparation method of 1.5mg/mL testosterone propionate diluent: take an appropriate amount of testosterone propionate and dilute it with high-pressure olive oil, and store it at room temperature (below 25°C).

2.4 Pentobarbital sodium: 25g/bottle, content ≥95.0%, batch number: 20201214, Merck packaging.

3. Main instruments

3.1 AG204-electronic analytical balance, METTLER, Switzerland;

3.2 MLS-3020 high pressure sterilizer, Sanyo Co., Ltd., Japan;

3.3 Nikon eclipse 80i microscope, Nikon Corporation;

4. Grouping and Dosing

Note: (1) The depilatory area of mice is uniformly 6 ^cm2 ; (2) 40 μL is administered per ^cm2 , and ² cm2 is applied to each mouse; (3) Frequency cycle BID*17 days; (4) Sample weight = concentration*volume; Example: To prepare 100 mL of 0.5% test compound solution, sample weight = 0.5%*100 mL = 0.5 g

5. Route and area of administration

Transdermal application, selection reason: consistent with the intended clinical route;

Dosing frequency and cycle: 2 times a day, for 17 consecutive days;

Dosing volume: 40 μL/cm ² ;

Method for locating the administration site: Use a marker pen to mark the 1×2 cm ² area on the shaved area at the back of the mouse as the administration area;

Dosing method: Before each administration, gently wipe the administration site with a cotton ball soaked in 0.9% sodium chloride injection at 37±0.5℃, and administer the drug after it dries; use a pipette to draw 80μL of the drug preparation of the corresponding concentration and apply it to the center of the marked area, and then use the gun tip to evenly spread the drug solution to the entire marked area; after administration, wait for the drug solution to dry before placing the mouse in the cage.

Note: Make sure the area for drug administration is the same every day. The first day of drug administration is defined as the first day of the trial.

6. Modeling method of androgenic seborrheic alopecia model

After the mice were numbered and weighed, they were anesthetized with sodium pentobarbital (0.3%, 0.25 mL/10 g). A 2cm×3cm experimental area was selected from the symmetrical parts of the body. Rosin and paraffin were mixed in a 1:1 ratio, heated in an electric furnace to melt into a liquid state, cooled to a temperature slightly higher than the mouse skin temperature, and about 1.41g was evenly applied to the experimental area. After the wax solidified, the condensed hair was gently torn off with tweezers, kept clean and dry, and confirmed that the hair growth of the unhaired mice was in the resting phase of the growth cycle, and the skin color was pink. On the second day after depilation, except for the mice in the normal control group, the depilated area of the mice in other groups was subcutaneously injected with testosterone propionate 5mg/kg (testosterone propionate was diluted with olive oil to a concentration of 1.5mg/mL, 33.3μL/10g) at multiple points, once a day, for 17 days, to create an androgenic alopecia model.

7. Observation indicators

7.1 Evaluation of Hair Growth Efficacy

7.1.1 Effects of the test compound on the grayscale rate of hair density in the administration area of androgenic alopecia model mice

The day of hair removal modeling was counted as day 0, and the drug administration started 24 hours after hair removal, which was counted as day 1. The skin of the hair removal area on the back of each group of mice was photographed on days 0, 2, 5, 9, 12, 14, and 16 of drug administration. Based on the photos taken, Adobe Photoshop CS6 software was used for analysis. The pictures were adjusted to grayscale mode, and the skin of the drug administration area of the mice was selected to calculate the grayscale rate of the skin of the drug administration area of each group of mice. Grayscale rate = grayscale value/255, grayscale value 255 is white. The lower the grayscale rate, the darker the color of the drug administration area and the denser the hair in the hair growth area.

Photographed animals: all surviving mice in each group; Photographing method: during the administration period, the mice were fixed in prone position by hand, and the animal numbers were marked above the mice. The camera was placed perpendicular to the back to take photos.

7.1.2 Effects of the test compounds on the time of skin discoloration and hair growth in the administration area of mice with androgenic alopecia

The skin color change time (the skin color of the dosing area changes from pink to gray) and hair growth time (the skin color of the dosing area changes from gray to black) of each mouse in each group were observed and recorded.

7.1.3 Effects of the test compounds on the length and weight of hair in the administration area of androgenic alopecia model mice

24 hours after the last administration on the 17th day, 5 areas were randomly selected from the administration area of each mouse, 5 tufts of hair were plucked with tweezers, and the length of hair growth in the administration area was measured. At the same time, the hair in the administration area was shaved with a shaver, and the weight and length of the hair were collected and weighed.

8. Data processing

SPSS 26.0 software was used for statistical analysis, and all data were expressed as mean ± standard error. It indicates that the ANOVA analysis of variance was used to evaluate the test results of the quantitative data, and the LSD test was used for pairwise comparison. The numerical values of the statistical results were retained to 1-2 decimal places.

9. Test results

Experimental results: Compared with the normal control group: *P<0.05, **P<0.01, ***P<0.001; compared with the model control group: ns

There was no statistically significant difference in the table, ^# P<0.05, ^## P<0.01, ^### P<0.001.

9.1 Effects of the test compounds on the time of skin discoloration and hair growth in the administration area of mice with androgenic alopecia model

Table 4 The skin color change time and hair growth start time of the hair removal drug administration area in each group of mice (d, n≥5)

As shown in Table 4, compared with the normal control group, the skin color change time and hair growth time of the administration area of the mice in the model control group were significantly prolonged, indicating that the model was successfully established. Compared with the model control group, the skin color change time and hair growth time of the administration area of the mice in the compound of the present invention group were significantly shortened.

9.2. Effects of the Test Compounds on Hair Length in the Administration Area of Mice with Androgenic Alopecia

Table 5 Hair length of the depilatory drug administration area of each group of mice (cm, n≥5)

As shown in Table 5, compared with the normal control group, the length of hair growth in the drug-administered area of the mice in the model control group was significantly decreased, indicating that the model was successfully established; compared with the model control group, the length of hair growth in the hair loss area of the mice in the compound of the present invention group was significantly increased.

9.3 Effects of the Test Compounds on Hair Weight in the Administration Area of Mice with Androgenic Alopecia

Table 6 Hair weight of the depilatory drug administration area of each group of mice (mg, n≥5)

As shown in Table 6, compared with the normal control group, the hair weight of the model control group mice in the administration area decreased significantly, indicating that the model was successfully established. Compared with the model control group, the hair weight of the depilatory area of the mice in the compound of the present invention group increased significantly.

9.4 Observation of skin hair follicle histomorphology and detection of hair follicle number

HE-stained skin longitudinal sections in the 100x microscope field of view show that the hair follicles in the normal group, minoxidil group, and test compound group are longer and in the thriving stage, with darker color, and the cells at the bottom of the hair follicles are fully developed, allowing the hair follicles to grow downward and wrap around the hair papilla. The hair parts are complete, with inner hair root sheaths, The melanin content was high. The hair follicles in the model control group were atrophic and in the resting stage, with a lighter color, apoptosis of the epithelial cells at the base of the hair follicles, and significant degeneration of the extracellular matrix of the hair papilla cells, most of which became black cell clusters, unclear hair papilla, no inner hair root sheath, and low melanin content.

Table 7 Detection of hair follicle numbers in the hair removal drug administration area of each group of mice (number, n≥5)

As shown in Table 7, there was no statistical difference in the total number of hair follicles in each group of HE-stained skin cross sections. The statistical analysis results of the ratio of terminal hair follicles to vellus hair follicles in HE-stained skin cross sections of each group showed that compared with the normal control group, the ratio of terminal hair follicles to vellus hair follicles in the model control group was significantly reduced, indicating that the model was successfully established. Compared with the model control group, the ratio of terminal hair follicles to vellus hair follicles in the administration area of mice in the compound of the present invention group was significantly increased.

10. Conclusion

The compound of the present invention has a therapeutic and improving effect on the pathological changes of C57BL/6 androgenic alopecia model mice.

Claims (11)

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
in, T is selected from CH and N; each R ₁ is independently selected from F, Cl, Br, I, C _1-3 alkyl and C _1-3 alkoxy, and the C _1-3 alkyl and C _1-3 alkoxy are each independently optionally substituted by 1, 2 or 3 R _a ; R ₂ is selected from H, C _1-4 alkyl and C _1-3 alkyl-S(═O) ₂ , wherein the C _1-4 alkyl and C _1-3 alkyl-S(═O) ₂ are each independently optionally substituted by 1, 2 or 3 R _b ; Ring A is selected from 5-8 membered bridged cycloalkyl groups, wherein the 5-8 membered bridged cycloalkyl groups are optionally substituted by 1, 2 or 3 R groups; each Ra _is independently selected from F, Cl, Br, I, OH, _NH2 and CN; each R _b and R is independently selected from F, Cl, Br, I, OH, CN, NH ₂ , C _1-3 alkyl and C _1-3 alkoxy; X is selected from O and NH; n is selected from 0, 1, 2 or 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein each Ra _is selected from F. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein each R ₁ is independently selected from F, OCH ₃ and CF ₃ . The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein each R _b is independently selected from F, Cl, Br and OH. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R ₂ is selected from CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH(CH ₃ ) ₂ , CH ₂ OH, CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ OH, CH ₂ CH(OH)CH ₂ (OH), CH ₂ CH(OH)CH ₃ and CH ₃ -S(＝O) ₂ . The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein each R is independently selected from F, CN, OH, CH ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ and OCH ₃ . The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein ring A is selected from According to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, the compound has a structure represented by the following formula (I-1):
T, X, R ₁ , R ₂ and n are as defined in any one of claims 1 to 6. The compound represented by the following formula or a pharmaceutically acceptable salt thereof,
Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases associated with androgen receptor antagonists. The use according to claim 10, characterized in that the disease associated with androgen receptor antagonists is androgenic alopecia.