CN108350024B - Substituted steroid compound and application thereof - Google Patents

Abstract

The invention provides a substituted steroid compound and application thereof. Specifically, the substituted steroid compound is a steroid compound shown in formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate thereof. The substituted steroid compound and the composition containing the compound disclosed by the invention have excellent inhibition on CYP17 enzyme and Androgen Receptor (AR), have better pharmacokinetic parameter characteristics, and can improve the drug concentration of the compound in an animal body so as to improve the drug curative effect and safety.

Description

Substituted steroid compound and application thereof

Technical Field

The invention belongs to the field of medicine. The invention relates to steroid compounds and application thereof, in particular to steroid compounds and application thereof as a CYP17 enzyme irreversible inhibitor and an Androgen Receptor (AR) antagonist, which can be used for treating and preventing diseases related to CYP17 enzyme and Androgen Receptor (AR).

Background

Prostate cancer (PCa) is a common malignancy of the male reproductive system. There were 903500 new cases of prostate cancer and 258400 deaths of prostate cancer worldwide in 2008. Wherein the new cases of prostate cancer account for 14 percent of all new cases of tumors in men, and the new cases of tumors in men are listed at the 2 nd position; prostate cancer deaths account for 6% of the cancer deaths in men, and the 6 th cancer deaths in men. Prostate cancer deaths account for 9% of men's cancer deaths, second only to lung cancer, and in the 2 nd of men's cancer deaths. With the recent increase in environmental pollution, the increase in aging speed of the population, the change in dietary patterns of people, and the like, the incidence and mortality of prostate cancer have rapidly increased, and thus, the disease has become one of the important diseases affecting the health of men in China.

The effect of androgens on the growth of Prostate cancer cells is mediated through the Androgen Receptor (AR) signaling pathway, and the progression of Prostate cancer in a patient is clinically diagnosed by observing the levels of Prostate Specific Antigen (PSA) in the patient through changes in AR signaling. Conventional castration therapy does not completely suppress androgen production or androgen receptor target gene expression, and when androgen-synthesizing biological enzymes are overexpressed, the level of androgen in the tumor is increased.

Cytochrome oxidase P450c17 (CYP17) is expressed in testis, adrenal gland and normal prostate tissue, as well as in prostate cancer cells. 17 alpha-hydroxylase and C17, 20-lyase in CYP17 are key enzymes in androgen biosynthesis, and can promote the conversion of steroid progestogen and pregnenolone into C19 androstenedione and dehydroepiandrosterone, respectively, which in turn converts testosterone and dihydrotestosterone.

Based on the research, the work of preventing and treating the prostate cancer is not slow, and the research and development of CYP17 enzyme inhibitors are important directions for the drug treatment of the prostate cancer. As a novel CYP17 enzyme inhibitor, abiraterone acetate was developed by Centocor Ortho for the treatment of prostate cancer. Abiraterone acetate was approved by the U.S. FDA on day 28/4/2011 and was used in combination with prednisone to treat castration-resistant prostate cancer, which is marketed under the trade name Zytiga. Year 2011, 28/7/t, Zytiga was approved by the health canada department. In prostate cancer patients, hormone testosterone can stimulate the growth of tumors, castration treatment including drug or surgical treatment can reduce the production of testosterone or block the action of testosterone, but the treatment cannot inhibit the production of androgen in other parts of the body, and prostate cancer can still continue to grow.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a substituted steroid compound, a composition containing the same, and uses thereof, which have better CYP17 enzyme and Androgen Receptor (AR) inhibitory activity and/or better pharmacodynamic/pharmacokinetic properties, and can be used for treating, preventing, and alleviating diseases mediated by CYP17 enzyme or Androgen Receptor (AR).

In contrast, the technical scheme of the invention is as follows:

a substituted steroid compound, which is a steroid compound shown in formula (I), or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvent compound thereof,

in the formula:

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³independently of each other, selected from the group consisting of "hydrogen (H), deuterium (D)";

X¹、X²independently of one another, from the group consisting of hydrogen (H), deuterium (D), methyl, CH₂D、CHD₂、CD₃、CH₂CH₃、CHDCH₃、CHDCH₂D、CHDCHD₂、CHDCD₃、CD₂CH₃、CD₂CH₂D、CD₂CHD₂、CD₂CD₃"a group consisting of;

y is selected from hydrogen (H), deuterium (D), hydroxyl, acetyl, and acetyl with one or more deuterations;

and the physiologically acceptable salts, prodrugs, metabolites, solvates, tautomers and stereoisomers thereof, including mixtures of these compounds in all ratios.

In another preferred embodiment, R¹、R²、R³、R⁴、R⁵Independently is deuterium or hydrogen.

In another preferred embodiment, Y is selected from hydrogen, deuterium, hydroxy, mono-or poly-deuterated acetyl.

In another preferred embodiment, X¹、X²Is a three-deuterated methyl group.

In another preferred embodiment, the compounds of the present invention are selected from the group consisting of substituted steroids, or pharmaceutically acceptable salts thereof, but are not limited to the following:

in another alternative, the compound does not include non-deuterated compounds.

As a further improvement of the present invention, the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³And X¹、X²The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

In another alternative, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³Preferably, at least one of R, preferably two of R, more preferably three of R, more preferably four of R, more preferably five of R, more preferably six of R, more preferably seven of R, more preferably eight of R, more preferably nine of R, more preferably ten of R, more preferably eleven of R, more preferably twelve of R, more preferably thirteen of R, more preferably fourteen of R, more preferably fifteen of R, more preferably sixteen of R, more preferably seventeen of R, more preferably eighteen of R, more preferably nineteen of R, more preferably twenty-one of R, more preferably twenty-two of R, more preferably twenty-three of R.

As a further improvement of the present invention, a pharmaceutically acceptable carrier is mixed with the substituted steroid compound as described above, or a crystalline form, a pharmaceutically acceptable salt, a prodrug, a metabolite, a stereoisomer, an isotopic variant hydrate or solvate thereof, to form the pharmaceutical composition.

The invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the substituted steroid compound, or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate, a stereoisomer, a prodrug or an isotopic variant thereof.

The invention also includes isotopically-labeled compounds, equivalent to those disclosed herein as the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

As a further improvement of the invention, the medicine also comprises other therapeutic drugs, and the therapeutic drugs are drugs for cancers, cell proliferative diseases, inflammations, infections, immunological diseases, organ transplantation, viral diseases, cardiovascular diseases or metabolic diseases.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-1000mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

The invention also discloses application of the substituted steroid compound or the crystal form, the pharmaceutically acceptable salt, the hydrate or the solvent compound thereof in preparing a pharmaceutical composition for treating, preventing and relieving diseases mediated by CYP17 enzyme and Androgen Receptor (AR).

Because the compound has excellent inhibitory activity on CYP17 enzyme and Androgen Receptor (AR), the compound and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound as a main active ingredient can be used for treating, preventing and relieving diseases mediated by CYP17 enzyme and Androgen Receptor (AR). According to the prior art, the compounds of the invention are useful for the treatment of the following diseases: prostate cancer, benign prostatic hyperplasia, hirsutism, alopecia, anorexia nervosa, breast cancer, male gonadal hyperactivity, etc.

The invention has the beneficial effects that:

the substituted steroid compound and the composition containing the compound have excellent inhibition on CYP17 enzyme and Androgen Receptor (AR), have better pharmacokinetic parameter characteristics, and can improve the drug concentration of the compound in animal bodies so as to improve the curative effect and safety of drugs; the substituted steroids and compositions containing them disclosed in this invention are useful in the treatment, prevention and alleviation of conditions mediated by the CYP17 enzyme and the Androgen Receptor (AR).

Detailed Description

The research of the inventor finds that the deuterated steroid compound and the pharmaceutically acceptable salt thereof have equivalent or better pharmacokinetic and/or pharmacodynamic properties than the non-deuterated compound, so that the deuterated steroid compound and the pharmaceutically acceptable salt thereof are suitable for being used as compounds for inhibiting CYP17 enzyme and Androgen Receptor (AR), and are further more suitable for preparing medicines for treating cancers and diseases related to CYP17 enzyme and Androgen Receptor (AR). The present invention has been completed based on this finding.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds in which the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and basic or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, the above salts can be prepared as follows: reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both; generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Suitable salts are described in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa, p.1418(1985), the disclosure of which is incorporated herein by reference.

For example, a salt of a compound of formula (I) may be formed as follows: the compound of formula (I) is reacted with, for example, an equivalent amount of an acid or base in a medium that allows the newly formed salt to precipitate out or be isolated by lyophilization. Exemplary acid salts that the compounds of formula (I) may form with inorganic and/or organic acids include, but are not limited to, for example, acetate, ascorbate, benzoate, benzenesulfonate, bisulfate, bitartrate, acid citrate, ethanesulfonate, formate, fumarate, gentisate, gluconate, glucarate, glutamate, hydrochloride, hydrobromide, hydroiodide, isonicotinate, maleate, methanesulfonate, nitrate, pantothenate, phosphate, acid phosphate, saccharate, salicylate, succinate, acid salt, tartrate, p-toluenesulfonate, trifluoroacetate, lactate and pamoate (i.e., 1' -methylene-bis (2-hydroxy-naphthalene-3-formate)). The above salts may be formed according to methods known to those skilled in the art.

Exemplary base salts that the compounds of formula (I) may form with inorganic and/or organic bases include, but are not limited to, for example, ammonium salts; alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as benzathine, dicyclohexylamine, 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tris, hydrabamines) (e.g., N-di (dehydroabietyl) ethylenediamine), N-methyl-D-glucamine, N-methyl-D-imidazolediamide, and t-butylamine, salts with amino acids such as arginine and lysine, and salts formed by quaternization of basic nitrogen-containing groups using agents such as lower alkyl halides (e.g., methyl chloride, methyl bromide, methyl iodide, ethyl chloride, ethyl bromide, ethyl iodide, propyl chloride, propyl bromide, propyl iodide, butyl chloride, butyl bromide, and butyl iodide), dialkyl sulfates (e.g., dimethyl sulfate, methyl iodide, ethyl chloride, ethyl bromide, ethyl iodide, propyl chloride, propyl bromide, butyl chloride, butyl bromide, and butyl iodide), and salts with basic nitrogen-containing groups, Diethyl sulfate, dibutyl sulfate, and diamyl sulfate), long-chain halides (e.g., decyl chloride, decyl bromide, decyl iodide, lauryl chloride, lauryl bromide, lauryl iodide, myristyl chloride, myristyl bromide, myristyl iodide, stearyl chloride, stearyl bromide, and stearyl iodide), and aralkyl halides (e.g., benzyl bromide and phenethyl bromide). The above salts may be formed according to methods known to those skilled in the art.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

In addition, the compound also comprises a prodrug of the steroid compound shown in the formula (I). The term "prodrug" includes a class of compounds which may be biologically active or inactive in nature and which, when administered by an appropriate method, undergo a metabolic or chemical reaction in the human body to convert the compound to formula (I), or a salt or solution of a compound of formula (I). The prodrugs include, but are not limited to, carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amide, carbamate, azo, phosphoramide, glucoside, ether, acetal forms of the compounds.

"therapeutically effective amount" is intended to include individual amounts of the compounds of the present invention or combined amounts of the claimed compounds or combined amounts of the compounds of the present invention and other active ingredients effective as antagonists of the CYP17 enzyme or effective in the treatment of cancer.

As used herein, "treatment" or "treatment" includes treatment of a disease state in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease state in a mammal, particularly when the mammal is predisposed to the disease state but has not yet been diagnosed with the disease state; (b) inhibiting, i.e. arresting the development of, said disease state; and/or (c) alleviating, i.e., allowing regression of, the disease state.

The compounds of the present invention may contain one or more additional asymmetric carbon atoms and may therefore exist in two or more stereoisomeric forms. The present invention includes all possible single stereoisomers, their single tautomeric forms and their mixtures. Diastereomers may be separated by conventional techniques, for example by fractional crystallization, chromatography or HPLC of a mixture of stereoisomers of the compounds of the invention or suitable salts or derivatives thereof. The single enantiomers of the compounds can also be prepared from the corresponding optically pure intermediates or as follows: the corresponding racemates are resolved (such as by HPLC) using a suitable chiral support or, where appropriate, the diastereomeric salts are fractionally crystallized, which are prepared by reacting the corresponding racemates with a suitable optically active acid or base. All stereoisomers (in mixture or pure or substantially pure form) of the compounds of the invention are included in the present invention.

The present invention also includes a class of pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as "carrier" materials) and optionally other active ingredients. The compounds of formula (I) may be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for the above route and in a dose effective for the desired treatment. For example, the compounds and compositions of the present invention may be administered orally, mucosally, or parenterally (including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, and infusion techniques) in dosage unit formulations containing conventional pharmaceutical carriers, adjuvants, and vehicles. For example, the pharmaceutical carrier may contain a mixture of mannitol or lactose and microcrystalline cellulose. The mixture may contain other components such as lubricants (e.g. magnesium stearate) and disintegrants (e.g. crospovidone). The carrier mixture may be filled into gelatin capsules or compressed into tablets.

The pharmaceutically active compounds of the present invention can be processed in accordance with conventional pharmaceutical procedures to prepare medicaments for administration to patients, including humans and other mammals.

For oral administration, the pharmaceutical composition may be in the form of: such as tablets, capsules, suspensions or liquid formulations. The pharmaceutical compositions are preferably prepared in dosage unit form containing specific amounts of the active ingredient. Examples of such dosage units are tablets or capsules.

The amount of compound administered and the dosage regimen for treating a condition with a compound and/or composition of the invention will depend upon a variety of factors including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration and the particular compound used. Thus, the dosage regimen may vary widely, but can be routinely determined using standard methods.

For therapeutic purposes, the active compounds according to the invention are usually combined with one or more adjuvants suitable for the intended route of administration. If administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose alkanoates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinyl alcohol and/or polyvinyl pyrrolidone, and then tableted or encapsulated for convenient administration. Such capsules or tablets may comprise a controlled release formulation which may be provided as a dispersion of the active compound in hydroxypropylmethyl cellulose.

The oily phase of the emulsion containing the compound of formula (I) may be constituted by known ingredients in a known manner. Although the phase may comprise only emulsifiers, it may comprise mixtures of at least one emulsifier with fats or oils or with fats and oils. Preferably, a hydrophilic emulsifier is included with a lipophilic emulsifier as a stabilizer. It is also preferred to include both oil and fat. In addition, emulsifiers (with or without stabilizers) constitute the so-called emulsifying waxes and said waxes, together with oils and fats, form the so-called emulsifying ointment base which forms the oily dispersed phase of the cream. Emulsifiers and emulsion stabilizers suitable for use in the formulations of the present invention include tween 60, span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate or glyceryl distearate, alone or with waxes or other materials known in the art.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations (such as sterilization) and/or may contain conventional adjuvants (such as preservatives, stabilizers, wetting agents, emulsifiers, buffers and the like). Tablets and pills may also be prepared with an enteric coating. The composition may also contain adjuvants such as wetting agents, sweeteners, flavoring agents and fragrances.

The pharmaceutical compositions of the present invention comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof and optionally other substances selected from any pharmaceutically acceptable carriers, adjuvants and vehicles. Alternatively, the compositions of the present invention comprise a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

The compounds of formula (I) are useful for treating cancer, for example cancer which relies on androgen receptor signalling. These compounds inhibit the activity of CYP17 enzymes, which are involved in androgen biosynthesis. Blocking this enzyme inhibits the production of gonads, adrenaline and tumor androgens and provides a new option for treating patients with cancers that depend on androgen receptor signaling, such as prostate cancer and estrogen receptor positive breast cancer. Accordingly, the treatment comprises administering to the patient a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In one embodiment, a method for treating cancer is provided, the method comprising administering to a mammal in need thereof a compound of formula (I). The methods of this embodiment may be used to treat a variety of cancers, including, but not limited to, bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreas/gall bladder cancer, prostate cancer, thyroid cancer, bone cancer, rhabdomyosarcoma, Malignant Fibrous Histiocytoma (MFH), fibrosarcoma, glioblastoma/astrocytoma, melanoma, and mesothelioma.

Preferably, the method of this embodiment is used to treat a variety of prostate cancers.

The amount of the compound of formula (I) administered and the dosage regimen for the treatment of a particular cancer will depend upon a variety of factors including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration and the particular compound used. Thus, the dosage regimen may vary widely, but can be routinely determined using standard methods. A daily dose of about 0.01 to 1500mg/kg body weight, preferably about 0.5 to about 50mg/kg body weight and most preferably about 0.1 to 20mg/kg body weight may be suitable. The daily dose may be administered from 1 to 4 times per day.

In the treatment of cancer, chemotherapeutic agents and/or other therapeutic (e.g., radiation therapy) combinations are often advantageous. The second (or third) agent may have the same or a different mechanism of action than the primary therapeutic agent. It may be particularly useful to use a combination of cytotoxic drugs wherein two or more drugs are administered that act in different ways or at different cell cycles, and/or wherein two or more drugs have overlapping toxicities or side effects, and/or wherein the drugs in combination each have a significant therapeutic effect in treating a particular disease state exhibited by a patient.

Thus, the compounds of formula (I) may be administered in combination with other anti-cancer treatments for the treatment of cancer or other proliferative diseases. The invention further includes the use of a compound of formula (I) in the manufacture of a medicament for the treatment of cancer, and/or a package comprising a compound of formula (I) herein together with instructions for use in combination with other anti-cancer or cytotoxic agents and in therapy for the treatment of cancer. The invention further includes the combination of a compound of formula (I) and one or more other agents in a kit, e.g., they are packaged together or placed in separate packages for sale together as a kit, or they are packaged for formulation together.

The additional anti-cancer agent may be selected from one or more of the following: alkylating agents (including nitrogen mustards, alkyl sulfonates, nitrosoureas, aziridine derivatives, and triazenes); anti-angiogenic agents (including matrix metalloproteinase inhibitors); antimetabolites (including adenosine deaminase inhibitors, folic acid antagonists, purine analogs, and pyrimidine analogs); antibiotics or antibodies (including monoclonal antibodies, CTLA-4 antibodies, anthracyclines); an aromatase inhibitor; a cell cycle response modifier; an enzyme; farnesyl-protein (farnesyl-protein) transferase inhibitors; hormones and anti-hormonal agents and steroids (including synthetic analogs, glucocorticoids, estrogens/anti-estrogens (e.g., SERMs), androgens/anti-androgens, progestins, progesterone receptor agonists, and luteinizing hormone releasing agonists and antagonists); insulin-like growth factor/insulin-like growth factor receptor system modulators; an integrin signaling inhibitor; kinase inhibitors (including multi-kinase inhibitors and/or Src kinase or Src/abl inhibitors), Cyclin Dependent Kinase (CDK) inhibitors, panHer, Her-1 and Her-2 antibodies, VEGF inhibitors (including anti-VEGF antibodies), EGFR inhibitors, mitogen-activated protein [ MAP ] inhibitors, MEK inhibitors, Aurora kinase inhibitors, PDGF inhibitors, and other tyrosine kinase inhibitors or serine/threonine kinase inhibitors); microtubule disruptors, such as ecteinascidins or their analogs and derivatives; microtubule stabilizing agents such as taxanes and naturally occurring epothilones and their synthetic and semi-synthetic analogs; microtubule binding and destabilizing agents (including vinca alkaloids); a topoisomerase inhibitor; prenyl protein transferase inhibitors; a platinum coordination complex; a signal transduction inhibitor; and other agents useful as anti-cancer and cytotoxic agents, such as biological response modifiers, growth factors, and immunomodulators.

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Parts and percentages are parts and percentages by weight unless otherwise indicated.

EXAMPLE 1 preparation of 3 beta-17- (1H-benzo [ d ]]Imidazol-2-d-1-yl) androsta-5, 16-dien-3-ol (compound) 7)

Step 1: synthesis of (3 beta) -3- (acetoxy) -17-chloroandrostane-5, 16-diene-16-carbaldehyde (Compound 2).

N, N-dimethylformamide (15mL, 192mmol) was slowly added dropwise to a solution of phosphorus oxychloride (15mL, 165mmol) in chloroform (45mL) under an ice bath and nitrogen blanket. After the addition, a solution of dehydroepiandrosterone acetate (3.00g, 9.00mmol) in chloroform (45mL) was slowly added dropwise. After the dropwise addition, the temperature was raised to room temperature and the reaction was refluxed for 5 hrs. The reaction mixture was concentrated under reduced pressure, and the residue was added to ice water, extracted with a mixed solvent of ether/ethyl acetate (8/2, v/v), and the organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure and purified by column chromatography to give 2.10g of an off-white solid, yield: 62.0 percent. LC-MS (APCI): 377.2[ M +1 ] M/z]⁺。

Step 2: synthesis of (3 β) -3- (acetoxy) -17- (1H-benzo [ d ] imidazol-1-yl) androsta-5, 16-diene-16-carbaldehyde (Compound 4).

Anhydrous N, N-dimethylformamide (10mL) was added to a mixture of (3 β) -3- (acetoxy) -17-chloroandrosta-5, 16-diene-16-carbaldehyde (2.10, 5.62mmol), benzimidazole (2.00g, 16.86mmol), and potassium carbonate (2.80g, 20.2mmol) at room temperature under nitrogen, and the reaction was stirred at 80 ℃ for 2.5 hours. The reaction was cooled to room temperature, added to ice water (250mL), the solid filtered, washed with water and dried in vacuo (60 ℃) to give the crude which was purified by column chromatography to give 2.10g of a yellow solid, yield: 80 percent. LC-MS (APCI): 459.3[ M +1 ] M/z]⁺。

And step 3: synthesis of (3 β) -3- (acetoxy) -17- (1H-benzo [ d ] imidazol-1-yl) androsta-5, 16-diene (Compound 5).

Palladium on carbon (10%, 1.2g) was added to (3 β) -3- (acetoxy) -17- (1H-benzo [ d ] at room temperature]Imidazol-1-yl) androsta-5, 16-diene-16-carbaldehyde (2.10mmol, 4.58mmol) in dry benzonitrile (12mL) was reacted under reflux for 24 hours. The reaction solution was cooled to the greenhouse, filtered through celite, and purified by column chromatography to give 1.48g of a yellow solid, yield: 75.0 percent. LC-MS (APCI): 431.3[ M +1 ] M/z]⁺。

And 4, step 4: synthesis of 3 beta-17- (1H-benzo [ d ] imidazol-1-yl) androsta-5, 16-dien-3-ol (Compound 6).

A10% solution of potassium hydroxide in methanol (10mL) was added to (3 β) -3- (acetoxy) -17- (1H-benzo [ d ] under nitrogen at room temperature]Imidazol-1-yl) androsta-5, 16-diene (1.45g, 3.36mmol) in methanol (20mL), and the reaction mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to about 10mL, added to ice water (40mL), the solid was filtered, washed with ice water, and dried under vacuum to give 1.20g of a beige solid, yield: 92.9%, purity: 98.64 percent. LC-MS (APCI): 389.3[ M +1 ] M/z]⁺；¹H NMR(300MHz，CDCl₃)(δ/ppm)8.14(s，1H)，7.66(dd，J＝6.8，1.8Hz，1H)，7.54(dd，J＝6.9，1.8Hz，1H)，7.36-7.23(m，2H)，6.04(dd，J＝2.8，1.6Hz，1H)，5.38(d，J＝5.0Hz，1H)，3.46-3.32(m，1H)，2.46-2.35(m，1H)，2.30-2.17(m，3H)，2.16-2.03(m，1H)，1.84-1.64(m，8H)，1.61-1.41(m，2H)，1.16-1.07(m，2H)，1.04(s，3H)，1.00(s，3H)。

And 5: synthesis of 3 beta-17- (1H-benzo [ d ] imidazol-2-d-1-yl) androsta-5, 16-dien-3-ol (Compound 7).

Sodium methoxide (90mg, 1.50mmol) was added to 17- (1H-benzo [ d ]]Imidazol-1-yl) androsta-5, 16-dien-3 β -ol (100mg, 0.25mmol) in deuterated methanol (CD)₃OD-d₄5mL) and the tube is sealed under nitrogen protection at 100 ℃ for reaction overnight. The reaction was quenched with heavy water (10mL), extracted with dichloromethane (10mLx4), the organic phase was washed with saturated brine (15mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was subjected to column separation to give a beige solid 50mg, yield: 50.0%, purity: 99.16 percent. LC-MS (APCI): m/z 390.0(M + 1);¹H NMR(300MHz，MeOD-d)(δ/ppm)8.19(s，0.04H)，7.76-7.65(m，1H)，7.59(dd，J＝6.8，1.8Hz，1H)，7.40-7.26(m，2H)，6.09(dd，J＝3.0，1.6Hz，1H)，5.43(d，J＝5.0Hz，1H)，3.50-3.37(m，1H)，2.52-2.41(m，1H)，2.35-2.22(m，3H)，2.21-2.09(m，1H)，1.92-1.69(m，8H)，1.66-1.43(m，2H)，1.22-1.12(m，2H)，1.09(s，3H)，1.05(s，3H)。

₃EXAMPLE 2 preparation of 3 beta-17- (1H-benzo [ d ]]Imidazole-5, 6, 7-d-1-yl) androsta-5, 16-dien-3-ol (Compound 13)

Adding anhydrous dimethyl sulfoxide (6mL) solution into 17-iodoandrost-5, 16-diene-3 beta-ol (300mg, 0.75mmol) and 1H-benzo [ d ] under nitrogen protection at room temperature]Imidazole-4, 5, 6, 7-d₄(110mg, 0.90mmol), L-proline (35mg, 0.3mmol), cuprous iodide (30mg, 0.15mmol) and potassium carbonate (259mg, 1.87mmol), and the reaction was allowed to proceed overnight at 120 ℃. Cooling to room temperature, adding water (25mL) to quench the reaction, filtering with celite, extracting the filtrate with ethyl acetate (30mLx3), combining the organic layers, washing the organic layer with saturated brine (30mL), drying over anhydrous sodium sulfate, concentrating the organic layer under reduced pressure, and purifying the concentrate by column chromatography to obtain 80mg of a white solidThe yield is as follows: 27.2%, purity: 99.37 percent. LC-MS (APCI): 393.0[ M +1 ] M/z]⁺；¹H NMR(300MHz，MeOD-d)(δ/ppm)8.18(s，1H)，6.08(dd，J＝3.1，1.7Hz，1H)，5.42(d，J＝5.1Hz，1H)，3.43(m，1H)，2.46(m，1H)，2.27(m，3H)，2.14(m，1H)，1.80(m，8H)，1.53(m，2H)，1.14(m，2H)，1.08(s，3H)，1.04(s，3H)。

₄EXAMPLE 3 preparation of 3 beta-17- (1H-benzo [ d ]]Imidazole-4, 5, 6, 7-d-1-yl) androsta-5, 16-dien-3-ol (Compound 15)

Adding anhydrous dimethyl sulfoxide (6mL) solution into 17-iodoandrost-5, 16-diene-3 beta-ol (300mg, 0.75mmol) and 1H-benzo [ d ] under nitrogen protection at room temperature]Imidazole-4, 5, 6, 7-d₄(110mg, 0.90mmol), L-proline (35mg, 0.3mmol), cuprous iodide (30mg, 0.15mmol) and potassium carbonate (259mg, 1.87mmol), and the reaction was allowed to proceed overnight at 120 ℃. After cooling to room temperature, the reaction was quenched with water (25mL), filtered through celite, the filtrate was extracted with ethyl acetate (30mLx3), the organic layers were combined, washed with saturated brine (30mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give 80mg of a white solid, yield: 27.2%, purity: 99.37 percent. LC-MS (APCI): 393.0[ M +1 ] M/z]⁺；¹H NMR(300MHz，MeOD-d)(δ/ppm)8.18(s，1H)，6.08(dd，J＝3.1，1.7Hz，1H)，5.42(d，J＝5.1Hz，1H)，3.43(m，1H)，2.46(m，1H)，2.27(m，3H)，2.14(m，1H)，1.80(m，8H)，1.53(m，2H)，1.14(m，2H)，1.08(s，3H)，1.04(s，3H)。

EXAMPLE 4 preparation of 17- (1H-benzo [ d ]]Imidazol-2-d-1-yl) androsta-5, 16-dien-3-one (Compound 16)

At room temperature, under the protection of nitrogen, reacting 17- (1H-benzo [ d ]]A mixture of imidazol-2-d-1-yl) androst-5, 16-dien-3 β -ol (130mg, 0.33mmol), anhydrous toluene (13mL) and N-methyl-4-piperidone (0.5mL) was added to a reflux apparatus with a water separator and the mixture was stirred at reflux for 1 hour (ca. distilling off 2mL of toluene). Aluminum isopropoxide (205mg, 1.05mmol) was added thereto, and the reaction mixture was stirred under reflux for reaction overnight. Cool to the greenhouse, quench the reaction with 20mL of water and 20mL of ethyl acetate, filter and extract the filtrate with ethyl acetate (20mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure and purified by column chromatography to give 100mg of an off-white solid, yield: 76.9%, purity: 97.73. LC-MS (APCI): 388.2(M + 1);¹H NMR(300MHz，MeOD-d)(δ/ppm)8.18(s，0.05H)，7.70(dd，J＝6.7，1.9Hz，1H)，7.58(dd，J＝6.8，1.8Hz，1H)，7.40-7.23(m，2H)，6.08(dd，J＝3.1，1.7Hz，1H)，5.75(s，1H)，2.63-2.18(m，6H)，2.12-1.89(m，3H)，1.87-1.53(m，6H)，1.28(s，3H)，1.25-1.11(m，2H)，1.07(s，3H)。

₃EXAMPLE 5 preparation of 17- (1H-benzo [ d ]]Imidazole-5, 6, 7-d-1-yl) androsta-5, 16-dien-3-one (compound Thing 17)

At room temperature, under the protection of nitrogen, reacting 17- (1H-benzo [ d ]]Imidazole-4, 5, 6, 7-d₄A mixture of-1-yl) androsta-5, 16-dien-3 β -ol (36mg, 0.09mmol), dry toluene (4mL) and N-methyl-4-piperidone (0.14mL) was added to a reflux apparatus with a water trap and the mixture was stirred at reflux for 1 hour (ca. 0.5mL of toluene was distilled off). Aluminum isopropoxide (60mg, 0.28mmol) was added thereto, and the reaction mixture was stirred under reflux for reaction overnight. Cool to the greenhouse, quench the reaction with 20mL of water and 20mL of ethyl acetate, filter and extract the filtrate with ethyl acetate (20mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressurePurification by liquid column chromatography gave 26mg of an off-white solid, yield: 72.2%, purity: 93.02 percent. LC-MS (APCI): m/z 391.2(M + 1);¹H NMR(300MHz，DMSO-d₆)(δ/ppm)8.25(s，1H)，6.05(s，1H)，5.66(s，1H)，2.44-1.52(m，15H)，1.18(s，3H)，1.08(M，2H)，0.98(s，3H)。

₄EXAMPLE 6 preparation of 17- (1H-benzo [ d ]]Imidazol-4, 5, 6, 7-d-1-yl) androsta-5, 16-dien-3-one Compound 18)

At room temperature, under the protection of nitrogen, reacting 17- (1H-benzo [ d ]]Imidazole-5, 6, 7-d₃A mixture of-1-yl) androsta-5, 16-dien-3 β -ol (140mg, 0.36mmol), dry toluene (14mL) and N-methyl-4-piperidone (0.54mL) was added to a reflux apparatus with a water trap and the mixture was stirred at reflux for 1 hour (approximately 3mL of toluene was distilled off). Aluminum isopropoxide (230mg, 1.09mmol) was added thereto, and the reaction mixture was stirred under reflux for reaction overnight. Cool to the greenhouse, quench the reaction with 20mL of water and 20mL of ethyl acetate, filter and extract the filtrate with ethyl acetate (20mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the organic layer was concentrated under reduced pressure and purified by column chromatography to give 29mg of an off-white solid, yield: 20.7%, purity: 94.44 percent. LC-MS (APCI): m/z 390.3(M + 1);¹H NMR(300MHz，CDCl₃)(δ/ppm)7.96(s，1H)，7.83(s，1H)，5.99(s，1H)，5.78(s，1H)，2.41(m，5H)，2.01(m，2H)，1.92-1.63(m，8H)，1.24(s，3H)，1.19-1.09(m，2H)，1.05(s，3H)。

biological activity assay

(1) CYP17 was measured in vitro.

The in vitro CYP17 inhibitory activity of this compound was evaluated using the rapid Acetic Acid Release Assay (AARA) using intact P450c 17-expressing e. Comprises using [21-3H ]]-17 alpha-hydroxypregnanolone as a substrate and labeling the form of acetic acid by a large amount of tritiumFormula (I) CYP17 activity was measured during cleavage of the C-21 side chain of the substrate. IC50 values were obtained directly from a plot correlating percent inhibition over the appropriate range versus inhibitory concentration (Grigoryev et al, Br. J. cancer, 1999, 81, 622-. Each compound was tested at a minimum of five different concentrations. Measured in triplicate, and IC₅₀The values are averaged over three experiments, where A represents IC₅₀Less than 0.1. mu.M, B represents an IC of 0.1. mu.M. or less₅₀Less than or equal to 0.5 mu M, C represents IC₅₀(> 0.5. mu.M) (as shown in Table 1 below).

(2) Inhibition of cellular PSA protein secretion.

The experimental steps are as follows: 1. replacing the original medium with medium containing 10% charcol strippod FBS, starving the cells for 24 hours in culture flasks; 2. digesting the cells, counting, inoculating LNcap cells into 96-well plates, 10,000/well, and culturing overnight; 3. adding DHT and a compound into the existing culture medium according to a set concentration, wherein the final concentration of the DHT is 1nM, the initial concentration of the compound is 50000nM, 5-fold dilution is carried out, 8 concentration gradients are carried out, and the culture is carried out for 48 hours; 4. cell culture supernatants were collected and PSA protein levels were detected according to ELISA kit instructions. IC was calculated from inhibition at each concentration using GraphPad Prism₅₀Wherein A represents IC₅₀Less than 0.1. mu.M, B represents an IC of 0.1. mu.M. or less₅₀Less than or equal to 0.5 mu M, C represents IC₅₀> 0.5. mu.M (as shown in Table 1 below)

TABLE 1 analysis of kinase inhibition of substituted steroids of examples 1-6

Example numbering	CYP17 enzyme IC50(μM)	PSA IC50(μM)
			Galeterone	B	-
Example 1	B	-
			Example 2	A	-
Example 3	A	-
			Galeterone metabolites	C	C
Example 4	B	B
			Example 5	B	C
Example 6	B	-

As shown in table 1, the compounds of the present invention have the CYP17 enzyme inhibitory activity of example 1 comparable to that of Galeterone, while the inhibitory activity of examples 2 and 3 is better than that of Galeterone, and the inhibitory activity of deuterated metabolites (examples 4, 5, 6) is also better than that of non-deuterated metabolites. In addition, the compound has stronger inhibitory effect on the secretion of PSA protein, so that the compound is more suitable for treating, preventing or eliminating various diseases related to CYP17 enzyme and androgen receptor, such as prostatic cancer, than Galeterone.

(3) Pharmacokinetic evaluation in rats.

8 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, divided into 2 groups of 4 rats each, given a single oral administration of 5mg/kg dose of (a) control group: 3 β -17- (1H-benzo [ d ] imidazol-1-yl) androsta-5, 16-dien-3-ol; (b) test groups: examples 1-3, the pharmacokinetic differences were compared.

Rats were fed with standard feed and given water. Fasting began 16 hours prior to the experiment. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300 μ L of blood was collected from the orbit into a test tube. There was 30 μ L of 1% heparin salt solution in the tube. Before use, the tubes were dried overnight at 60 ℃. After completion of blood sample collection at a subsequent time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted at least 5 times to ensure mixing and then placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the plasma from the erythrocytes. Pipette out 100 μ L of plasma into a clean plastic centrifuge tube, indicating the name of the compound and the time point. Plasma was stored at-80 ℃ before analysis. The concentration of the compounds of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The results of the experiments show that the compounds of the invention have better pharmacokinetics and thus better pharmacodynamics and therapeutic effects in animals compared to the control compounds.

(4) And (5) evaluating the metabolic stability.

Microsome experiment: human liver microsomes: 0.5mg/mL, Xenotech; rat liver microsomes: 0.5mg/mL, Xenotech; coenzyme (NADPH/NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

Preparing a stock solution: an amount of the powder of the compound example was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to be put into a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30 min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CLint, where V/M is equal to 1/protein concentration.

The compounds of the examples were analyzed according to the above procedure, and the results are shown in Table 2.

TABLE 2 Experimental results for the metabolic stability of substituted steroids of examples 1-6

As shown in table 2, the compounds of the present invention have an extended half-life and a reduced clearance rate, indicating that the compounds of the present invention can significantly improve metabolic stability.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (4)

1. A compound, or a pharmaceutically acceptable salt or tautomer thereof, selected from the following compounds:

2. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of claim 1, or a pharmaceutically acceptable salt or tautomer thereof.

3. A method for preparing a pharmaceutical composition, comprising: mixing a pharmaceutically acceptable carrier with the compound of claim 1 or a pharmaceutically acceptable salt or tautomer thereof to obtain a pharmaceutical composition.

4. Use of a compound of claim 1 or a pharmaceutically acceptable salt or tautomer thereof, or a pharmaceutical composition of claim 2, for the manufacture of a medicament for the treatment and/or prevention of disorders associated with the CYP17 enzyme and the androgen receptor.