WO2017140183A1 - 一种取代的甾体类化合物及其应用 - Google Patents

一种取代的甾体类化合物及其应用 Download PDF

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Publication number
WO2017140183A1
WO2017140183A1 PCT/CN2016/112946 CN2016112946W WO2017140183A1 WO 2017140183 A1 WO2017140183 A1 WO 2017140183A1 CN 2016112946 W CN2016112946 W CN 2016112946W WO 2017140183 A1 WO2017140183 A1 WO 2017140183A1
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Prior art keywords
compound
pharmaceutically acceptable
compounds
cancer
formula
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PCT/CN2016/112946
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English (en)
French (fr)
Inventor
王义汉
任兴业
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深圳市塔吉瑞生物医药有限公司
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Priority to CN202110328854.2A priority Critical patent/CN112851741B/zh
Priority to CN201680065029.5A priority patent/CN108350024B/zh
Publication of WO2017140183A1 publication Critical patent/WO2017140183A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/007—Steroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • the invention belongs to the field of medicine.
  • the present invention relates to steroidal compounds and uses thereof, and more particularly to steroidal compounds and their use as irreversible inhibitors of CYP17 enzyme and androgen receptor (AR) antagonists for the treatment and prevention of CYP17 Enzymes and androgen receptor (AR) related diseases.
  • AR androgen receptor
  • Prostate cancer (PCa in English) is a common malignant tumor of the male reproductive system.
  • 2008 there were 903,500 new cases of prostate cancer and 258,400 cases of prostate cancer deaths worldwide.
  • new cases of prostate cancer accounted for 14% of all new cases of male tumors, ranking second in new cases of male tumors; prostate cancer deaths accounted for 6% of male cancer deaths, ranking sixth among male cancer deaths.
  • Prostate cancer deaths accounted for 9% of male cancer deaths, second only to lung cancer, and ranked second among male cancer deaths.
  • the incidence and mortality of prostate cancer have risen rapidly, and it has become one of the important diseases affecting the health of Chinese men.
  • the effect of androgen on the growth of prostate cancer cells is mediated by the androgen receptor (AR) signaling pathway.
  • AR androgen receptor
  • PSA prostate specific antigen
  • Traditional castration therapy does not completely inhibit the production of androgen or the expression of the androgen receptor target gene. When the enzyme synthesis of androgen is overexpressed, it will increase the level of androgen in the tumor.
  • Cytochrome oxidase P450c17 (CYP17) is expressed in testis, adrenal gland and normal prostate tissue, and it is also expressed in prostate cancer cells. 17 ⁇ -hydroxylase and C17,20-lyase in CYP17 are key enzymes in androgen biosynthesis, which can promote the conversion of steroid progesterone and pregnenolone to C19 androstenedione and dehydroepiandrosterone, respectively. Both in turn re-transform testosterone and dihydrotestosterone.
  • CYP17 enzyme inhibitor is an important direction of prostate cancer drug treatment.
  • abiraterone acetate was developed by Centocor Ortho for the treatment of prostate cancer.
  • Abiraterone acetate was approved by the US FDA on April 28, 2011, and was combined with prednisone to treat castration-resistant prostate cancer.
  • the trade name is Zytiga. On July 28, 2011, Zytiga was approved by the Health Canada.
  • hormone testosterone can stimulate tumor growth, and castration therapy, such as drugs or surgery, can reduce testosterone production or block testosterone, but this treatment can not inhibit the production of androgens in other parts of the body. Prostate cancer can continue to grow.
  • the present invention discloses a substituted steroidal compound and a composition comprising the same and use thereof, which have better CYP17 enzyme and androgen receptor (AR) inhibitory activity and/or have more Good pharmacodynamic/pharmacokinetic properties for the treatment, prevention, and alleviation of diseases mediated by the CYP17 enzyme or androgen receptor (AR).
  • a substituted steroidal compound such as a steroid compound of formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 And R 18 , R 19 , R 20 , R 21 , R 22 and R 23 are each independently selected from the group consisting of "hydrogen (H), hydrazine (D)";
  • X 1 and X 2 are independently selected from the group consisting of "hydrogen (H), hydrazine (D), methyl, CH 2 D, CHD 2 , CD 3 , CH 2 CH 3 , CHDCH 3 , CHDCH 2 D, CHDCHD 2 , CHDCD 3, CD 2 CH 3, CD 2 CH 2 D, CD 2 CHD 2, CD 2 CD 3 " from the group consisting of;
  • Y is selected from "hydrogen (H), hydrazine (D), hydroxy, acetyl, one or more deuterated acetyl groups";
  • R 1 , R 2 , R 3 , R 4 , R 5 are independently hydrazine or hydrogen.
  • Y is selected from the group consisting of hydrogen, deuterium, hydroxyl, and one or more deuterated acetyl groups.
  • X 1 and X 2 are three deuterated methyl groups.
  • the compound of the present invention is selected from the group consisting of substituted steroidal compounds or pharmaceutically acceptable salts thereof, but is not limited to the following compounds:
  • the compound does not include a non-deuterated compound.
  • the cerium isotope content of cerium in the deuterated position is at least 0.015%, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than the natural strontium isotope content. More than 95%, more preferably more than 99%.
  • the osmium isotope content of each of R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 and X 1 , X 2 in the deuterated position is at least 5%, preferably greater than 10 More preferably, more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more preferably more than 40%, more preferably more 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%, and even more preferably greater than 80%, More preferably greater than 85%, more preferably greater
  • the three Rs contain ruthenium, more preferably four R ⁇ , more preferably five R ⁇ , more preferably six R ⁇ , more preferably seven R ⁇ , more preferably eight R ⁇ , preferably nine R ⁇ , more preferably ten R ⁇ , more preferably eleven R ⁇ , more preferably twelve R ⁇ , more preferably thirteen R ⁇ More preferably, fourteen Rs contain yttrium, more preferably fifteen R ⁇ , more preferably sixteen R ⁇ , more preferably seventeen R ⁇ ,
  • a pharmaceutically acceptable carrier and a substituted steroidal compound as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a metabolite, a stereoisomer, an isotope is mixed to form a pharmaceutical composition.
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a substituted steroidal compound as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, A pharmaceutical composition of a stereoisomer, prodrug or isotopic variation.
  • the invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein.
  • isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, respectively. , 31 P, 32 P, 35 S, 18 F and 36 Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention .
  • isotopically-labeled compounds of the present invention such as the radioisotopes of 3 H and 14 C, are also among them, useful in tissue distribution experiments of drugs and substrates. ⁇ , ie 3 H and carbon-14, ie 14 C, are easier to prepare and detect and are preferred in isotopes.
  • Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.
  • it further comprises other therapeutic agents, which are drugs for cancer, cell proliferative diseases, inflammation, infection, immune diseases, organ transplantation, viral diseases, cardiovascular diseases or metabolic diseases. .
  • compositions of the present invention comprise a safe or effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical compositions contain from 1 to 2000 mg of the compound of the invention per agent, more preferably from 10 to 1000 mg of the compound of the invention per agent.
  • the "one dose" is a capsule or tablet.
  • the present invention also discloses the use of a substituted steroidal compound as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, for the preparation of a treatment, prevention and amelioration by the CYP17 enzyme and the male A pharmaceutical composition of a hormone receptor (AR) mediated disease.
  • a substituted steroidal compound as described above or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, for the preparation of a treatment, prevention and amelioration by the CYP17 enzyme and the male A pharmaceutical composition of a hormone receptor (AR) mediated disease.
  • AR hormone receptor
  • the compound of the present invention has excellent inhibitory activity against the CYP17 enzyme and the androgen receptor (AR), the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and A pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for the treatment, prevention, and alleviation of diseases mediated by the CYP17 enzyme and the androgen receptor (AR).
  • the compounds of the present invention are useful for the treatment of diseases such as prostate cancer, benign prostatic hyperplasia, hirsutism, alopecia, anorexia nervosa, breast cancer, and male hypergonadism.
  • Substituted steroidal compounds disclosed in the present invention and compositions comprising the same for CYP17 enzyme and male The receptor (AR) has excellent inhibitory properties and has better pharmacokinetic parameter characteristics, which can increase the drug concentration of the compound in the animal to improve the efficacy and safety of the drug; the substituted steroids disclosed in the present invention
  • the compounds and compositions comprising the compounds are useful for treating, preventing, and ameliorating diseases mediated by the CYP17 enzyme and the androgen receptor (AR).
  • the deuterated steroidal compounds of the present invention and pharmaceutically acceptable salts thereof have equivalent or superior pharmacokinetic and/or pharmacodynamic properties compared to undeuterated compounds. Therefore, it is suitable as a compound which inhibits the CYP17 enzyme and the androgen receptor (AR), and is more suitable for the preparation of a medicament for treating cancer and CYP17 enzyme and androgen receptor (AR)-related diseases.
  • the present invention has been completed on this basis.
  • pharmaceutically acceptable salt refers to a derivative of the compound wherein the parent compound is modified by the preparation of its acid or base salt.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids.
  • Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or 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 containing a basic or acidic moiety by conventional chemical methods.
  • the above salts can be prepared by reacting these compounds in the form of the free acid or base with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two; usually, a nonaqueous medium such as diethyl ether or ethyl acetate Esters, ethanol, isopropanol or acetonitrile are preferred.
  • a nonaqueous medium such as diethyl ether or ethyl acetate Esters, 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.
  • a salt of a compound of formula (I) can be formed by reacting a compound of formula (I) with, for example, an equivalent amount of an acid or base in a medium which allows the newly formed salt to be precipitated, for example, by precipitation or by lyophilization. .
  • Exemplary acid salts of the compounds of formula (I) which may be formed with inorganic acids and/or organic acids include, but are not limited to, for example, acetates, ascorbates, benzoates, besylate, hydrogen sulfate, hydrogen tartrate, Acid citrate, citrate, ethanesulfonate, formate, fumarate, gentisate, gluconate, glucarate, glutamate, hydrochloride , hydrobromide, hydroiodide, isonicotinic acid salt, maleate, methanesulfonate, methanesulfonate, nitrate, pantothenate, phosphate, acid phosphate, sugar diacid salt , salicylate, succinate, acid salt, tartrate, p-toluenesulfonate, trifluoroacetate, lactate and pamoate (ie 1,1'-methylene-di(2-) Hydroxy-naphthalene-3-formate)
  • Exemplary base salts of the compounds of formula (I) which may be formed with inorganic bases and/or organic bases include, but are not limited to, for example, ammonium salts; alkali metal salts such as sodium, lithium and potassium; alkaline earth metal salts such as calcium salts and a magnesium salt; a salt formed with an organic base such as benzathine, dicyclohexylamine, 2-amino-2-(hydroxymethyl)propane-1,3-diol (trishydroxyl) Aminomethane, hydrabamines (eg N,N-di(dehydroabietic) ethylenediamine), N-methyl-D-glucosamine, N-methyl-D-imidazole diamide and uncle a butylamine; a salt formed with an amino acid such as arginine and lysine; and a salt formed by quaternizing a basic nitrogen-containing group by using, for example, a lower alkyl group Halogen (eg methyl chloride,
  • solvate refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio.
  • Hydrophilate means a complex formed by the coordination of a compound of the invention with water.
  • the compound of the present invention further includes a prodrug of a steroidal compound represented by the formula (I).
  • prodrug includes a compound of the formula (I) which may be biologically active or inactive, which, when taken by a suitable method, is metabolized or chemically reacted in the human body, or a salt or solution of a compound of formula (I).
  • the prodrug includes, but is not limited to, a carboxylic acid ester, a carbonate, a phosphate, a nitrate, a sulfate, a sulfone ester, a sulfoxide, an amino compound, a carbamate, an azo compound of the compound. , phosphoramide, glucoside, ether, acetal and the like.
  • “Therapeutically effective amount” is intended to include the combined amount of the compound of the present invention alone or the claimed compound or the combined amount of a compound of the present invention with other active ingredients effective for use as a CYP17 enzyme antagonist or to effectively treat cancer.
  • treating 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 the disease state, ie preventing its progression; and/or (c) mitigating the disease state, ie, The disease state has subsided.
  • the compounds of the invention may contain one or more additional asymmetric carbon atoms and may therefore exist in two or more stereoisomeric forms.
  • the invention includes all possible single stereoisomers, their individual tautomeric forms, and mixtures thereof. Diastereomers can be separated by conventional techniques, for example by fractional crystallization, chromatography or HPLC on a mixture of stereoisomers of a compound of the invention or a suitable salt or derivative thereof.
  • the single enantiomer of the compound can also be prepared from the corresponding optically pure intermediate or prepared by resolution of the corresponding racemate using a suitable chiral carrier (such as by HPLC) or, where appropriate, The diastereomeric salts are subjected to fractional crystallization, which is prepared by reacting the corresponding racemate 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 invention.
  • the invention also includes a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants (in the present application) Collectively referred to as "carrier” materials) and optionally other active ingredients.
  • the compound of formula (I) can be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for the above routes, and in a dosage effective for the desired treatment.
  • the compounds and compositions of the present invention can be administered orally, mucosally or parenterally (including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular) in dosage unit formulations containing conventional pharmaceutical carriers, excipients, and vehicles.
  • the pharmaceutical carrier can contain mannitol or a mixture of lactose and microcrystalline cellulose.
  • the mixture may contain other components such as a lubricant (such as magnesium stearate) and a disintegrant (such as crospovidone).
  • the carrier mixture can be filled into gelatin capsules or compressed into tablets.
  • the pharmaceutically active compounds of the present invention can be processed according to conventional pharmaceutical methods to prepare a medicament for administration to a patient, including humans and other mammals.
  • the pharmaceutical composition may be in the form of, for example, a tablet, a capsule, a suspension or a liquid preparation.
  • the pharmaceutical composition is prepared in the form of a dosage unit containing a particular amount of active ingredient. Examples of such dosage units are tablets or capsules.
  • the amount of the compound administered and the dosage regimen for treating the condition with the compounds and/or compositions of the invention will depend on a variety of factors, including the subject's age, weight, sex and medical condition, type of disease, severity of the disease. , route of administration and frequency, and the particular compound employed. Thus, the dosage regimen can vary widely, but can be routinely determined using standard methods.
  • the active compounds of the invention are usually combined with one or more excipients suitable for the particular route of administration.
  • the compound can be combined with lactose, sucrose, starch powder, cellulose alkanoate, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, phosphoric acid and sodium sulphate.
  • the salt is mixed with calcium salt, gelatin, gum arabic, sodium alginate, polyvinyl alcohol and/or polyvinylpyrrolidone and then tableted or encapsulated for ease of administration.
  • the above capsules or tablets may include a controlled release formulation which may be provided as a dispersion of the active compound in hydroxypropylmethylcellulose.
  • the oil phase of the emulsion containing the compound of formula (I) can be constituted by known ingredients in a known manner.
  • the phase may comprise only an emulsifier, it may comprise at least one emulsifier in combination with a fat or oil or with a fat and an oil.
  • the hydrophilic emulsifier is included with a lipophilic emulsifier as a stabilizer. It is also preferred to include both oil and fat.
  • emulsifiers (with or without stabilizers) constitute the so-called emulsifying wax and together with the oil and fat form a so-called emulsifying ointment base which forms the oil-dispersed phase of the cream.
  • Emulsifiers and emulsion stabilizers suitable for use in the formulations of the invention include Tween 60, Span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate or distearate Glycerides, these materials are used alone or in combination with waxes or other materials known in the art.
  • compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional excipients such as preservatives, stabilizers, wetting agents, emulsifying and buffering agents and the like. Tablets and pills can also be prepared with enteric coatings.
  • the compositions may also contain adjuvants such as wetting agents, sweetening, flavoring, and perfuming agents.
  • compositions of the present invention comprise a compound of formula (I), or a pharmaceutically acceptable salt thereof, and optionally other materials selected from any of the pharmaceutically acceptable carriers, adjuvants and vehicles.
  • Alternative compositions of the invention comprise a compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein, and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • the compounds of formula (I) are useful in the treatment of cancer, such as cancers that are dependent on androgen receptor signaling. These compounds inhibit the activity of the CYP17 enzyme, which is involved in the biosynthesis of androgens. Blocking this enzyme inhibits the production of gonadal, adrenaline and tumor androgen, and provides a new option for patients with cancers that rely 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.
  • a method for treating cancer comprising administering a compound of formula (I) to a mammal in need thereof.
  • the method of this embodiment can be used to treat a variety of cancers including, but not limited to, bladder cancer, breast cancer, colorectal cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreas/ gallbladder cancer, prostate cancer, thyroid Cancer, osteosarcoma, rhabdomyosarcoma, malignant fibrous histiocytoma (MFH), fibrosarcoma, glioblastoma/alstrocytoma, melanoma, and mesothelioma.
  • bladder cancer breast cancer, colorectal cancer, stomach cancer, head and neck cancer
  • kidney cancer liver cancer
  • lung cancer ovarian cancer
  • pancreas/ gallbladder cancer prostate cancer
  • thyroid Cancer osteosarcoma
  • rhabdomyosarcoma malignant fibrous histiocytoma (
  • the method of this embodiment is for treating a variety of prostate cancers.
  • the amount and dosage regimen of the compound of formula (I) administered for the treatment of a particular cancer depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, administration Route and frequency and the specific compound used. Thus, the dosage regimen can vary widely, but can be routinely determined using standard methods.
  • a daily dose of from about 0.01 to 1500 mg/kg body weight, preferably from about 0.5 to about 50 mg/kg body weight and most preferably from about 0.1 to 20 mg/kg body weight may be suitable.
  • the daily dose can be administered from 1 to 4 times per day.
  • Combinations of chemotherapeutic agents and/or other treatments are often advantageous when treating cancer.
  • the second (or third) formulation 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 in which two or more drugs that act in different ways or in different cell cycles are administered, and/or two or more of which have overlapping toxicity or side effects, And/or wherein the drugs combined in treating the particular disease state exhibited by the patient each have a significant therapeutic effect.
  • the compound of formula (I) can be used in combination with other anticancer treatments for the treatment of cancer or other proliferative diseases.
  • the invention further comprises the use of a compound of formula (I) for the manufacture of a medicament for the treatment of cancer, and/or a package comprising a compound of formula (I) according to the present specification, wherein said compound is used in combination with other anticancer agents or cells Combination of toxin agents and in the treatment of cancer.
  • the invention further comprises a combination of a compound of formula (I) and one or more other pharmaceutical agents in kit form, for example, they are packaged together or placed in separate packages for sale as a kit, or they are packaged for formulation together.
  • anticancer agents may be selected from one or more of the group consisting of alkylating agents (including nitrogen mustard, alkyl sulfonate, nitrosourea, aziridine derivatives, and triazene); anti-angiogenic agents ( Including matrix metalloproteinase inhibitors; antimetabolites (including adenosine deaminase inhibitors, folate antagonists, purine analogs, and pyrimidine analogs); antibiotics or antibodies (including monoclonal antibodies, CTLA-4 antibodies, anthracyclines) Antibiotics; aromatase inhibitors; cell cycle response modifiers; enzymes; farnesyl-protein transferase inhibitors; hormones and antihormonal agents, and steroids (including synthetic analogues, glucocorticoids, females) Hormone/antiestrogens (eg SERMs), androgen/antiandrogen, progesterone, progesterone receptor agonists and luteinizing hormone releasing agonists and antagonists; insulin
  • Step 1 Synthesis of (3 ⁇ )-3-(acetoxy)-17-chloroandrost-5,16-diene-16-carbaldehyde (Compound 2).
  • Step 2 Synthesis of (3 ⁇ )-3-(acetoxy)-17-(1H-benzo[d]imidazol-1-yl)androst-5,16-diene-16-carbaldehyde (Compound 4) .
  • Step 3 Synthesis of (3 ⁇ )-3-(acetoxy)-17-(1H-benzo[d]imidazol-1-yl)androst-5,16-diene (Compound 5).
  • Step 4 Synthesis of 3 ⁇ -17-(1H-benzo[d]imidazol-1-yl)androst-5,16-dien-3-ol (Compound 6).
  • Step 5 Synthesis of 3 ⁇ -17-(1H-benzo[d]imidazol-2-d-1-yl)androst-5,16-dien-3-ol (Compound 7).
  • the in vitro CYP17 inhibitory activity of this compound was evaluated using the rapid acetate release assay (AARA) using intact P450c17-expressing E. coli as the enzyme source. Included is the use of [21-3H]-17 ⁇ -hydroxypregnenolone as a substrate and the measurement of CYP17 activity by a large amount of europium-labeled acetic acid during the C-21 side chain splitting of the substrate.
  • AARA rapid acetate release assay
  • A represents a IC 50 ⁇ 0.1 ⁇ M
  • B represents 0.1 ⁇ M ⁇ IC 50 ⁇ 0.5 ⁇ M
  • C represents the IC 50> 0.5 ⁇ M (as shown in Table 1) .
  • IC 50 The inhibition rate at each concentration, IC 50 was calculated using GraphPad Prism, wherein, A represents a IC 50 ⁇ 0.1 ⁇ M, B represents 0.1 ⁇ M ⁇ IC 50 ⁇ 0.5 ⁇ M, C represents the IC 50> 0.5 ⁇ M (Table 1 below)
  • Example 1 the CYP17 enzyme inhibitory activity of Example 1 was comparable to Galeterone compared to Galeterone, while the inhibitory activities of Examples 2 and 3 were better than Galeterone, and deuterated metabolites (Examples) The inhibitory activities of 4, 5, and 6) are also better than those of non-deuterated metabolites.
  • the inhibition of PSA protein secretion is also stronger, and thus the compounds of the present invention are more suitable than Galeterone for the treatment, prevention or elimination of various conditions associated with CYP17 enzymes and androgen receptors, such as prostate cancer.
  • control group 3 ⁇ -17-(1H- Benzo[d]imidazol-1-yl)androst-5,16-dien-3-ol
  • Test group Examples 1-3, comparing their pharmacokinetic differences.
  • Rats were fed a standard diet and given water. Fasting began 16 hours before the test.
  • the drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.
  • Rats were briefly anesthetized after inhalation of ether, and 300 ⁇ L of blood samples were collected from the eyelids in test tubes. There was 30 ⁇ L of 1% heparin salt solution in the test tube. The tubes were dried overnight at 60 ° C before use. After the blood sample collection was completed at a later time point, the rats were anesthetized with ether and sacrificed.
  • Plasma samples were centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate plasma from red blood cells. Pipette 100 ⁇ L of plasma into a clean plastic centrifuge tube to indicate the name and time point of the compound. Plasma was stored at -80 °C prior to analysis. The concentration of the compound 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 experimental results show that the compound of the present invention has better pharmacokinetics in animals relative to the control compound, and thus has better pharmacodynamics and therapeutic effects.
  • Microsomal experiments human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).
  • Preparation of stock solution A certain amount of the powder of the compound example was accurately weighed and dissolved to 5 mM with DMSO, respectively.
  • phosphate buffer 100 mM, pH 7.4.
  • the pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.
  • NADPH regeneration system containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride was prepared and placed on wet ice before use.
  • Formulation stop solution acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 ⁇ L of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 ⁇ L of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 ⁇ L of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 ⁇ L of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.
  • the reaction of the corresponding compound is concentrated The degree was 1 ⁇ M and the protein concentration was 0.5 mg/mL.
  • 100 ⁇ L of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min.
  • the plate was centrifuged at 5000 x g for 10 min at 4 °C.
  • 100 ⁇ L of the supernatant was taken into a 96-well plate to which 100 ⁇ L of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

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Abstract

本发明提供了一种取代的甾体类化合物及其应用。具体地，本发明所述取代的甾体类化合物为如式(I)所示的甾体化合物，或其晶型、药学上可接受的盐、前药，立体异构体、水合物或溶剂化合物。本发明公开的取代的甾体类化合物及包含该化合物的组合物对CYP17酶及雄激素受体(AR)具有优异的抑制性，同时具有更好的药代动力学参数特性，能够提高化合物在动物体内的药物浓度，以提高药物疗效和安全性。

Description

一种取代的甾体类化合物及其应用 技术领域
本发明属于医药领域。具体地，本发明涉及甾体类化合物及其用途，更具体地是，涉及甾体类化合物及其作为CYP17酶不可逆抑制剂及雄激素受体(AR)拮抗剂，可用于治疗和预防与CYP17酶及雄激素受体(AR)相关疾病。
背景技术
前列腺癌(prostate cancer，英文简写为PCa)是男性生殖系统常见的恶性肿瘤。2008年全球有903500例前列腺癌新发病例和258400例前列腺癌死亡病例。其中前列腺癌新发病例占男性所有肿瘤新发病例的14％,位列男性肿瘤新发病例第2位；前列腺癌死亡病例占男性癌症死亡病例的6％，位列男性癌症死亡病例第6位。前列腺癌死亡病例占男性癌症死亡病例的9％，仅次于肺癌，位列男性癌症死亡病例第2位。随着近些年环境污染加重，人口老龄化速度加快以及人们生活饮食方式的改变等原因，前列腺癌的发病率及死亡率快速上升，现已成为影响中国男性健康的重要疾病之一。
雄激素对前列腺癌细胞生长的影响是通过雄激素受体(androgen receptor,AR)信号通路介导的，临床上通过AR信号的变化来观察患者体内前列腺特异性抗原(Prostate Specific Antigen,PSA)水平，从而诊治患者前列腺癌的进展状况。传统的去势治疗不能彻底地抑制雄激素的产生或雄激素受体目标基因的表达,当合成雄激素的生物酶过度表达时，就会增加肿瘤内雄激素的水平。
细胞色素氧化酶P450c17(CYP17)在睾丸，肾上腺和正常前列腺组织都有表达，同时它也在前列腺癌细胞中表达。CYP17中的17α-羟化酶和C17,20-裂解酶是雄激素生物合成中的关键酶，可以促使类固醇孕激素和孕烯醇酮分别转化为C19雄烯二酮和去氢表雄酮，两者进而再转化睾酮和二氢睾酮。
基于上述研究，防治前列腺癌的工作已刻不容缓，而研究开发CYP17酶抑制剂是前列腺癌药物治疗的重要方向。作为新型CYP17酶抑制剂，醋酸阿比特龙被Centocor Ortho公司研发出来用于治疗前列腺癌。醋酸阿比特龙于2011年4月28日经美国FDA批准上市，与泼尼松联用治疗去势抵抗性前列腺癌，其商品名为Zytiga。2011年7月28日，Zytiga获加拿大卫生部批准。对于前列腺癌患者，荷尔蒙睾丸激素可刺激肿瘤的生长，药物或手术治疗在内的去势治疗可减少睾丸激素的生成或阻断睾丸激素的作用，但这种治疗无法抑制身体其它部位产生雄激素，前列腺癌仍可以继续增长。
发明内容
针对以上技术问题，本发明公开了一种取代的甾体类化合物及包含该化合物的组合物及其用途，其具有更好的CYP17酶及雄激素受体(AR)抑制活性和/或具有更好药效学/药代动力学性能，可用于治疗、预防以及缓解由对CYP17酶或雄激素受体(AR)介导的疾病。
对此，本发明的技术方案为：
一种取代的甾体类化合物，如式(I)所示的甾体化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物，
Figure PCTCN2016112946-appb-000001
式中：
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23相互独立地选自由“氢(H)、氘(D)”组成的组；
X1、X2相互独立地选自由“氢(H)、氘(D)、甲基、CH2D、CHD2、CD3、CH2CH3、CHDCH3、CHDCH2D、CHDCHD2、CHDCD3、CD2CH3、CD2CH2D、CD2CHD2、CD2CD3”组成的组；
Y选自“氢(H)、氘(D)、羟基、乙酰基，一次或多次氘代的乙酰基”；
及其生理学上可接受的盐、前药、代谢物、溶剂化物、互变异构体和立体异构体，包括这些化合物以所有比例形成的混合物。
在另一优选例中，R1、R2、R3、R4、R5自独立地为氘或氢。
在另一优选例中，Y选自氢、氘、羟基、一次或多次氘代的乙酰基。
在另一优选例中，X1、X2为三次氘代的甲基。
在另一优选例中，本发明化合物选自以下组内的取代的甾体类化合物或其药学上可接受的盐，但不局限于如下化合物：
Figure PCTCN2016112946-appb-000002
Figure PCTCN2016112946-appb-000003
在另一选例中，所述化合物不包括非氘代化合物。
作为本发明的进一步改进，氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量0.015％，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。
具体地说，在本发明中R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23及X1、X2各氘代位置中氘同位素含量至少是5％，较佳地大于10％，更佳地大于15％，更佳地大于20％，更佳地大于25％，更佳地大于30％，更佳地大于35％，更佳地大于40％，更佳地大于45％，更佳地大于50％，更佳地大于55％，更佳地大于60％，更佳地大于65％，更佳地大于70％，更佳地大于75％，更佳地大于80％，更佳地大于85％，更佳地大于90％，更佳地大于95％，更佳地大于99％。
在另一选例中，式(I)中化合物的R1、R2、R3、R4、R5、R6、R7、R8、R9、 R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23，至少其中一个R含氘，更佳地两个R含氘，更佳地三个R含氘，更佳地四个R含氘，更佳地五个R含氘，更佳地六个R含氘，更佳地七个R含氘，更佳地八个R含氘，更佳地九个R含氘，更佳地十个R含氘，更佳地十一个R含氘，更佳地十二个R含氘，更佳地十三个R含氘，更佳地十四个R含氘，更佳地十五个R含氘，更佳地十六个R含氘，更佳地十七个R含氘，更佳地十八个R含氘，更佳地十九个R含氘，更佳地二十个R含氘，更佳地二十一个R含氘，更佳地二十二个R含氘，更佳地二十三个R含氘。
作为本发明的进一步改进，将药学上可接受的载体与如上所述的取代的甾体类化合物，或其晶型、药学上可接受的盐、前药、代谢物、立体异构体、同位素变体水合物或溶剂合物进行混合，从而形成药物组合物。
本发明还公开了一种药物组合物，其含有药学上可接受的载体和如上所述的取代的甾体类化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药或同位素变体的药物组合物。
本发明还包括同位素标记的化合物，等同于原始化合物在此公开。可以列为本发明的化合物同位素的例子包括氢，碳，氮，氧，磷，硫，氟和氯同位素，分别如2H，3H，13C，14C，15N，17O，18O，31P，32P，35S，18F以及36Cl。本发明中的化合物，或对映体，非对映体，异构体，或药学上可接受的盐或溶剂化物，其中含有上述化合物的同位素或其他其他同位素原子都在本发明的范围之内。本发明中某些同位素标记化合物，例如3H和14C的放射性同位素也在其中，在药物和底物的组织分布实验中是有用的。氚，即3H和碳-14，即14C，它们的制备和检测比较容易，是同位素中的首选。同位素标记的化合物可以用一般的方法，通过用易得的同位素标记试剂替换为非同位素的试剂，用示例中的方案可以制备。
作为本发明的进一步改进，其还包含其他治疗药物，所述治疗药物为癌症、细胞增殖性疾病、炎症、感染、免疫性疾病、器官移植、病毒性疾病、心血管疾病或代谢性疾病的药物。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-1000mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
本发明还公开了如上所述的取代的甾体类化合物，或其晶型、药学上可接受的盐、水合物或溶剂化合物的用途，用于制备治疗、预防以及缓解由对CYP17酶及雄激素受体(AR)介导的疾病的药物组合物。
由于本发明化合物对CYP17酶及雄激素受体(AR)具有优异的抑制活性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解由对CYP17酶及雄激素受体(AR)介导的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：前列腺癌、良性前列腺增生症、多毛症、脱发、神经性厌食、乳腺癌和男性性腺机能亢进等。
本发明的有益效果为：
本发明公开的取代的甾体类化合物及包含该化合物的组合物对CYP17酶及雄激 素受体(AR)具有优异的抑制性，同时具有更好的药代动力学参数特性，能够提高化合物在动物体内的药物浓度，以提高药物疗效和安全性；本发明公开的取代的甾体类化合物及包含该化合物的组合物可用于治疗、预防以及缓解由CYP17酶及雄激素受体(AR)介导的疾病。
具体实施方式
本发明人研究发现，本发明的氘代的甾体类化合物及其药学上可接受的盐与未氘代的化合物相比，具有等效或者更优异的药物动力学和/或药效学性能，因此适合作为抑制CYP17酶及雄激素受体(AR)的化合物，进而更适用于制备治疗癌症以及CYP17酶和雄激素受体(AR)相关疾病的药物。在此基础上完成了本发明。
本申请使用的“药用盐”是指所述化合物的衍生物，其中母体化合物通过制备其酸盐或碱盐来改性。药用盐的实例包括但不限于碱性残基诸如胺的无机酸盐或有机酸盐；及酸性残基诸如羧酸的碱盐或有机盐。药用盐包括例如由无毒无机酸或有机酸形成的母体化合物的常规无毒盐或季铵盐。本发明药用盐可由含有碱性或酸性部分的母体化合物通过常规化学方法来合成。通常，上述盐可如下制备：使呈游离酸或碱形式的这些化合物与化学计量的适当碱或酸在水或有机溶剂或这两者的混合物中反应；通常，非水介质例如乙醚、乙酸乙酯、乙醇、异丙醇或乙腈为优选的。适当的盐参见Remington’s Pharmaceutical Sciences，17th Edition,Mack Publishing Company,Easton,PA,p.1418(1985)，将其公开的内容引入本申请作为参考。
例如，式(I)化合物的盐可如下形成：使式(I)化合物与例如等量的酸或碱在以下介质中反应，所述介质允许新形成的盐例如沉淀析出或通过冻干来分离。式(I)化合物可与无机酸和/或有机酸形成的示例性酸盐包括但不限于例如乙酸盐、抗坏血酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、酒石酸氢盐、酸式枸橼酸盐、枸橼酸盐、乙磺酸盐、甲酸盐、富马酸盐、龙胆酸盐、葡糖酸盐、葡糖二酸盐、谷氨酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、异烟酸盐、马来酸盐、甲磺酸盐、甲磺酸盐、硝酸盐、泛酸盐、磷酸盐、酸式磷酸盐、糖二酸盐、水杨酸盐、琥珀酸盐、酸盐、酒石酸盐、对甲苯磺酸盐、三氟乙酸盐、乳酸盐和扑酸盐(即1,1’-亚甲基-二(2-羟基-萘-3-甲酸盐))。上述盐可按照本领域技术人员已知的方法来形成。
式(I)化合物可与无机碱和/或有机碱形成的示例性碱盐包括但不限于例如铵盐；碱金属盐，例如钠盐、锂盐和钾盐；碱土金属盐，例如钙盐和镁盐；与有机碱形成的盐，所述有机碱为例如苄星(benzathine)、二环己基胺、2-氨基-2-(羟基甲基)丙-1,3-二醇(三羟甲基氨基甲烷、哈胺(hydrabamines)(例如N,N-二(脱氢松香基)乙二胺)、N-甲基-D-葡糖胺、N-甲基-D-咪唑二酰胺和叔丁基胺；与氨基酸(例如精氨酸和赖氨酸)形成的盐；和通过使用以下试剂以对碱性含氮基团进行季铵化而形成的盐，所述试剂为例如低级烷基卤(例如甲基氯、甲基溴、甲基碘、乙基氯、乙基溴、乙基碘、丙基氯、丙基溴、丙基碘、丁基氯、丁基溴和丁基碘)、硫酸二烷基酯(例如硫酸二甲酯、硫酸二乙酯、硫酸二丁酯和硫酸二戊酯)、长链卤化物(例如癸基氯、癸基溴、癸基碘、月桂基氯、月桂基溴、月桂基碘、肉豆蔻基氯、肉豆蔻基溴、肉豆蔻基碘、硬脂基氯、硬脂基溴和硬脂基碘)和芳烷基卤(例如苄基溴和苯乙基溴)。上述盐可按照本领域技术人员已知的方法来形成。
术语“溶剂合物”指本发明化合物与溶剂分子配位形成特定比例的配合物。“水合物”是指本发明化合物与水进行配位形成的配合物。
此外，本发明化合物还包括式(I)所示的甾体类化合物的前药。术语“前药”包括其本身可以是具有生物学活性的或非活性的，当用适当的方法服用后，其在人体内进行代谢或化学反应而转变成式(I)的一类化合物，或式(I)的一个化合物所组成的盐或溶液。所述的前药包括(但不局限于)所述化合物的羧酸酯、碳酸酯、磷酸酯、硝酸酯、硫酸酯、砜酯、亚砜酯、氨基化合物、氨基甲酸盐、偶氮化合物、磷酰胺、葡萄糖苷、醚、乙缩醛等形式。
“治疗有效量”意在包括本发明化合物的单独量或要求保护的化合物的组合量或本发明化合物与有效用作CYP17酶拮抗剂或有效治疗癌症的其它活性成分的组合量。
本申请使用的“治疗(treating)”或“治疗(treatment)”包括在哺乳动物特别是人类中对疾病状态进行的治疗并包括：(a)在哺乳动物中预防所述疾病状态发生，特别是当所述哺乳动物易患所述疾病状态但尚未确诊患上所述疾病状态时；(b)抑制所述疾病状态，即阻止其发展；和/或(c)缓解所述疾病状态，即令所述疾病状态消退。
本发明化合物可含有一个或多个额外的不对称碳原子，因此可按两种或多种立体异构形式存在。本发明包括所有可能的单一立体异构体、它们的单一互变异构形式及它们的混合物。非对映异构体可通过常规技术来分离，例如通过对本发明化合物或其合适的盐或衍生物的立体异构体混合物进行分级结晶、色谱或HPLC。所述化合物的单一对映异构体也可由相应的光学纯的中间体来制备或如下制备：使用合适的手性载体对相应的外消旋体进行拆分(诸如通过HPLC)或当合适时，对非对映异构体盐进行分级结晶，所述非对映异构体盐通过使相应的外消旋体与合适的光学活性酸或碱反应来制备。本发明化合物的所有立体异构体(呈混合物或纯或基本纯形式)均包括在本发明中。
本发明还包括一类药物组合物，所述药物组合物包含式(I)化合物或其药用盐及一种或多种无毒药用载体和/或稀释剂和/或辅料(在本申请中统称为“载体”材料)及任选的其它活性成分。式(I)化合物可通过任何合适的途径优选以适于上述途径的药物组合物形式且以就所期望的治疗而言有效的剂量来给药。例如，本发明化合物和组合物可按含有常规药用载体、辅料和媒介物的剂量单位制剂形式来口服给药、粘膜给药或肠胃外(包括血管内、静脉内、腹膜内、皮下、肌内、胸骨内和输注技术)给药。例如，所述药物载体可含有甘露醇或乳糖和微晶纤维素的混合物。所述混合物可含有其它组分，诸如润滑剂(例如硬脂酸镁)和崩解剂(例如交聚维酮)。可将所述载体混合物填充到明胶胶囊中或压制成片剂。
本发明药物活性化合物可按照常规药学方法来加工以制备用于向患者(包括人类及其它哺乳动物)给药的药物。
对于口服给药，所述药物组合物可呈以下形式：例如片剂、胶囊剂、混悬剂或液体制剂。优选将所述药物组合物制备成含有特定量的活性成分的剂量单位形式。所述剂量单位的实例为片剂或胶囊剂。
所给药的化合物量和用本发明化合物和/或组合物对病症进行治疗的给药方案取决于多种因素，包括受试者的年龄、体重、性别和医学状态、疾病类型、疾病严重性、给药途径和频率及所使用的具体化合物。因此，所述给药方案可变化很大，但可使用标准方法来常规确定。
出于治疗目的，通常将本发明活性化合物与一种或多种适于指定给药途径的辅料组合。若口服给药，则可将所述化合物与乳糖、蔗糖、淀粉粉末、烷酸纤维素酯、纤维素烷基酯、滑石、硬脂酸、硬脂酸镁、氧化镁、磷酸和硫酸的钠盐和钙盐、明胶、阿拉伯胶、海藻酸钠、聚乙烯醇和/或聚乙烯吡咯烷酮混合，然后压片或包囊以方便给药。上述胶囊剂或片剂可包括控释制剂，所述控释制剂可按活性化合物在羟丙基甲基纤维素中的分散体形式来提供。
含有式(I)化合物的乳剂的油相可由已知成分按已知方式来构成。尽管所述相可仅包含乳化剂，但其可包含至少一种乳化剂与脂肪或油或与脂肪和油的混合物。优选地，亲水性乳化剂与作为稳定剂的亲脂性乳化剂包含在一起。既包含油又包含脂肪也是优选的。另外，乳化剂(带有或不带有稳定剂)构成所谓的乳化蜡且所述蜡与油和脂肪一起形成所谓的乳化软膏基质，所述乳化软膏基质形成乳膏剂的油分散相。适于在本发明制剂中使用的乳化剂和乳剂稳定剂包括吐温60、司盘80、鲸蜡硬脂醇、肉豆蔻醇、单硬脂酸甘油酯、月桂基硫酸钠或二硬脂酸甘油酯，这些物质单独使用或与蜡或本领域公知的其它材料一起使用。
所述药物组合物可经历常规制药操作(诸如灭菌)和/或可含有常规辅料(诸如防腐剂、稳定剂、润湿剂、乳化剂和缓冲剂等)。片剂和丸剂还可用肠溶衣制备。所述组合物还可包含辅料，诸如润湿剂、甜味剂、矫味剂和芳香剂。
本发明药物组合物包含式(I)化合物或其药用盐及任选的其它物质，所述其它物质选自任何药用载体、辅料和媒介物。可选择的本发明组合物包含本申请所述的式(I)化合物或其药用盐及药用载体、辅料或媒介物。
式(I)化合物用于治疗癌症，例如依赖于雄激素受体信号传导的癌症。这些化合物抑制CYP17酶的活性，所述CYP17酶参与雄激素的生物合成。阻断该酶会抑制性腺、肾上腺素和肿瘤雄激素的生成，并且提供一种治疗依赖于雄激素受体信号传导的癌症(诸如前列腺癌和雌激素受体阳性的乳腺癌)患者的新选择。因此，所述治疗包括给予所述患者式(I)化合物或其药用盐。
在一个实施方案中，提供一种用于治疗癌症的方法，所述方法包括给予需要的哺乳动物式(I)化合物。该实施方案的方法可用于治疗多种癌症，包括但不限于膀胱癌、乳腺癌、结肠直肠癌、胃癌、头颈癌、肾癌、肝癌、肺癌、卵巢癌、胰腺/胆囊癌、前列腺癌、甲状腺癌、骨肉癌、横纹肌肉瘤、恶性纤维组织细胞瘤(MFH)、纤维肉瘤、成胶质细胞瘤/星形细胞瘤、黑素瘤和间皮瘤。
优选地，该实施方案的方法用于治疗多种前列腺癌。
用于治疗特定癌症的所给药的式(I)化合物的量和给药方案取决于多种因素，包括受试者的年龄、体重、性别和医学状态、疾病类型、疾病严重性、给药途径和频率及所使用的具体化合物。因此，所述给药方案可变化很大，但可使用标准方法来常规确定。约0.01至1500mg/kg体重、优选为约0.5至约50mg/kg体重且最优选为约0.1至20mg/kg体重的日剂量可为合适的。所述日剂量可按1-4次/日来给药。
治疗癌症时，化学治疗剂和/或其它治疗(例如放射治疗)组合往往是有利的。第二(或第三)制剂可具有与主要治疗剂相同或不同的作用机理。使用细胞毒类药物组合可能是特别有用的，其中给予两种或多种以不同方式或在不同的细胞周期起作用的药物，和/或其中两种或多种药物具有重叠的毒性或副作用，和/或其中在治疗患者表现出的特定疾病状态时组合的药物各自具有明显的疗效。
因此，式(I)化合物可与其它用于治疗癌症或其它增殖性疾病的抗癌治疗 组合给予。本发明进一步包括式(I)化合物在制备用于治疗癌症的药物中的用途，和/或包括本申请式(I)化合物与说明书的包装，其中所述化合物用在与其它抗癌剂或细胞毒素剂组合和用于治疗癌症的治疗中。本发明进一步包括式(I)化合物和一个或多个其它药剂以试剂盒形式的组合，例如它们被包装在一起或置于分开的包装中作为试剂盒一起销售，或它们被包装从而一起配制。
其它的抗癌剂可选自以下的一个或多个：烷化剂(包括氮芥、烷基磺酸酯、亚硝基脲、氮丙啶衍生物和三氮烯)；抗血管生成剂(包括基质金属蛋白酶抑制剂)；抗代谢物(包括腺苷脱氨酶抑制剂、叶酸拮抗剂、嘌呤类似物和嘧啶类似物)；抗生素或抗体(包括单克隆抗体、CTLA-4抗体、蒽环类抗生素)；芳香酶抑制剂；细胞周期反应调节剂；酶；法呢基-蛋白(farnesyl-protein)转移酶抑制剂；激素和抗激素剂以及类固醇(包括合成类似物、糖皮质激素、雌激素/抗雌激素(例如SERMs)、雄激素/抗雄激素、孕激素、孕酮受体激动剂以及促黄体生成激素释放激动剂和拮抗剂)；胰岛素样生长因子/胰岛素样生长因子受体系统调节剂；整联蛋白信号传导抑制剂；激酶抑制剂(包括多激酶抑制剂和/或Src激酶或Src/abl抑制剂)、细胞周期蛋白依赖性激酶(CDK)抑制剂、panHer、Her-1和Her-2抗体、VEGF抑制剂(包括抗VEGF抗体)、EGFR抑制剂、促分裂原活化蛋白[MAP]抑制剂、MEK抑制剂、Aurora激酶抑制剂、PDGF抑制剂和其它酪氨酸激酶抑制剂或丝氨酸/苏氨酸激酶抑制剂)；微管干扰剂，诸如海鞘素类化合物或它们的类似物和衍生物；微管稳定剂诸如紫杉烷类和天然存在的埃坡霉素和它们的合成和半合成的类似物；微管结合和去稳定剂(包括长春花生物碱)；拓扑异构酶抑制剂；异戊二烯基蛋白转移酶抑制剂；铂配位络合物；信号转导抑制剂；和其它用作抗癌和细胞毒性剂的制剂，诸如生物反应调节剂、生长因子和免疫调节剂。
下面更具体地描述本发明式(I)结构化合物的制备方法，但这些具体方法不对本发明构成任何限制。本发明化合物还可以任选将在本说明书中描述的或本领域已知的各种合成方法组合起来而方便地制得，这样的组合可由本发明所属领域的技术人员容易地进行。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比为重量份和重量百分比。
实施例1制备3β-17-(1H-苯并[d]咪唑-2-d-1-基)雄甾-5,16-二烯-3-醇(化合物 7)
Figure PCTCN2016112946-appb-000004
步骤1：(3β)-3-(乙酰氧基)-17-氯雄甾-5,16-二烯-16-甲醛(化合物2)的合成。
冰浴下，氮气保护下将N,N-二甲基甲酰胺(15mL,192mmol)缓慢滴加到三氯氧磷(15mL,165mmol)的氯仿(45mL)溶液中。滴加完毕后，将醋酸去氢表雄酮(3.00g,9.00mmol)的氯仿(45mL)溶液缓慢滴入。滴加完毕，升温至室温，回流反应5hrs。减压浓缩反应液，残渣加入到冰水中，用乙醚/乙酸乙酯(8/2，v/v)混合溶剂萃取，合并有机层，用饱和食盐水洗涤，无水硫酸钠干燥。减压浓缩有机层，浓缩液柱层析纯化得到2.10g米白色固体，产率：62.0％。LC-MS(APCI):m/z＝377.2[M+1]+。
步骤2：(3β)-3-(乙酰氧基)-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯-16-甲醛(化合物4)的合成。
室温下，氮气保护下将无水N,N-二甲基甲酰胺(10mL)加入到(3β)-3-(乙酰氧基)-17-氯雄甾-5,16-二烯-16-甲醛(2.10,5.62mmol)，苯并咪唑(2.00g,16.86mmol)和碳酸钾(2.80g,20.2mmol)的混合物中，反应液在80℃下反应搅拌2.5小时。反应液冷却至室温，加入到冰水(250mL)中，固体过滤，水洗，真空干燥(60℃)得到粗品，粗品柱层析纯化得到2.10g黄色固体，产率：80％。LC-MS(APCI):m/z＝459.3[M+1]+。
步骤3：(3β)-3-(乙酰氧基)-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯(化合物5)的合成。
室温下，将钯碳(10％，1.2g)加入到(3β)-3-(乙酰氧基)-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯-16-甲醛(2.10mmol,4.58mmol)的无水苯甲腈(12mL)，回流反应24小时。反应液冷却至温室，硅藻土过滤，柱层析纯化得到1.48g黄色固体，产率：75.0％。LC-MS(APCI):m/z＝431.3[M+1]+。
步骤4：3β-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯-3-醇(化合物6)的合成。
室温下，氮气保护下，将10％的氢氧化钾的甲醇溶液(10mL)加入到(3β)-3-(乙酰氧基)-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯(1.45g,3.36mmol)的甲醇溶液(20mL)，反应液在室温下搅拌反应2小时。减压浓缩至大约10mL的反应液，加入到冰水(40mL)中，固体过滤，用冰水洗涤，真空干燥得到1.20g米黄色固体，收率：92.9％，纯度：98.64％。LC-MS(APCI):m/z＝389.3[M+1]+；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)。
步骤5：3β-17-(1H-苯并[d]咪唑-2-d-1-基)雄甾-5,16-二烯-3-醇(化合物7)的合成。
将甲醇钠(90mg，1.50mmol)加入到17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯-3β-醇(100mg，0.25mmol)的氘代甲醇(CD3OD-d4,5mL)，氮气保护下封管100℃反应过夜。加重水(10mL)淬灭反应，用二氯甲烷萃取(10mL x 4)，有机相用饱和食盐水(15mL)洗涤，无水硫酸钠干燥，减压浓缩，浓缩液进行柱分离，得到米黄色固体50mg，收率：50.0％，纯度：99.16％。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)。
实施例2制备3β-17-(1H-苯并[d]咪唑-5,6,7-d3-1-基)雄甾-5,16-二烯-3-醇(化 合物13)
Figure PCTCN2016112946-appb-000005
室温下，氮气保护将无水二甲亚砜(6mL)液加入到17-碘雄甾-5,16-二烯-3β-醇(300mg,0.75mmol),1H-苯并[d]咪唑-4,5,6,7-d4(110mg,0.90mmol),L-脯氨酸(35mg,0.3mmol)，碘化亚铜(30mg,0.15mmol)和碳酸钾(259mg,1.87mmol)的混合物中，反应液在120℃下过夜。冷却至室温，加水(25mL)淬灭反应，硅藻土过滤，滤液用乙酸乙酯萃取(30mL x 3)，合并有机层，有机层用饱和的食盐水(30mL)洗涤，无水硫酸钠干燥，减压浓缩有机层，浓缩液柱层析纯化得到80mg白色固体，收率：27.2％，纯度：99.37％。LC-MS(APCI):m/z＝393.0[M+1]+；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)。
实施例3制备3β-17-(1H-苯并[d]咪唑-4,5,6,7-d4-1-基)雄甾-5,16-二烯-3-醇(化 合物15)
Figure PCTCN2016112946-appb-000006
室温下，氮气保护将无水二甲亚砜(6mL)液加入到17-碘雄甾-5,16-二烯-3β-醇(300mg,0.75mmol),1H-苯并[d]咪唑-4,5,6,7-d4(110mg,0.90mmol),L-脯氨酸(35mg,0.3mmol)，碘化亚铜(30mg,0.15mmol)和碳酸钾(259mg,1.87mmol)的混合物中，反应液在120℃下过夜。冷却至室温，加水(25mL)淬灭反应，硅藻土过滤，滤液用乙酸乙酯萃取(30mL x 3)，合并有机层，有机层用饱和的食盐水(30mL)洗涤，无水硫酸钠干燥，减压浓缩有机层，浓缩液柱层析纯化得到80mg白色固体，收率：27.2％，纯度：99.37％。LC-MS(APCI):m/z＝393.0[M+1]+；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)。
实施例4制备17-(1H-苯并[d]咪唑-2-d-1-基)雄甾-5,16-二烯-3-酮(化合物16)
Figure PCTCN2016112946-appb-000007
室温下，将氮气保护下将17-(1H-苯并[d]咪唑-2-d-1-基)雄甾-5,16-二烯-3β-醇(130mg，0.33mmol)，无水甲苯(13mL)和N-甲基-4-哌啶酮(0.5mL)的混合物加入到带有分水器的回流装置中，混合物在回流搅拌1小时(大约蒸出2mL的甲苯)。再加入异丙醇铝(205mg,1.05mmol)，反应液在回流下搅拌反应过夜。冷却至温室，加入20mL的水和20mL的乙酸乙酯淬灭反应，过滤，滤液用乙酸乙酯(20mL x 3)萃取。合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩有机层，浓缩液柱层析纯化得到100mg米白色固体，产率：76.9％，纯度：97.73。LC-MS(APCI):m/z＝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)。
实施例5制备17-(1H-苯并[d]咪唑-5,6,7-d3-1-基)雄甾-5,16-二烯-3-酮(化合 物17)
Figure PCTCN2016112946-appb-000008
室温下，将氮气保护下将17-(1H-苯并[d]咪唑-4,5,6,7-d4-1-基)雄甾-5,16-二烯-3β-醇(36mg，0.09mmol)，无水甲苯(4mL)和N-甲基-4-哌啶酮(0.14mL)的混合物加入到带有分水器的回流装置中，混合物在回流搅拌1小时(大约蒸出0.5mL的甲苯)。再加入异丙醇铝(60mg,0.28mmol)，反应液在回流下搅拌反应过夜。冷却至温室，加入20mL的水和20mL的乙酸乙酯淬灭反应，过滤，滤液用乙酸乙酯(20mL x 3)萃取。合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩有机层，浓缩液柱层析纯化得到26mg米白色固体，产率：72.2％，纯度：93.02％。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)。
实施例6制备17-(1H-苯并[d]咪唑-4,5,6,7-d4-1-基)雄甾-5,16-二烯-3-酮(化 合物18)
Figure PCTCN2016112946-appb-000009
室温下，将氮气保护下将17-(1H-苯并[d]咪唑-5,6,7-d3-1-基)雄甾-5,16-二烯-3β-醇(140mg，0.36mmol)，无水甲苯(14mL)和N-甲基-4-哌啶酮(0.54mL)的混合物加入到带有分水器的回流装置中，混合物在回流搅拌1小时(大约蒸出3mL的甲苯)。再加入异丙醇铝(230mg,1.09mmol)，反应液在回流下搅拌反应过夜。冷却至温室，加入20mL的水和20mL的乙酸乙酯淬灭反应，过滤，滤液用乙酸乙酯(20mL x 3)萃取。合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩有机层，浓缩液柱层析纯化得到29mg米白色固体，产率：20.7％，纯度：94.44％。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)。
生物活性测试
(1)CYP17体外测定。
该化合物的体外CYP17抑制活性使用快速醋酸释放测定(AARA)来评价，利用完整的P450c17-表达E.coli作为酶源。包括用[21-3H]-17α-羟基孕烯醇酮作为底物并且通过大量氚标记醋酸的形式测量CYP17活性，在底物的C-21侧链分裂期间。IC50值从超过适当范围的抑制百分比与抑制浓度的关联曲线直接获得(Grigoryev等，Br.J.Cancer,1999,81,622-630)。每一个化合物在五个不同浓度的最小值实验。测量三份，并且IC50值取三次实验的平均值，其中，A表示IC50<0.1μM，B表示0.1μM≤IC50≤0.5μM，C表示IC50>0.5μM(如下表1所示)。
(2)细胞PSA蛋白分泌的抑制作用。
实验步骤：1.用含10％Charcoal Stripped FBS的培养基替换原来培养基，使细胞在培养瓶中饥饿24小时；2.消化细胞，计数，将LNcaP细胞接种到96孔板中，10,000/well，培养过夜；3.将DHT和化合物按照设定浓度添加到现有培养基中，DHT终浓度为1nM,化合物起始浓度为50000nM，5倍稀释，8个浓度梯度，培养48小时；4.收集细胞培养上清，按照ELISA试剂盒说明书检测PSA蛋白水平。根据各浓度的抑制率，用GraphPad Prism计算IC50，其中，A表示IC50<0.1μM，B表示0.1μM≤IC50≤0.5μM，C表示IC50>0.5μM(如下表1所示)
表1实施例1～6的取代的甾体类化合物的激酶抑制作用分析表
实施例编号 CYP17酶IC50(μM) PSA IC50(μM)
Galeterone B -
实施例1 B -
实施例2 A -
实施例3 A -
Galeterone代谢物 C C
实施例4 B B
实施例5 B C
实施例6 B -
如表1所示，本发明化合物与Galeterone相比，实施例1的CYP17酶抑制活性与Galeterone相当，而实施例2和3的抑制活性比Galeterone更好，而且，氘代的代谢物(实施例4、5、6)的抑制活性也比未氘代的代谢物更好。此外，对PSA蛋白分泌的抑制作用也更强，因此本发明化合物比Galeterone更适于用于治疗、预防或消除各种与CYP17酶及雄激素受体相关的病症，如前列腺癌。
(3)大鼠中的药代动力学评价。
8只雄性Sprague-Dawley大鼠，7-8周龄，体重约210g，分成2组，每组4只，单次口服给予5mg/kg剂量的(a)对照组：3β-17-(1H-苯并[d]咪唑-1-基)雄甾-5,16-二烯-3-醇；(b)试验组：实施例1-3，比较其药代动力学差异。
大鼠采用标准饲料饲养，给予水。试验前16小时开始禁食。药物用PEG400和二甲亚砜溶解。眼眶采血，采血的时间点为给药后0.083小时，0.25小时、0.5小时、1小时、2小时、4小时、6小时、8小时、12小时和24小时。
大鼠吸入乙醚后短暂麻醉，眼眶采集300μL血样于试管。试管内有30μL1％肝素盐溶液。使用前，试管于60℃烘干过夜。在随后一个时间点血样采集完成之后，大鼠乙醚麻醉后处死。
血样采集后，立即温和地颠倒试管至少5次，保证混合充分后放置于冰上。血样在4℃5000rpm离心5分钟，将血浆与红细胞分离。用移液器吸出100μL血浆到干净的塑料离心管中，表明化合物的名称和时间点。血浆在进行分析前保存在-80℃。用LC-MS/MS测定血浆中本发明化合物的浓度。药代动力学参数基于每只动物在不同时间点的血药浓度进计算。
实验结果表明，相对于对照化合物，本发明化合物在动物体内具有更好的药物动力学，因而具有更好的药效学和治疗效果。
(4)代谢稳定性评价。
微粒体实验：人肝微粒体：0.5mg/mL，Xenotech；大鼠肝微粒体：0.5mg/mL，Xenotech；辅酶(NADPH/NADH)：1mM，Sigma Life Science；氯化镁：5mM，100mM磷酸盐缓冲剂(pH为7.4)。
储备液的配制：精密称取一定量的化合物实施例的粉末，并用DMSO分别溶解至5mM。
磷酸盐缓冲液(100mM，pH7.4)的配制：取预先配好的0.5M磷酸二氢钾150mL和700mL的0.5M磷酸氢二钾溶液混合，再用0.5M磷酸氢二钾溶液调节混合液pH值至7.4，使用前用超纯水稀释5倍，加入氯化镁，得到磷酸盐缓冲液(100mM)，其中含100mM磷酸钾，3.3mM氯化镁，pH为7.4。
配制NADPH再生系统溶液(含有6.5mM NADP，16.5mM G-6-P，3U/mL G-6-P D，3.3mM氯化镁)，使用前置于湿冰上。
配制终止液：含有50ng/mL盐酸普萘洛尔和200ng/mL甲苯磺丁脲(内标)的乙腈溶液。取25057.5μL磷酸盐缓冲液(pH7.4)至50mL离心管中，分别加入812.5μL人肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。取25057.5μL磷酸盐缓冲液(pH 7.4)至50mL离心管中，分别加入812.5μL SD大鼠肝微粒体，混匀，得到蛋白浓度为0.625mg/mL的肝微粒体稀释液。
样品的孵育：用含70％乙腈的水溶液将相应化合物的储备液分别稀释至0.25mM，作为工作液，备用。分别取398μL的人肝微粒体或者大鼠肝微粒体稀释液加入96孔孵育板中(N＝2)，分别加入2μL 0.25mM的的工作液中，混匀。
代谢稳定性的测定：在96孔深孔板的每孔中加入300μL预冷的终止液，并置于冰上，作为终止板。将96孔孵育板和NADPH再生系统置于37℃水浴箱中，100转/分钟震荡，预孵5min。从孵育板每孔取出80μL孵育液加入终止板，混匀，补充20μL NADPH再生系统溶液，作为0min样品。再向孵育板每孔加入80μL的NADPH再生系统溶液，启动反应，开始计时。相应化合物的反应浓 度为1μM，蛋白浓度为0.5mg/mL。分别于反应10、30、90min时，各取100μL反应液，加入终止板中，涡旋3min终止反应。将终止板于5000×g，4℃条件下离心10min。取100μL上清液至预先加入100μL蒸馏水的96孔板中，混匀，采用LC-MS/MS进行样品分析。
数据分析：通过LC-MS/MS系统检测相应化合物及内标的峰面积，计算化合物与内标峰面积比值。通过化合物剩余量的百分率的自然对数与时间作图测得斜率，并根据以下公式计算t1/2和CLint，其中V/M即等于1/蛋白浓度。
对实施例的化合物按照上述步骤进行分析，结果如表2所示。
表2实施例1～6的取代的甾体类化合物的代谢稳定性的实验结果
Figure PCTCN2016112946-appb-000010
如表2所示，本发明化合物半衰期延长，清除率降低，说明本发明化合物能够显著改善代谢稳定性。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (11)

  1. 一种取代的甾体类化合物，其特征在于：如式(I)所示的甾体类化合物：
    Figure PCTCN2016112946-appb-100001
    式中：
    R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23相互独立地选自由“氢(H)、氘(D)”组成的组；
    X1、X2相互独立地选自由“氢(H)、氘(D)、甲基、CH2D、CHD2、CD3、CH2CH3、CHDCH3、CHDCH2D、CHDCHD2、CHDCD3、CD2CH3、CD2CH2D、CD2CHD2、CD2CD3”组成的组；
    Y选自“氢(H)、氘(D)、羟基、乙酰基，一次或多次氘代的乙酰基”；
    及其生理学上可接受的盐、前药、代谢物、溶剂化物、互变异构体和立体异构体，包括这些化合物以所有比例形成的混合物；
    附加条件是，所述的化合物不包括非氘代的化合物。
  2. 如权利要求1所述化合物，其特征在于，R1、R2、R3、R4、R5自独立地为氘或氢。
  3. 如权利要求1所述化合物，其特征在于，Y选自氢、氘、羟基、一次或多次氘代的乙酰基。
  4. 如权利要求1所述化合物，其特征在于，X1、X2为三次氘代的甲基。
  5. 如权利要求1所述化合物，其特征在于，选自以下组内的取代的甾体类化合物或其药学上可接受的盐，但不局限于如下化合物：
    Figure PCTCN2016112946-appb-100002
    Figure PCTCN2016112946-appb-100003
  6. 一种药物组合物，其特征在于：其含有药学上可接受的载体和如权利要求1～5任意一项所述的取代的甾体化合物，或其晶型、药学上可接受的盐、水合物或溶剂合物、立体异构体、前药、代谢物、同位素变体或其混合物。
  7. 根据权利要求6所述的药物组合物，其特征在于：其还包含其他治疗药物，所述治疗药物为癌症、细胞增殖性疾病、炎症、感染、免疫性疾病、器官移植、病毒性疾病、心血管疾病或代谢性疾病的药物。
  8. 一种如权利要求6或7所述的药物组合物的制备方法，其特征在于：将药学上可接受的载体与如权利要求1～5任意一项所述的取代的甾体类化合物，或其晶型、药学上可接受的盐、前药、代谢物、立体异构体、同位素变体水合物或溶剂合物进行混合，得到药物组合物。
  9. 一种如权利要求1～5任意一项所述的取代的甾体类化合物的用途，其特征在于：可以用于治疗、预防或消除各种CYP17酶及雄激素受体(AR)相关病症(如前列腺癌)；包含这些化合物的药物组合物用于在不同治疗领域诸如癌症中治疗、预防疾病或障碍或减慢所述疾病或障碍进程。
  10. 一种在受试者中治疗和/或预防与CYP17酶及雄激素受体(AR)相关的疾病的方法，所述方法包括向所述受试者给药如权利要求1～5任意一项所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者权利要求6或7中任一项的药物组合物。
  11. 根据权利要求1～5任意一项所述的式(I)化合物或其多晶型、药学上可接受的盐、前药、立体异构体、同位素变体、水合物或溶剂化合物，或者权利要求6或7中任一项的药物组合物，其用于治疗和/或预防与CYP17酶及雄激素受体(AR)相关的疾病。
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