WO2020007273A1 - 新型雄激素受体抑制剂及其合成方法和应用 - Google Patents

新型雄激素受体抑制剂及其合成方法和应用 Download PDF

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WO2020007273A1
WO2020007273A1 PCT/CN2019/094301 CN2019094301W WO2020007273A1 WO 2020007273 A1 WO2020007273 A1 WO 2020007273A1 CN 2019094301 W CN2019094301 W CN 2019094301W WO 2020007273 A1 WO2020007273 A1 WO 2020007273A1
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余江
陈元伟
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Sichuan University
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Definitions

  • the present invention relates to the field of medicine, and in particular, the present invention relates to the synthesis of a novel androgen receptor inhibitor.
  • Prostate cancer is one of the more common malignant tumors of the male genitourinary system. It is the most common malignant tumor in men in Western countries and the third leading cause of death in cancer. In 2012, the incidence of prostate cancer in tumor registration areas in China was 9.92 per 100,000, ranking sixth among male malignancies. In 2015, there were approximately 60,300 new cases of prostate cancer and 26,600 deaths in China, which has become one of the important reasons threatening the health of elderly men in China. According to the 2014 edition of the Chinese Urology Guidelines, the standard treatment for advanced prostate cancer is androgen deprivation therapy (ADT). In the early stages of androgen deprivation therapy, symptoms can be alleviated to some extent, but about 40% of patients will develop castration-resistant prostate cancer (CRPC).
  • ADT androgen deprivation therapy
  • the reason is related to the reactivation of androgen receptor signaling pathways, including androgens secreted by the adrenal glands, androgens secreted by tumors, mutations in androgen receptors, and effects of steroid hormone-related signaling pathways.
  • the median survival of CRPC patients is only 12-18 months.
  • the androgen receptor (AR) belongs to the nuclear receptor superfamily, which contains 918 amino acids, and has a similar structure and function to other nuclear receptors. It is composed of three important domains, namely DNA binding domain (DBD), ligand binding domain (LBD), and nitrogen terminal domain (NTD), DBD and The LBDs are connected by a hinge region (Hinge).
  • the LBD existing at the carbon end of AR is the site where AR binds to the ligand, which determines the specificity of binding of the ligand to AR, and the ligand binds to LBD to activate AR.
  • testosterone is mainly produced by the testes, and in addition the adrenal glands can secrete a small amount.
  • DHT dihydrotestosterone
  • Hsp90 heat shock protein
  • the AR-androgen complex thus formed enters the nucleus to recognize the corresponding androgen response element, recruits other co-regulatory proteins, forms an active transcription complex, and initiates the transcription of AR target genes.
  • Antagonists competitively bind AR with endogenous androgens, but do not cause transcription of related genes and subsequent biological effects.
  • Androgen receptor plays a very important role in the occurrence and development of prostate cancer. Studies show that castration-resistant prostate cancer still depends on the role of AR. Therefore, targeting AR signaling pathways remains important.
  • AR inhibitors can be divided into steroidal AR antagonists and non-steroidal AR antagonists.
  • Cyproterone acetate is a representative of steroidal AR antagonists. Due to the poor oral bioavailability and selectivity of this class of drugs, it is prone to cross effects and adverse reactions, which limits its clinical application.
  • Nonsteroidal AR inhibitors have relatively high oral bioavailability and high selectivity for AR, so they have become the main direction for research and development of antiandrogens.
  • AR inhibitors that have been marketed in the treatment of prostate cancer include first-generation flutamide, R-bicalutamide, nilutamide, and second-generation anthracene.
  • Enzalutamide all act on LDB of AR.
  • ARN509 which has a very similar structure to anthazalamide, and small molecule inhibitors such as ODM-201 developed by ORION.
  • the first-generation anti-androgens have a lower affinity for AR than the endogenous ligand DHT.
  • Long-term use of anti-androgens can cause antagonism to agonism, leading to aggravation of the disease.
  • This phenomenon may be caused by mutations in certain sites of AR, such as the T877A and W741C mutations that have been reported to cause AR to switch to an activated conformation after interacting with the antagonist.
  • early anti-androgen therapy is effective.
  • traditional AR antagonists cannot continue to be used. This is related to AR overexpression, AR mutations, and intra-tumor androgen biosynthesis. And other factors.
  • the second-generation AR inhibitor anthrazelamide (Figure 1) was approved for marketing by the FDA. Its affinity for AR is 8 times that of carumamine, and its IC50 value is 36nmol / L, which is higher than that of carumamine. After 3 to 5 times, the compound can inhibit the transfer of AR from the cytoplasm to the nucleus and prevent the binding of AR and DNA. However, the results of clinical studies have shown that anthrazamide may induce epilepsy, with an incidence of about 0.6%. At the same time, AR-F876L mutations have been found to be resistant to anthrazelamide.
  • the present invention provides a compound represented by the general formula (I), which has far higher affinity for AR than the first-generation androgen receptor inhibitor and the first-generation AR inhibitor hydroxyflutamide.
  • the T877A mutation and the F876L mutation produced by the second-generation AR inhibitor anthranilamide were effective.
  • these compounds have a similar chemical structure to ODM201, which can hardly cross the blood-brain barrier and reduce the side effects of compounds caused by epilepsy.
  • the compounds provided by the present invention can not only competitively bind AR with natural androgens but also down-regulate the expression of AR and its splice mutants, thereby better inhibiting abnormal activation of the androgen signaling pathway.
  • the present invention first provides a compound represented by Formula I or a stereoisomer, a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • Z 1 and Z 2 are each independently C or N;
  • Ring A is a C 5 to C 12 carbocyclic ring, heterocyclic ring, imidazole dione, aromatic ring, aromatic heterocyclic ring, benzocarbon ring, benzoheterocyclic ring, wherein the carbon on the benzene ring may be substituted by one or more heteroatoms ;
  • Ring B is a C 5 to C 12 carbocyclic ring, heterocyclic ring, fused ring, bridged ring, spiro ring, aromatic ring, aromatic heterocyclic ring or benzoaromatic ring, benzoaromatic ring, benzocarbocyclic ring or benzoheterocyclic ring Where the carbon on the benzene ring may be substituted by one or more heteroatoms;
  • R 1 and R 2 are each independently hydrogen, halogen, CHF 2 , CF 3 , cyano, nitro, amino, hydroxylamino, hydroxyl, mercapto, sulfonic acid, C 1 to C 6 alkyl, olefin, Alkynyl, alkoxy, cycloalkyl, heterocyclyl or aryl, heteroaryl, and -R 9 C (O) R 10- , -R 9 CO (O) R 10- , -R 9 (O) COR 10- , -R 9 NHC (O) R 10 -or -R 9 C (O) NHR 10- , wherein hydroxyl, mercapto, amino, alkyl, alkenyl, alkynyl, alkoxy, aryl, Heteroaryl, cycloalkyl, heterocyclyl may be further substituted by one or more halogen, cyano, amino, hydroxylamino, mercapto, hydroxyl, alkyl
  • R 3 is hydrogen, halogen, cyano, nitro, amino, hydroxyl, hydroxylamine, mercapto, sulfonic acid, carboxyl, aldehyde, carbonyl, ester, amide bond, or C 1 to C 6 alkyl, alkoxy Alkyl, cycloalkyl, heterocyclyl, aryl, arylheteroyl, in which alkyl, alkynyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl are further substituted by one or Multiple halogen, cyano, amino, hydroxylamino, mercapto, hydroxyl, alkyl, alkoxy, carbonyl, aldehyde, carboxyl, ester, amide bond, aryl, arylhetero group substitution;
  • R 1 and R 2 and R 3 may be connected to each other by a single bond, a double bond, or a triple bond to form a ring;
  • R 4 , R 4 ′, R 5, and R 5 ′ each independently represent hydrogen, hydroxyl, halogen, amino, hydroxylamino, cyano, nitro, alkenyl, alkynyl, C 1 to C 6 alkyl, or cycloalkane Group, alkoxy group, carbonyl group, thiocarbonyl group, aldehyde group, carboxyl group, ester group, and amide group, wherein the C 1 to C 6 alkyl group, alkoxy group, alkenyl group, alkynyl group, ester group, and amide group may be Each independently further substituted with one or more groups selected from the group consisting of halogen, hydroxy, cyano, nitro, hydroxylamine, amino, hydroxylamine, alkoxy, and cycloalkyl;
  • R 5 , R 5 ′, R 4 , R 4 ′ may be connected to each other to form a bond
  • R 7 , R 7 ′, R 6 , R 6 ′, and R 8 each independently represent hydrogen, hydroxyl, hydroxylamino, amino, nitro, cyano, halogen, C 1 to C 6 alkyl, alkoxy, Alkoxy-substituted alkyl, cycloalkyl, carbonyl, aldehyde, carboxy, ester, and amide groups;
  • R 7 , R 7 ′, R 6 , R 6 ′ may be connected to each other to form a bond
  • R 11 and R 12 each independently represent hydrogen, a hydroxyl group, an amino group, a hydroxylamine group, a nitro group, a sulfonic acid group, a cyano group, a halogen group, a C 1 to C 6 alkyl group, an alkoxy group, a cycloalkyl group, or an aryl group; Wherein, the C 1 to C 6 alkyl, alkoxy, cycloalkyl, amino, and aryl are each independently optionally further selected from one or more of halogen, hydroxyl, cyano, nitro, alkyl, and cyclic Substituted by alkyl, alkoxy, aryl groups;
  • R 9 and R 10 are each independently represented as hydrogen, C 1 to C 6 straight or branched chain alkyl, alkoxy, and amino, wherein the alkyl and alkoxy may be further substituted by one or more halogen or cyano , Hydroxyl, nitro, amino.
  • the ring A of the compound has a structure as shown in Ia:
  • M is CH or O; the dashed line indicates an optional double bond; the asterisk indicates the position connected to the ring;
  • R 4 and R 5 have the same definitions as the above structure I.
  • the compound has a structure represented by Formula II:
  • R 1 and R 2 are each independently hydrogen, halogen, CHF 2 , CF 3 , cyano, nitro, amino, hydroxylamino, hydroxyl, mercapto, sulfonic acid, C 1 to C 6 alkyl, and olefin.
  • Alkynyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl or aryl, heteroaryl; hydroxy, mercapto, amino, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl Group, cycloalkyl group, heterocyclic group may be further substituted by one or more halogen, cyano, amino, hydroxylamino, mercapto, hydroxyl, alkyl, alkoxy, carbonyl, aldehyde, carboxyl, ester, amide bond, Aryl, arylheteroaryl substitution;
  • the ring B is a C5 to C12 carbocyclic ring, a heterocyclic ring, an aromatic ring, an aromatic heterocyclic ring, or a benzoaromatic ring, a benzoheterocyclic ring, or a benzoaromatic heterocyclic ring, wherein the carbon on the benzene ring may be substituted by one or more heterocyclic rings.
  • R 11 and R 12 each independently represent hydrogen, a hydroxyl group, an amino group, a hydroxylamine group, a nitro group, a sulfonic acid group, a cyano group, a halogen group, a C 1 to C 6 alkyl group, an alkoxy group, a cycloalkyl group, or an aryl group; Wherein, the C 1 to C 6 alkyl, alkoxy, cycloalkyl, amino, and aryl are each independently optionally further selected from one or more of halogen, hydroxyl, cyano, nitro, alkyl, and cyclic Substituted by alkyl, alkoxy, aryl groups.
  • the compound has a structure represented by Formula III:
  • the compound has a structure represented by Formula IV:
  • the ring B is an N-containing cyclic ring, an N-containing aromatic heterocyclic ring, a benzo N-containing heterocyclic ring, a benzo N-containing aromatic heterocyclic ring, or a purine.
  • the compound is one of the following:
  • the present invention also provides the aforementioned compounds or stereoisomers, pharmaceutically acceptable salts, hydrates or solvates thereof.
  • the reaction route is as follows:
  • E is halogen Cl, Br, I;
  • the present invention also provides the use of the aforementioned compound or a stereoisomer thereof, a pharmaceutically acceptable salt, a hydrate or a solvate in the preparation of an androgen receptor inhibitor.
  • the androgen receptor includes a mutant of the androgen receptor.
  • the androgen receptor mutant T877A mutant, W741C mutant and / or AR-F876L mutant Please explain the three mutations in detail, indicating the location of the mutations and how to make them.
  • the inhibitor is an inhibitor of androgen receptor expression and / or an androgen receptor pathway.
  • the inhibition of the androgen receptor pathway is to inhibit the transcription of genes downstream of the androgen receptor PSA and FKBP5.
  • the present invention also provides the use of the aforementioned compound or a stereoisomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof in the preparation of a medicament for androgen receptor-related diseases.
  • the medicine is a medicine for treating androgen receptor positive prostate cancer, benign prostatic hyperplasia, hirsutism, hair loss, anorexia nervosa, breast cancer, acne, male sexual dysfunction or AIDS.
  • the invention also provides a novel androgen receptor inhibitor, which uses the aforementioned compound or a stereoisomer thereof, a pharmaceutically acceptable salt, a hydrate or a solvate as an active ingredient, plus Formulations prepared from acceptable excipients.
  • the term "compound of the invention” refers to a compound of formula I.
  • the term also includes stereoisomers, tautomers, isotopic substitution forms, mixtures and pharmaceutically acceptable salts of the compounds of formula I, as well as their metabolites and metabolic precursors or prodrugs.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention and an acid or base suitable for use as a medicament.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • the pharmaceutically acceptable excipient has certain physiological activity, but the addition of the ingredient will not change the dominance of the above-mentioned pharmaceutical composition in the course of disease treatment, but only exerts auxiliary effects, and these auxiliary effects are only for the component
  • auxiliary effects are only for the component
  • the use of known activities is a common adjuvant therapy in the medical field. If the above auxiliary ingredients are used in combination with the pharmaceutical composition of the present invention, they should still fall within the protection scope of the present invention.
  • stereoisomers includes the presence of stereocenters (such as carbons with 4 different substituents), axial asymmetry such as critical, planar asymmetry, and mixtures thereof.
  • Substitute refers to the replacement of a hydrogen atom in a molecule with another different atom or molecule.
  • alkyl is a hydrocarbon group obtained by missing one hydrogen atom in an alkane molecule.
  • C1 to C6 alkyl groups refer to C1, C2, C3, C4, C5, and C6 alkyl groups.
  • a straight or branched alkyl group of one carbon atom such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, and the like.
  • Alkynyl refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond.
  • the alkynyl group may be linear or branched.
  • the alkynyl group is preceded by a limited number of carbon atoms (such as a C 2-8 alkynyl group), for example, the term “C 2-8 alkynyl group” refers to a straight or branched chain alkynyl group having 2 to 8 carbon atoms.
  • Alkenyl refers to an aliphatic hydrocarbon group having at least one carbon-carbon double bond.
  • the alkenyl group may be linear or branched.
  • the alkenyl has a carbon number limitation (such as a C 2-8 alkenyl), for example, the term “C 2-8 alkenyl” refers to a straight-chain or branched alkenyl having 2-8 carbon atoms.
  • Halogen is fluorine, chlorine, bromine or iodine.
  • Aryl refers to a full-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups, such as phenyl and naphthyl, having a conjugated pi-electron system.
  • the aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur.
  • the point connecting the parent must be in a conjugated ⁇ -electron system On a carbon atom.
  • Aryl may be substituted or unsubstituted.
  • Heteroaryl refers to a heteroaromatic group containing one to more heteroatoms.
  • the heteroatoms referred to here include oxygen, sulfur and nitrogen.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.
  • Heteroaryl groups can be optionally substituted or unsubstituted.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic.
  • C 3-8 cycloalkyl refers to a cycloalkyl group having 3 to 8 carbon atoms.
  • Heterocyclyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic and carry at least one cycloalkyl selected from O, S or a substituted nitrogen atom, and the rest The ring atom is carbon.
  • C 3-8 heterocyclyl refers to a heterocyclic group having 3 to 8 carbon atoms and heteroatoms in total.
  • the compound of the present invention can effectively inhibit AR. Specifically, it can not only inhibit the protein expression of AR and its mutants by down-regulating the mRNA, but also inhibit the transcription of genes downstream of AR and its mutants PSA and FKBP5.
  • the drug-resistant AR-F876L still has an inhibitory effect, indicating that the compounds of the present invention can effectively inhibit the AR signaling pathway.
  • the compounds of the present invention can effectively inhibit the proliferation of AR-positive prostate cancer cells, and have good cell selectivity, providing a new option for clinical screening and / or preparation of drugs for treating prostate cancer. Application prospects.
  • E is halogen Cl, Br, I
  • Triphenylphosphine (8.4 g, 30 mmol) was added to a round-bottomed flask, N 2 was drawn through, and dry THF (25 ml) was added. Stir with an ice bath until the triphenylphosphine is completely dissolved, slowly add DIAD (6.4 g, 30 mmol), and precipitate a white solid after the addition.
  • DIAD 6.4 g, 30 mmol
  • (S) -tert-butyl (1-hydroxyprop-2-yl) carbamate (5.6 g, 30 mmol) was dissolved in dry THF (20 ml) and slowly added to the reaction mixture.
  • 6-chloropurine 160mg, 0.92mmol
  • ethanol 5ml
  • (S) -4- (1- (2-aminopropyl) -1H-pyrazol-3-yl) -2-chlorobenzonitrile was added (240 mg, 0.92 mmol) and triethylamine (72 mg, 0.79 mmol), heated to 90 ° C. and stirred for 48 h.
  • the title compound was prepared by the method described in Example 10 to obtain 80 mg of a colorless, thick, SKLB-C4667.
  • This title compound was prepared by the method described in Example 5 using 6-bromoisoquinoline (118.6 mg, 0.57 mmol) and (S) -4- (1- (2-aminopropyl) -1H-pyrazol-3-yl ) -2-chlorobenzonitrile (100 mg, 0.38 mmol) was prepared as a raw material to obtain 80 mg of a pale yellow solid, namely SKLB-C4674.
  • RIPM 1640 medium (Hyclone, SH30809.01), DMEM high sugar medium (Hyclone, SH30243.01), fetal bovine serum, FBS (BI, 04-001-1ACS), cyano-streptomycin (Hyclone, SV30010) .
  • Complete cell culture medium RIPM 1640 medium / DMEM high sugar medium, 10% FBS, 1% penicillin-streptomycin.
  • LNCap / AR, 22RV1, PC-3 were cultured in 1640 complete medium, VCap was cultured in DMEM high-sugar complete medium, and cultured in a 37 ° C incubator with 5% carbon dioxide. The cells were seeded in a 96-well plate, with two sub-wells, 100 microliters per well, and the number of seeded cells were LNCap / AR2000 / well, 22RV13000 / well, VCap 20000 / well, and PC-3 1000 / well.
  • Drugs were prepared with DMSO (dimethyl sulfoxide) into a 30 mM stock solution. The plate was diluted to 600 ⁇ M with complete medium the next day after plating, and then diluted 10-fold with complete medium, and a 3-fold gradient dilution was started from 60 ⁇ M for a total of 9 concentrations. . 100 ⁇ L of the diluted compound was added to the cell culture wells, and the blank control group was added with 100 ⁇ L of culture medium, that is, the drug concentration was up to 30 ⁇ M.
  • DMSO dimethyl sulfoxide
  • the above experimental data show that the compound of the present invention has a better inhibitory effect on AR-positive prostate cancer cells LNCap / AR, 22RV1, LNCap (containing the T877A mutation) and VCap, especially on prostate cancer cells 22RV1 and AR-V7 mutation-containing prostate cancer cells.
  • the VCap inhibitory activity is better than that of the marketed drug anthrazamide and the clinical phase III drug ODM201.
  • the poor PC-3 inhibition effect on AR-negative prostate cancer cells indirectly indicates that the compound of the present invention inhibits the AR signal pathway, has good cell selectivity, and has good safety.
  • Test Example 2 Evaluation of biological activity for suppressing wild-type AR transcriptional activity
  • DMEM medium Gibco, 31053028
  • androgen-free fetal bovine serum Biological Industries, 04-011-1A
  • HEK293 cells provided by WuXi PharmaTech New Drug Development Co., Ltd.
  • transfection reagents Promega, E2311
  • wt-AR expression plasmid Origene, RC235415
  • reporter gene plasmid Promega, E1360
  • Steady-Glo detection reagent Promega, E2550
  • HEK293 cells were seeded in a 96-well plate with a complete medium containing 89% DMEM medium, 1% GlutaMax, and 10% fetal bovine serum to remove androgens by dialysis.
  • 5 ng wt-AR plasmid was transfected using a transfection reagent according to the instructions. Cotransfect into cells with 100ng reporter gene plasmid. After 24 hours, cells were treated with compound or DMSO and 2nM testosterone. After a 24 hour incubation, the luminescence signal was measured on an Envision instrument using Steady-Glo reagent. The experiment was completed by Shanghai Wuxi Kangde New Drug Development Co., Ltd.
  • This result shows that the compounds of the present invention SKLB-C4651, SKLB-C4652, SKLB-C4664, SKLB-C4670, SKLB-C4669, SKLB-C4674, SKLB-C4682, SKLB-C4535, SKLB-C4661, SKLB-C4686 can inhibit androgen activation Transcription of wild-type AR, and the activity of the compound is basically consistent with the cell proliferation inhibitory activity.
  • RIPM 1640 medium (Hyclone, SH30809.01), fetal bovine serum, FBS (BI, 04-001-1ACS), penicillin-streptomycin (Hyclone, SV30010).
  • Complete cell culture medium RIPM 1640 medium, 10% FBS, 1% penicillin-streptomycin.
  • LNCap and 22RV1 were cultured in 1640 complete medium, VCap was cultured in DMEM high-sugar complete medium, and cultured in a 37 ° C incubator with 5% carbon dioxide. On the first day, cells were seeded in 6-well plates, the number of LNCap and 22RV1 plates was 700,000 / well, and the VCap was 1 million / well.
  • the drug was added.
  • the drug was formulated into a 30 mM stock solution with DMSO (dimethyl sulfoxide), diluted with complete medium, and the blank group was added with complete medium containing the same concentration of DMSO.
  • DMSO dimethyl sulfoxide
  • the medium was aspirated, washed with pre-chilled PBS once, blotted dry, 60 ⁇ l of RIPA lysate per well, lysed on ice for 5 min, collected by cell scraping and transferred to a centrifuge tube. Centrifuge at 4 ° C for 10 min, 13000 rpm. Transfer the supernatant to a new centrifuge tube, use the BCA protein quantification kit for protein quantification, then add 1/4 volume of 5X loading buffer, boil it in boiling water for 5 minutes, cool on ice, and then transfer to -20 ° C for storage.
  • a 10% polyacrylamide gel kit was used to make the gel, and 30 ⁇ g of protein was loaded. The volume was filled with 1X loading buffer. The gel was concentrated with 80V and the gel was separated with 100V. Wet transfer film, 300mA, 1h, and then block with 5% skim milk for 2h at room temperature. Primary antibody Tublin was diluted 1: 5000, AR and PSA were diluted 1: 2000 and 1: 1000, respectively, and incubated at 4 ° C with shaking overnight.
  • results of FIG. 2 show that 10 ⁇ M of the present compound 4682 (SKLB-C4682) and 20 ⁇ M of the present compound 4535 (SKLB-C4535) can significantly inhibit the AR protein level in LNCap cells, and have a significant concentration dependence.
  • both the marketed drug anthrazamide and the clinical phase III drug ODM201 cannot down-regulate AR protein levels at a concentration of 20 ⁇ M.
  • results in FIG. 3 show that 20 ⁇ M of the compounds 4535 (SKLB-C4535) and 4682 (SKLB-C4682) of the present invention can significantly inhibit the protein levels of AR and its mutants in VCap cells, and at the same time have a significant concentration dependence and activity Better than the reported compound galaterone.
  • both the marketed drug anthrazamide and the clinical phase III drug ODM201 failed to down-regulate AR protein levels at a concentration of 20 ⁇ M.
  • results of FIG. 4 show that 20 ⁇ M of the present compound 4682 (SKLB-C4682) and 30 ⁇ M of the present compound 4682 can significantly inhibit the protein levels of AR and its shear mutant AR-V7 in 22RV1 cells, and have significant concentrations.
  • Dependence However, both the marketed drug anthrazamide and the clinical phase III drug ODM201 cannot down-regulate AR protein levels at a concentration of 30 ⁇ M.
  • the above experimental results show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can down-regulate the expression of AR and its mutants in prostate cancer cells.
  • Overexpression of AR in cancer cells is one of the reasons leading to drug resistance in patients with prostate cancer. Therefore, while inhibiting AR activity and down-regulating the protein level of AR can better inhibit the androgen signaling pathway, the present invention can well inhibit the abnormal activation of the androgen signaling pathway.
  • Test Example 4 Evaluation of mRNA biological activity of reducing PSA, FKBP5 and AR in prostate cancer cells
  • RIPM 1640 medium (Hyclone, SH30809.01), DMEM high sugar medium (Hyclone, SH30243.01), bovine serum, FBS (BI, 04-001-1ACS), androgen-free fetal bovine serum, CSS-FBS (Given by Sichuan Kangcheng Biotechnology Co., Ltd.), cyano-streptomycin (Hyclone, SV30010).
  • Complete cell culture medium RIPM 1640 medium / DMEM high sugar medium, 10% FBS or 5% CSS, 1% penicillin-streptomycin.
  • LNCap and 22RV1 were cultured in 1640 complete medium, VCap was cultured in DMEM high-sugar complete medium, and cultured in a 37 ° C incubator with 5% carbon dioxide. On the first day, cells were seeded in a 6-well plate with a medium containing 5% CSS and 1% double antibody. The number of LNCap and 22RV1 plates was 700,000 / well, and the VCap was 1.2 million / well.
  • the drug was added.
  • the drug was prepared with DMSO (dimethyl sulfoxide) into a 30 mM stock solution.
  • R1881 was prepared with DMSO into a 5 ⁇ M stock solution, diluted with 5% CSS complete medium, and the blank group was added with the same concentration of DMSO. Complete medium.
  • RNA concentration was measured on ice.
  • reverse transcription kit instructions to perform reverse transcription (25 ° C for 10min, 42 ° C for 15min, and 85 ° C for 5min) to obtain cDNA, store at -20 ° C, and store the RNA in a refrigerator at -80 ° C.
  • FIG. 5 show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can inhibit the transcription of the AR downstream genes PSA and FKBP5 activated by androgen R1881 in LNCap cells.
  • the results of FIG. 6 show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can inhibit the transcription of the AR downstream genes PSA and FKBP5 activated by androgen R1881 in VCap cells, and the inhibitory effect is concentration-dependent.
  • the inhibitory activity and the positive compounds ODM201 and Anthranilamide is equivalent.
  • results of FIG. 7 show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can inhibit the transcription of the AR downstream gene FKBP5 activated by androgen R1881 in 22RV1 cells, and the activity is better than that of the positive compounds ODM201 and anthranulamide.
  • results of FIG. 8 show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can inhibit the mRNA of AR and its splicing mutant AR-V7 in LNCap cells, indicating that the reason for the decrease in AR protein is that the compounds reduce the androgen receptor. mRNA.
  • the compounds SKLB-C4682 and SKLB-C4535 of the present invention down-regulate the protein expression of AR by reducing androgen receptor mRNA.
  • the compound of the present invention can inhibit the transcription of the downstream genes PSA and FKBP5 of AR, especially the inhibitory effect of FKBP5 in 22RV1 cells is significantly better than that of anthranilamide and ODM201. .
  • DME / F12 medium (Hyclone, SH30023.01), fetal bovine serum, FBS (BI, 04-001-1ACS), androgen-free fetal bovine serum, CSS-FBS (Given by Sichuan Kangcheng Biotechnology Co., Ltd. ), Cyano-streptomycin (Hyclone, SV30010).
  • Complete cell culture medium DME / F12 medium, 10% FBS or 5% CSS, 1% penicillin-streptomycin (Millipore, SM-2001-C).
  • PC-3 cells Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee, SCSP-532
  • transfection reagent EndoFectinTM-Max GeneCopoeia, EF003
  • AR-F876L plasmid constructed by Biotech Biotechnology Shanghai Co., Ltd.
  • Cignal Androgen Receptor Reporter (luc) Kit QIAGEN, CCS-1019L
  • synthetic androgen R1881 Neanjing Kangmanlin Chemical Industry Co., Ltd., CAS: 965-93-5
  • dual luciferase detection kit promega, E1910
  • Multifunctional microplate reader BioTek, Synergy H1.
  • PC-3 cells were cultured in DME / F12 complete medium and cultured in a 37 ° C incubator with 5% carbon dioxide. On the first day, the cells were seeded in a 96-well plate with a medium containing 5% CSS and no double antibody, and the number of plates was 10,000 / well.
  • the medium was aspirated and discarded, and the luminescence signal was detected by a multi-functional microplate reader according to the instructions of the dual luciferase detection kit.
  • FIG. 9 show that the compounds SKLB-C4682 and SKLB-C4535 of the present invention can inhibit the transcriptional activity of AR-F876L activated by 1nM synthetic androgen R1881, and have a significant concentration dependence. In contrast, with the increase of anthrazelamide concentration, the transcriptional activity of AR-F876L was enhanced, showing resistance.
  • Test example 6 in vivo antitumor activity test
  • mice Male Balb / c nude mice (Beijing Huafukang Biotechnology Co., Ltd.), Matrigel (BD, 354234)
  • the tumor tissue was individually sacrificed and isolated for 15 days after oral administration of the same volume of blank solvent.
  • the other experimental groups were sacrificed by intraperitoneal injection of paraformaldehyde after 40 days of administration. weight. All animal experiments were approved by the Animal Protection and Therapy Committee of Sichuan University, China (IACUC number: 20100318).
  • the compounds of the present invention can not only inhibit the protein expression of AR and its mutants by down-regulating the mRNA, but also inhibit the transcription of the genes downstream of AR and its mutants PSA and FKBP5, and at the same time AR-resistant to AR- F876L still has an inhibitory effect, indicating that the compounds of the present invention can effectively inhibit the AR signaling pathway.
  • the compounds of the present invention can effectively inhibit the proliferation of AR-positive prostate cancer cells, and have good cell selectivity, providing a new option for clinical screening and / or preparation of drugs for treating prostate cancer. Application prospects.

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Abstract

本发明公开了式(I)所示的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物。本发明还公开了前述化合物的制备方法和用途。本发明式(I)所示的化合物是优良的AR抑制剂，为临床上筛选和/或制备治疗前列腺癌的药物提供了一种新的选择，具有良好的应用前景。

Description

新型雄激素受体抑制剂及其合成方法和应用 技术领域
本发明涉及医药领域，具体地，本发明涉及种新型的雄激素受体抑制剂的合成。
背景技术
前列腺癌是男性泌尿生殖系统较为普遍的恶性肿瘤之一。在西方国家是男性最常见的恶性肿瘤，是第三大癌症导致死亡的病因。2012年我国肿瘤登记地区的前列腺癌发病率为9.92/10万，在男性恶性肿瘤中居第6位。2015年我国前列腺癌新发病例约60300例，死亡病例约26600例，已成为威胁我国老年男性健康的重要原因之一。根据2014版的中国泌尿外科诊疗指南指出，晚期前列腺癌的标准治疗方案为雄激素剥夺治疗(androgen deprivation therapy,ADT)。在雄激素剥夺治疗的早期，症状能得到一定程度的缓解，但约40％的患者会发展成为去势抵抗型前列腺癌(castration-resistant prostate cancer,CRPC)。究其原因，与雄激素受体信号通路再激活有关，包括肾上腺分泌的雄激素、肿瘤内分泌的雄激素、雄激素受体的突变、甾醇类激素相关信号通路的效应等。CRPC患者的中位生存期仅为12～18个月。
雄激素受体(androgen receptor，AR)属于核受体超家族,包含918个氨基酸，与其他核受体具有相似的结构和功能。它由三个重要的结构域组成，分别是DNA结合域(DNA binding domain，DBD)、配体结合域(ligand binding domain，LBD)和氮端结合域(N-terminal domain，NTD)，DBD和LBD之间通过一个铰链区(Hinge)相连。存在于AR碳端的LBD是AR与配体结合的位点，决定了配体与AR结合的特异性，配体与LBD结合从而激活AR。男性的睾酮主要由睾丸分泌，此外肾上腺也能分泌少量。在前列腺组织中，90％的睾酮被5α-还原酶催化生成二氢睾酮(DHT)，其生物活性比睾酮更强，对前列腺的生长和发育起着重要的作用。在正常情况下，雄激素受体存在于胞质中并与热休克蛋白(Hsp90)等结合形成稳定的适合于雄激素结合的三级结构。当内源性雄激素出现时Hsp90被释放，AR的LBD与雄激素结合后发生构象的改变，并伴随着AR的二聚化和磷酸化，导致AR被激活。由此形成的AR-雄激素复合物进入细胞核识别相应的雄激素响应元件，募集其他共调控蛋白，形成有活性的转录复合物， 启动AR靶基因的转录。拮抗剂与内源性的雄激素竞争性地结合AR，但不会引起相关基因的转录以及后续的生物学效应。
雄激素受体(androgen receptor,AR)在前列腺癌的发生、发展中起着非常重要作用，研究表明去势抵抗型前列腺癌仍然依赖AR的作用。因此，靶向AR信号通路仍然有重要意义。
根据化学结构的不同，可将AR抑制剂分为甾体类AR拮抗剂和非甾体类AR拮抗剂。醋酸环丙孕酮是甾体类AR拮抗剂的代表，由于该类药物的口服生物利用度和选择性较差，易产生交叉作用引起不良反应，从而限制了其临床应用。而非甾体AR抑制剂的口服生物利用度相对较高，并且对AR有较高的选择性，因此成为研究开发抗雄激素的主要方向。
目前，在治疗前列腺癌的药物中已上市的AR抑制剂有第一代的氟他胺(flutamide)、比卡鲁胺(R-bicalutamide)和尼鲁米特(nilutamide)和第二代的蒽杂鲁胺(enzalutamide)，均作用于AR的LDB。处于临床研究阶段的还有与蒽杂鲁胺结构极其相似的ARN509以及ORION公司研发的ODM-201等小分子抑制剂。
第一代抗雄激素对AR的亲和力低于内源性配体DHT，长期服用会出现拮抗作用转化为激动作用的现象，从而导致病情加重。这种现象可能是由AR的某些位点突变引起的，如已经报道的T877A和W741C突变导致AR与拮抗剂作用之后转变为激活的构象。部分前列腺癌患者初期使用抗雄激素疗法有效，但若转变为去势抵抗型前列腺癌，传统的AR拮抗剂也无法继续使用，这与AR过表达、AR突变以及瘤内雄激素的生物合成增加等因素有关。
2012年，第二代AR抑制剂蒽杂鲁胺(图1)被FDA批准上市，它对AR的亲和力是比卡鲁胺的8倍，其IC50值为36nmol/L，较比卡鲁胺提高了3～5倍,该化合物能抑制AR从胞质向细胞核的转移，阻止AR与DNA的结合。但是，临床研究结果显示蒽杂鲁胺有诱发癫痫的可能，发病率约0.6％。同时，目前已经发现AR-F876L突变对蒽杂鲁胺产生耐药性。研究表明由mRNA剪切产生的一类缺少配体结合区的AR突变体，例如临床上发现的AR-V7亚型，在CRPC患者中表达增加，对CRPC的发生发展起着重要作用。蒽杂鲁胺以及处于临床III期的AR抑制剂ODM201主要作用于AR配体结合区，对这种缺少配体结合 区的突变型AR无效。
因此，研发新的有效药物显得十分必要和迫切。
发明内容
为了解决上述问题，本发明提供一种通式(I)所示的化合物，对AR的亲和力远高于第一代雄激素受体抑制剂，对第一代AR抑制剂羟基氟他胺所产生的T877A突变以及第二代AR抑制剂蒽杂鲁胺产生的F876L突变有效。同时此类化合物与ODM201有着相似的化学结构，几乎不能通过血脑屏障，降低化合物致癫痫的副作用。本发明提供的化合物不仅可以与天然雄激素竞争性结合AR还能下调AR及其剪切突变体的表达，从而更好的抑制雄激素信号通路的异常激活。
本发明首先提供了式I所示的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物：
Figure PCTCN2019094301-appb-000001
Z 1、Z 2各自独立的为C或者N；
环A为C 5～C 12碳环、杂环、咪唑二酮、芳环、芳杂环、苯并碳环、苯并杂环，其中苯环上的碳可被一个或多个杂原子取代；
环B为C 5～C 12碳环、杂环、并环、桥环、螺环、芳环、芳杂环或苯并芳环、苯并芳杂环、苯并碳环或苯并杂环，其中苯环上的碳可被一个或多个杂原子取代；
R 1和R 2相互独立地为氢、卤素、CHF 2、CF 3、氰基、硝基、氨基、羟胺基、羟基、巯基、磺酸基、C 1～C 6的烷基、烯烃基、炔烃基、烷氧基、环烷基、杂环基或芳基、杂芳基以及-R 9C(O)R 10-、-R 9CO(O)R 10-、-R 9(O)COR 10-、-R 9NHC(O)R 10-或者-R 9C(O)NHR 10-，其中的羟基、巯基、氨基、烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基可进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
R 3为氢、卤素、氰基、硝基、氨基、羟基、羟胺基、巯基、磺酸基、羧基、醛基、羰基、酯基、酰胺键或C 1～C 6的烷基、烷氧基、环烷基、杂环基、芳基、芳杂基，其中的烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
R 1和R 2和R 3可以相互以单键、双键或三键连接成环；
R 4、R 4’、R 5和R 5’各自独立地表示氢、羟基、卤素、氨基、羟胺基、氰基、硝基、烯基、炔基、C 1～C 6烷基、环烷基、烷氧基、羰基、硫羰基、醛基、羧基、酯基、酰胺基,其中所述C 1～C 6的烷基、烷氧基、烯基、炔基、酯基、酰胺基可以各自独立地进一步被一个或者多个选自卤素、羟基、氰基、硝基、羟胺基、氨基、羟胺基、烷氧基、环烷基的基团所取代；
R 5、R 5’、R 4、R 4’可以相互连接成键；
R 7、R 7’、R 6、R 6’、R 8各自独立地表示氢、羟基、羟胺基、氨基、硝基、氰基、卤素、C 1～C 6的烷基、烷氧基、烷氧基取代的烷基、环烷基、羰基、醛基、羧基、酯基、酰胺基；
R 7、R 7’、R 6、R 6’可以相互连接成键；
R 11、R 12各自独立地表示氢、羟基、氨基、羟胺基、硝基、磺酸基、氰基、卤素、C 1～C 6的烷基、烷氧基、环烷基、芳基；其中所述C 1～C 6烷基、烷氧基、环烷基、氨基、芳基各自独立的任选进一步被一个或者多个选自卤素、羟基、氰基、硝基、烷基、环烷基、烷氧基、芳基的基团所取代；
R 9、R 10各自独立地表示为氢、C 1～C 6直链或者支链烷基、烷氧基、氨基，其中的烷基、烷氧基可进一步被一个或者多个卤素、氰基、羟基、硝基、氨基取代。
优选地，所述化合物的环A具有如Ia所示结构：
Figure PCTCN2019094301-appb-000002
其中,R为O，N，C＝O或者C＝S；
X为C或N；Y为C或N；G为CH，C＝O或者C＝S；
M为CH或者O；虚线表示可选择的双键；星号表示与环连接的位置；
R 4和R 5定义同上述结构I。
优选地，所述化合物具有式Ⅱ所示结构：
Figure PCTCN2019094301-appb-000003
其中，R 1和R 2相互独立地为氢、卤素、CHF 2、CF 3、氰基、硝基、氨基、羟胺基、羟基、巯基、磺酸基、C 1～C 6的烷基、烯烃基、炔烃基、烷氧基、环烷基、杂环基或芳基、杂芳基；其中的羟基、巯基、氨基、烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基可进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
其中，环B为C5～C12碳环、杂环、芳环、芳杂环或苯并芳环、苯并杂环、苯并芳杂环，其中苯环上的碳可被一个或多个杂原子取代；
R 11、R 12各自独立地表示氢、羟基、氨基、羟胺基、硝基、磺酸基、氰基、卤素、C 1～C 6的烷基、烷氧基、环烷基、芳基；其中所述C 1～C 6烷基、烷氧基、环烷基、氨基、芳基各自独立的任选进一步被一个或者多个选自卤素、羟基、氰基、硝基、烷基、环烷基、烷氧基、芳基的基团所取代。
优选地，所述化合物具有式Ⅲ所示结构：
Figure PCTCN2019094301-appb-000004
优选地，所述化合物具有式Ⅳ所示结构：
Figure PCTCN2019094301-appb-000005
优选地，所述环B为含N并环、含N芳杂环、苯并含N杂环、苯并含N芳杂环或者嘌呤。
最优选地，所述化合物为如下化合物之一：
Figure PCTCN2019094301-appb-000006
Figure PCTCN2019094301-appb-000007
Figure PCTCN2019094301-appb-000008
本发明还提供了前述化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物方法，其反应路线如下所示：
Figure PCTCN2019094301-appb-000009
E为卤素Cl、Br、I；
或者，
Figure PCTCN2019094301-appb-000010
或者，
Figure PCTCN2019094301-appb-000011
或者，
Figure PCTCN2019094301-appb-000012
本发明还提供了前述化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物在制备雄激素受体抑制剂中的用途。
其中，所述雄激素受体包括雄激素受体的突变体。
其中，所述雄激素受体的突变体T877A突变体、W741C突变体和/或AR-F876L突变体。请对这三个突变进行具体解释，指出其突变的位置以及如何突变。
其中，所述抑制剂是抑制雄激素受体表达和/或雄激素受体通路的抑制剂。
其中，所述抑制雄激素受体通路是抑制雄激素受体下游基因PSA和FKBP5的转录。
本发明还提供了前述化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物在制备雄激素受体相关疾病的药物中的用途。
其中，所述药物是治疗雄激素受体表达阳性的前列腺癌、前列腺增生、多毛症、脱发、精神性厌食症、乳腺癌、痤疮、男性性功能障碍或艾滋病的药物。
本发明还提供了一种新型的雄激素受体抑制剂，它是以前述化合物或其立体 异构体、药学上可接受的盐、水合物或溶剂合物为活性成分，再加上药学上可接受的辅料制备而成的制剂。
活性成分
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式I化合物的立体异构体、互变异构体、同位素替代形式、混合物形式和药学上可接受的盐以及其代谢产物和代谢前体或前药。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。
药学上可接受的辅料
所述药学上可接受的辅料，它具有一定生理活性，但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位，而仅仅发挥辅助功效，这些辅助功效仅仅是对该成分已知活性的利用，是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明药物组合物配合使用，仍然应属于本发明保护的范围。
关于本发明的使用术语的定义：除非另有说明，本文中基团或者术语提供的初始定义适用于整篇说明书的该基团或者术语；对于本文没有具体定义的术语，应该根据公开内容和上下文，给出本领域技术人员能够给予它们的含义。
术语“立体异构体”包括立体中心(例如带有4个不同取代基的碳)、轴不对称例如有关键、平面不对称及其混合物的存在。
“取代”是指分子中的氢原子被其它不同的原子或分子所替换。
本发明中，“烷基”是烷烃分子中少掉一个氢原子而成的烃基，C1～C6的烷基是指C1、C2、C3、C4、C5、C6的烷基，即具有1～6个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、仲丁基、戊基、己基等等。
“炔基”是指具有至少一个碳‐碳三键的脂肪族碳氢基团。所述的炔基可以是直链或支链的。当炔基前具有碳原子数限定(如C 2‐8炔基)时，例如，术语“C 2‐8炔基”指具有2‐8个碳原子的直链或支链炔基。
“烯基”是指具有至少一个碳‐碳双键的脂肪族碳氢基团。所述的烯基可以是直链或支链的。当烯基前具有碳原子数限定(如C 2‐8烯基)时，例如，术语“C 2‐8烯基”指具有2‐8个碳原子的直链或支链烯基。
卤素为氟、氯、溴或碘。
“芳基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，例如苯基和萘基。所述芳基环可以稠合于其它环状基团(包括饱和和不饱和环)，但不能含有杂原子如氮，氧，或硫，同时连接母体的点必须在具有共轭的π电子体系的环上的碳原子上。芳基可以是取代的或未取代的。
“杂芳基”指包含一个到多个杂原子的杂芳族基团。这里所指的杂原子包括氧、硫和氮。例如呋喃基、噻吩基、吡啶基、吡唑基、吡咯基、N‐烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是任选取代的或未取代的。
“环烷基”指饱和或不饱和的环状烃取代基；环状烃可以是单环也可以是多环。例如，“C 3‐8环烷基”指碳原子数为3～8的环烷基。
“杂环基”指饱和或不饱和的环状烃取代基；环状烃可以是单环也可以是多环，且携带至少一个选自O、S或取代的氮原子的环烷基，其余环原子为碳，例如，“C 3‐8杂环基”指碳原子数和杂原子数共为3～8的杂环基。
本发明化合物可以有效抑制AR，具体地，不仅能够通过下调AR及其突变体的mRNA来抑制其蛋白表达，还能抑制AR及其突变体下游基因PSA和FKBP5的转录，同时对蒽杂鲁胺耐药的AR-F876L仍然有抑制作用，说明本发明化合物能够有效抑制AR信号通路。在细胞水平，本发明化合物能有效抑制AR阳性的前列腺癌细胞的增殖，且具有良好的细胞选择性，为临床上筛选和/或制备治疗前列腺癌的药物提供了一种新的选择，具有良好的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1.化合物对野生型AR转录活性的影响
图2.化合物对LNCap细胞中AR蛋白水平的影响
图3.化合物对VCap细胞中AR及其突变体蛋白水平的影响
图4.化合物对22RV1细胞中AR及其突变体蛋白水平影响
图5.化合物对LNCap细胞中PSA和FKBP5基因mRNA的影响
图6.化合物对VCap细胞中PSA和FKBP5基因mRNA的影响
图7.化合物对22RV1细胞中FKBP5基因mRNA的影响
图8.化合物对LNCap细胞中AR和AR-V7基因mRNA的影响
图9.化合物对AR-F876L突变体转录活性的影响
图10.化合物的体内抗肿瘤活性。
具体实施方式
本发明化合物与其编号见表1。
表1.本发明化合物编号
Figure PCTCN2019094301-appb-000013
Figure PCTCN2019094301-appb-000014
通用合成路线一：
Figure PCTCN2019094301-appb-000015
E为卤素Cl、Br、I
通用合成路线二：
Figure PCTCN2019094301-appb-000016
通用合成路线三：
Figure PCTCN2019094301-appb-000017
通用合成路线四：
Figure PCTCN2019094301-appb-000018
实施例1本发明中化合物SKLB-C4534的合成
Figure PCTCN2019094301-appb-000019
a)合成2-氯-4-(1-(四氢-2H-吡喃-2-基)-1H-吡唑-5-基)苯腈
将4-溴-2-氯苯腈(2g,8mol)和1-(四氢-2H-吡喃-2-基)-1H-吡唑-5-硼酸频那酯(4.45g,16mmol)溶于THF(22ml)。加入二(三苯基膦)二氯化钯(II)(281mg,0.4mmol)、碳酸钠(2g，19.2mmol)和水(6ml)，抽换N 2三次。混合物加热至45℃，搅拌过夜。
蒸发溶剂，硅藻土过滤除去不溶物，加水稀释并用乙酸乙酯萃取2次，无水硫酸钠干燥后浓缩，硅胶柱纯化(PE:EA＝5:1)得淡黄色固体2.4g。
b)合成2-氯-4-(1H-吡唑-5-基)苯腈
将2-氯-4-(1-(四氢-2H-吡喃-2-基)-1H-吡唑-5-基)苯腈(8.3mmol，2.4g)和10％HCl/EtOH(80ml)加入烧瓶中，回流搅拌过夜。
停止加热，冷至室温，蒸发溶剂，用氢氧化钠和饱和碳酸氢钠调pH至8。加二氯甲烷萃取3次，无水硫酸钠干燥后浓缩，硅胶柱纯化(PE:EA＝2:1)，得1.7g白色固体。
c)合成(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈
将三苯基膦(8.4g，30mmol)加入圆底烧瓶中，抽换N 2，加入干燥THF(25ml)。用冰浴搅拌至三苯基膦完全溶解，缓慢加入DIAD(6.4g，30mmol)，加毕混合物析出白色固体。将(S)-叔-丁基(1-羟基丙-2-基)氨基甲酸酯(5.6g，30mmol)溶于干燥THF(20ml)，缓慢加入反应混合液中。加毕，将2-氯-4-(1H-吡唑-5-基)苯腈(3.3g，15mmol)溶于干燥THF(30ml)，缓慢加入反应混合物中。加毕，将混合物移至室温并搅拌4h。
将水(25ml)和浓盐酸(10ml)加入混合物中，室温搅拌2天期间补浓盐酸5ml。至原料基本反应完全，蒸发有机溶剂，加入DCM萃取2次，有机相合并用1M盐酸水溶液洗两次，水相合并，用50％NaOH调pH至12。加二氯甲烷萃取3次，有机层合并，无水硫酸钠干燥后浓缩，得2.8g白色固体。
d)合成SKLB-C4534
2,6-二氯嘌呤(110mg，0.52mmol)溶于正丁醇(3ml)，加入(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(98mg，0.52mmol)和三乙胺(72mg,0.79mmol)，加热至110℃搅拌过夜。
停止加热，冷至室温，呈白色浑浊，沉淀过滤，用水、乙酸乙酯、二氯甲烷和甲醇洗涤，得白色固体30mg白色固体，即SKLB-C4534。
1H NMR(400MHz,DMSO)δ13.01(s,1H),8.14(s,2H),8.03–7.90(m,2H),7.90–7.78(m,2H),6.87(s,1H),4.75(s,1H),4.37(d,J＝5.5Hz,2H),1.24(d,J＝6.4Hz,3H).MS(ESI):413.07[M+H +]
实施例2本发明中化合物SKLB-C4535的合成
Figure PCTCN2019094301-appb-000020
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
6-氯嘌呤(160mg，0.92mmol)溶于乙醇(5ml)，加入(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(240mg，0.92mmol)和三乙胺(72mg,0.79mmol)，加热至90℃搅拌48h。
停止加热，冷至室温，呈白色浑浊。加水稀释后用DCM萃取2次，有机层合并，浓缩。干法上样，硅胶柱纯化，先用3％MeOH/DCM洗脱约3个柱体积，再换纯EA洗脱，得白色固体30mg，即SKLB-C4535。
1H NMR(400MHz,DMSO)δ12.86(s,1H),8.12(d,J＝8.0Hz,2H),8.04(s,1H),7.96(d,J＝8.1Hz,1H),7.89(d,J＝8.1Hz,1H),7.84(d,J＝1.8Hz,1H),7.62(s,1H),6.89(d,J＝1.8Hz,1H),4.85(s,1H),4.64–4.19(m,2H),1.22(d,J＝4.2Hz,4H).MS(ESI):401.1[M+Na +]
实施例3本发明中化合物SKLB-C4535的合成
Figure PCTCN2019094301-appb-000021
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
2-氯-4甲基嘧啶(51.4mg，0.4mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(86mg，0.31mmol)至于微波管中，加入三乙胺(47.1mg，0.465mmol)和乙醇(0.5ml)。微波加热至130℃，反应60min。
冷至室温，用厚硅胶板分离纯化，以PE:EA＝1:1为展开剂，刮板后直接干法上样，硅胶柱纯化得白色固体20mg，即SKLB-C4537。
1H NMR(400MHz,CDCl3)δ8.13(d,J＝4.6Hz,1H),7.95(s,1H),7.77(d,J＝8.0Hz,1H),7.66(d,J＝8.1Hz,1H),7.45(s,1H),6.57(s,1H),6.44(d,J＝4.8Hz,1H),5.16(d,J＝7.1Hz,1H),4.51(m,1H),4.35(m,2H),2.32(s,3H),1.24(d,J＝7.1Hz,3H).MS(ESI):353.1[M+H +]
实施例4本发明中化合物SKLB-C4538的合成
Figure PCTCN2019094301-appb-000022
该标题化合物利用实施例3所述方法用2-氯嘧啶(50mg，0.4mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(86mg，0.31mmol)作为原料制备，得25mg白色固体，即SKLB-C4538。
1H NMR(400MHz,CDCl3)δ8.28(s,2H),7.95(s,1H),7.77(s,1H),7.67(s,1H),7.45(s,1H),6.58(s,2H),4.51(s,1H),4.36(s,2H),1.26(s,3H).MS(ESI):339.1[M+H +]
实施例5本发明中化合物SKLB-C4651的合成
Figure PCTCN2019094301-appb-000023
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
碘苯(100mg，0.48mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104.4mg，0.4mmol)、碳酸铯(260.6mg，0.8mmol)、盐酸二甲双胍(13.2mg，0.08mmol)和乙醇(2ml)加入反应管中，抽换氮气3次，加热至80℃搅拌过夜。
冷至室温，呈棕色浑浊。加乙酸乙酯稀释，水洗、饱和氯化钠洗，25％乙酸乙酯/石油醚过柱纯化，得90mg黄色粘稠状物，即SKLB-C4651。
1H NMR(400MHz,CDCl3)δ7.96(s,1H),7.78(d,J＝7.2Hz,1H),7.67(d,J＝7.3Hz,1H),7.42(s,1H),7.18(s,2H),6.72(s,1H),6.70–6.32(m,3H),4.25(s,2H),4.00(s,1H),3.80(s,1H),1.23(d,J＝5.2Hz,3H).MS(ESI):337.1[M+H +]
实施例6本发明中化合物SKLB-C4652的合成
Figure PCTCN2019094301-appb-000024
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
3-溴吡啶(182mg，1.16mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(200mg，0.76mmol)、碳酸铯(496mg，1.52mmol)、盐酸二甲双胍(25.2mg，0.152mmol)和乙醇(2ml)加入反应管中，抽换氮气3次，加热至85℃反应48小时。
冷至室温，硅胶柱纯化，纯EA洗脱得90mg淡黄色固体。再用反相制备柱以甲醇/水体系梯度洗脱，得60mg白色固体即SKLB-C4652。
1H NMR(400MHz,CDCl3)δ8.02(d,J＝2.8Hz,1H),8.01–7.88(m,2H),7.77(dd,J＝8.1,1.5Hz,1H),7.68(d,J＝8.1Hz,1H),7.42(d,J＝2.3Hz,1H),7.05(dd,J＝8.3,4.7Hz,1H),6.84(dd,J＝8.3,1.6Hz,1H),6.57(d,J＝2.3Hz,1H),4.29(dd,J＝13.8,4.9Hz,1H),4.21(dd,J＝13.8,5.4Hz,1H),4.07–3.96(m,1H),3.95(d,J＝8.3Hz,1H),1.26(d,J＝6.3Hz,3H).MS(ESI):338.1[M+H +]
实施例7本发明中化合物SKLB-C4660的合成
Figure PCTCN2019094301-appb-000025
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
3-碘吡啶(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)、叔丁醇钠(48mg，0.52mmol)、BINAP(17.2mg，0.028mmol)、醋酸钯(6mg，0.028mmol)和甲苯(2.5ml)加入反应管中，抽换氮气3次，加热至115℃反应48小时。
冷至室温，硅藻土过滤后加硅胶拌样，干法上样用硅胶柱纯化，以 DCM:EA＝1:1加5％三乙胺为展开剂，纯化得白色固体30mg，即SKLB-C4660。
1H NMR(400MHz,CDCl3)δ8.14(d,J＝4.2Hz,2H),7.95(s,1H),7.76(d,J＝8.0Hz,1H),7.69(d,J＝7.9Hz,1H),7.43(s,1H),6.57(s,1H),6.43(d,J＝4.4Hz,2H),4.80(s,1H),4.31(dd,J＝14.5,3.5Hz,1H),4.23(dd,J＝12.8,4.0Hz,1H),4.08(m,1H),1.27(d,J＝6.6Hz,3H).MS(ESI):338.1[M+H +]
实施例8本发明中化合物SKLB-C4659的合成
Figure PCTCN2019094301-appb-000026
该标题化合物利用实施例5所述方法用2-溴吡啶(95mg，0.6mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得30mg淡黄色固体，即SKLB-C4539。
1H NMR(400MHz,CDCl3)δ8.09(dd,J＝5.0,1.1Hz,1H),7.95(d,J＝1.4Hz,1H),7.77(dd,J＝8.1,1.5Hz,1H),7.67(d,J＝8.1Hz,1H),7.43(d,J＝2.3Hz,1H),7.38(m,1H),6.64–6.51(m,2H),6.35(d,J＝8.4Hz,1H),4.57(d,J＝7.3Hz,1H),4.49–4.21(m,3H),1.25(d,J＝6.5Hz,3H).
MS(ESI):338.1[M+H +]
实施例9本发明中化合物SKLB-C4662的合成
Figure PCTCN2019094301-appb-000027
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
2，6-二氯嘧啶(68.5mg，0.46mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)溶于乙醇(1ml)，加入DIPEA(59mg，0.46mmol)，加热至80℃，搅拌过夜。
冷至室温，乙醇浓缩后，硅胶柱纯化，用60％EA+30％PE洗脱，得60mg白色固体，即SKLB-C4662。
1H NMR(400MHz,CDCl3)δ7.96(d,J＝5.9Hz,1H),7.93(d,J＝1.4Hz,1H),7.75(dd,J＝8.1,1.5Hz,1H),7.68(d,J＝8.1Hz,1H),7.46(d,J＝2.2Hz,1H),6.59(d,J＝2.3Hz,1H),6.18(d,J＝5.2Hz,1H),5.55(d,J＝8.0Hz,1H),4.53(s,1H),4.41(dd,J＝14.0,4.1Hz,1H),4.21(dd,J＝13.9,5.5Hz,1H),1.27(d,J＝6.7Hz,3H).MS(ESI):395.0[M+Na +]
实施例10本发明中化合物SKLB-C4661的合成
Figure PCTCN2019094301-appb-000028
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
4-氯喹唑啉(75mg，0.46mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)和三乙胺(46.5mg，0.46mmol)溶于乙腈(2ml)，加热至90℃，搅拌过夜。
冷至室温，浓缩溶剂，加5％DCM/MeOH溶解，湿法上样，用硅胶柱纯化，先用纯EA洗脱分离出杂质再换2％DCM/MeOH洗脱，得20mg白色固体，即SKLB-C4661。
1H NMR(400MHz,CDCl3)δ8.66(s,1H),8.04(d,J＝1.3Hz,1H),7.85(d,J＝8.2Hz,1H),7.77(d,J＝7.6Hz,2H),7.74(d,J＝1.4Hz,1H),7.70(d,J＝8.1Hz,1H),7.52(d,J＝2.3Hz,1H),7.50(d,J＝7.4Hz,1H),7.05(d,J＝7.5Hz,1H),6.65(d,J＝2.3Hz,1H),4.99–4.81(m,1H),4.59(dd,J＝14.0,3.7Hz,1H),4.35(dd,J＝14.0,4.6Hz,1H),1.31(d,J＝6.7Hz,3H).MS(ESI):389.1[M+H +]
实施例11本发明中化合物SKLB-C4664的合成
Figure PCTCN2019094301-appb-000029
该标题化合物利用实施例5所述方法用5-溴嘧啶(95mg，0.6mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料，85℃反应48小时制备得28mg淡黄色固体，即SKLB-C4664。
1H NMR(400MHz,DMSO)δ8.30(s,1H),8.08(d,J＝1.2Hz,1H),8.07(s,2H),7.98(d,J＝8.2Hz,1H),7.92(dd,J＝8.2,1.4Hz,1H),7.84(d,J＝2.3Hz,1H),6.90(d,J＝2.3Hz,1H),6.05(d,J＝8.8Hz,1H),4.26(dd,J＝13.7,5.7Hz,1H),4.19(dd,J＝13.7,6.7Hz,1H),4.12–3.96(m,1H),1.16(d,J＝6.5Hz,3H).MS(ESI):361.1[M+Na +]
实施例12本发明中化合物SKLB-C4667的合成
Figure PCTCN2019094301-appb-000030
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-三氟甲基苯腈利用实施例1(c)所述的方法用2-三氟甲基-4溴苯腈为原料制备。
该标题化合物利用实施例10所述方法制备，得80mg无色粘稠，即SKLB-C4667。
1H NMR(400MHz,CDCl3)δ8.65(s,1H),8.32(s,1H),8.02(dd,J＝8.1,1.1Hz,1H),7.94–7.80(m,2H),7.80–7.67(m,2H),7.55(d,J＝2.3Hz,1H),7.51–7.39(m,1H),6.93(d,J＝7.4Hz,1H),6.71(d,J＝2.4Hz,1H),5.02–4.84(m,1H),4.60(dd,J＝14.0,3.6Hz,1H),4.37(dd,J＝14.0,4.7Hz,1H),1.31(d,J＝6.7Hz,3H).MS(ESI):423.1[M+H +]
实施例13本发明中化合物SKLB-C4669的合成
Figure PCTCN2019094301-appb-000031
该标题化合物利用实施例5所述方法用1-溴萘(118mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料，加热至90℃反应48h，所得棕红色混合物加EA稀释，水洗、饱和氯化钠洗、无水硫酸钠干燥后浓缩，用硅胶柱纯化，PE:EA＝4:1洗脱，得40mg淡黄色固体，即SKLB-C4669。
1H NMR(400MHz,CDCl3)δ8.02(d,J＝1.3Hz,1H),7.91–7.73(m,3H),7.68(d,J＝8.1Hz,1H),7.57–7.38(m,3H),7.34(t,J＝7.9Hz,1H),7.23(s,1H),6.65(d,J＝7.5Hz,1H),6.59(d,J＝2.3Hz,1H),5.10(s,1H),4.46(dd,J＝13.8,4.5Hz,1H),4.36(dd,J＝13.8,5.2Hz,1H),4.26–4.09(m,1H),1.33(d,J＝6.4Hz,3H).MS(ESI):387.1[M+H +]
实施例14本发明中化合物SKLB-C4670的合成
Figure PCTCN2019094301-appb-000032
该标题化合物利用实施例5所述方法2-溴萘(118mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得48mg纯白色固体，即SKLB-C4670。
1H NMR(400MHz,CDCl3)δ7.97(s,1H),7.78(d,J＝7.3Hz,1H),7.74–7.58(m,3H),7.55(d,J＝8.2Hz,1H),7.41(d,J＝2.1Hz,1H),7.35(t,J＝7.4Hz,1H),7.20(t,J＝7.4Hz,1H),6.84(dd,J＝8.7,2.1Hz,1H),6.80(s,1H),6.55(d,J＝2.2Hz,1H),4.36(dd,J＝13.8,5.0Hz,1H),4.29(dd,J＝13.8,5.1Hz,1H),4.21–4.06(m,1H),3.98(d,J＝7.4Hz,1H),1.30(d,J＝6.5Hz,3H).MS(ESI):387.1[M+H +]
实施例15本发明中化合物SKLB-C4671的合成
Figure PCTCN2019094301-appb-000033
该标题化合物利用实施例5述方法用5-溴-1甲基-1H-吲哚(115mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得40mg淡黄色固体，即SKLB-C4671。
1H NMR(400MHz,CDCl3)δ7.98(d,J＝1.4Hz,1H),7.79(dd,J＝8.1,1.4Hz,1H),7.67(d,J＝8.1Hz,1H),7.43(d,J＝2.3Hz,1H),7.16(d,J＝8.7Hz,1H),6.97(d,J＝3.0Hz,1H),6.86(d,J＝2.0Hz,1H),6.63(dd,J＝8.7,2.1Hz,1H),6.57(d,J＝2.3Hz,1H),6.30(d,J＝2.6Hz,1H),4.35–4.20(m,2H),4.05–3.95(m,1H),3.73(s,3H),1.27(d,J＝7.1Hz,3H).MS(ESI):390.1[M+H +]
实施例16本发明中化合物SKLB-C4673的合成
Figure PCTCN2019094301-appb-000034
该标题化合物利用实施例5所述方法用6-溴喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得45mg淡绿色固体，即SKLB-C4673。
1H NMR(400MHz,CDCl3)δ8.62(dd,J＝4.2,1.3Hz,1H),7.96(d,J＝1.1Hz,1H),7.87(d,J＝9.0Hz,1H),7.83(d,J＝8.1Hz,1H),7.77(dd,J＝8.1,1.3Hz,1H),7.69(d,J＝8.1Hz,1H),7.42(d,J＝2.2Hz,1H),7.26–7.20(m,1H),7.04(dt,J＝17.2,8.6Hz,1H),6.68(d,J＝2.5Hz,1H),6.54(d,J＝2.3Hz,1H),4.37(dd,J＝13.7,4.1Hz,1H),4.28(dd,J＝13.8,4.7Hz,1H),4.15(d,J＝3.1Hz,1H),1.32(s,3H).MS(ESI):388.1[M+H +]
实施例17本发明中化合物SKLB-C4674的合成
Figure PCTCN2019094301-appb-000035
该标题化合物利用实施例5所述方法用6-溴异喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得80mg淡黄色固体，即SKLB-C4674。
1H NMR(400MHz,CDCl3)δ8.93(s,1H),8.29(d,J＝5.6Hz,1H),7.96(s,1H),7.77(d,J＝7.9Hz,1H),7.71(dd,J＝17.9,11.4Hz,2H),7.42(d,J＝1.5Hz,1H),7.30(d,J＝5.6Hz,1H),6.89(d,J＝7.8Hz,1H),6.63(s,1H),6.55(d,J＝1.6Hz,1H),4.49(d,J＝7.5Hz,1H),4.38(dd,J＝13.7,4.7Hz,1H),4.28(dd,J＝13.8,5.3Hz,1H),4.18(m,1H),1.32(d,J＝6.4Hz,3H).MS(ESI):388.1[M+H +]
实施例18本发明中化合物SKLB-C4678的合成
Figure PCTCN2019094301-appb-000036
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
8-溴喹啉(125mg，0.6mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)、叔丁醇钠(96mg，1mmol)、三(二亚苄基丙酮)钯(II)(24mg，0.028mmol)和davephos(11.2mg，0.028mmol)加入微波中，再加入1，4-二氧六环(2ml)，氮气吹3次。微波加热至150℃反应30分钟。
冷至室温，硅藻土过滤，加硅胶拌样，用硅胶柱纯化，以15％EA/PE洗脱，得41mg淡黄色固体，即SKLB-C4678。
1H NMR(400MHz,CDCl3)δ8.75(dd,J＝4.2,1.7Hz,1H),8.06(dd,J＝8.3,1.6Hz,1H),8.01(d,J＝1.4Hz,1H),7.79(dd,J＝8.1,1.5Hz,1H),7.67(d,J＝8.1Hz,1H),7.43(d,J＝2.3Hz,1H),7.39(dd,J＝8.3,4.2Hz,1H),7.33(t,J＝7.9Hz,1H),7.05(dd,J＝8.2,0.8Hz,1H),6.65(d,J＝7.6Hz,1H),6.52(d,J＝2.3Hz,1H), 6.48(d,J＝8.4Hz,1H),4.47–4.29(m,2H),4.21(tq,J＝12.1,6.2Hz,1H),1.36(d,J＝6.5Hz,3H).MS(ESI):388.1[M+H +]
实施例19本发明中化合物SKLB-C4679的合成
Figure PCTCN2019094301-appb-000037
该标题化合物利用实施例5所述方法用4-溴异喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得15mg淡黄色固体，即SKLB-C4679。
1H NMR(400MHz,CDCl3)δ8.68(s,1H),8.01(d,J＝1.4Hz,1H),7.92(d,J＝8.6Hz,2H),7.85(d,J＝8.4Hz,1H),7.76(dd,J＝8.1,1.5Hz,1H),7.72–7.64(m,2H),7.61(t,J＝7.2Hz,1H),7.49(d,J＝2.3Hz,1H),6.62(d,J＝2.3Hz,1H),5.24(s,1H),4.52(dd,J＝13.9,4.1Hz,1H),4.36(dd,J＝13.9,5.3Hz,1H),4.21(s,1H),1.34(d,J＝6.4Hz,3H).MS(ESI):388.1[M+H +]
实施例20本发明中化合物SKLB-C4682的合成
Figure PCTCN2019094301-appb-000038
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
5-溴喹啉(125mg，0.6mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)、叔丁醇钠(96mg，1mmol)、三(二亚苄基丙酮)钯(II)(24mg，0.028mmol)和x-phos(13.3mg，0.028mmol)加入反应管中，再加入甲苯(3ml)，抽换氮气3次。加热至100℃反应过夜。
冷至室温，硅藻土过滤，浓缩溶剂后用厚硅胶板分离纯化，以PE:EA＝2:1为展开剂，得48g橘红色固体。再用C18键合硅胶柱纯化，以甲醇/水体系梯度洗脱得30mg淡黄色固体，即SKLB-C4682。
1H NMR(400MHz,CDCl3)δ8.88(d,J＝2.7Hz,1H),8.20(d,J＝8.3Hz,1H), 8.01(s,1H),7.75(d,J＝7.9Hz,1H),7.68(d,J＝7.9Hz,1H),7.66–7.35(m,3H),7.33(dd,J＝8.2,3.9Hz,1H),6.68(d,J＝7.3Hz,1H),6.60(s,1H),5.25(d,J＝5.9Hz,1H),4.48(dd,J＝13.7,3.7Hz,1H),4.34(dd,J＝13.8,4.9Hz,1H),4.16(s,1H),1.32(d,J＝6.2Hz,3H).MS(ESI):388.1[M+H +]
实施例21本发明中化合物SKLB-C4676的合成
Figure PCTCN2019094301-appb-000039
该标题化合物利用实施例20所述方法用8-溴异喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得35mg淡黄色固体，即SKLB-C4676。
1H NMR(400MHz,CDCl3)δ9.29(s,1H),8.48(d,J＝5.5Hz,1H),7.92(s,1H),7.81(d,J＝8.2Hz,1H),7.71(d,J＝8.0Hz,1H),7.66–7.33(m,3H),7.14(d,J＝8.0Hz,1H),6.70(d,J＝7.7Hz,1H),6.59(s,1H),5.26(d,J＝5.4Hz,1H),4.44(dd,J＝13.8,4.6Hz,1H),4.37(dd,J＝13.9,5.0Hz,1H),4.23(m,1H),1.37(d,J＝6.3Hz,3H).MS(ESI):388.1[M+H +]
实施例22本发明中化合物SKLB-C4677的合成
Figure PCTCN2019094301-appb-000040
该标题化合物利用实施例20所述方法用5-溴异喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得40mg白色固体，即SKLB-C4677。
1H NMR(400MHz,CDCl3)δ9.16(s,1H),8.48(d,J＝5.7Hz,1H),8.01(s,1H),7.76(d,J＝7.7Hz,1H),7.69(d,J＝8.0Hz,1H),7.59(d,J＝5.7Hz,1H),7.44(d,J＝9.5Hz,2H),7.33(d,J＝7.9Hz,1H),6.80(d,J＝7.5Hz,1H),6.60(s,1H),5.18(d,J＝6.3Hz,1H),4.46(dd,J＝13.7,3.8Hz,1H),4.35(dd,J＝13.9,4.9Hz,1H),4.18(s,1H),1.33(d,J＝6.2Hz,3H).MS(ESI):388.1[M+H +]
实施例23本发明中化合物SKLB-C4675的合成
Figure PCTCN2019094301-appb-000041
该标题化合物利用实施例20所述方法用7-溴喹啉(118.6mg，0.57mmol)和(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(100mg，0.38mmol)作为原料制备，得30mg白色固体，即SKLB-C4675。
1H NMR(400MHz,CDCl3)δ8.72(s,1H),7.94(s,2H),7.76(d,J＝6.4Hz,1H),7.66(d,J＝6.9Hz,1H),7.57(d,J＝7.6Hz,1H),7.43(s,1H),7.10(s,2H),6.87(d,J＝7.4Hz,1H),6.56(s,1H),4.35(m,3H),4.16(s,1H),1.29(d,J＝4.1Hz,3H).MS(ESI):388.1[M+H +]
实施例24本发明中化合物SKLB-C4684的合成
Figure PCTCN2019094301-appb-000042
中间体(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈利用实施例1(c)所述的方法合成。
2-溴喹啉(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)、叔丁醇钠(48mg，0.52mmol)、BINAP(17.2mg，0.028mmol)、三(二亚苄基丙酮)二钯(25.6mg，0.028mmol)和甲苯(2.5ml)加入反应管中，抽换氮气3次，加热至110℃反应20小时。
冷至室温，硅藻土过滤后加硅胶拌样，干法上样用硅胶柱纯化，以PE:EA＝1:1洗脱，得淡黄色固体70mg，即SKLB-C4684。
1H NMR(400MHz,CDCl3)δ7.94(d,J＝1.3Hz,1H),7.80(d,J＝8.8Hz,1H),7.75(dd,J＝8.1,1.4Hz,1H),7.68(t,J＝9.1Hz,2H),7.59(d,J＝8.0Hz,1H),7.57–7.50(m,1H),7.46(d,J＝2.3Hz,1H),7.25–7.18(m,1H),6.56(s,2H),4.85(d,J＝6.7Hz,1H),4.79–4.58(m,1H),4.51(dd,J＝13.7,5.0Hz,1H),4.40(dd,J＝13.7, 4.9Hz,1H),1.33(d,J＝6.7Hz,3H).MS(ESI):388.1[M+H +]
实施例25本发明中化合物SKLB-C4685的合成
Figure PCTCN2019094301-appb-000043
该标题化合物利用实施例7所述方法用3-溴异喹啉(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)作为原料制备，的45mg淡绿色固体，即SKLB-C4685。
1H NMR(400MHz,CDCl3)δ8.82(s,1H),7.95(d,J＝1.5Hz,1H),7.80–7.69(m,2H),7.67(d,J＝8.1Hz,1H),7.51–7.34(m,3H),7.21(ddd,J＝8.0,6.3,1.5Hz,1H),6.50(d,J＝2.3Hz,1H),6.46(s,1H),4.65(d,J＝5.9Hz,1H),4.44–4.22(m,3H),1.33(d,J＝6.3Hz,3H).MS(ESI):388.1[M+H +]
实施例26本发明中化合物SKLB-C4686的合成
Figure PCTCN2019094301-appb-000044
该标题化合物利用实施例7所述方法用1-溴异喹啉(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)作为原料制备，的75mg淡绿色固体，即SKLB-C4686。
1H NMR(400MHz,CDCl3)δ8.01(d,J＝1.5Hz,1H),7.98(d,J＝5.8Hz,1H),7.80(d,J＝8.3Hz,1H),7.75(dd,J＝8.1,1.5Hz,1H),7.71–7.64(m,2H),7.64–7.56(m,1H),7.55–7.42(m,2H),6.95(d,J＝5.8Hz,1H),6.61(d,J＝2.3Hz,1H),6.10(d,J＝7.2Hz,1H),4.88–4.76(m,1H),4.58(dd,J＝13.8,4.1Hz,1H),4.46–4.32(m,1H),1.31(d,J＝6.7Hz,3H).MS(ESI):388.1[M+H +]
实施例27本发明中化合物SKLB-C4681的合成
Figure PCTCN2019094301-appb-000045
该标题化合物利用实施例7所述方法用1-溴异喹啉(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)作为原料制备，的25mg淡黄色固体，即SKLB-C4681。
1H NMR(400MHz,CDCl3)δ8.50(d,J＝5.5Hz,1H),8.05(s,1H),8.02(d,J＝8.4Hz,1H),7.88(d,J＝8.3Hz,1H),7.78(d,J＝8.1Hz,1H),7.75–7.60(m,2H),7.58–7.39(m,2H),6.62(d,J＝2.2Hz,1H),6.53(d,J＝5.8Hz,1H),6.44(d,J＝5.5Hz,1H),4.55(dd,J＝13.9,3.8Hz,1H),4.38(dd,J＝14.0,5.2Hz,1H),4.25(m,1H),1.34(d,J＝6.4Hz,3H).MS(ESI):388.1[M+H +]。
实施例28本发明中化合物SKLB-C4683的合成
Figure PCTCN2019094301-appb-000046
该标题化合物利用实施例7所述方法用1-溴异喹啉(108mg，0.52mmol)、(S)-4-(1-(2-氨基丙基)-1H-吡唑-3-基)-2-氯苯腈(104mg，0.4mmol)作为原料制备，的61mg淡黄色固体，即SKLB-C4683。
1H NMR(400MHz,CDCl3)δ8.39(d,J＝2.8Hz,1H),8.02–7.85(m,2H),7.75(dd,J＝8.1,1.4Hz,1H),7.72–7.63(m,1H),7.54–7.45(m,1H),7.46–7.33(m,3H),6.96(d,J＝2.7Hz,1H),6.52(d,J＝2.3Hz,1H),4.38(dd,J＝13.8,4.6Hz,1H),4.25(dd,J＝13.8,5.8Hz,1H),4.18–4.07(m,1H),1.33(t,J＝6.7Hz,3H).MS(ESI):388.1[M+H +]。
以下用试验例的方式来验证本发明的有益效果：
试验例1对LNCap/AR、VCap、22RV1和PC-3细胞的增殖抑制活性评价
1、材料及仪器：
RIPM 1640培养基(Hyclone,SH30809.01)、DMEM高糖培养基(Hyclone, SH30243.01)、胎牛血清、FBS(BI,04-001-1ACS)、青-链霉素(Hyclone,SV30010)。
细胞完全培养液：RIPM 1640培养基/DMEM高糖培养基，10％FBS，1％青-链霉素。
胰酶(Millipore,SM-2001-C)、LNCap/AR细胞(高表达AR的LNCap细胞，通过逆转录病毒转染方法构建，由四川康城生物科技有限公司赠予)、VCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu220)、22RV1细胞(中国科学院典型培养物保藏委员会细胞库，TCHu100)、LNCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu173)、PC-3细胞(中国科学院典型培养物保藏委员会细胞库，SCSP-532)、细胞计数试剂CCK-8(SAB,CP002)、ThermoMultiskan MK3酶标仪。
2、方法：
将LNCap/AR,22RV1,PC-3培养于1640完全培养基中，VCap培养于DMEM高糖完全培养基中，置于5％二氧化碳的37℃培养箱中培养。将细胞接种于96孔板中，2个副孔，每孔100微升，接种细胞数分别为LNCap/AR 2000/孔、22RV13000/孔、VCap 20000/孔、PC-3 1000/孔。
药物用DMSO(二甲亚砜)配置成30mM的储备液，铺板后第二天用完全培养基稀释成600μM,再用完全培养基稀释10倍，从60μM开始3倍梯度稀释，共9个浓度。取100μL稀释后的化合物加入细胞培养孔中，空白对照组加100μL培养基，即药物作用浓度最高30μM。
药物作用6天后，吸走90μL含药培养基，每孔加入10μL CCK-8，37℃避光孵育1-2h后，在酶标仪上，450nM，测定各孔的OD值。按照CCK-8试剂盒说明书计算IC 50(见表2)。
表2.
Figure PCTCN2019094301-appb-000047
Figure PCTCN2019094301-appb-000048
Figure PCTCN2019094301-appb-000049
上述实验数据表明，本发明化合物对AR阳性的前列腺癌细胞LNCap/AR,22RV1，LNCap(含T877A突变)和VCap有较好的抑制作用，尤其是对含AR-V7突变的前列腺癌细胞22RV1和VCap抑制活性优于上市药物蒽杂鲁胺和临床III期药物ODM201。而对AR阴性的前列腺癌细胞PC-3抑制作用差，间接说明本发明化合物抑制AR信号通路，且具有良好的细胞选择性，安全性好。
试验例2、抑制野生型AR转录活性的生物活性评价
1、材料仪器
DMEM培养基(Gibco,31053028)、去除雄激素的胎牛血清(Biological Industries,04-011-1A)、HEK293细胞(由药明康德新药开发有限公司提供)、转染试剂(Promega,E2311)、wt-AR表达质粒(Origene,RC235415)、报告基因质粒(Promega,E1360)、Steady-Glo检测试剂(Promega,E2550)
2、方法
用含89％DMEM培养基、1％GlutaMax和10％经透析去除雄激素的胎牛血清的完全培养基将HEK293细胞接种在96孔板中，同时根据说明书使用转染试剂将5ng wt-AR质粒和100ng报道基因质粒共转染入细胞。24小时后，加入化合物或DMSO以及2nM睾酮处理细胞。孵育24小时后，使用Steady-Glo试剂在Envision仪上测量发光信号。该实验由上海药明康德新药开发有限公司完成。
由图1所示，测试结果表示为平均值±SD(n＝3)。该结果说明本发明化合物SKLB-C4651、SKLB-C4652、SKLB-C4664、SKLB-C4670、SKLB-C4669、SKLB-C4674、SKLB-C4682、SKLB-C4535、SKLB-C4661、SKLB-C4686能抑制雄激素激活的野生型AR的转录，且化合物的活性与细胞增殖抑制活性基本一致。
上述实验表明，本发明化合物可以有效抑制野生型AR的转录活性，从而阻 断前列腺中异常激活的AR信号。
试验例3下调LNCap、VCap和22RV1细胞中AR表达的生物活性评价
1、材料仪器
RIPM 1640培养基(Hyclone,SH30809.01)、胎牛血清、FBS(BI,04-001-1ACS)、青-链霉素(Hyclone,SV30010)。
细胞完全培养液：RIPM 1640培养基，10％FBS，1％青-链霉素。
胰酶(Millipore,SM-2001-C)、VCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu220)、22RV1细胞(中国科学院典型培养物保藏委员会细胞库，TCHu100)、LNCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu173)、RIPA裂解液(Beyotime,P0013B)、BCA蛋白定量试剂盒(Beyotime,P0012)、5X loading buffer(Beyotime,P0015)、10％聚丙烯酰胺凝胶试剂盒(佰和,PG112)、PVDF膜(Immobilon-PSQ,ISEQ00010)、Tublin Antibody(Zen BioScience,200608)、Androgen Receptor Antibody(CST,3202S)、PSA/KLK3(D11E1)
Figure PCTCN2019094301-appb-000050
Rabbit mAb(CST,D11E1)、敏ECL化学发光液(四正柏,4AW011-50)、学发光仪(上海勤翔,Serial NO.810060)。
2、方法
将LNCap，22RV1培养于1640完全培养基中，VCap培养于DMEM高糖完全培养基中，置于5％二氧化碳的37℃培养箱中培养。第一天将细胞接种于6孔板中，LNCap和22RV1铺板数为70万/孔，VCap为100万/孔。
第三天加药，药物用DMSO(二甲亚砜)配置成30mM的储备液，用完全培养基稀释，空白组加含等量浓度DMSO的完全培养基。
药物作用24h后，吸弃培养基，用预冷的PBS洗一次，吸干，每孔60μl RIPA裂解液，冰上裂解5min，用细胞刮收集并转移至离心管中。4℃离心10min，13000rpm。将上清液转移至新的离心管中，用BCA蛋白定量试剂盒进行蛋白定量，然后加入1/4体积的5X loading buffer，沸水煮5min后放冰上降温，再转移至-20℃保存。
用10％的聚丙烯酰胺凝胶试剂盒制胶，蛋白上样30μg，体积用1X loading buffer补齐，用80V跑浓缩胶，100V跑分离胶。湿法转膜，300mA，1h，然后用5％脱脂牛奶室温封闭2h。一抗Tublin按1:5000稀释，AR和PSA分别按1:2000和1:1000稀释，4℃摇床孵育过夜。
TBS/T洗膜10min*3次，二抗按1:5000稀释，室温孵育2h,TBS/T洗膜10min*3次,加超敏TCL化学发光液曝光。
由图2结果表明，10μM的本发明化合物4682(SKLB-C4682)和20μM的本发明化合物4535(SKLB-C4535)均能明显抑制LNCap细胞中AR的蛋白水平，同时具有明显的浓度依赖性。但已上市药物蒽杂鲁胺和临床III期药物ODM201在20μM的浓度下均不能下调AR的蛋白水平。
由图3结果表明，20μM的本发明化合物4535(SKLB-C4535)和4682(SKLB-C4682)均能明显抑制VCap细胞中AR及其突变体的蛋白水平，同时具有明显的浓度依赖性，且活性优于已报道化合物galaterone。但已上市药物蒽 杂鲁胺和临床III期药物ODM201在20μM的浓度下均不能下调AR的蛋白水平。
由图4结果表明，20μM的本发明化合物4682(SKLB-C4682)和30μM的本发明化合物4682均能显著抑制22RV1细胞中AR及其剪切突变体AR-V7的蛋白水平，且具有明显的浓度依赖性。但已上市药物蒽杂鲁胺和临床III期药物ODM201在30μM的浓度下均不能下调AR的蛋白水平。
上述实验结果表明，本发明化合物SKLB-C4682、SKLB-C4535能够下调前列腺癌细胞中AR及其突变体的表达，由于癌细胞中AR过表达是导致前列腺癌患者产生耐药性的原因之一，因此抑制AR活性的同时下调AR的蛋白水平能更好的抑制雄激素信号通路，因此，本发明能够很好地抑制雄激素信号通路的异常激活。
试验例4降低前列腺癌细胞中PSA、FKBP5和AR的mRNA生物活性评价
1、材料仪器
RIPM 1640培养基(Hyclone,SH30809.01)、DMEM高糖培养基(Hyclone,SH30243.01)、牛血清、FBS(BI,04-001-1ACS)、去雄激素的胎牛血清、CSS-FBS(由四川康城生物科技有限公司赠予)、青-链霉素(Hyclone,SV30010)。
细胞完全培养液：RIPM 1640培养基/DMEM高糖培养基，10％FBS或者5％CSS，1％青-链霉素。
胰酶(Millipore,SM-2001-C)、VCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu220)、22RV1细胞(中国科学院典型培养物保藏委员会细胞库，TCHu100)、LNCap细胞(中国科学院典型培养物保藏委员会细胞库，TCHu173)、人工合成雄激素R1881(南京康满林化工实业有限公司,CAS:965-93-5)、Trizol(ambion，15596026)、氯仿(CAS:67-66-3)、异丙醇(天津市致远化学试剂有限公司，CAS:67-63-0)、无水乙醇(川东化工)、RNase-free water(biosharp，BL510A)、测RNA浓度的仪器(Thermo，NANODROP 2000)、逆转录试剂盒(abm，G492)、逆转录仪(LongGene，型号：Mini1620；规格：DC 20V 160W；序号：022-00001)、QPCR试剂盒(abm，MaterMix-S)、QPCR仪(BIO-RAD，CFX96TMOptics Module)、PSA引物(GeneCopoeia，HQP009633)、FKBP5引物(GeneCopoeia，HQP057374)、AR-full引物序列(f-ACCCTGCAAATGCTCTTCTTGTCAC；r-CTTTAGGCTTTGCCGCTGAAG)、AR-V7引物序列(f-TGACTCAGGCGGATGCAGTGAAG；r-ACAGTTGCTTCTTGTCCAAGGTGAC)。
2、方法
将LNCap，22RV1培养于1640完全培养基中，VCap培养于DMEM高糖完全培养基中，置于5％二氧化碳的37℃培养箱中培养。第一天用含5％CSS和1％双抗的培养基将细胞接种于6孔板中，LNCap和22RV1铺板数为70万/孔，VCap为120万/孔。
第三天加药，药物用DMSO(二甲亚砜)配置成30mM的储备液，R1881用DMSO配成5μM的储备液，用5％CSS完全培养基稀释，空白组加含等量浓度DMSO的完全培养基。
药物作用24h后，吸弃培养基，加500μl Trizol，用力摇晃15秒，室温放置3分钟。然后4℃，12000g离心15分钟。吸取上层水相至新离心管中，加250μl异丙醇颠倒混匀，室温放置10分钟后4℃，12000g离心10分钟。弃上清，加500μl 75％乙醇洗涤，温和振荡。然后4℃，7500g离心5分钟，弃尽上清，室温晾干5-10min。用30μl RNase-free水溶解RNA后，放置冰上测RNA浓度。按照逆转录试剂盒说明书操作进行逆转录(25℃10min，42℃15min，85℃5min)得到cDNA，-20℃储存，RNA放置-80℃冰箱储存。
由图5结果表明，本发明化合物SKLB-C4682、SKLB-C4535能够抑制LNCap细胞中由雄激素R1881激活的AR下游基因PSA和FKBP5的转录，活性与阳性化合物ODM201相当，且抑制作用具有浓度依赖性。
由图6结果表明，本发明化合物SKLB-C4682、SKLB-C4535能够抑制VCap细胞中由雄激素R1881激活的AR下游基因PSA和FKBP5的转录，且抑制作用具有浓度依赖性，抑制活性与阳性化合物ODM201和蒽杂鲁胺相当。
由图7结果表明，本发明化合物SKLB-C4682、SKLB-C4535能够抑制22RV1细胞中由雄激素R1881激活的AR下游基因FKBP5的转录，且活性优于阳性化合物ODM201和蒽杂鲁胺。
由图8结果表明，本发明化合物SKLB-C4682、SKLB-C4535能抑制LNCap细胞中AR及其剪切突变体AR-V7的mRNA，说明导致AR蛋白减少的原因是化合物减少了雄激素受体的mRNA。
上述实验结果表明，本发明化合物SKLB-C4682、SKLB-C4535通过减少雄激素受体mRNA来下调AR的蛋白表达量。同时本发明化合物能抑制AR下游基因PSA和FKBP5的转录，尤其是对22RV1细胞中FKBP5的抑制作用明显优于蒽杂鲁胺和ODM201，说明本发明化合物能够有效抑制前列腺癌细胞中的AR信号通路。
试验例5抑制AR-F876L转录活性的生物活性评价
1、材料仪器
DME/F12培养基(Hyclone,SH30023.01)、胎牛血清，FBS(BI,04-001-1ACS)、去雄激素的胎牛血清，CSS-FBS(由四川康城生物科技有限公司赠予)、青-链霉素(Hyclone,SV30010)。
细胞完全培养液：DME/F12培养基，10％FBS或者5％CSS，1％青-链霉素胰酶(Millipore,SM-2001-C)。
PC-3细胞(中国科学院典型培养物保藏委员会细胞库，SCSP-532)、转染试剂EndoFectinTM-Max(GeneCopoeia，EF003)、AR-F876L质粒(由生工生物 工程上海股份有限公司构建)、Cignal Androgen Receptor Reporter(luc)Kit(QIAGEN,CCS-1019L)、人工合成雄激素R1881(南京康满林化工实业有限公司,CAS:965-93-5)、双荧光素酶检测试剂盒(promega，E1910)、多功能酶标仪(BioTek，Synergy H1)。
2、方法
将PC-3细胞培养于DME/F12完全培养基中，置于5％二氧化碳的37℃培养箱中培养。第一天用含5％CSS且不含双抗的培养基将细胞接种于96孔板中，铺板数为1万/孔。
24小时内按转染试剂说明书将50ng AR-F876L和50ng AR-reporter一并转染进细胞。第三天加药，药物用DMSO(二甲亚砜)配置成30mM的储备液，R1881用DMSO配成3μM的储备液，用5％CSS完全培养基稀释，阳性对照组合阴性对照组加含等量浓度的DMSO和R1881。
药物作用24h后，吸弃培养基，按双荧光素酶检测试剂盒说明书操作，用多功能酶标仪检测发光信号。
由图9结果表明，本发明化合物SKLB-C4682、SKLB-C4535能抑制由1nM人工合成雄激素R1881所激活的AR-F876L的转录活性，且具有明显的浓度依赖性。相反，随着蒽杂鲁胺浓度的增加，AR-F876L的转录活性增强，表现出抗性。
上述实验表明，本发明化合物可以很好的抑制AR-F876L的转录活性，而AR-F876L是临床上发现对恩杂鲁胺产生耐药性的一种AR突变体，说明本发明化合物对蒽杂鲁胺耐药的AR-F876L仍然有效。
试验例6、体内抗肿瘤活性测试
1、材料
雄性Balb/c裸鼠(北京华阜康生物科技股份有限公司)、基质胶(BD,354234)
2、方法
1×10 7个VCaP细胞经皮下接种到5-6周龄的雄性Balb/c上，待肿瘤长至150mm 3左右则在异戊巴比妥麻醉的条件下实施睾丸切除术即物理去势并保留一组未切除睾丸的假手术对照组。术后肿瘤体积有缩小的迹象然后缓慢生长，待肿瘤重新生长至200-350mm 3则随机分组，即完成了CRPC肿瘤模型的建立。然后开始一天一次口服给药或空白溶剂(10％DMA+90％PEG200)，每两天测量肿瘤体积和裸鼠体重。由于假手术组的肿瘤生长明显更快，仅口服等体积的空白溶剂15天就单独处死并分离出肿瘤组织，其他实验组均给药40天后腹腔注射多聚甲醛处死，然后分离肿瘤组织并称重。所有动物实验均经中国四川大学动物保护与治疗专业委员会批准(IACUC编号:20100318)。
如图10结果显示在去势抵抗的VCaP前列腺癌模型中，30mg/kg和60mg/kg化合物SKBL-C4535(图中简写为4535)能够显著抑制肿瘤的生长，抑制率分别为64.7％(30mg/kg)和77.8％(60mg/kg)，活性优于已上市药物恩杂鲁胺(图中用Enza表示)。
上述实验表明，本发明化合物可以有效抑制去势抵抗型前列腺肿瘤的生长。
综上，本发明化合物不仅能够通过下调AR及其突变体的mRNA来抑制其蛋白表达，还能抑制AR及其突变体下游基因PSA和FKBP5的转录，同时对蒽杂鲁胺耐药的AR-F876L仍然有抑制作用，说明本发明化合物能够有效抑制AR信号通路。在细胞水平，本发明化合物能有效抑制AR阳性的前列腺癌细胞的增殖，且具有良好的细胞选择性，为临床上筛选和/或制备治疗前列腺癌的药物提供了一种新的选择，具有良好的应用前景。

Claims (16)

  1. 式I所示的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物：
    Figure PCTCN2019094301-appb-100001
    Z 1、Z 2各自独立的为C或者N；
    环A为C 5～C 12碳环、杂环、咪唑二酮、芳环、芳杂环、苯并碳环、苯并杂环，其中苯环上的碳可被一个或多个杂原子取代；
    环B为C 5～C 12碳环、杂环、并环、桥环、螺环、芳环、芳杂环或苯并芳环、苯并芳杂环、苯并碳环或苯并杂环，其中苯环上的碳可被一个或多个杂原子取代；
    R 1和R 2相互独立地为氢、卤素、CHF 2、CF 3、氰基、硝基、氨基、羟胺基、羟基、巯基、磺酸基、C 1～C 6的烷基、烯烃基、炔烃基、烷氧基、环烷基、杂环基或芳基、杂芳基以及-R 9C(O)R 10-、-R 9CO(O)R 10-、-R 9(O)COR 10-、-R 9NHC(O)R 10-或者-R 9C(O)NHR 10-，其中的羟基、巯基、氨基、烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基可进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
    R 3为氢、卤素、氰基、硝基、氨基、羟基、羟胺基、巯基、磺酸基、羧基、醛基、羰基、酯基、酰胺键或C 1～C 6的烷基、烷氧基、环烷基、杂环基、芳基、芳杂基，其中的烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
    R 1和R 2和R 3可以相互以单键、双键或三键连接成环；
    R 4、R 4’、R 5和R 5’各自独立地表示氢、羟基、卤素、氨基、羟胺基、氰基、硝基、烯基、炔基、C 1～C 6烷基、环烷基、烷氧基、羰基、硫羰基、醛基、羧基、酯基、酰胺基,其中所述C 1～C 6的烷基、烷氧基、烯基、炔基、酯基、酰胺基可 以各自独立地进一步被一个或者多个选自卤素、羟基、氰基、硝基、羟胺基、氨基、羟胺基、烷氧基、环烷基的基团所取代；
    R 5、R 5’、R 4、R 4’可以相互连接成键；
    R 7、R 7’、R 6、R 6’、R 8各自独立地表示氢、羟基、羟胺基、氨基、硝基、氰基、卤素、C 1～C 6的烷基、烷氧基、烷氧基取代的烷基、环烷基、羰基、醛基、羧基、酯基、酰胺基；
    R 7、R 7’、R 6、R 6’可以相互连接成键；
    R 11、R 12各自独立地表示氢、羟基、氨基、羟胺基、硝基、磺酸基、氰基、卤素、C 1～C 6的烷基、烷氧基、环烷基、芳基；其中所述C 1～C 6烷基、烷氧基、环烷基、氨基、芳基各自独立的任选进一步被一个或者多个选自卤素、羟基、氰基、硝基、烷基、环烷基、烷氧基、芳基的基团所取代；
    R 9、R 10各自独立地表示为氢、C 1～C 6直链或者支链烷基、烷氧基、氨基，其中的烷基、烷氧基可进一步被一个或者多个卤素、氰基、羟基、硝基、氨基取代；
    R8可与R 7、R 7’、R 6、R 6’以及R 11、R 12相互连接成键。
  2. 根据权利要求1所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述化合物的环A具有如Ia所示结构：
    Figure PCTCN2019094301-appb-100002
    其中,R为O，N，C＝O或者C＝S；
    X为C或N；Y为C或N；G为CH，C＝O或者C＝S；
    M为CH或者O；虚线表示可选择的双键；星号表示与环连接的位置；
    R 4和R 5定义同上述结构I。
  3. 根据权利要求1或2所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述化合物具有式Ⅱ所示结构：
    Figure PCTCN2019094301-appb-100003
    其中，R 1和R 2相互独立地为氢、卤素、CHF 2、CF 3、氰基、硝基、氨基、羟胺基、羟基、巯基、磺酸基、C 1～C 6的烷基、烯烃基、炔烃基、烷氧基、环烷基、杂环基或芳基、杂芳基；其中的羟基、巯基、氨基、烷基、烯烃基、炔烃基、烷氧基、芳基、杂芳基、环烷基、杂环基可进一步被一个或多个卤素、氰基、氨基、羟胺基、巯基、羟基、烷基、烷氧基、羰基、醛基、羧基、酯基、酰胺键、芳基、芳杂基取代；
    其中，环B为C5～C12碳环、杂环、芳环、芳杂环或苯并芳环、苯并杂环、苯并芳杂环，其中苯环上的碳可被一个或多个杂原子取代；
    R 11、R 12各自独立地表示氢、羟基、氨基、羟胺基、硝基、磺酸基、氰基、卤素、C 1～C 6的烷基、烷氧基、环烷基、芳基；其中所述C 1～C 6烷基、烷氧基、环烷基、氨基、芳基各自独立的任选进一步被一个或者多个选自卤素、羟基、氰基、硝基、烷基、环烷基、烷氧基、芳基的基团所取代。
  4. 根据权利要求3所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述化合物具有式Ⅲ所示结构：
    Figure PCTCN2019094301-appb-100004
  5. 根据权利要求4所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述化合物具有式Ⅳ所示结构：
    Figure PCTCN2019094301-appb-100005
  6. 根据权利要求3～5任意一项所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述环B为含N并环、含N芳杂环、苯并含N杂环、苯并含N芳杂环或者嘌呤。
  7. 根据权利要求6所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物，其特征在于：所述化合物为如下化合物之一：
    Figure PCTCN2019094301-appb-100006
    Figure PCTCN2019094301-appb-100007
    Figure PCTCN2019094301-appb-100008
  8. 一种制备权利要求1～7任意一项所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物方法，其特征在于：其反应路线如下所示：
    Figure PCTCN2019094301-appb-100009
    E为卤素Cl、Br、I；
    或者，
    Figure PCTCN2019094301-appb-100010
    或者，
    Figure PCTCN2019094301-appb-100011
    或者，
    Figure PCTCN2019094301-appb-100012
  9. 权利要求1～7任一项所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物在制备雄激素受体抑制剂中的用途。
  10. 根据权利要求9所述的用途，其特征在于：所述雄激素受体包括雄激素受体的突变体。
  11. 根据权利要求10所述的用途，其特征在于：所述雄激素受体的突变体T877A突变体、W741C突变体和/或AR-F876L突变体。
  12. 根据权利要求9～11任意一项所述的用途，其特征在于：所述抑制剂是抑制雄激素受体表达和/或雄激素受体通路的抑制剂。
  13. 根据权利要求12所述的用途，其特征在于：所述抑制雄激素受体通路是抑制雄激素受体下游基因PSA和FKBP5的转录。
  14. 权利要求1～7任一项所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物在制备雄激素受体相关疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其特征在于：所述药物是治疗雄激素受 体表达阳性的前列腺癌、前列腺增生、多毛症、脱发、精神性厌食症、乳腺癌、痤疮、男性性功能障碍或艾滋病的药物。
  16. 一种新型的雄激素受体抑制剂，其特征在于：它是以权利要求1～7任一项所述的化合物或其立体异构体、药学上可接受的盐、水合物或溶剂合物为活性成分，再加上药学上可接受的辅料制备而成的制剂。
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US12084453B2 (en) 2021-12-10 2024-09-10 Incyte Corporation Bicyclic amines as CDK12 inhibitors
RU2843187C1 (ru) * 2020-05-11 2025-07-08 Орион Корпорейшн Способ получения антагонистов андрогенных рецепторов и их промежуточных соединений

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