Pharmaceutical composition for the treatment of prostate cancer or breast cancer

Abstract

The purpose of the present invention is to provide: a therapeutic drug for therapy-resistant cancer resistant to hormone therapy; and a therapeutic method.　This pharmaceutical composition is for treatment of prostate cancer and contains, as an active ingredient, a compound represented by formula (1) of the present invention or a salt thereof, or a compound represented by formula (2) or (3) or a salt thereof. Furthermore, the pharmaceutical composition according to the present invention is not limited to hormone therapy-resistant prostate cancer or breast cancer, and can effectively treat prostate cancer or breast cancer that is not resistant to hormone therapy, and therapy-resistant prostate cancer or breast cancer that involve other mechanisms.

Description

[Technical Field]

[0001] This invention relates to a pharmaceutical composition for the treatment of prostate cancer or breast cancer. According to this invention, refractory prostate cancer or breast cancer can be treated. [Background technology]

[0002] Prostate cancer and breast cancer are the most common cancers affecting men and women in Western countries. In Japan, the number of prostate cancer patients has increased dramatically due to the Westernization of dietary habits and the aging of the population. Generally, the growth of prostate cancer is stimulated by androgens, which are male hormones. On the other hand, in breast cancer, estrogen, a female hormone, promotes its growth. Therefore, in the treatment of prostate cancer and breast cancer, hormone therapy that inhibits the production and function of androgens and estrogens is often chosen. Although the effect is very good, prostate cancer recurs within a few years as androgen-independent prostate cancer (castration-resistant prostate cancer, CRPC). Furthermore, the acquisition of hormone therapy resistance is also a problem in breast cancer (Patent Documents 1 and 2). Therefore, controlling treatment-resistant cancers that are resistant to hormone therapy is the most important challenge. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2014 / 188775 [Patent Document 2] Japanese Patent Publication No. 2013-17414 [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, the object of the present invention is to provide a therapeutic agent and a treatment method for hormone therapy-resistant cancer. [Means for solving the problem]

[0005] As a result of intensive research on therapeutic agents and treatment methods for hormone therapy-resistant treatment-resistant cancer, the present inventor surprisingly found that two groups of compounds having a specific chemical structure can effectively treat hormone therapy-resistant treatment-resistant cancer. The present invention is based on such findings. Therefore, the present invention provides [1] The following formula (1):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0006] The pharmaceutical composition of the present invention can effectively treat hormone therapy-resistant prostate cancer or hormone therapy-resistant breast cancer. Furthermore, the pharmaceutical composition of the present invention is not limited to hormone therapy-resistant prostate cancer or breast cancer, but can also effectively treat prostate cancer or breast cancer that is not hormone therapy-resistant, or other treatment-resistant prostate cancer or breast cancer due to other mechanisms. [Brief explanation of the drawing]

[0007] [Figure 1] This graph shows the growth inhibitory effects of compounds 10-0, 10-1, 10-3, 14-0, and 14-5 on hormone therapy-resistant prostate cancer model cell line 22Rv1, hormone therapy-resistant model LTAD (long time androgen deprived) cells, and VCaP cells. [Figure 2] This graph shows the binding ability of compounds 10-0, 10-1, 10-2, 10-3, 10-4, 14-0, and 14-5 to PSF, measured by the RNA pulldown method. [Figure 3] This graph shows the cell proliferation inhibitory effects of compounds 10-0, 10-1, 10-3, 10-4, 14-0, and 14-5 using 22Rv1 cells (prostate cancer), MCF7 cells (breast cancer), and OHTR cells (breast cancer). [Figure 4] This graph shows the suppression of PSF target genes AR, AR-V7, SchLaP1, FKBP5, and ACSL3 in 22Rv1 cells (prostate cancer) by compounds 10, 10-1, and 10-3. [Figure 5] This graph shows the repression of compound 10, 10-1, and 10-3 on the binding of PSF target genes ERα, SCFD2, TRA2B, and the control GAPDH to mRNA in OHT-TamR cells (breast cancer). [Figure 6] This figure shows the in vivo anticancer effects of compound 10-3 using a mouse subcutaneous transplantation model of AR-positive CRPC tumors (22Rv1 cells). [Figure 7] These are photographs and graphs showing the expression of Ki67 and AR in cancer tissue treated with compound 10-3. [Figure 8] This graph shows the cell proliferation inhibitory effects of compounds 10-1 and 10-3 using AR-negative, treatment-resistant prostate cancer model cells (DU145 cells) with mutations in the tumor suppressor p53. [Figure 9] This figure shows the in vivo anticancer effects of compound 10-3 using a mouse subcutaneous transplantation model of AR-negative CRPC tumors (DU145 cells). [Figure 10]This figure shows the binding sites of compound 10-3 to its target protein PSF, PSF, analyzed by docking analysis. The binding of three mutants (mut#1: K516I, D517V, mut#2: Y490H, mut#1+2: Y490H, K516I, D517V) with PSF amino acids predicted to interact with compound 10-3, as well as wild-type PSF, to the RNA strand of CTBP1-AS, confirmed by RNA pulldown. [Modes for carrying out the invention]

[0008] The pharmaceutical composition for the treatment of prostate cancer or breast cancer of the present invention is the following formula (1): [ka] (In the formula, R 1 R is a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon atoms, and R 2 is a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, or a heterocyclic alkyl group, or R 1 and R 2 Together, they form a 5-7 membered heterocycle which may have substituents, the substituents being a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkoxy group, a C6-C12 phenylalkyl group, and R 3 (These are a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, or an alkoxy group having 1 to 6 carbon atoms.) A compound represented by (hereinafter sometimes referred to as Compound A) or a salt thereof, Formula (2) or (3) below: [ka] (In the formula, R 4 Each of these independently consists of a hydrogen atom, an aldehyde group, a hydroxyl group, a C1-C6 alkyl group, or an alkyl ester alkylene group, or two R 4 Together they form an oxygen atom, R 5Each of these independently consists of a hydrogen atom, an aldehyde group, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkyl ester group, or an alkyl ester alkylene group, or two R 4 Together they form an oxygen atom, R 6 R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and 7 The active ingredient is a compound represented by a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 alkyl ester group, or a C1-C6 alkoxy group (hereinafter sometimes referred to as Compound B) or a salt thereof.

[0009] Examples of C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secondary butyl, tertiary butyl, n-pentyl, isopentyl, neopentyl, tertiary pentyl, n-hexyl, and isohexyl groups. C1-C3 alkyl groups are preferred.

[0010] Examples of alkoxy groups having 1 to 6 carbon atoms include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, secondary butoxy, tertiary butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, n-hexyloxy, or isohexyloxy groups. Alkoxy groups having 1 to 3 carbon atoms are preferred.

[0011] A heterocyclic alkyl group is a group in which an alkylene group is bonded to a heterocycle group (a monovalent group formed by the removal of one hydrogen atom bonded to a ring of a heterocyclic compound). The heterocycle group is not limited, but is preferably a saturated or unsaturated 5 or 6-membered ring, more preferably a saturated 5 or 6-membered ring, and examples include pyrrolidinyl group, piperidyl group, piperidino group, morpholinyl group, morpholino group, piperazinyl group, pyrrolyl group, imidazolyl group, or pyridyl group. The alkyl portion (alkylene group) is not limited, but is preferably an alkylene group having 1 to 6 carbon atoms, more preferably an alkylene group having 1 to 3 carbon atoms. Specifically, examples include piperidinomethyl group, pyrrolidinylmethyl group, piperidylmethyl group, morpholinylmethyl group, morpholinomethyl group, piperazinylmethyl group, pyrrolylmethyl group, imidazolylmethyl group, pyridylmethyl group, (1-pyrrolidinyl)butyl group, morpholinopropyl group, 1,1-dimethyl-2-(1-pyrrolidinyl)ethyl group, 1,1-dimethyl-2-piperidinoethyl group, 1,1-dimethyl-3-(imidazole-1-yl)propyl group, (2,6-dimethylpiperidino)methyl group, (2,6-dimethylpiperidino)ethyl group, and (2,6-dimethylpiperidino)propyl group.

[0012] The 5- to 7-membered heterocycle, which may have substituents, is preferably a 5 or 6-membered heterocycle, and most preferably a 6-membered heterocycle. The heterocycle is not limited to saturated or unsaturated heterocycles, and is preferably a saturated heterocycle. Examples of 5- to 7-membered heterocycles include morpholine (tetrahydro-1,4-oxazine), piperazine, pyrrolidine, imidazolidine, pyrazolidine, piperidine, imidazole, pyridine, tetrahydro-1,3-oxazine, or tetrahydro-1,2-oxazine. Examples of substituents include hydroxyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, or C6-C12 phenylalkyl groups. C6-C12 phenylalkyl groups are groups in which an alkylene group is bonded to a phenyl group. The alkyl portion (alkylene group) is not limited, but is preferably a C1-C6 alkylene group, and more preferably a C1-C3 alkylene group.

[0013] An aldehyde group is a group represented by -CHO. A hydroxyl group is a group represented by -OH.

[0014] A C1-C6 alkyl ester group is a group in which an ester group (-COO-) is bonded to an alkyl group having 1-C6 atoms. The C1-C6 alkyl groups are as described above.

[0015] Alkyl ester alkylene groups are groups in which an ester group (-COO-) is bonded to an alkyl group having 1 to 6 carbon atoms, and then an alkylene group having 1 to 6 carbon atoms is bonded to that group. The alkyl groups having 1 to 6 carbon atoms are as described above. Examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, t-butylene, n-pentylene, 2-methylbutylene, 2,2-dimethylpropylene, n-hexylene, or heptylene.

[0016] Compound A is a derivative of 4-phenylcoumarin. 4-phenylcoumarin and its derivatives can be synthesized by various methods. Compound B is a derivative of indole or indoline. Indole or indoline and their derivatives can also be synthesized by various methods. For example, indole can be synthesized from aniline as a starting material through a gas-phase reaction of aniline and ethylene glycol in the presence of a catalyst. These compounds can also be purchased commercially.

[0017] Compound A is not limited to the following compounds, but examples include compounds 10-0, 10-1, 10-2, 10-3, 10-4, and 10-5, which are represented by the following formulas. [ka]

[0018] Compound B is not limited to, but includes compounds 14-0, 14-1, 14-2, 14-3, 14-4, 14-5, 14-6, and 14-7, which are represented by the following formulas. [ka]

[0019] Salts of compound A and compound B are pharmaceutically acceptable salts and may form acid addition salts or salts with bases depending on the type of substituent. Specifically, examples include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyl tartaric acid, ditoluyl tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid; salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine, and ornithine; and salts with various amino acids and amino acid derivatives such as acetylleucine, as well as ammonium salts.

[0020] Furthermore, the active ingredients used in the present invention also include compound A or compound B, as well as various hydrates and solvates of their salts, and crystalline polymorphs. The present invention also includes compounds labeled with various radioactive or non-radioactive isotopes.

[0021] The prostate cancer targeted by the pharmaceutical composition of the present invention is a cancer that occurs in the prostate gland, and hormone therapy, including the administration of anti-androgen agents, is sometimes used. The type of prostate cancer targeted for treatment is not particularly limited, but it is particularly effective against androgen-independent prostate cancer (CRPC) that has acquired resistance to hormone therapy. As shown in the examples described below, the pharmaceutical composition of the present invention shows remarkable anticancer activity against AR-positive and AR-negative CRPC, and can be effectively used against CRPC that has acquired resistance to hormone therapy. However, it can also treat prostate cancer that is not resistant to hormone therapy and other treatment-resistant prostate cancers due to other mechanisms.

[0022] The breast cancer targeted by the pharmaceutical composition of this invention is a carcinoma that develops in breast tissue, and hormone therapy, such as the administration of anti-estrogen agents, is sometimes used. The type of breast cancer targeted for treatment is not particularly limited, but it is especially effective for estrogen-independent breast cancer that has acquired resistance to hormone therapy. However, it can also treat breast cancer that is not resistant to hormone therapy and other treatment-resistant breast cancers due to other mechanisms.

[0023] The inventors have discovered that in prostate cancer or breast cancer that has acquired resistance to hormone therapy, the RNA-binding protein PSF (PTB-Associated Splicing Factor) regulates factors involved in cancer malignancy. For example, PSF increases the expression of AR or its variant AR-V7, which is increased in castration-resistant prostate cancer (CRPC). In addition, PSF expression is elevated in OHTR cells of breast cancer tissue that has become resistant to hormone therapy. Compounds A and B used in the present invention can bind to PSF. As shown in the examples, the expression of AR, AR-V7, and SchLaP1, which are RNA-level target genes of PSF, can be suppressed in 22Rv1 cells, a hormone-resistant prostate cancer model cell. Furthermore, the expression of ERα and SCFD2, which are downstream genes of PSF, can be suppressed in OHT-TamR cells, a hormone-resistant breast cancer model cell. In other words, compounds A and B are thought to be effective in treating hormone-resistant prostate cancer or breast cancer by inhibiting the function of PSF through binding to it.

[0024] A pharmaceutical composition containing one or more of the aforementioned compound A or compound B, or salts thereof, as an active ingredient can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. Administration may be in any form, including oral administration in the form of tablets, pills, capsules, granules, powders, or liquids, or parenteral administration in the form of injections such as intra-articular, intravenous, or intramuscular injections, suppositories, eye drops, eye ointments, transdermal solutions, ointments, transdermal patches, transmucosal solutions, transmucosal patches, or inhalants.

[0025] For oral administration, solid compositions such as tablets, powders, and granules are used. In such solid compositions, one or more active ingredients are mixed with at least one inert excipient, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, and / or magnesium aluminometasilicate. The composition may also contain inert additives according to conventional methods, such as lubricants like magnesium stearate, disintegrants like sodium carboxymethyl starch, stabilizers, and solubilizers. Tablets or pills may be coated with sugar or a gastric-soluble or enteric-soluble film as needed. Liquid compositions for oral administration include pharmaceutically acceptable emulsifiers, solutions, suspensions, syrups, or elixirs, and commonly used inert diluents, such as purified water or ethanol. In addition to the inert diluent, the liquid composition may also contain auxiliary agents such as solubilizers, wetting agents, and suspensions, as well as sweeteners, flavoring agents, fragrances, and preservatives.

[0026] Injectable preparations for parenteral administration contain sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Aqueous solvents include, for example, distilled water for injection or physiological saline. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, or vegetable oils such as olive oil, alcohols such as ethanol, or polysorbate 80 (pharmacopoeia name). Such compositions may further contain isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, or solubilizers. These are sterilized, for example, by filtration through a bacterial retention filter, incorporation of a bactericide, or irradiation. Alternatively, sterile solid compositions may be prepared and dissolved or suspended in sterile water or sterile solvent for injection before use.

[0027] External preparations include ointments, plasters, creams, gels, poultices, sprays, lotions, eye drops, and eye ointments. They also contain commonly used ointment bases, lotion bases, aqueous or non-aqueous solutions, suspensions, emulsions, etc. For example, ointment or lotion bases include polyethylene glycol, propylene glycol, white petrolatum, bleached beeswax, polyoxyethylene hydrogenated castor oil, glyceryl monostearate, stearyl alcohol, cetyl alcohol, lauromacrogol, and sorbitan sesquioleate.

[0028] Inhalants and nasal preparations, and other transmucosal preparations, are used in solid, liquid, or semi-solid form and can be manufactured according to conventionally known methods. For example, known excipients, as well as pH adjusters, preservatives, surfactants, lubricants, stabilizers, and thickeners, may be added as appropriate. Administration can be carried out using a suitable inhalation or blowing device. For example, known devices such as metered-dose inhalation devices or sprayers can be used to administer the compound alone or as a powder in a formulated mixture, or as a solution or suspension in combination with a pharmaceutically acceptable carrier. Dry powder inhalers may be for single or multiple doses, and can utilize dry powder or powder-containing capsules. Alternatively, they may be in the form of a pressurized aerosol spray using a suitable excipient, such as a suitable gas like chlorofluoroalkane, hydrofluoroalkane, or carbon dioxide.

[0029] The dosage varies depending on the individual patient, including the type of disease, symptoms, age, and sex. However, for oral administration, the usual dose for adults is approximately 0.001 mg / kg to 500 mg / kg per day, administered once or divided into two to four doses. For injection, adults receive approximately 0.0001 mg / kg to 10 mg / kg per day, administered once or twice by rapid intravenous injection or intravenous drip infusion. For inhalation, adults receive approximately 0.0001 mg / kg to 10 mg / kg per day, administered once or multiple times. For transdermal formulation, adults receive approximately 0.01 mg / kg to 10 mg / kg per day, applied once or twice daily.

[0030] Compound A or Compound B, or a salt thereof, can be used in combination with various therapeutic or prophylactic agents for diseases in which Compound A or Compound B, or a salt thereof, is considered effective. This combination may be administered simultaneously, separately and consecutively, or at desired time intervals. The simultaneously administered formulation may be a combination formulation or formulated separately.

[0031] Treatment methods for prostate cancer or breast cancer Compound A or compound B can be used in a method for treating prostate cancer or breast cancer. That is, this specification discloses a method for treating prostate cancer or breast cancer, comprising the step of administering a therapeutically effective amount of the compound represented by formula (1) or a salt thereof, or the compound represented by formula (2) or (3) or a salt thereof, to a patient with prostate cancer or breast cancer.

[0032] Compound A or Compound B for use in the treatment of prostate cancer or breast cancer. Compound A or Compound B can be used in a method for treating prostate cancer or breast cancer. Specifically, a compound represented by formula (1) or a salt thereof, or a compound represented by formula (2) or (3) or a salt thereof, is disclosed for use in a method for treating prostate cancer or breast cancer.

[0033] Use of Compound A or Compound B in the manufacture of pharmaceutical compositions Compound A or Compound B can be used in the manufacture of a pharmaceutical composition for the treatment of prostate cancer or breast cancer. That is, this specification discloses the use of the compound represented by formula (1) or a salt thereof, or the compound represented by formula (2) or (3) or a salt thereof, in the manufacture of a pharmaceutical composition for the treatment of prostate cancer or breast cancer.

[0034] 《Action》 Although the mechanism by which compound A or compound B is effective in treating prostate cancer or breast cancer has not been analyzed in detail, it can be presumed as follows: Compound A is a derivative of 4-phenylcoumarin, and the 4-phenylcoumarin skeleton is thought to be important for binding to PSF (anti-cancer effect). Furthermore, the presence of an oxygen atom at the 7th position of the coumarin is thought to enhance the anti-cancer effect. Compound B is a derivative of indole or indoline, and the indole or indoline skeleton is thought to be important for binding to PSF (anti-cancer effect). [Examples]

[0035] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0036] Example 1 In this example, the effects of compounds A and B on the proliferative capacity of hormone therapy-resistant prostate cancer model cell line 22Rv1 and hormone therapy-resistant model LTAD (long time androgen deprived) cells were investigated. Place each cell in a 96-well plate in a 3x10⁶ 3 Seeds were seeded in the following manner, and compounds 10-0 and 14-0 were added at a concentration of 30 μM. After 3 days, cell proliferation ability was evaluated by MTS assay. The MTS assay was performed as follows. The cells were reacted for 1 hour using Cell titer 96 (Promega). The MTS assay is based on a reduction reaction that converts the tetrazolium salt [MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] to the chromogenic formazan product via PES (phenazine ethosulfate), and measures the number of viable cells. After the reaction, cell proliferation was measured using a microplate reader at 490 nm absorbance. Furthermore, MST assays were performed similarly using LTAD cells and VCaP cells, with compounds 10-0, 10-1, 10-3, 14-0, and 14-5 added at concentrations of 1 μM or 10 μM. As a result, all of compounds 10-0, 10-1, 10-3, 14-0, and 14-5 significantly suppressed cell proliferation (Figure 1).

[0037] Example 2 In this example, the binding ability of compounds 10-0, 10-1, 10-2, 10-3, 10-4, 14-0, and 14-5 to PSF was examined using the RNA pulldown method. We prepared a biotin-labeled CTBP1-AS probe using Biotin RNA Labeling Mix (Roche). A DNA strand incorporating 1.5kb of CTBP1-AS into the T7 promoter was then processed using T7 RNA polymerase. template RNA probes were prepared using the following method. RNA was mixed with nuclear extracts obtained from cells using RIP buffer and rotated at 4°C for 8 hours to allow binding. Avidin beads were then added and the mixture was rotated for 2 hours to collect the biotinylated RNA probes. The beads were collected by centrifugation, washed three times with buffer, and then SDS sample buffer was added to the beads. The bound proteins were analyzed by Western blotting. The 22Rv1 cells used were obtained in the same manner as in Example 1, except that the concentration of the added compounds was varied. As shown in Figure 2, all compounds inhibited the binding of PSF to RNA, but compound 10-3 showed a particularly strong inhibitory effect (IC50 = 0.22 pM).

[0038] Example 3 In this example, the effects of compounds 10-0, 10-1, 10-3, 14-0, and 14-5 on cell proliferation were investigated using hormone therapy-resistant prostate cancer model cell line 22Rv1, breast cancer cells MCF7, and hormone therapy-resistant model OHTR cells established from MCF7 cells. The MTS assay was performed in the same manner as in Example 1. As shown in Figure 3, each compound inhibited cell proliferation in 22Rv1 cells. Furthermore, compound 10-3 showed a stronger inhibitory effect on hormone therapy-resistant OHTR cells than on normal breast cancer cells (MCF7).

[0039] Example 4 In this example, using 22Rv1, the expression levels of PSF target genes AR, AR-V7, SchLaP1, and downstream signals of AR, FKBP5 and ACSL3, were analyzed by adding compounds 10-0, 10-1, and 10-3. Compounds 10-0, 10-1, and 10-3 were added to 22Rv1 cells, and RNA was collected after 48 hours. Changes in mRNA expression of downstream genes were then examined. Expression was measured by quantitative real-time PCR as follows. Using ISOGEN (NIPPON Gene), total RNA was recovered from cells, and cDNA was synthesized using the Prime script RT reagent kit (TaKaRa Bio). mRNA expression levels of each gene and the internal control, GAPDH, were measured using Step one real-time PCR (Applied biosystem) and the KAPA SYBR Fast PCR kit (NIPPON Genetics). The expression level for GAPDH was calculated from the number of cycles using the ΔΔCt method. Administration of compounds 10, 10-1, and 10-3 (10 μM) suppressed the expression of AR, AR-V7, SchLaP1, FKBP5, and ACSL3 mRNA (Figure 4).

[0040] Example 5 In this example, OHT-TamR cells, a hormone therapy-resistant model of breast cancer, were used, and compounds 10-0, 10-1, and 10-3 were added. After 48 hours of culture, the cells were harvested, pulverized, and immunoprecipitation was performed using a PSF antibody to extract total RNA that binds to the PSF protein. Quantitative PCR was used to quantify and analyze the amount of RNA binding to PSF for its target genes at the RNA level: ERα, SCFD2, and TRA2B. As a control, the binding RNA of GAPDH was also quantified. Compounds 10-0, 10-1, and 10-3 were added to OHT-TamR cells, and RNA was collected from the cells after 48 hours. The amount of RNA bound to each target gene and the control GAPDH was measured. Quantitative real-time PCR was performed in the same manner as in Example 4. Administration of compound 10-3 (10 μM) significantly inhibited the binding of ERα, SCFD2, and TRA2B to PSF proteins compared to GAPDH, while administration of compound 10-1 significantly inhibited the binding of ERα and SCFD2 (Figure 5).

[0041] Example 6 In this example, the anticancer effect of compound 10-3 in vivo was investigated using a mouse subcutaneous tumor transplantation model. 1 × 10⁶ 22Rv1 cells for tumor transplantation 7 The solution was adjusted, mixed with Matrigel in a 1:1 ratio, and subcutaneously injected into 6-week-old male nude mice (BALB / cAJcl-nu / nu). Five to eight weeks after subcutaneous injection, the tumor volume was 100-200 mm². 3 At a certain stage, orchiectomy was performed to create a model for the efficacy of treatment for refractory CRPC. Six mice were intraperitoneally injected with compound 10-3 at a concentration of 1 mg / kg. Controls were administered Vehicle (DMSO). Treatment was repeated 5 times / week for 2 weeks, and tumor size was measured (Figure 6). As shown in Figure 6, tumor growth was significantly suppressed in mice administered compound 10-3. On the other hand, no significant decrease in mouse body weight was observed with the administration of No. 10-3. Furthermore, administration of compound 10-3 reduced the number of cells expressing Ki67 and AR in cancer tissue (Figure 7).

[0042] Example 7 In this example, the effects of compounds 10-1 and 10-3 on cell proliferation were investigated using an AR-negative, treatment-resistant prostate cancer cell line (DU145 cells). The MTS assay was performed in the same manner as in Example 1. As shown in Figure 8, each compound inhibited cell proliferation in DU145 cells.

[0043] Example 8 In this example, we used a mouse subcutaneous transplantation model of AR-negative CRPC tumors (DU145 cells) to investigate the anticancer effects of compound 10-3 in vivo. DU145 cells 1 × 10⁶ for tumor transplantation 7 The solution was adjusted, mixed with Matrigel in a 1:1 ratio, and subcutaneously injected into 6-week-old male nude mice (BALB / cAJcl-nu / nu). Five to eight weeks after subcutaneous injection, the tumor volume was 100-200 mm². 3Once the mice reached a certain stage, compound 10-3 was injected intraperitoneally at a concentration of 5 mg / kg into eight mice (the photo shows four to five mice). The control group was administered Vehicle (DMSO). The treatment was repeated five times a week for three weeks, and the tumor size was measured (Figure 8). As shown in Figure 8, tumor growth was significantly suppressed in mice administered compound 10-3. On the other hand, no significant decrease in mouse body weight was observed with the administration of No. 10-3. The compounds of the present invention can suppress AR expression in cancer cells, but they also exhibit anticancer effects against AR-negative cancer cells. Therefore, it is thought that they exert anticancer effects through pathways other than the suppression of AR expression.

[0044] Example 9 In this example, the binding site of compound 10-3 used in the present invention with the target protein PSF was analyzed by docking analysis. Furthermore, mutations were introduced into amino acids of PSF that were predicted to interact with compound 10-3, and the binding of compound 10-3 was confirmed. The crystal structure of human PSF (276-535 amino acids) was downloaded from the Protein Data Bank (PDB). Volume and shape analysis of the binding cavity was performed using the SiteFinder module of the Molecular Engineering Environment (MOE) program. As a result, as shown in Figure 10, compound 10-3 was estimated to bind to the Coild-Coil site from NOPS in PSF. Mutations were introduced in the following combinations at the estimated site: tyrosine (Y) at position 490, lysine (K) at position 516, and aspartic acid (D) at position 517. mut#1:K516I, D517V mut#2:Y490H mut#1+2:Y490H, K516I, D517V The binding of mutated PSF (mut#1, mut#2, mut#1+2, or wild-type) to the RNA strand of CTBP1-AS was verified using the RNA pull-down method. The binding inhibitory effect of compound 10-3 was measured by adding compound 10-3. Compared to the wild type, the mut#1, mut#2, or mut#1+2s mutants showed reduced binding inhibitory effect by compound 10-3. Therefore, it was considered that compound 10-3 is likely to bind to tyrosine (Y) at position 490, lysine (K) at position 516, and aspartic acid (D) at position 517 in the region spanning from NOPS to Coil-Coil in PSF. [Industrial applicability]

[0045] The pharmaceutical composition of the present invention can be effectively used in the treatment of prostate cancer and breast cancer.

Claims

[Claim 1] the below described 【Chemistry 1】 A compound or salt thereof represented by a formula selected from the group consisting of the following: A pharmaceutical composition for the treatment of hormone therapy-resistant prostate cancer, comprising as an active ingredient.

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