Autophagy Inhibitors

Abstract

To provide an autophagy inhibitor having sufficiently high autophagy inhibitory activity.SOLUTION: The present invention discloses a compound represented by general formula (1) or a salt thereof (where n is an integer of 2-20); an autophagy inhibitor containing the compound represented by the general formula (1) or a salt thereof as an active ingredient; an Atg4B inhibitor containing the compound represented by the general formula (1) or a salt thereof as an active ingredient; and an activity enhancer for anticancer drug, containing the autophagy inhibitor or the Atg4B inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an autophagy inhibitor, and more particularly to a novel compound, an autophagy inhibitor, an Atg4B inhibitor, and an activity enhancer for an anticancer agent, a pharmaceutical composition, and a kit for cancer treatment, each of which has excellent autophagy inhibitory activity. [Background technology]

[0002] Induction of autophagy in tumors is involved in tumor malignancy and the acquisition of anticancer drug resistance by suppressing the accumulation of abnormal proteins and abnormal organelles, and the resulting oxidative stress. Therefore, autophagy inhibitors are expected to be new anticancer drugs that enhance the effectiveness of existing anticancer drugs. Although there are no cases yet in which autophagy inhibitors have been used clinically as anticancer drugs, immunotherapy drugs such as Opdivo (nivolumab) are examples of drugs that can similarly enhance the effects of existing anticancer drugs. The market size of cancer immunotherapy drugs is large and is expected to continue to grow. However, although cancer immunotherapy drugs have shown excellent treatment results in clinical trials, it is said that only about 20% of patients are effective, and the development of drugs that can enhance the effects of existing anticancer drugs through other mechanisms of action is desired.

[0003] Furthermore, research on autophagy is on the rise, with over 5,000 related papers published in 2017 (see, for example, Non-Patent Documents 1-3, etc.), and this trend is still on the rise. However, the only autophagy inhibitors used as research reagents are PI3 kinase inhibitors and lysosomal inhibitors, neither of which are selective inhibitors of autophagy. In order to correctly understand the phenomenon of autophagy, it is necessary to develop selective inhibitors of autophagy, and it is expected that their use will be widespread even at the reagent level. It is known that some existing pharmaceuticals have autophagy inhibitory activity (see Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-002619 A [Non-patent literature]

[0005] [Non-Patent Document 1] Autophagy: from phenomenology to molecular understanding in less than a decade. Klionsky DJ. Nat Rev Mol Cell Biol. 2007 Nov;8(11):931-7. Review. [Non-Patent Document 2] The exponential growth of autophagy-related research: from the humble yeast to the Nobel Prize. Mizushima N. FEBS Lett. 2017 Mar;591(5):681-689. [Non-Patent Document 3] Development of autophagy inducers in clinical medicine. Levine B, Packer M, Codogno P. J Clin Invest. 2015 Jan;125(1):14-24. Summary of the Invention [Problem to be solved by the invention]

[0006] It has been believed that some intractable tumors, including KRAS mutant tumors, survive and progress in an autophagy-dependent manner, and that autophagy inhibitors can contain the tumors. For this reason, clinical trials have been conducted on combination therapy with existing anticancer drugs, such as chloroquine and hydroxychloroquine, which are known to be autophagy inhibitors that inhibit lysosomes. However, a recent report has shown that autophagy is not involved in the anticancer activity of chloroquine, calling for a reconsideration of the significance of autophagy in cancer. The background to this situation is thought to be that selective autophagy inhibitors have not yet been developed. Therefore, the development of selective autophagy inhibitors is one of the most important issues in elucidating the physiological and pathophysiological significance of autophagy.

[0007] Under these circumstances, a main object of the present invention is to provide an autophagy inhibitor having excellent autophagy inhibitory activity. [Means for solving the problem]

[0009] That is In the present invention, First, a compound represented by the following general formula (1) or a salt thereof is contained as an active ingredient. Autophagy inhibitors, or The compound represented by the following general formula (1) or a salt thereof is contained as an active ingredient. Anticancer drug activity enhancer, including Atg4B inhibitor of provide. [ka] (In general formula (1), n ​​is an integer of 2 to 20.) Furthermore, the present invention provides a pharmaceutical composition comprising the anticancer agent and the activity enhancer for the anticancer agent. The present invention also provides a kit for cancer treatment comprising the anticancer agent activity enhancer and an anticancer agent. Effect of the Invention

[0010] According to the present invention, an autophagy inhibitor having excellent autophagy inhibitory activity can be provided. Note that the effects described here are not necessarily limited to those described herein, and may be any of the effects described in this specification. [Brief description of the drawings]

[0011] [Figure 1] FIG. 13 is a diagram showing the results of Experimental Example 2. [Diagram 2] FIG. 13 is a diagram showing the results of Experimental Example 3. [Diagram 3] FIG. 13 is a diagram showing the results of Experimental Example 4. [Figure 4] FIG. 13 is a diagram showing the results of Experimental Example 5. [Diagram 5] FIG. 13 is a diagram showing the results of Experimental Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Preferred embodiments for carrying out the present invention will now be described. The embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not to be construed as being narrow.

[0013] 1. A compound represented by the above general formula (1) or a salt thereof The compound according to the present invention is a compound represented by the above general formula (1) or a salt thereof. As shown in the Examples below, the compound according to the present invention significantly suppressed starvation-induced autophagy at the cellular level in a study using a prostate cancer cell line. In addition, the compound was also found to have an effect of enhancing the action of existing anticancer drugs. Therefore, the compound according to the present invention can be put to practical use as a cancer adjuvant drug that can enhance the action of existing anticancer drugs, and as an autophagy inhibitor that inhibits Atg4B as its action site.

[0014] In the above general formula (1), n ​​is an integer of 2 to 20, preferably, n is 2, 4, 6, 8 to 12, 14, 16 to 18, or 20, more preferably, n is 4, 6, 8 to 12, 14, 16 to 18, or 20, even more preferably, n is 6, 8 to 12, 17, or 18, even more preferably, n is 9, 10, 11, or 17, and particularly preferably, n is 10.

[0015] Specifically, the salt is, for example, a "pharmaceutical acceptable salt", which refers to an inorganic or organic acid or base addition salt that has the biological effectiveness of the parent compound (salt-free compound) and is biologically non-toxic or has low biological toxicity. Examples of the salt include inorganic acid addition salts with hydrochloric acid, sulfuric acid, etc.; organic acid addition salts with formic acid, acetic acid, trifluoroacetic acid, tartaric acid, etc.; alkali metal salts with sodium, potassium, etc.; alkaline earth metal salts with calcium, magnesium, etc.; organic amine salts with methylamine, ethylamine, diethanolamine, etc.

[0016] 2. Autophagy inhibitors, Atg4B inhibitors The autophagy inhibitor according to the present invention contains the compound represented by the above general formula (1) or a salt thereof as an active ingredient. The autophagy agent according to the present invention contains the compound represented by the above general formula (1) or a salt thereof, and is not particularly limited as long as it does not impair the effects of the present invention, and may contain a pharma- ceutically acceptable carrier described below, other known or unknown active ingredients, and the like.

[0017] As used herein, the term "autophagy inhibitor" refers to an agent that inhibits or suppresses autophagy.

[0018] The autophagy inhibitor according to the present invention can be used as a preventive or therapeutic agent for various diseases in which autophagy is involved. It can also be used as a research reagent, diagnostic agent, etc.

[0019] The Atg4B inhibitor according to the present invention contains the compound represented by the above general formula (1) or a salt thereof as an active ingredient. The Atg4B inhibitor according to the present invention contains the compound represented by the above general formula (1) or a salt thereof, and is not particularly limited as long as it does not impair the effects of the present invention, and may contain a pharma- ceutically acceptable carrier described below, other known or unknown active ingredients, and the like.

[0020] As used herein, the term "Atg4B inhibitor" refers to an agent that inhibits or suppresses the activity of Atg4B. Atg4B, also known as Autophagin-1, is a cysteine ​​protease essential for the maturation of LC3, a component of the autophagosomal membrane. It is known that autophagy is not completed unless Atg4B functions normally. Therefore, inhibition of Atg4B is thought to be a rational point of action for inhibiting autophagy.

[0021] The Atg4B inhibitor according to the present invention can be used as a preventive or therapeutic agent for various diseases in which Atg4B is involved. In addition, it can be used as a research reagent, as well as a diagnostic agent, etc.

[0022] The subjects to which the autophagy inhibitors and Atg4B inhibitors according to the present invention are administered are usually humans; however, the present invention also includes mammals other than humans, for example pet animals such as dogs and cats, and livestock such as cows, sheep, and pigs.

[0023] The administration form of the autophagy inhibitor and Atg4B inhibitor according to the present invention may be either a solid preparation or a liquid preparation, and examples of such preparations include injections, oral preparations (tablets, granules, powders, capsules), ointments, creams, patches, suppositories, etc.

[0024] To obtain the form of these pharmaceutical compositions, they can be formulated with a pharma- ceutically acceptable carrier. Examples of such carriers include, but are not limited to, lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, starch, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, ethanol, carboxymethylcellulose, carboxymethylcellulose calcium salt, magnesium stearate, talc, acetylcellulose, sucrose, titanium oxide, benzoic acid, paraoxybenzoic acid ester, sodium dehydroacetate, gum arabic, tragacanth, methylcellulose, egg yolk, surfactant, sucrose, simple syrup, citric acid, distilled water, ethanol, glycerin, propylene glycol, macrogol, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, glucose, sodium chloride, phenol, thimerosal, paraoxybenzoic acid ester, sodium hydrogen sulfite, etc.

[0025] The content of the active ingredient in the pharmaceutical composition is not particularly limited since it varies greatly depending on the form of the preparation, but can be, for example, 0.001 to 100% by mass based on the total amount of the pharmaceutical composition.

[0026] 3. Anticancer drug activity enhancer The activity enhancer according to the present invention includes the above-mentioned autophagy inhibitor or Atg4B inhibitor. The activity enhancer according to the present invention is not particularly limited as long as it contains the above-mentioned autophagy inhibitor or Atg4B inhibitor and does not impair the effects of the present invention, and may contain the above-mentioned pharma- ceutically acceptable carrier, other known or unknown active ingredients, and the like.

[0027] Generally, autophagy is induced as a defense mechanism against cancer. Therefore, when cells are treated with anticancer drugs, apoptosis is induced toward cell death, but autophagy is also induced to protect the cells. This induction of autophagy is also known to cause a decrease in sensitivity to anticancer drugs, and it is thought that the combined use of autophagy inhibitors with anticancer drugs can enhance the effects of the anticancer drugs.

[0028] Cancer is the main cause of death worldwide, and there is an urgent need to develop new anticancer drugs aimed at preventing and curing cancer. In recent years, anticancer drugs with excellent therapeutic response rates have been launched on the market, but most of them are antibody drugs. Although antibody drugs are highly effective because they act directly on cancer-causing proteins, their high drug prices due to the difficulty of mass production and other factors are a problem, and many patients cannot choose treatment for financial reasons. Therefore, the development of small molecule drugs that are easy to mass-produce and relatively inexpensive is desired. In contrast, the activity enhancer of the present invention is easy to chemically synthesize and is expected to withstand mass production, so if it is adapted to cancer treatment, it is thought that it will contribute to improving patients' treatment opportunities and ultimately reducing national medical expenses.

[0029] In the present specification, the term "cancer" is broadly interpreted and is used interchangeably with "malignant tumor". In addition, before a diagnosis is confirmed pathologically, that is, before a tumor is confirmed to be benign or malignant, it may collectively include benign tumors, benign / malignant borderline lesions, and malignant tumors. The cancers that are the target of the activity enhancer according to the present invention are not particularly limited, and examples thereof include head and neck cancer, esophageal cancer, gastric cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer, gallbladder / bile duct cancer, biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, blood cancer, multiple myeloma, skin cancer, brain tumor, mesothelioma, blood cancer, and the like. In the present invention, prostate cancer is particularly preferred, and castration-resistant prostate cancer (CRPC) is particularly preferred.

[0030] Although hormone therapy is effective for the treatment of hormone-dependent cancer, hormone therapy is completely ineffective for castration-resistant prostate cancer (CRPC) in which hormone sensitivity has been lost congenitally or during the course of treatment, and therefore the development of a new anticancer drug is urgently needed. As shown in the examples below, the activity enhancer of the present invention is very useful because it enhances the activity of a CRPC therapeutic drug.

[0031] In the present specification, the term "anticancer agent" refers to a drug that exhibits a therapeutic or preventive effect against cancer, which is a target disease or pathology. The therapeutic effect also includes alleviating (alleviating) symptoms characteristic of cancer or associated symptoms, and preventing or delaying the worsening of symptoms. The anticancer agent that is the target of the activity enhancer of the present invention is not particularly limited, and examples thereof include antiandrogens such as flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, canrenone, drospirenone, ketoconazole, topirutamide (fluridil), and cimetidine; CYP17 lyase inhibitors such as abiraterone; and taxane anticancer agents such as docetaxel and cabazitaxel. In the present invention, castration-resistant prostate cancer (CRPC) therapeutic agents are particularly preferred, and among these, abiraterone, apalutamide, enzalutamide, and cabazitaxel are particularly preferred.

[0032] 4. Pharmaceutical Compositions The pharmaceutical composition according to the present invention contains the above-mentioned activity enhancer and anticancer agent. The pharmaceutical composition according to the present invention is not particularly limited as long as it contains the above-mentioned activity enhancer and anticancer agent and does not impair the effects of the present invention, and may contain the above-mentioned pharma- ceutical acceptable carriers, other known or unknown active ingredients, and the like.

[0033] The pharmaceutical composition according to the present invention can be prepared as a pharmaceutical composition for cancer treatment by combining the above-mentioned activity enhancer with, for example, one of the above-mentioned anticancer agents, and administered to a subject. When multiple anticancer agents are approved for clinical use against a specific cancer, an anticancer composition can be prepared by combining the activity enhancer according to the present invention with them and used. In addition, when multiple anticancer agents are administered against two or more different types of cancer, the activity enhancer according to the present invention can also be combined with them.

[0034] When the activity enhancer of the present invention is used together with an anticancer drug, it can be combined with other cancer treatment methods, and more effective therapeutic effects can be expected than when each treatment method is performed alone.For example, when combined with surgical resection, chemotherapy can be continued regardless of whether it is before or after surgery.It can also be combined with radiation therapy.

[0035] The dose of the activity enhancer of the present invention to be combined with an anticancer drug can be appropriately selected depending on the age, symptoms, etc. of the patient, and is not particularly limited as long as it is an amount that enhances the anticancer activity of the anticancer drug. In reality, it can vary within a wide range depending on the predetermined dose of the anticancer drug.

[0036] The activity enhancer and the anticancer agent according to the present invention are preferably mixed together and then administered to a subject as a single mixed preparation (anticancer composition). However, the same therapeutic effect can be expected even if they are administered via different administration routes (e.g., intravenous administration of the activity enhancer and oral administration of the anticancer agent).

[0037] When the activity enhancer according to the present invention and an anticancer agent are administered separately, the respective dosages can be determined according to the type of anticancer agent to be administered, the condition of the subject, etc. The activity enhancer according to the present invention can be administered either before or after the administration of the anticancer agent, but it is preferable that the administration interval is not too long.

[0038] 5. Cancer treatment kit The cancer treatment kit according to the present invention includes the above-mentioned activity enhancer and an anticancer drug. The cancer treatment kit according to the present invention includes the above-mentioned activity enhancer and an anticancer drug, and may include other substances without being particularly limited as long as the effects of the present invention are not impaired.

[0039] The cancer treatment kit according to the present invention may, for example, comprise a first storage section containing the activity enhancer according to the present invention and a second storage section containing an anticancer drug. The "storage section" in the kit is not particularly limited as long as it is in an effective form in which the respective drugs are present independently without being mixed, and may be, for example, a container or an individual package, or may be in the form of an independently divided region in a sheet.

[0040] In the cancer treatment kit of the present invention, each drug may be administered alone, or when necessary, the activity enhancer of the present invention and the anticancer drug may be removed from their respective containers and mixed to prepare a formulation (anticancer composition). EXAMPLES

[0041] The present invention will now be described in further detail with reference to examples. The embodiment described below is merely a representative example of the present invention, and the scope of the present invention should not be construed as being narrow.

[0042] <Experimental Example 1> Synthesis of Compounds [ka]

[0043] Under Ar atmosphere, maleic anhydride (25 mg, 0.252 mmol) and aluminum chloride (67 mg, 0.504 mmol) were added to a solution of alkylbenzene (1a-n, 0.252 mmol) in CHCl (1.5 mL) at room temperature, and the mixture was stirred at room temperature for 1 hour. Water (1.5 mL) was added under ice cooling, and the CHCl layer was separated. The aqueous layer was extracted three times with CHCl (1.5 mL) and combined with the previous CHCl layer. The combined CHCl layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The residue was purified by silica gel column chromatography (SiO: 7 g, n-hexane:acetone = 3:1 to 1:1) to obtain the AUP derivative as white crystals.

[0044] [ka] (E)-4-Oxo-4-(4-propylphenyl)but-2-enoic acid (AUP06) 1 H-NMR (400 MHz, CDCl3) δ: 0.96 (3H, t, J = 7.2 Hz), 1.68 (2H, sext, J = 7.2 Hz), 2.67 (2H, t, J= 7.2 Hz), 6.88 (1H, d, J = 16.0 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.99 (1H, d, J = 16.0 Hz)

[0045] [ka] (E)-4-Oxo-4-(4-pentylphenyl)but-2-enoic acid (AUP07) 1 H-NMR (400 MHz, CDCl3) δ: 0.90 (3H, t, J = 7.6 Hz), 1.25 - 1.38 (4H, m), 1.65 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.99 (1H, d, J = 15.2 Hz)

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[0057] <Example 2> Evaluation of Atg4B inhibitory activity and PLA2 inhibitory activity of each compound

[0058] [Method: Evaluation of Atg4B inhibitory activity] LC3-GST, which is made by fusing glutathione-S-transferase to the C-terminus of purified LC3, was mixed with Atg4B and an inhibitor, and incubated at 37°C for 3 hours. SDS-PAGE was then performed and stained to observe the amount of conversion from LC3-GST to LC3 and GST. Each band was quantitatively analyzed using Image J. As a control, a sample was prepared to which dimethylsulfoxide (DMSO) was added. The amount of conversion and conversion rate (%) were calculated using the following formula. The conversion rate (%) was calculated assuming the control group to be 100%. Conversion rate = (GST + LC3) / (LC3-GST + GST ​​+ LC3) Conversion rate (%) = (amount converted when inhibitor was added) / (amount converted when DMSO was added) × 100

[0059] [Method: PLA2 inhibitory activity evaluation] The PLA2 inhibitory activity was evaluated using the EnzChek Phospholipase A2 Assay Kit (@Thermofisher) according to the experimental protocol provided. The 50% inhibitory concentration was calculated from the inhibition percentage of the control group activity of samples containing various concentrations of compounds, using a sample containing DMSO as the control group.

[0060] [Results and Discussion] The 50% inhibitory concentration for Atg4B decreased with increasing alkyl chain length, being the smallest when the alkyl chain length was 11, and AUP01 with an alkyl chain length of 11 most potently inhibited Atg4B. Furthermore, the activity decreased with an increase in the number of carbon atoms, but the activity increased again when the alkyl chain length was 18 or 19. This is thought to be because the inhibitory activity increased due to increased interaction with the enzyme with increasing alkyl chain length, but as the alkyl chain length increased to 15 and 17, the activity decreased due to decreased solubility due to increased lipid solubility, and further, at alkyl chain lengths of 18 and 19, the inhibitory activity was enhanced by the acquisition of new interactions.

[0061] In addition, the 50% inhibitory concentrations for PLA2 of alkyl chain lengths 18 and 19 were 0.049±0.013 μM and 0.35±0.13 μM, respectively, while the 50% inhibitory concentration for PLA2 of AUP01 was 3.5±0.3 μM, suggesting that AUP01 has higher selectivity for Atg4B than these.

[0062] <Experimental Example 3> Evaluation of autophagy inhibitory activity of AUP01

[0063] [method] Prostate cancer LNCaP cells were cultured in a carbon dioxide incubator at 37°C under 5% CO2 conditions. The growth medium used was RPMI1640 containing 5% (v / v) FBS, 100 U / ml penicillin-G potassium, 100 μg / ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0). Both cells were cultured at 1 × 10 6 The cells were seeded on a 6 cm dish at 100 cells / dish, and after 24 hours, the growth medium was replaced with one containing 2% FBS, and compound AUP01 or compound 17 was added. After 2 hours, the medium was replaced with amino acid-depleted medium, and after further culturing for 3 hours, the cells were harvested with DPBS and subjected to Western blotting and DAPGreen staining as follows.

[0064] [Method: Western blot (Figure 2A)] After the treatment, the cells were washed three times with DPBS and detached using a cell scraper. The collected cells were washed with Dulbecco's phosphate buffered saline (DPBS) and suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membrane was disrupted by sonication. The cell lysate was centrifuged (15,000 xg, 15 min, 4°C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using 12.5% ​​polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA and reacted with primary antibodies against p62 and β-actin and horseradish peroxidase-conjugated secondary antibodies in sequence. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0065] [Method: DAPGreen staining (Figure 2B)] Cells were suspended in growth medium and plated at 2 × 10 4 Cells / 500 μL were seeded and cultured at 37 °C, 5% CO2 for 24 hours, after which DAPGreen was added and incubated for 30 minutes. The medium was replaced with a growth medium containing 2% FBS, and compound AUP01 was added. After 2 hours, the medium was replaced with an amino acid-depleted medium, and after further culture for 3 hours, the cells were washed twice with DPBS, excess water was removed, and a cover glass was fixed on a slide glass using a mounting agent (fluoromount-G). The cover glass was placed on a confocal laser microscope LSM700 (Carl Zeiss) and fluorescence observation was performed. Images were captured using a 40x oil immersion objective lens of an inverted fluorescence microscope.

[0066] [Results and Discussion] Treatment with amino acid-depleted medium for 3 hours increased DAPGreen dot-like fluorescence due to the degradation of p62 and the generation of autophagosomes, indicating the induction of autophagy. Pretreatment with AUP01 increased p62 expression in a concentration-dependent manner, and its effect was stronger than that of lead compound 17. DAPGreen fluorescence induced by amino acid-depleted medium was also reduced. These results indicate that AUP01 inhibits autophagy at the cultured cell level.

[0067] <Experimental Example 4> Evaluation of induction of apoptosis and autophagy by abiraterone

[0068] [Method: Viable cell count measurement] LNCaP cells suspended in growth medium were plated in a 96-well multiplate at 2 × 10 4 Cells / 200 μL were seeded and cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS, and the samples were added to the medium and cultured for an additional 24 hours. As a control, cells were prepared with the addition of dimethylsulfoxide (DMSO). Next, the medium was replaced with one without serum or phenol red, 40 μM resazurin was added, and the medium was cultured at 37°C for 2-4 hours. The absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad). Cell viability (%) was calculated using the following formula: Cell viability (%) = (SA) / (BA) × 100 S: Absorbance of the well to which the sample and cells were added A: Absorbance of the well to which only medium was added B: Absorbance of wells containing DMSO and cells

[0069] [Method: Western blot] Prostate cancer LNCaP cells were cultured in a carbon dioxide incubator at 37°C under 5% CO2 conditions. The growth medium used was RPMI1640 containing 5% (v / v) FBS, 100 U / ml penicillin-G potassium, 100 μg / ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0). Both cells were cultured at 1 × 10 6 Cells / dish were seeded on a 6 cm dish, and after 24 hours, the medium was replaced with 10 μM abiraterone-containing medium with 2% FBS concentration. After another 24 hours of culture, the cells were harvested with DPBS and subjected to Western blotting as follows. The cells treated with various reagents were washed three times with DPBS and detached using a cell scraper. The harvested cells were washed with Dulbecco's phosphate buffered saline (DPBS), suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membrane was destroyed by sonication. The cell lysate was centrifuged (15,000 xg, 15 min, 4 °C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using 12.5% ​​polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 1% BSA, the PVDF membrane was reacted sequentially with primary antibodies against PARP, p62, p-p62 (Ser403), LC3, and β-actin, and horseradish peroxidase-conjugated secondary antibodies. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0070] [Results and Discussion] Abiraterone, a drug used to treat castration-resistant prostate cancer (CRPC), was shown to produce cleaved PARP in a dose-dependent manner in LNCaP cells, inducing apoptotic cell death. At the same time, p-p62 and LC3-II were produced, indicating that autophagy was also induced.

[0071] <Experimental Example 5> Combination effect of abiraterone and AUP01

[0072] [Method: DAPGreen staining (Figure 4A)] Cells were suspended in growth medium and plated at 2 × 10 4 Cells / 500 μL were seeded and cultured at 37 °C, 5% CO2 for 24 hours, after which DAPGreen was added and incubated for 30 minutes. The growth medium was replaced with one containing 2% FBS, and compound AUP01 was added. After 2 hours, the medium was replaced with one containing 10 μM abiraterone, and after further culture for 24 hours, the cells were washed twice with DPBS, excess water was removed, and a cover glass was fixed on a slide glass using a mounting agent (fluoromount-G). The cover glass was placed on a confocal laser microscope LSM700 (Carl Zeiss) and fluorescence observation was performed. Images were captured using a 40x oil immersion objective lens of an inverted fluorescence microscope.

[0073] [Method: Immunofluorescence staining (Figure 4B)] Cells were suspended in growth medium and plated at 2 × 10 4Cells were seeded at 500 μL per well and cultured for 24 hours at 37°C, 5% CO2, then replaced with growth medium with 2% FBS concentration and compound AUP01 was added. After 2 hours, the medium was replaced with 10 μM abiraterone-containing medium, and after further culture for 24 hours, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes, 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the cells were left to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, and the cells were blocked for 1 hour. After washing twice with DPBS, the cells were incubated overnight at 4°C in a solution of primary antibody (anti-Caspase-3 antibody) diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated in a solution of Alexa Fluoro-488-labeled rabbit secondary antibody diluted 500:1 in DPBS for 1 hour at room temperature in the dark. After washing twice with PBS, excess water was removed and a cover glass was fixed on a slide glass using a mounting agent (DAPI fluoromount-G). The fluorescently immunostained cells were placed on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence. Images were captured using a 40x oil immersion objective lens on an inverted fluorescence microscope.

[0074] [Method: Western blot (Figure 4CD)] Prostate cancer LNCaP cells were cultured in a carbon dioxide incubator at 37°C under 5% CO2 conditions. The growth medium used was RPMI1640 containing 5% (v / v) FBS, 100 U / ml penicillin-G potassium, 100 μg / ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0). Both cells were cultured at 1 × 10 6The cells were seeded on a 6 cm dish at 1000 x g, and the growth medium was replaced with one containing 2% FBS. Compound AUP01 or wortmannin (Wo) was added. After 2 hours, the medium was replaced with 10 μM abiraterone. After 24 hours of culture, the cells were washed twice with DPBS and subjected to Western blotting as follows. The cells treated with various reagents were washed three times with DPBS and then detached using a cell scraper. The collected cells were washed with Dulbecco's phosphate buffered saline (DPBS), suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membrane was destroyed by sonication. The cell lysate was centrifuged (15,000 x g, 15 minutes, 4 °C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using a 12.5% ​​polyacrylamide gel, proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 1% BSA, the PVDF membrane was reacted sequentially with primary antibodies against PARP and β-actin and horseradish peroxidase-conjugated secondary antibodies. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0075] [Method: Viable cell count (Figure 4D)] LNCaP cells suspended in growth medium were plated in a 96-well multiplate at 2 × 10 4Cells were seeded at 200 μL each and cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS, and AUP01 (1 μM; △, 2 μM; ▲) was added 2 hours later, 5 and 10 μM abiraterone was added and cultured for another 24 hours. As a control, cells were prepared with the addition of dimethylsulfoxide (DMSO). Next, the medium was replaced with serum-free and phenol red-free medium, 40 μM resazurin was added, and the cells were cultured at 37°C for 2-4 hours, after which the absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad).

[0076] [Results and Discussion] Autophagy induced by abiraterone treatment was suppressed by pretreatment with AUP01, and apoptotic cell death was enhanced by pretreatment with AUP01. Thus, AUP01 enhanced apoptosis by inhibiting autophagy induced by abiraterone treatment.

[0077] <Experimental Example 6> Enhancement of anticancer activity of CRPC therapeutic drug by AUP01

[0078] [Method: Viable cell count measurement] LNCaP cells suspended in growth medium were plated in a 96-well multiplate at 2 × 10 4 Cells were seeded at 200 μL per well and cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS, and 5 μM AUP01 was added 2 hours later. Apalutamide (50, 100 μM), cabazitaxel (10, 20 μM) or enzalutamide (20, 50 μM) were added and cultured for another 24 hours. As a control, cells were prepared with dimethylsulfoxide (DMSO). Next, the medium was replaced with serum-free and phenol red-free medium, and 40 μM resazurin was added. After 2-4 hours of culture at 37°C, the absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad).

[0079] [Results and Discussion] AUP01 also significantly enhanced the anticancer activity of three castration-resistant prostate cancer (CRPC) drugs: apalutamide, cabazitaxel, and enzalutamide. [Industrial Applicability]

[0080] According to the present invention, it is possible to provide a novel compound, an autophagy inhibitor, an Atg4B inhibitor, and an activity enhancer of an anticancer agent using the same, as well as a pharmaceutical composition and a cancer treatment kit, all of which have excellent autophagy inhibitory activity. The autophagy inhibitors and the like according to the present invention can be easily chemically synthesized and are suitable for mass production. Therefore, it is believed that application of the autophagy inhibitors and the like according to the present invention to cancer treatment and the like can improve patients' opportunities for treatment and ultimately contribute to reducing national medical expenses.

Claims

1. An autophagy inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient, or an Atg4B inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient, an agent for enhancing the activity of an anticancer agent. 【Chemistry 1】 (In general formula (1), n ​​is an integer from 2 to 20.)

2. A pharmaceutical composition comprising the anticancer agent activity enhancer according to claim 1 and an anticancer agent.

3. A cancer treatment kit comprising the anticancer agent activity enhancer according to claim 1 and an anticancer agent.

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