Compound, adjuvant composition, and pharmaceutical composition

Abstract

To provide an alternative technique for the treatment or prevention of melanoma.SOLUTION: The adjuvant composition contains a compound represented by a specific structural formula and is used in combination with a BRAF inhibitor for administration to a subject.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present disclosure relates to adjuvant compositions. [Background technology]

[0002] Melanoma is known as a type of intractable cancer, and BRAF inhibitors are sometimes used as therapeutic agents (for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Proietti, I. et al. Mechanisms of acquired BRAF inhibitor resistance in melanoma: a systematic review. Cancers 12, 2801 (2020). Summary of the Invention [Problem to be solved by the invention]

[0004] As described in Non-Patent Document 1, monotherapy with BRAF inhibitors has the problem of developing resistance within about six months. Furthermore, monotherapy with BRAF inhibitors carries the risk of developing other skin cancers. Therefore, multidrug combination therapy is sometimes used, and in Japan, MEK inhibitors, which inhibit RAS / MEK / ERK signaling, are currently approved for use in combination with immune checkpoint inhibitors. However, MEK inhibitors and immune checkpoint inhibitors are often unable to be used in many cases due to the risk of serious side effects such as liver dysfunction, interstitial lung disease, and hypersensitivity reactions. Furthermore, the 5-year survival rate for stage IV melanoma, for which drug therapy is primarily used, is only 10%. Therefore, the development of other technologies for the treatment or prevention of melanoma is needed. [Means for solving the problem]

[0005] The present invention can be realized as the following aspects.

[0006] (1) According to one aspect of the present invention, there is provided a compound or a salt thereof, which is represented by the following structural formula (1): [ka] This form of the compound or a salt thereof has AKR1B10 inhibitory activity and can therefore be used to treat or prevent melanoma.

[0007] (2) According to another aspect of the present invention, there is provided an adjuvant composition comprising the compound according to (1) above or a salt thereof, which is administered to a subject in combination with a BRAF inhibitor. The adjuvant composition of this aspect can enhance the effect of the BRAF inhibitor when used in combination with the BRAF inhibitor.

[0008] (3) In the adjuvant composition described in (2) above, the BRAF inhibitor may comprise Vemurafenib. This form of the adjuvant composition can enhance the effect of Vemurafenib.

[0009] (4) Another aspect of the present invention provides a pharmaceutical composition for use in treating or preventing melanoma, comprising a BRAF inhibitor and a compound represented by the following structural formula (1) or a salt thereof: [ka] This form of pharmaceutical composition can enhance the action of a BRAF inhibitor through inhibition of AKR1B10, and can therefore be used to treat or prevent melanoma.

[0010] The present disclosure can be realized in various forms, for example, a method for producing a compound represented by the above structural formula (1) or a salt thereof, a method for producing an adjuvant composition, a method for producing a pharmaceutical composition used in the treatment or prevention of melanoma, an AKR1B10 inhibitor, a method for producing an AKR1B10 inhibitor, a compound that enhances the activity of a BRAF inhibitor, a method for producing a compound that enhances the activity of a BRAF inhibitor, a compound that enhances the activity of Vemurafenib, a method for producing a compound that enhances the activity of Vemurafenib, a therapeutic or preventive agent for melanoma, a method for producing a therapeutic or preventive agent for melanoma, use of a compound represented by the above structural formula (1) or a salt thereof for producing an AKR1B10 inhibitor, use of a compound represented by the above structural formula (1) or a salt thereof for producing an adjuvant composition, use of a compound represented by the above structural formula (1) or a salt thereof for producing a pharmaceutical composition used in the treatment or prevention of melanoma, a method for treating melanoma, a method for preventing melanoma, etc. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is an explanatory diagram showing the results of Vemurafenib sensitivity evaluation. [Figure 2] FIG. 1 is an explanatory diagram showing the results of AKR1B10 gene expression analysis using quantitative PCR. [Figure 3] FIG. 1 is an explanatory diagram showing the results of AKR1B10 expression analysis using Western blotting. [Figure 4] FIG. 1 is an explanatory diagram showing the results of Vemurafenib sensitivity evaluation. [Figure 5] FIG. 1 is an explanatory diagram showing combination indexes. [Figure 6] FIG. 1 is an explanatory diagram showing the measurement results of the amount of ROS produced. [Figure 7] FIG. 1 is an explanatory diagram showing a comparison of the amount of ROS produced in each treatment group. [Figure 8] FIG. 1 is an explanatory diagram showing the results of Western blotting. [Figure 9] FIG. 1 is an explanatory diagram showing the quantification results of each protein. [Figure 10]FIG. 1 is an explanatory diagram showing the results of fluorescent immunostaining. DETAILED DESCRIPTION OF THE INVENTION

[0012] According to one aspect of the present disclosure, there is provided a compound represented by the following structural formula (1) or a salt thereof:

[0013] [ka]

[0014] The compound represented by the structural formula (1) is 5,7-Dihydroxy-2-[(1E)-2-(2-chloro-4-hydroxyphenyl)ethenyl]-8-methyl-4H-1-benzopyran-4-one. In the following description, the compound represented by the structural formula (1) is also referred to as "Compound A" for convenience. Compound A has AKR1B10 inhibitory activity, as shown in the examples below. Therefore, according to another aspect of the present disclosure, an AKR1B10 inhibitor comprising Compound A or a salt thereof is provided.

[0015] The main roles of AKR1B10 in cancer include promoting proliferation by activating Ras / MAPK signaling via prenylation, controlling differentiation by suppressing retinoic acid synthesis through the reduction of retinal, and suppressing oxidative stress by reducing and detoxifying lipid peroxide-derived aldehydes. AKR1B10 has been suggested to be involved in cancer progression and anticancer drug resistance. AKR1B10 is highly expressed in many cancer types (lung cancer, breast cancer, pancreatic cancer, liver cancer, etc.) and has attracted attention as a diagnostic marker. It is known that its expression is further upregulated with anticancer drug resistance [Endo et al., Metabolites, 11, 332 (2021)]. Therefore, AKR1B10 inhibitors are thought to suppress cancer cell proliferation and survival, and are expected to be used as adjuvant drugs to enhance the efficacy of anticancer drugs and overcome resistance.

[0016] Compound A may be in the form of a salt, more specifically, a pharmacologically acceptable salt. In the present disclosure, the term "pharmacologically acceptable salt" refers to a salt of an active compound that is a relatively non-toxic acid or base addition salt. Examples of acid addition salts include, but are not limited to, hydrochloride, hydrobromide, nitrate, carbonate, phosphate, acetate, propionate, isobutyrate, maleate, malonate, benzoate, succinate, fumarate, lactate, benzenesulfonate, p-toluenesulfonate, citrate, tartrate, oxalate, and methanesulfonate. In the present disclosure, the term "pharmacologically acceptable salt" also includes a pharmacologically acceptable solvate or a salt thereof. A solvate is a stoichiometric complex of a molecule with one or more solvent molecules. Examples of pharmacologically acceptable solvates include, but are not limited to, water, methanol, ethanol, dimethyl sulfoxide, acetate, and the like. Solvates that contain water as the solvent are equivalent to hydrates.

[0017] Compound A includes isomers such as optical isomers, geometric isomers, and tautomers. Compound A also includes hydrates, solvates, and crystalline polymorphs thereof. Compound A also includes pharmacologically acceptable prodrugs. In the present disclosure, a "pharmacologically acceptable prodrug" means a compound that is converted to compound A by a reaction with an enzyme, gastric acid, or the like under physiological conditions in a living body. In other words, it includes compounds that are converted to compound A by enzymatic oxidation, reduction, hydrolysis, or the like, and compounds that are converted to compound A by hydrolysis, or the like, with gastric acid, or the like.

[0018] The following describes a method for producing Compound A. Compound A can be synthesized, for example, by the reaction steps shown below.

[0019] [ka]

[0020] Compounds 1 and 2 in the above reaction steps can be synthesized, for example, with reference to a report by Yin, Huanhuan et al. (European Journal of Medicinal Chemistry (2019), 180, 350-366). The following synthesis method can be used as an example. Under an argon atmosphere, a solution of methylchromone compound 2 (100 mg, 0.43 mmol, 1.0 equiv.) in methanol (5 mL) is added to the corresponding aldehyde compound 3 (0.52 mmol, 1.2 equiv.) and sodium methoxide (116 mg, 2.15 mmol, 5.0 equiv.) under ice cooling. The reaction solution is then stirred at 70°C for 42 hours. After cooling, the reaction is stopped by adding 10% hydrochloric acid. The mixture is then extracted with dichloromethane (3 mL x 3), and the resulting organic layer is dried over sodium sulfate. After the solvent is removed using a rotary evaporator, the resulting residue may be used directly in the next reaction without purification. The resulting residue is dissolved in 1,2-dichloroethane (5 mL) under an argon atmosphere, and boron tribromide (1 M in CHCl, 3.44 mL, 3.44 mmol, 8.0 equiv.) is added under ice cooling. The reaction solution is then stirred at 80°C for 17 hours. After cooling, the reaction solution is diluted with methanol, and the solvent is removed using a rotary evaporator. The resulting residue can be purified by silica gel column chromatography to obtain compound A.

[0021] According to another aspect of the present disclosure, there is provided an adjuvant composition comprising Compound A or a salt thereof, which is administered to a subject in combination with a BRAF inhibitor.

[0022] In the present disclosure, the term "adjuvant composition" refers to a composition that can enhance the effect of another substance when administered in combination with that substance. The adjuvant composition of the present disclosure enhances the effect of the BRAF inhibitor when administered to a subject in combination with the BRAF inhibitor. The adjuvant composition of the present disclosure can be used for the treatment or prevention of melanoma (malignant melanoma).

[0023] In the present disclosure, the term "BRAF inhibitor" refers to a composition capable of inhibiting the activity of mutant BRAF, a type of protein kinase. Examples of BRAF inhibitors include Vemurafenib.

[0024] "Treatment" in this disclosure includes producing at least one effect from the following: extension of progression-free survival (PFS), extension of overall survival (OS), extension of disease-free survival (DFS), extension of time to progression (TTP), extension of event-free survival (EFS), extension of recurrence-free survival (RFS), reduction in cancer cell count, reduction in tumor size, inhibition (delay or halt) of tumor growth, inhibition (delay or halt) of tumor metastasis, inhibition (prevention or delay) of recurrence, and alleviation of one or more symptoms associated with cancer. Furthermore, "prevention" in this disclosure includes prevention of melanoma onset and delay of melanoma onset.

[0025] The subjects of the present disclosure include, for example, melanoma patients and those suspected of having melanoma. Melanoma patients may be patients with multiple types of cancer, patients either before or after chemotherapy with a BRAF inhibitor or other anticancer agent, patients either before or after surgery, patients either before or after immunotherapy, or patients either before or after radiation therapy. The gender and age of the subjects are not particularly limited. The target animal species are primarily mammals. Examples of such mammals include, but are not limited to, humans, primates such as chimpanzees, dogs, cats, rabbits, and the like.

[0026] The term "administered in combination" includes simultaneous administration of the compounds in the same or different dosage forms, or separate administration (e.g., sequential administration) of the compounds. More specifically, the compounds may be administered in the form of a combined preparation in which multiple components are combined in a single formulation, or as separate formulations. Separate administration includes simultaneous administration and administration at different times. Staggered administration may involve administering the adjuvant composition first and the BRAF inhibitor later, or the BRAF inhibitor first and the adjuvant composition later. The administration methods may be the same or different. The adjuvant composition of the present disclosure may be administered before or after the development of resistance to the BRAF inhibitor in a subject. The adjuvant composition of the present disclosure is suitable for treating or preventing melanoma after the development of resistance to a BRAF inhibitor, and is particularly suitable for treating or preventing melanoma after the development of resistance to vemurafenib.

[0027] The concentration of the adjuvant composition of the present disclosure is not particularly limited, but may be, for example, 5 μM to 10 μM relative to 1 μM to 10 μM of Vemurafenib administered in combination.

[0028] The adjuvant composition of the present disclosure may be formulated by appropriately blending Compound A with a pharmaceutically acceptable carrier or additive. Specifically, the composition may be in the form of oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, or emulsions, or parenteral preparations such as injections, infusions, suppositories, ointments, or patches. The blending ratio of the carrier or additive may be appropriately set based on the range commonly used in the pharmaceutical field. The carrier or additive is not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0029] According to another aspect of the present disclosure, there is provided a pharmaceutical composition for use in the treatment or prevention of melanoma. This pharmaceutical composition comprises a BRAF inhibitor and compound A or a salt thereof. This pharmaceutical composition corresponds to a combination of the BRAF inhibitor and the aforementioned compound A or a salt thereof. In this pharmaceutical composition, the BRAF inhibitor and the aforementioned compound A or a salt thereof may be formulated separately as two separate drugs, or may be mixed together and formulated as a single drug. The formulation may be as described above for the formulation of the adjuvant composition.

[0030] Compound A or a salt thereof, adjuvant composition, and pharmaceutical composition of the present disclosure can be used for the treatment or prevention of melanoma. More specifically, because compound A has AKR1B10 inhibitory activity, it can enhance the action of a BRAF inhibitor through AKR1B10 inhibition. As a result, it can be used for the treatment or prevention of melanoma. Compound A or a salt thereof of the present disclosure can also be used to overcome resistance to BRAF inhibitors. Furthermore, because compound A or a salt thereof, adjuvant composition, and pharmaceutical composition of the present disclosure are small molecule drugs, they are easier to produce than antibody drugs, and increases in production costs can be suppressed. As a result, drug prices can be reduced compared to antibody drugs, contributing to improved treatment opportunities for patients. [Example]

[0031] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0032] 1. Method (1) Synthesis of Compound A Compound A was synthesized according to the reaction steps described above. Under an argon atmosphere, a solution of methylchromone compound 2 (100 mg, 0.43 mmol, 1.0 equiv.) in methanol (5 mL) was added to the corresponding aldehyde compound 3 (0.52 mmol, 1.2 equiv.) and sodium methoxide (116 mg, 2.15 mmol, 5.0 equiv.) under ice cooling. The reaction solution was then stirred at 70 °C for 42 hours. After cooling, the reaction was terminated by adding 10% hydrochloric acid. The mixture was then extracted with dichloromethane (3 mL x 3), and the resulting organic layer was dried over sodium sulfate. The solvent was removed using a rotary evaporator, and the resulting residue was used directly in the next reaction without purification. The resulting residue was dissolved in 1,2-dichloroethane (5 mL) under an argon atmosphere, and boron tribromide (1 M in CHCl, 3.44 mL, 3.44 mmol, 8.0 equiv.) was added under ice cooling. The reaction solution was then stirred at 80°C for 17 hours. After cooling, the reaction solution was diluted with methanol, and the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography to obtain compound A. 87% yield in 2 steps (yellow solid); 1 H-NMR (500 MHz, acetone-d6) δ: 9.42 (br, 1H), 8.02 (d, 1H, J = 16.0 Hz), 7.84 (d, 1H, J = 8.5 Hz), 7.04 (d, 1H, J = 16.0 Hz), 7.00 (s, 1H), 6.92 (dd, 1H, J = 8.5 Hz, 2.0 Hz), 6.31 (s, 1H), 6.24 (s, 1H), 2.29 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ: 182.0, 162.2, 161.9, 159.9, 158.9, 154.3, 134.7, 131.2, 128.6, 123.2, 119.3, 116.2, 115.6, 107.9, 104.0, 101.7, 98.1, 7.3; IR (KBr) 3429, 3221, 2924, 1693, 1655, 1543, 1522, 1420, 1259, 1169 cm -1 ; mp: 295-297 ℃; HRMS (ESI) : Calcd for C 18 H 13 O5Cl 345.0524 ([M+H] + ), Found 345.0514.

[0033] (2) Evaluation of AKR1B10 inhibitory activity The dehydrogenase activity of AKR1B10 was determined by spectrophotometrically measuring the rate of NADPH decomposition (340 nm) in the following standard reaction system: 0.1 M potassium phosphate buffer (pH 7.4), 0.1 mM NADPH, 0.2 mM pyridine-3-aldehyde, and enzyme in a total volume of 2.0 mL. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of NADPH per minute at 25°C.

[0034] (3) Measurement of cell viability Cell viability was measured using the Alamar blue assay. Cells suspended in growth medium were plated at 2 × 10 cells per well in a 96-well multiplate. 4Cells were seeded at 200 μL per well and then cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS (Sigma-Aldrich), after which the samples were added and cultured for an additional 24 hours. As a control, cells were prepared with DMSO (dimethylsulfoxide). The medium was then replaced with serum-free and phenol red-free medium, and 50 μM resazurin (Sigma-Aldrich) was added. After culturing at 37°C for 2-4 hours, the absorbance was measured at a wavelength of 570 nm and a reference wavelength of 595 nm using an iMark microplate reader (BIO RAD). Cell viability (%) was calculated using the following formula: Cell viability (%)=(SA) / (BA)×100 (In the formula, S is the absorbance of the well to which the sample and cells were added, A is the absorbance of the well to which only the medium was added, and B is the absorbance of the well to which DMSO and cells were added.)

[0035] (4) Quantitative PCR Total RNA was extracted using RNAzol RT Reagent (Molecular Research Center Inc.). The extracted total RNA was reverse-transcribed using a ReverTra Ace qPCR RT kit (Toyobo Co., Ltd.) by incubation at 37°C for 30 minutes and then at 98°C for 5 minutes. Single-stranded complementary DNA (cDNA) was prepared by reverse transcription. Quantitative PCR was performed using the prepared cDNA (1 μg) as a template and Thunderbird SYBR qPCR Mix (Toyobo Co., Ltd.). The sequences of the specific primers used for quantitative PCR are listed in Table 1 below, along with their sequence numbers. PCR was performed using a quantitative PCR system, CFX96 Deep Well Real-Time System (BIO RAD). The PCR reaction temperature conditions were a 95°C / 30-second heat treatment followed by 40 cycles of 95°C / 15 seconds and 55°C / 1 minute. Human β-actin cDNA was amplified as an internal standard.

[0036] [Table 1]

[0037] (5) Western blot analysis The treated cells were washed twice with DPBS (Dulbecco's phosphate-buffered saline, Nissui Pharmaceutical Co., Ltd.) (pH 7.4) and then detached using a cell scraper. DPBS contains 0.25% trypsin and 0.02% EDTA. After washing with DPBS, the collected cells were suspended in 50 mM sodium phosphate buffer (pH 7.4) containing 8 M urea and 10 mM tris (hydroxymethyl) aminomethane, followed by sonication to disrupt the cell membrane. The cell lysate was centrifuged (12,000 × g, 10 min, 4°C), and the supernatant was used as the cell extract. Proteins were separated by SDS-PAGE using 10%, 12.5%, or 15% polyacrylamide gels, and then electrophoretically transferred to a PVDF (polyvinylidene difluoride) membrane (Merck Millipore). After blocking with 1% BSA (bovine serum albumin), the PVDF membrane was sequentially reacted with each primary antibody and a horseradish peroxidase (HRP)-conjugated mouse secondary antibody (#1031-05, SouthernBiotech). Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). The luminescence intensity of each band was quantified using image analysis software Image J (National Institutes of Health).

[0038] (6) Measurement of Reactive Oxygen Species (ROS) production After removing the medium from the variously stimulated cells, 1 ml of RPMI medium containing 5% (v / v) FBS, 100 U / ml penicillin-G potassium, and 100 μg / ml streptomycin sulfate, and 2 μL of 10 mM 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA) were added and incubated for 20 minutes. Cells were detached using 0.083% trypsin and collected by centrifugation (1,000 × g, 5 minutes, 16°C). After removing the supernatant, 1 ml of 4% paraformaldehyde phosphate buffer solution was added and incubated for 30 minutes. The cells were then centrifuged (1,000 × g, 5 minutes, 16°C), the supernatant was removed, and the cells were washed twice with DPBS and resuspended in 1 ml of DPBS. Just before measurement, the samples were passed through a nylon filter and subjected to flow cytometry. ROS production was measured using a BD FACS Verse Flow Cytometer.

[0039] (7) Fluorescent immunostaining Cells suspended in growth medium were placed in a 24-well multiplate at 1.5 × 10 5Cells were seeded at 500 μL per well and cultured overnight at 37 °C under 5% CO₂. The medium was replaced with a medium containing antibiotics and 2% FBS, and the sample was added to the medium and cultured for an additional 24 hours. As a control group, cells with DMSO added were prepared. After removing the medium, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added and fixed for 10 minutes. 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added and left standing for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added and blocked for 1 hour. After washing twice with DPBS, it was replaced with a primary antibody (anti-Ki67 antibody) solution diluted 300:1 in DPBS and incubated overnight at 4 °C. After washing twice with PBS, it was replaced with a DyLight594-labeled mouse secondary antibody solution diluted 500:1 in DPBS and incubated for 1 hour in the dark at room temperature. After washing twice with DPBS, excess moisture was removed, and a coverslip was fixed on a slide glass using a mounting agent (DAPI fluoromount-G). The cells subjected to fluorescence immunostaining were set on a confocal laser microscope LSM700 (manufactured by Carl Zeiss), and fluorescence observation was performed. Images were captured using a 40× oil immersion objective lens of an inverted fluorescence microscope.

[0040] 2. Experimental content and results <Evaluation of vemurafenib sensitivity and analysis of AKR1B10 expression in vemurafenib-resistant melanoma C32TG cells> Vemurafenib-resistant melanoma C32TG cells were established. More specifically, melanoma C32TG cells (hereinafter also referred to as "C32TG cells") were made less sensitive to Vemurafenib by gradually increasing the Vemurafenib concentration over a period of about half a year, thereby establishing Vemurafenib-resistant melanoma C32TG cells (hereinafter also referred to as "C32TG / Vem cells"). Using C32TG and the established C32TG / Vem, Vemurafenib sensitivity evaluation and AKR1B10 expression analysis were performed. The Vemurafenib sensitivity evaluation was evaluated by measuring the cell viability in the presence of 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM Vemurafenib. As the AKR1B10 expression analysis, gene expression analysis using quantitative PCR and protein expression analysis using Western blotting were performed.

[0041] Figure 1 is an explanatory diagram showing the results of the Vemurafenib sensitivity evaluation. In Figure 1, the vertical axis indicates the percentage of viable cell numbers, and "**" indicates that there is a significant difference at the 1% significance level compared to C32TG cells. In C32TG cells, the viability decreased with Vemurafenib concentrations of 1 μM or higher, whereas in C32TG / Vem cells, no significant decrease in viability was confirmed up to a concentration of 10 μM.

[0042] Figure 2 is an explanatory diagram showing the results of the AKR1B10 gene expression analysis using quantitative PCR. Figure 3 is an explanatory diagram showing the results of the AKR1B10 expression analysis using Western blotting. As shown in Figures 2 and 3, high expression of AKR1B10 was confirmed in both the mRNA amount and protein amount of AKR1B10 in C32TG / Vem cells.

[0043] <Evaluation of AKR1B10 inhibitory activity> The inhibitory activity of compound A on AKR1B10 was evaluated. The IC of compound A 50The value was calculated from the inhibition rates when compounds A with five different concentrations were added to the above-mentioned standard reaction system. These inhibition constants were expressed as the average value ± standard deviation of at least three measurements. The IC 50 of compound A against AKR1B10 was 14 ± 1.9 nM. Therefore, it was revealed that compound A exhibits strong inhibitory activity against AKR1B10.

[0044] <Confirmation of the effect of compound A on the vemurafenib sensitivity of C32TG / Vem cells> The vemurafenib sensitivity of C32TG / Vem cells was evaluated by measuring the cell viability in the presence of 0 μM, 1 μM, 2 μM, 5 μM, 10 μM vemurafenib and 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM compound A. In addition, the synergy of the combined use of vemurafenib and compound A was evaluated using the CompuSyn program.

[0045] Figure 4 is an explanatory diagram showing the results of the vemurafenib sensitivity evaluation. In Figure 4, the vertical axis indicates the percentage of viable cells, and the horizontal axis indicates the concentration of vemurafenib (μM). Figure 5 is an explanatory diagram showing the combination index. As shown in Figure 5, since it was confirmed that the combination index (CI) was less than 1, it was revealed that there is a synergistic effect by the combined use of vemurafenib and compound A. That is, it was shown that compound A can enhance the anticancer activity of vemurafenib.

[0046] <Confirmation of the combined effect of vemurafenib and compound A in C32TG / Vem cells> The combined effects of Vemurafenib and Compound A on C32TG / Vem cells were investigated. Reactive oxygen species (ROS) production was assessed in 10 μM Vemurafenib monotherapy and in 10 μM Vemurafenib and 20 μM Compound A monotherapy, and the effect on apoptosis was also examined. A control group was treated with neither Vemurafenib nor Compound A. ROS production was measured using flow cytometry, and the effect on apoptosis was assessed by Western blotting of apoptosis-related protein expression and immunofluorescence staining using anti-Ki67 antibody. 20 μM Compound A monotherapy was also evaluated by immunofluorescence staining.

[0047] FIG. 6 is an explanatory diagram showing the measurement results of the amount of ROS produced. In FIG. 6, the vertical axis represents the number of cells, and the horizontal axis represents DCF fluorescence intensity. FIG. 7 is an explanatory diagram showing a comparison of the amount of ROS produced in each treatment group. In FIG. 7, the amount of ROS produced in each treatment group in the region indicated by M1 in FIG. 6 is shown relative to the control, with the control being set at 100%. Note that "**" indicates a significant difference at the 1% significance level compared to the control, and "##" indicates a significant difference at the 1% significance level compared to the group treated with Vemurafenib alone. As shown in FIGS. 6 and 7, it was found that the amount of reactive oxygen induced by Vemurafenib was significantly increased by the combined use of Compound A.

[0048] Figure 8 is an explanatory diagram showing the results of Western blot. Figure 9 is an explanatory diagram showing the quantification results of each protein. In Figure 9, the MEK phosphorylation level (%), p53 phosphorylation level (%), Bax / Bcl-2 expression level (%), and PARP cleavage level (%) are shown relative to the control, with the control set at 100%. "**" and "*" indicate significant differences from the control at the 1% and 5% significance levels, respectively, and "##" and "#" indicate significant differences from Vemurafenib at the 1% and 5% significance levels, respectively. In addition, "NS" in the Vemurafenib-only treatment group indicates a significant difference from the control at the 5% significance level, and "NS" in the combination treatment group indicates a significant difference from the Vemurafenib-only treatment group at the 5% significance level.

[0049] The results shown in Figures 8 and 9 reveal the following. Specifically, because Vemurafenib is a BRAF inhibitor, it significantly inhibited the phosphorylation of MEK, which is located downstream of BRAF. Furthermore, the combined use of Compound A did not change the MEK phosphorylation level, but reduced the total MEK protein amount. The combined use of Vemurafenib and Compound A significantly increased p53 phosphorylation, Bax / Bcl-2 expression ratio, and PARP cleavage, suggesting an enhancement of apoptotic cell death.

[0050] Figure 10 is an explanatory diagram showing the results of fluorescent immunostaining. Figure 10 shows images stained with DAPI and Ki67, as well as a merged image of each stained image. As shown in Figure 10, it was found that the combined use of Vemurafenib and Compound A significantly reduced the expression level of Ki67, a proliferation marker.

[0051] These results suggest that Compound A can be used as an adjuvant drug to enhance the activity of the BRAF inhibitor Vemurafenib by inhibiting AKR1B10. Furthermore, the use of Compound A as an adjuvant drug can help overcome resistance to the BRAF inhibitor Vemurafenib.

[0052] The present invention is not limited to the above-described embodiments and can be realized in various configurations without departing from the spirit of the present invention. For example, the technical features in the embodiments and examples corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A compound represented by the following structural formula (1) or a salt thereof: 【Chemical 1】

2. An adjuvant composition comprising the compound of claim 1 or a salt thereof, It is administered to a subject in combination with a BRAF inhibitor. Adjuvant compositions.

3. The adjuvant composition according to claim 2, the BRAF inhibitor comprises vemurafenib; Adjuvant compositions.

4. A pharmaceutical composition for use in treating or preventing melanoma, comprising: A pharmaceutical composition comprising a BRAF inhibitor and a compound represented by the following structural formula (1) or a salt thereof: 【Chemistry 2】

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