Phenylfurocoumarin derivatives or salts thereof, inhibitors of ABC transporter function, and pharmaceuticals for enhancing the antitumor effect of anticancer agents.

Abstract

To provide a novel agent for inhibiting the functions of ABC transporter, and a pharmaceutical for enhancing the antitumor effect of an anticancer drug.SOLUTION: The present invention discloses an agent for inhibiting the functions of ABC transporter, containing a phenyl furocoumarin derivative of a specific structure or a salt thereof, and a pharmaceutical for enhancing the antitumor effect of an anticancer drug.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to phenylfurocoumarin derivatives or salts thereof, inhibitors of ABC transporter function, and pharmaceuticals for enhancing the antitumor effect of anticancer agents. [Background technology]

[0002] Chemotherapy is one of the treatment options for cancer, but cancer cells can develop resistance to anticancer drugs, which often becomes a problem in clinical practice. One mechanism of anticancer drug resistance is the overexpression of ABC (ATP binding cassette) transporters in cancer cells. ABC transporters are membrane transport proteins that use ATP as energy to expel anticancer drugs from inside the cell to outside (Non-Patent Documents 1-3). In particular, ABCG2, along with ABCBl and ABCC1, is representative of multidrug resistance to anticancer drugs. It is thought that if the efflux function of ABCG2 could be suppressed, it would be possible to overcome anticancer drug resistance, but no inhibitors with clinical applicability have been developed to date (Non-Patent Documents 4-7). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Gottesman, MM; Fojo, T.; Bates, SE, Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002, 2 (1), 48-58. [Non-Patent Document 2] Ueda, K., ABC proteins protect the human body and maintain optimal health. Biosci Biotechnol Biochem 2011, 75 (3), 401-9. [Non-Patent Document 3] Shukla, S.; Ohnuma, S.; Ambudkar, S. V., Improving cancer chemotherapy with modulators of ABC drug transporters. Curr Drug Targets 2011, 12 (5), 621-630.

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

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[0004] The present invention provides an inhibitor of the function of a novel ABC transporter. In another embodiment, the present invention provides a medicament for enhancing the antitumor effect of an anticancer agent. [Means for Solving the Problems]

[0005] Under such circumstances, as a result of intensive studies on various compounds, the present inventors have found that a phenylfurocoumarin derivative represented by the general formula (I) or a salt thereof exhibits an inhibitory effect on the function of an ABC transporter. Further, the present inventors have found that the above-mentioned phenylfurocoumarin derivative or a salt thereof that exhibits an inhibitory effect on the function of an ABC transporter enhances the antitumor effect of an anticancer agent. The present invention is based on such new findings.

[0006] Therefore, the present invention provides the following items: Item 1. An inhibitor of the function of an ABC transporter, comprising a phenylfurocoumarin derivative represented by the following general formula (I) or a salt thereof:

[0007] [Chemical formula]

[0008] [In the formula, l represents an integer of 1 to 3. m represents an integer of 0 to 2. n represents an integer of 0 to 5. R represents an alkyl group, an alkoxy group or a halogen atom. When n is 2 or more, a plurality of R may be the same or different.].

[0009] Item 2. Pharmaceuticals for enhancing the antitumor effect of anticancer agents, comprising phenylfurocoumarin derivatives represented by the following general formula (I) or salts thereof:

[0010] [ka]

[0011] [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 0 to 5. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

[0012] Item 3. Pharmaceuticals comprising phenyl furocoumarin derivatives represented by the following general formula (I) or salts thereof, and anticancer agents:

[0013] [ka]

[0014] [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 0 to 5. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

[0015] Section 4. Group

[0016] [ka]

[0017] [In the formula, n and R are the same as above.] However,

[0018] [ka]

[0019] [In the formula, n and R are the same as above.] The functional inhibitor described in item 1, or the pharmaceutical product described in item 2 or 3.

[0020] Item 5. A functional inhibitor as described in Item 1, a pharmaceutical as described in Item 2 or Item 3, or a functional inhibitor or pharmaceutical as described in Item 4, wherein n is an integer from 1 to 3.

[0021] Item 6. Phenylfurocoumarin derivatives or salts thereof represented by the following general formula (I'):

[0022] [ka]

[0023] [In the formula, l' represents an integer from 1 to 3. m' represents an integer from 0 to 2. n' represents an integer from 0 to 5. R' represents an alkyl group, alkoxy group, or halogen atom. If n' is 2 or greater, multiple R's may be the same or different. However,

[0024] [ka]

[0025] [Excluding...]

[0026] Section 7. Group

[0027] [ka]

[0028] [In the formula, n' and R' are the same as above.] However,

[0029] [ka]

[0030] [In the formula, n' and R' are the same as above.] The phenylfurocoumarin derivative or salt thereof as described in item 6.

[0031] Item 8. A phenylfurocoumarin derivative or salt thereof according to item 6 or 7, wherein n' is an integer from 1 to 3. [Effects of the Invention]

[0032] According to the present invention, a phenylfurocoumarin derivative represented by general formula (I) or a salt thereof exerts an inhibitory effect on the function of ABC transporters. Furthermore, a phenylfurocoumarin derivative represented by general formula (I) or a salt thereof can enhance the antitumor effect of anticancer drugs. This enhancement of the antitumor effect is thought to be due to the inhibition of the function of ABC transporters, which expel anticancer drugs from inside the cell to outside the cell. [Brief explanation of the drawing]

[0033] [Figure 1] This document outlines the efflux test of pheopheorbide a (PhA, a fluorescent substrate of ABCG2) using the cell line HCT-116 / BCRP. [Figure 2a-1] The results of the PhA efflux test using flow cytometry are shown. Figures 2a1-2: Histograms. [Figure 2a-2] The results of the PhA efflux test using flow cytometry are shown. Figures 2a1-2: Histograms. [Figure 2b] The results of the PhA efflux test by flow cytometry are shown. Figure 2b: Graph of fluorescence intensity at 10 μM (ratio of each compound to positive control). [Figure 3] Figure 3A: Structural formula of phenylfurocoumarin derivative (PFC, compound 14). Figure 3B: Structural formula of oxypoisedanine. [Figure 4] Figure 4A: Results of cytotoxicity tests of PFC against HCT-116 / BCRP. Figure 4B: Results of cytotoxicity tests of PFC against HCT-116. [Figure 5] The results of the ATP hydrolysis test using PFCs are shown. [Figure 6] The graph shows the ABCG2 expression levels of HCT-116 / BCRP in the presence of HCT-116 / BCRP and PFCs. [Figure 7AB] The results of animal experiments using a mouse subcutaneous tumor model are shown. Figure 7A: Changes in tumor volume. Figure 7B: Changes in mouse body weight. [Figure 7C] The results of animal experiments using a mouse subcutaneous tumor model are shown. [Modes for carrying out the invention]

[0034] Inhibitors of ABC transporter function The present invention provides an inhibitor of ABC transporter function comprising a phenylfurocoumarin derivative represented by the following general formula (I) or a salt thereof:

[0035] [ka]

[0036] [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 0 to 5. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

[0037] In the present invention, the phenyl furocoumarin derivative represented by general formula (I) may also be simply referred to as compound (I).

[0038] In the present invention, unless otherwise specified, "alkyl group" refers to a linear or branched saturated hydrocarbon group, such as a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Specifically, alkyl groups include, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a t-butyl group, an n-pentyl group, an n-hexyl group, and the like.

[0039] In the present invention, unless otherwise specified, "alkoxy group" refers to an alkoxy in which the alkyl portion is the above-mentioned "alkyl group," and examples include alkoxy groups in which the alkyl portion is a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Specifically, alkoxy groups include, for example, methyloxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, t-butoxy group, n-pentyloxy group, n-hexyloxy group, and the like.

[0040] In the present invention, unless otherwise specified, examples of "halogen atoms" include fluorine, chlorine, bromine, iodine, etc., with chlorine being preferred.

[0041] In general formula (I), the alkoxy group is preferably an alkoxy group in which the alkyl portion is a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, more preferably an alkoxy group in which the alkyl portion is a linear or branched saturated hydrocarbon group having 1 to 3 carbon atoms, and even more preferably a methoxy group.

[0042] In general formula (I), l is 1 to 3, preferably 1 to 2, and more preferably 1.

[0043] In general formula (I), m is between 0 and 2, preferably between 0 and 1, and more preferably 0.

[0044] In general formula (I), n is 0 to 5, preferably 0 to 3, and more preferably 0 to 2. When R is an alkoxy group, n is preferably 1 to 2, and more preferably 2.

[0045] In general formula (I), when n is 1 to 5, the position of R relative to the phenyl group is not particularly limited, but it is preferable that R be present at least at the para position. Furthermore, when n is 2 or greater, it is preferable that R be present at least at the para and ortho positions.

[0046] Of compound (I),

[0047] [ka]

[0048] All other compounds are novel.

[0049] Accordingly, in another embodiment, the present invention provides a phenylfurocoumarin derivative or a salt thereof represented by the following general formula (I'):

[0050] [ka]

[0051] [In the formula, l' represents an integer from 1 to 3. m' represents an integer from 0 to 2. n' represents an integer from 0 to 5. R' represents an alkyl group, alkoxy group, or halogen atom. If n' is 2 or greater, multiple R's may be the same or different. However,

[0052] [ka]

[0053] [Excluding...]

[0054] In the present invention, the phenylfurocoumarin derivative represented by general formula (I') may also be simply referred to as compound (I').

[0055] In general formula (I'), the alkoxy group is preferably an alkoxy group in which the alkyl portion is a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, more preferably an alkoxy group in which the alkyl portion is a linear or branched saturated hydrocarbon group having 1 to 3 carbon atoms, and even more preferably a methoxy group.

[0056] In general formula (I'), l' is 1 to 3, preferably 1 to 2, and more preferably 1.

[0057] In general formula (I'), m' is between 0 and 2, preferably between 0 and 1, and more preferably 0.

[0058] In general formula (I'), n' is 0 to 5, preferably 0 to 3, and more preferably 0 to 2. In a typical embodiment, in general formula (I'), n' is 1. When R' is an alkoxy group, n is preferably 1 to 2.

[0059] In general formula (I'), when n' is 1 to 5, the position of R' relative to the phenyl group is not particularly limited, but it is preferable that R' be present at least at the para position. Furthermore, when n' is 2 or greater, it is preferable that R' be present at least at the para and ortho positions.

[0060] Compound (I) can be produced, for example, by the production methods described below or by the methods shown in the examples. However, the production methods for compound (I) of the present invention are not limited to these reaction examples.

[0061] [ka]

[0062] [In the formula, l', m', n', and R' are as described above.] In the above reaction equation, first, compound (i) is reacted with N-bromosuccinimide to produce compound (ii). In this reaction, the ratio of compound (i) to N-bromosuccinimide used is not particularly limited, but can be appropriately set in the range of 1:1 to 1:3, preferably 1:1 to 1:1.5, in terms of molar ratio. The reaction temperature is also not particularly limited, but can be appropriately set in the range of -80 to 50°C, preferably -20 to 30°C. The reaction time is also not limited, but can be appropriately set in the range of 1 to 12 hours, preferably 2 to 5 hours. As the reaction solvent, for example, acetonitrile, methylene chloride, N,N-dimethylformamide, etc. can be used. These solvents can be used individually or in combination of several types. Acetic acid, phosphoric acid, etc. may be used as a catalyst. When these catalysts are used, their concentration is not limited, but can be appropriately set in the range of 0.5 to 3 M, preferably 1 to 2 M. When these catalysts are used, they can be used individually or in combination of several types.

[0063] Next, compound (ii) and compound (iii) are reacted to produce compound (I'). In this reaction, the ratio of compound (ii) to compound (iii) used is not particularly limited, but can be appropriately set in the range of 1:1 to 1:3, preferably 1:1 to 1:1.5, in terms of molar ratio. The reaction temperature is also not particularly limited, but can be appropriately set in the range of 0 to 120°C, preferably 50 to 100°C. The reaction time is also not limited, but can be appropriately set in the range of 2 to 24 hours, preferably 6 to 12 hours. As the reaction solvent, for example, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, etc. can be used. These solvents can be used individually or in combination of several types. Furthermore, as a catalyst, tetrakis(triphenylphosphine)palladium(O), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·dichloromethane complex, etc. may be used. When using these catalysts, their concentrations are not limited, but can be appropriately set within the range of, for example, 0.001 to 0.05 M, preferably 0.002 to 0.01 M. When using these catalysts, they can be used individually or in combination of multiple types.

[0064] The salts of compound (I), which is the active ingredient of the present invention, include salts of acid addition salts and bases. Specific examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, and phosphate; organic acid salts such as oxalate, malonate, succinate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate; and acidic amino acid salts such as glutamate and aspartate. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, or calcium salt; salts with organic bases such as pyridine salt and triethylamine salt; and salts with basic amino acids such as lysine and arginine.

[0065] Since compound (I), which is the active ingredient of the present invention, and its salts may also exist in the form of hydrates or solvates, these hydrates and solvates are also included in the compound that is the active ingredient of the present invention.

[0066] Examples of solvents that form solvates include alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, and DMSO (dimethyl sulfoxide).

[0067] Examples of ABC transporters targeted by the inhibitor of the present invention include ABCG2, ABCBl, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, and others, with ABCG2 being a typical example.

[0068] In the present invention, compound (I), which is the active ingredient of the present invention, or its salt itself may be used as an inhibitor of ABC transporter function, or it may be used as a composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.).

[0069] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0070] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, as well as ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid.

[0071] Examples of stabilizers include sodium bisulfite. Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.

[0072] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and other quaternary ammonium salts, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0073] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

[0074] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol.

[0075] Examples of viscosity-concentrating agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0076] In embodiments of the composition, the content of compound (I) or its salt in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc., in terms of the content of compound (I).

[0077] In embodiments of the composition, the content of compound (I) or its salt in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc., in terms of the content of compound (I).

[0078] Pharmaceuticals to enhance the antitumor effect of anticancer drugs In another embodiment, the present invention provides a pharmaceutical agent comprising compound (I) or a salt thereof for enhancing the antitumor effect of an anticancer agent.

[0079] Details regarding the compound (I) and its salt in this embodiment are as described above. In this embodiment as well, the compound (I) or its salt, which is the active ingredient of the present invention, may be used as a pharmaceutical, or it may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.).

[0080] In embodiments of the pharmaceutical composition, the content of compound (I) or its salt in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc., in terms of the content of compound (I).

[0081] The formulation form is not particularly limited and can include various formulation forms such as oral preparations like tablets, pills, capsules, powders, granules, and syrups; and parenteral preparations such as injections (intravenous injection, intramuscular injection, local injection, etc.), mouthwashes, drips, topical preparations (ointments, creams, patches, inhalants), and suppositories. Among the above formulation forms, preferred ones include, for example, oral preparations (tablets, pills, capsules, powders, granules, syrups, etc.).

[0082] In the present invention, the dosage of compound (I) or its salt varies depending on the route of administration, the patient's age, weight, symptoms, etc., and cannot be specified in general terms. However, the dosage of compound (I) for adults should be such that the daily dose is usually about 5000 mg or less, preferably about 1000 mg or less. Furthermore, according to the present invention, compound (I) is effective even at low doses, so the daily dose of compound (I) for adults may be about 100 mg or less, about 10 mg or less, about 8 mg or less, about 5 mg or less, etc. The lower limit of the dosage of compound (I) or its salt is not particularly limited, and for example, the daily dose of compound (I) for adults can be appropriately set in the range of usually 0.1 mg or more, preferably 0.5 mg or more. If administered once a day, this amount should be contained in one formulation, and if administered three times a day, one-third of this amount should be contained in one formulation.

[0083] The pharmaceutical agent for enhancing the antitumor effect of the anticancer agent of the present invention is administered to patients such as mammals. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cattle, horses, and sheep, with humans being preferred.

[0084] In the embodiment for enhancing the antitumor effect, the anticancer agent to be combined with compound (I) or a salt thereof is not particularly limited, but typically includes those that are excreted extracellularly by ABC transporters. Those that can be excreted extracellularly by ABC transporters are preferred. From the viewpoint of ease of excretion into extracellular space via ABC transporters, antitumor agents with a molecular weight of 300 to 2000 da are preferred, and those with a molecular weight of 400 da or more are more preferred. Furthermore, from the viewpoint of ease of excretion into extracellular space via ABC transporters, antitumor agents with relatively high lipid solubility are preferred. For example, those with a log P value of 2.9 or higher are preferred, and those with a log P value of 5 or higher are more preferred (Non-patent documents 8, 9). Specific examples of anticancer agents include chemotherapy anticancer agents such as irinotecan, SN-38, doxorubicin, daunorubicin, etoposide, mitoxantrone, and topotecan; and molecular targeted drugs such as imatinib, nilotinib, dasatinib, and gefitinib.

[0085] In this embodiment, examples of cancers that can be treated include solid tumors such as breast cancer, colorectal cancer, adrenal cancer, liver cancer, and lung cancer; and blood cancers such as leukemia and lymphoma.

[0086] In this embodiment, the ratio of compound (I) or its salt to the anticancer agent is not limited, but can be appropriately set in the range of 0.0001 to 10000 parts by mass, preferably 0.001 to 1000 parts by mass, more preferably 0.01 to 100 parts by mass, and even more preferably 0.1 to 10 parts by mass, per 1 part by mass of the former.

[0087] Pharmaceuticals, including anticancer drugs In another embodiment, a pharmaceutical product comprising compound (I) or a salt thereof and an anticancer agent is provided. In the present invention, "a pharmaceutical product comprising compound (I) or a salt thereof and an anticancer agent" includes not only embodiments in which compound (I) or a salt thereof and an anticancer agent are contained in a single formulation, but also embodiments in which compound (I) or a salt thereof and an anticancer agent are contained in separate formulations. In this embodiment, a pharmaceutical product comprising compound (I) or a salt thereof and an anticancer agent can be used for the prevention or treatment of cancer.

[0088] Details regarding the contents of compound (I) and its salt, as well as the method of use, in such embodiments are as described above. [Examples]

[0089] Example 1 8-(3,4-dimethoxyphenyl)oxypoisedanine (compound 14)

[0090] [ka]

[0091] Oxypoisedanine (910 mg, 3.18 mmol) was dissolved in acetonitrile (1.0 mL) and acetic acid (0.1 mL), and the mixture was cooled to 0°C and stirred. Subsequently, N-bromosuccinimide (700 mg, 3.93 mmol) was added and the mixture was stirred for 4 hours. Water (10 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (15 mL). The resulting ethyl acetate layers were combined, washed with water (25 mL) and saturated saline (25 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluted with hexane-ethyl acetate (3:2)) to obtain 8-bromooxypoisedanine (501 mg, 1.37 mmol, yield 43%). Under an argon atmosphere, 8-bromooxypoisedanine (52.0 mg, 0.143 mmol), 3,4-dimethoxyphenylboronic acid (30.5 mg, 0.167 mmol), potassium carbonate (56.4 mg, 0.408 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·dichloromethane complex (8.9 mg, 0.011 mmol) were mixed with tetrahydrofuran (2.0 mL). After heating under reflux for 12 hours, the mixture was cooled to room temperature and ethyl acetate (5.0 mL) was added. The reaction mixture was filtered through Celite, and the filtrate was removed by reduced pressure. The residue was purified by silica gel column chromatography (eluted with hexane-ethyl acetate (1:1)) to obtain 8-(3,4-dimethoxyphenyl)oxypoisedanine (37.3 mg, 0.088 mmol, yield 62%). The products were analyzed by mass spectrometry using EIMS (electron impact mass spectrometry) and NMR. The EIMS and NMR results are shown below. EIMS m / z (rel. int.)422 [M] + (100), 337(94), 306(45) HREIMS m / z 422.1354 [M] + (C 24 H 22 The calculated value for O7 is 422.1364. 1 1H NMR (CDCl3, 400 MHz) δ 8.25 (1H, d, J = 9.8 Hz), 7.61 (1H, d, J = 2.3 Hz), 7.25 (1H, dd, J = 8.4, 2.0 Hz), 7.20 (1H, d, J = 2.0 Hz), 7.01 (1H, d, J = 8.4 Hz), 6.98 (1H, d, J = 2.3 Hz), 6.33 (1H, d, J= 9.8 Hz), 4.60 (1H, dd, J = 11.0, 4.3 Hz), 4.43 (1H, dd, J = 11.0, 6.6 Hz), 3.93 (3H, s), 3.90 (3H, s), 3.24 (1H, dd, J = 6.6, 4.3 Hz), 1.40 (3H, s), 1.33 (3H, s).

[0092] Example 2 8-Phenyloxypoisedenine (Compound A)

[0093] [Chemical formula]

[0094] The title compound was synthesized according to the same production method as in Example 1, except that phenylboronic acid was used instead of dimethoxyphenylboronic acid.

[0095] EIMS m / z (rel. int.)362 [M] + (78), 278(100), 249(29), 85(98) HREIMS m / z 362.1177 [M] + (C 22 H 18 O5 calculated value 362.1154). 1 H NMR (CDCl3, 400 MHz) δ 8.26 (1H, d, J = 9.4 Hz), 7.67 (2H, d, J = 7.8 Hz), 7.61 (1H, d, J = 2.0 Hz), 7.48-7.52 (2H, m), 7.43 (1H, d, J= 7.8 Hz), 6.98 (1H, d, J = 2.0 Hz), 6.33 (1H, d, J = 9.4 Hz), 4.64 (1H, dd, J = 11.2, 3.8 Hz), 4.43 (1H, dd, J = 11.2, 7.2 Hz), 3.26 (1H, dd, J = 7.2, 3.8 Hz), 1.43 (3H, s), 1.37 (3H, s).

[0096] Example 3 8-(4-chlorophenyl)oxypoisedanine (compound B)

[0097] [ka]

[0098] The indicated compound was synthesized according to the same method as in Example 1, except that 4-chlorophenylboronic acid was used instead of dimethoxyphenylboronic acid.

[0099] EIMS m / z (rel. int.)398 [M+2] + (20), 396 [M] + (59), 312(84), 85(100) HREIMS m / z 396.0754 [M] + (C 22 H 17 The calculated value for O5Cl is 396.0765. 1 H NMR (CDCl3, 400 MHz) δ 8.26 (1H, d, J = 9.1 Hz), 7.63 (2H, d, J = 7.4 Hz), 7.61 (1H, d, J = 1.9 Hz), 7.46 (2H, d, J = 7.4 Hz), 7.00 (1H, d, J = 1.9 Hz), 6.33 (1H, d, J = 9.1 Hz), 4.64 (1H, dd, J= 10.8, 4.0 Hz), 4.45 (1H, dd, J = 10.8, 6.6 Hz), 3.27 (1H, dd, J= 6.6, 4.0 Hz), 1.43 (3H, s), 1.37 (3H, s).

[0100] Example 4 8-(4-methylphenyl)oxypoisedanine (compound C)

[0101] [ka]

[0102] The indicated compound was synthesized according to the same method as in Example 1, except that 4-methylphenylboronic acid was used instead of dimethoxyphenylboronic acid.

[0103] EIMS m / z (rel. int.)376 [M] + (79), 292(100), 263(34), 85(60) HREIMS m / z 376.1304 [M] + (C 23 H 20 O5's calculated value is 376.1311). 1 H NMR (CDCl3, 400 MHz) δ 8.26 (1H, d, J = 8.9 Hz), 7.60 (1H, d, J = 2.4 Hz), 7.56 (2H, d, J = 7.5 Hz), 7.33 (2H, d, J = 7.5 Hz), 6.97 (1H, d, J = 2.4 Hz), 6.32 (1H, d, J = 8.9 Hz), 4.62 (1H, dd, J= 11.2, 4.0 Hz), 4.44 (1H, dd, J = 11.2, 7.0 Hz), 3.27 (1H, dd, J= 7.0, 4.0 Hz), 2.42 (3H, s), 1.44 (3H, s), 1.37 (3H, s).

[0104] Example 5 8-(4-methoxyphenyl)oxypoisedanine (compound D)

[0105] [ka]

[0106] Compound D was synthesized according to the same procedure as in Example 1, except that 4-methoxyphenylboronic acid was used instead of dimethoxyphenylboronic acid. EIMS m / z (rel. int.)392 [M] + (86), 308(100), 279(44) HREIMS m / z 392.1239 [M] + (C 23 H 20 The calculated value for O6 is 392.1260. 1 H NMR (CDCl3, 400 MHz) δ 8.28 (1H, d, J = 9.2 Hz), 7.63 (2H, d, J = 8.4 Hz), 7.61 (1H, d, J = 2.1 Hz), 7.02 (2H, d, J = 8.4 Hz), 6.98 (1H, d, J = 2.1 Hz), 6.33 (1H, d, J = 9.2 Hz), 4.63 (1H, dd, J= 11.0, 4.0 Hz), 4.43 (1H, dd, J = 11.0, 6.8 Hz), 3.87 (3H, s), 3.28 (1H, dd, J = 6.8, 4.0 Hz), 1.42 (3H, s), 1.35 (3H, s).

[0107] Test example Test method I. Cell culture Human colorectal cancer cell lines HCT-116 and HCT-116 / BCRP were used. HCT-116 / BCRP is a resistant strain of HCT-116 obtained by introducing the ABCG2 gene using a retrovirus and overexpressing it. HCT-116 and HCT-116 / BCRP cells were provided by Professor Yoshikazu Sugimoto of the Department of Chemotherapy, Graduate School of Pharmaceutical Sciences, Keio University. These cell lines were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Sigma) at 37°C under 5% carbon dioxide. 10% FBS (fetal bovine serum; Sigma) and 1% penicillin-streptomycin (Sigma) were added to the culture medium.

[0108] II. Compounds In this study, compounds 1-14 were selected from the compound library managed by the Graduate School of Pharmaceutical Sciences, Tohoku University. The structures of each compound are described in Tables 1-1 to 1-2 below. Each compound was dissolved in DMSO to a concentration of 2 μM and provided in 2 μL portions in 384-well plates.

[0109] III. PhA efflux testing by flow cytometry Flow cytometry was used to investigate the inhibitory effect of each compound on ABCG2 function. The cell line used was HCT-116 / BCRP. Pheopheorbide a (PhA), a fluorescent substrate of ABCG2, is taken up into HCT-116 cells, increasing its intracellular fluorescence intensity. However, in HCT-116 / BCRP cells that overexpress ABCG2, pheopheorbide a is exported to the extracellular space by transporters, resulting in decreased intracellular fluorescence intensity. When an ABCG2 function inhibitor is used in combination, drug export to the extracellular space is suppressed, leading to accumulation within the cell and increased fluorescence intensity. Therefore, it is possible to evaluate the inhibition of ABCG2 function by measuring the fluorescence intensity of PhA (Figure 1).

[0110] Cultured HCT-116 / BCRP cells 3 × 10 5 Cells were prepared in IMDM (Iscove's Modified Dulbecco's Medium, Thermo Fisher Scientific) at a cell concentration of cells / ml and added to FACS tubes (Becton, Dickinson and Company). Pheophorbide a was added to the culture medium to a concentration of 10 μM, and a tube containing 1 μM of Ko143 (Tocris bioscience), a known inhibitor of the ABCG2 transporter, was used as a positive control. Compound 1 was added to the other tubes at concentrations of 1, 5, and 10 μM, respectively, and incubated in a 37°C incubator under light protection for 30 minutes. After incubation, the cultures were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and each was resuspended in 1 mL of IMDM. Subsequently, without administering pheophorbide a, Ko143 and each candidate drug were added in the same manner as before, and incubated in a 37°C incubator under light protection for 1 hour. After incubation, the pellet was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the pellet was resuspended in 300 μl of PBS (-). Fluorescence intensity was measured using BD FACS Verse (Becton, Dickinson and Company), with a PerCP-Cy5.5 filter (excitation wavelength 488 nm, maximum fluorescence wavelength 690 nm). The inhibitory effect of each compound on ABCG2 function was measured in the same manner as above, except that compounds 2-14 were used instead of compound 1.

[0111] IV. Cytotoxicity Test Cytotoxicity tests (MTS assays) were performed on compounds 1-14 using CellTiter 96 AQueous One Solution Reagent (Promega). First, HCT-116 / BCRP cells were seeded into 96-well plates. At that time, the number of cells per well (100 μL) was 1 × 10⁶. 4 The cells were adjusted to a specific concentration. After seeding, they were cultured for 24 hours at 37°C in 5% carbon dioxide. The supernatant was then removed, and culture media with varying concentrations of SN-38 (an active metabolite of the anticancer drug irinotecan) were added to each well. As a positive control, Ko143 was added to each well at a concentration of 1 μM, or compound 1 at 5 or 10 μM, both adjusted with PBS (-). The volume per well was 100 μL. The plates administered with the compounds were cultured for 72 hours under the same conditions as above. After 72 hours, the culture medium was replaced with 100 μL, and 20 μL of MTS solution was added. After 1 hour of culture, the absorbance (492 nm) was measured using a Multiskan FC plate reader (Thermo Fisher Scientific). IC25 of compound 1 50 The inhibition concentration (50%) was calculated using JMP Pro 14.0 (SAS International INC.). To confirm the effects of the candidate drugs identified in this study and Ko143 on parental strains that do not express ABCG2, cytotoxicity tests were performed using HCT-116 with the same method as above. Furthermore, to evaluate the toxicity of compound 1, cytotoxicity tests were performed using culture media with gradually varied concentrations of the candidate drugs and Ko143, with the same method as above.

[0112] Cytotoxicity tests for each compound were performed in the same manner as described above, except that compounds 2-14 were used instead of compound 1.

[0113] V. ATP hydrolysis test When a substrate binds to the substrate binding site of ABCG2, the transporter is activated using the energy of ATP, and the substrate is expelled from the cell. In this process, ATP is hydrolyzed into ADP and inorganic phosphate (Non-Patent Literature 10). In this experiment, by measuring the amount of inorganic phosphate produced in a color reaction, it is possible to determine the effect of the target drug on ATP hydrolysis (Non-Patent Literature 10, 11).

[0114] In this study, we used cell membrane proteins extracted from insect cells that had been infected with baclovirus genetically modified with human ABCG2 cDNA (Non-Patent Literature 12). These proteins were provided by Dr. Suresh V. Ambudkar. 2×ATPase buffer and H2O were added to ATPase assay tubes, and 6 μg of the cell membrane protein was added. Compound 14, adjusted to various concentrations (final concentrations ranging from 0.15 to 5.0 μM), was then added to each tube. Two types of tubes were prepared for each concentration: one with 0.3 mM orthovanadic acid (Vi) (Sigma) added (Vi(+)) and one without (Vi(-)). After adding 5 mM ATP, the mixtures were allowed to stand in a 37°C incubator for 20 minutes. Subsequently, 5% SDS (sodium dodecyl sulfate) was added to stop the reaction. To investigate the degree of the ATP reaction by measuring the amount of inorganic phosphate produced by ATP hydrolysis, inorganic phosphate reagent and 1% ascorbic acid were added, a colorimetric reaction was performed, and the mixture was allowed to stand at room temperature for 10 minutes. Afterward, the absorbance was measured at 880 nm using an absorbance reader, Ultraspec 3100 pro (Amersham Biosciences). Since Vi inhibits the ATP reaction by ABCG2, the ATP hydrolysis by ABCG2 was calculated by determining the difference between the reaction in the Vi (-) tube (ATP reaction of the entire protein) and the reaction in the Vi (+) tube (ATP reaction other than ABCG2). The EC of each reaction was measured. 50 The half maximal effective concentration (50% effective concentration) was calculated using JMP Pro 14.0 (SAS International INC.).

[0115] VI. Confirmation of ABCG2 expression (RT-qPCR) Quantitative real-time PCR was performed to confirm the degree of ABCG2 mRNA expression in the HCT-116 / BCRP cell line and the effect of compound 14 on ABCG2 expression. HCT-116 cells, HCT-116 / BCRP cells, and HCT-116 / BCRP cells cultured for 3 days in the presence of compound 14 (10 μM) were used. Total RNA was extracted from the cell lines using the RNeasy Mini Kit (QIAGEN), and sufficient concentration was confirmed using NanoDrop2000 (Thermo Scientific). The extracted total RNA was reverse transcribed using the PrimeScript RT reagent Kit (TakaraBIO) to synthesize cDNA (complementary DNA). Real-time PCR reactions were performed using the StepOnePlus Real Time PCR System (Life Technologies), SYBR Premix Ex Taq II (Tli RNaseH Plus), and ROX plus (TAKARA BIO), and the ΔΔCt was calculated using the 2-ΔΔCt method. The reaction conditions consisted of 40 cycles, each cycle being 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the endogenous control. mRNA expression levels were quantified relative to GAPDH and expressed as a percentage of the control. The measured mRNA levels were expressed as relative amounts to HCT-116 cells.

[0116] The sequence information of the primers used in RT-qPCR is shown below. ABCG2 (forward):5'-GGTCAGAGTGTGGTTTCTGTAGCA-3'(Sequence ID 1) ABCG2 (reverse):5'-GTGAGAGATCGATGCCCTGCTTTA-3' (Sequence ID 2) GAPDH (forward): 5'-GCACCGTCAAGGCTGAGAAC-3' (Sequence ID 3) GAPDH (reverse):5'-TGGTGAAGACGCCAGTGGA-3 (Sequence ID 4)

[0117] VII. Animal Experiment Models A subcutaneous tumor transplantation model was created using 8-10 week old female BALB / c nu-nu mice, and animal experiments were conducted to investigate the effect of compound 14 on the sensitivity of anticancer drugs. In this study, an animal experiment protocol approved by the Tohoku University Animal Experiment Center was used (approval number: 2019 Ido-164), and the "Regulations and Commentary on Animal Experiments, etc. at Tohoku University, National University Corporation, 14th Edition" was followed. The cell line used was HCT-116 / BCRP, which overexpresses ABCG2, and 2 × 10⁶ 7 Cells were suspended in PBS (-) to a concentration of cells / mL. This cell suspension was then mixed with Matrigel matrix (Corning) in a 2:1 ratio on ice, and 200 μL was dispensed into 1 mL syringes. Subcutaneous transplantation was performed on the left and right backs of mice under a three-part mixed anesthesia (medetomidine hydrochloride + midazolam + butorphanol tartrate). After subcutaneous transplantation, the engraftment status of the tumors was checked every 1-2 days, and drug administration was performed when the tumor diameter reached 5-10 mm in its longest diameter. For the anticancer drug, based on previous reports, irinotecan at a concentration of 30 mg / kg was used for intraperitoneal administration (Non-Patent Literature 13, 14). Furthermore, since there were no previous reports on the concentration of candidate drugs, it was decided to administer them intraperitoneally at a concentration of 10 mg / kg, referring to the previously reported Ko143, which was used as a positive control in in vitro experiments (Non-Patent Literature 15). The mice were then divided into the following four groups (n=5) for drug administration. a) 400 μL of physiological saline was administered intraperitoneally, b) 30 mg / kg of irinotecan was diluted in physiological saline and administered intraperitoneally in 400 μL, c) 10 mg / kg of the candidate drug and 30 mg / kg of irinotecan were diluted in physiological saline and administered intraperitoneally in 400 μL, and d) 10 mg / kg of the candidate drug was diluted in physiological saline and administered intraperitoneally in 400 μL. For drug preparation, the candidate drug was dissolved in DMSO to a concentration of 5% w / v and then diluted in physiological saline, while irinotecan, which was obtained in solution form, was diluted directly in physiological saline. The first administration day was designated as day 0, and a total of 7 administrations were performed every 3 days on days 3, 6, 9, 12, 15, and 18. Body weight and tumor diameter were measured on the day of drug administration, and the calculation was 0.5 × (long diameter) × (short diameter). 2 The tumor volume was calculated. Then, on day 21, after measuring body weight and tumor diameter, the mice were euthanized by deep anesthesia and cervical dislocation. The subcutaneous tumor was removed and its weight was measured.

[0118] VIII. Statistical analysis Due to the limited volume of screening compounds, fluorescent dye efflux tests using flow cytometry were performed only once per compound. In subsequent experiments, three or more independent tests were conducted, and the mean, standard deviation, and standard error were calculated. Student's t-test was used to test the mean. JMP Pro 14.0 (SAS International INC.) was used for statistical analysis, and P < 0.05 was considered statistically significant.

[0119] Test results I. PhA efflux testing by flow cytometry Flow cytometry was performed on compounds 1-14 shown in Tables 1-1 to 1-2 below. The histograms of the results are shown in Figure 2a, 1-2. Although there were differences in degree, concentration-dependent enhancement of fluorescence intensity was observed for most compounds. Figure 2b shows the ratio of fluorescence intensity at 10 μM for each compound to that of the positive control. This was calculated using the median fluorescence intensity in flow cytometry. Six compounds showed less than 30% of the positive control's fluorescence intensity at 10 μM, and these were considered to have a low inhibitory effect on ABCG2. The following cytotoxicity tests were performed on the remaining 16 compounds.

[0120] [Table 1-1]

[0121] [Table 1-2]

[0122] II. Cytotoxicity Tests (1) Effects on sensitivity to anticancer drugs Cytotoxicity studies (MTS assays) against HCT-116 / BCRP were performed using seven compounds that showed effective inhibitory effects in flow cytometry analysis. The results are shown in Table 2.

[0123] [Table 2]

[0124] Compounds No. 7 and No. 11 have strong cytotoxicity, IC 50 It was not possible to evaluate it. No. 14 is a phenylfurocoumarin derivative. * IC without Ko143 and candidate compounds (Medium) 50 The relative susceptibility enhancement rate is shown with the value set to 1.0.

[0125] Positive control Ko143 1 μM IC 50 Compound 14 showed an IC50 of 0.26 ± 0.027, while compound 14 showed an IC50 of 5 μM. 50 IC was 0.36 ± 0.11 μM and 10 μM. 50 The value was 0.19 ± 0.018 μM. Relative susceptibility enhancement rate (fold reversal: IC under conditions without candidate drug and positive control) 50 The fold reversal (calculated as a ratio) was 10.1 times at 1 μM for Ko143, 7.3 times at 5 μM for No. 22, and 13.8 times at 10 μM for No. 22, with compound 14 at 10 μM showing the greatest enhancement of SN-38 sensitivity. This compound 14 (in this test example, compound 14 may be referred to as a phenylfurocoumarin derivative, PFC) was considered to be the most effective ABCG2 inhibitor and was used in subsequent experiments. The structural formula of PFC is shown in Figure 3A, and the results of the cytotoxicity test are shown in Figure 4A. Furthermore, a similar cytotoxicity test was performed on oxypoisedanin (Figure 3B, dotted line indicates common structure), which has a similar structure to this compound. The fold reversal of oxypoisedanin was 2.35 times at 5 μM and 3.0 times at 10 μM, suggesting that its ABCG2 inhibitory effect was weaker compared to PFC and Ko143.

[0126] (2) Evaluation of cytotoxicity of HCT-116 A similar MTS assay was performed to evaluate the cytotoxicity of PFCs to HCT-116. The results are shown in Table 3 and Figure 4B.

[0127] [Table 3]

[0128] As shown in Table 3 and Figure 4B, informed consent for chemotherapy alone 50 This is 0.11 ± 0.0037 μM, IC in the presence of 1 μM Ko143. 50 IC in the presence of 0.11 ± 0.0036 μM of PFC and 5 μM of PFC. 50 This is 0.077 ± 0.022 μM, IC in the presence of 10 μM PFC. 50 The concentration was 0.057 ± 0.013 μM. PFC administration resulted in a slight increase in IC (immediately impaired concentration). 50 Although a decrease was observed, it was not as pronounced as the results with HCT-116 / BCRP.

[0129] (3) Toxicity assessment of the compound Toxicity evaluations of Ko143 and PFC against HCT-116 and HCT-116 / BCRP were performed on each individual. The results are shown in Table 4.

[0130] [Table 4]

[0131] The Ko143 1 μM and PFC 10 μM used in previous experiments were suggested to have no toxicity significant enough to affect the experimental results.

[0132] III. ATP Hydrolysis Test The effect of PFCs on ATP hydrolysis was evaluated. The results are shown in Figure 5. As shown in Figure 5, PFCs promote ATP hydrolysis in a concentration-dependent manner, and EC 50 The concentration was 0.038 ± 0.0064 μM. This suggests that PFC is a compound that promotes ATP hydrolysis in ABCG2, that is, a compound that acts on the substrate binding site of ABCG2.

[0133] IV. Effects on ABCG2 expression RT-qPCR was performed to evaluate ABCG2 expression in HCT-116 / BCRP and the effect of PFCs on ABCG2 expression. The ABCG2 expression levels in HCT-116 / BCRP and HCT-116 / BCRP in the presence of PFCs were calculated relative to the ABCG2 expression level in the parental HCT-116 strain, and the results are shown in Figure 6. There was no significant difference between the two, suggesting that PFCs do not affect ABCG2 expression.

[0134] V. Animal experiments using a mouse subcutaneous tumor model We investigated whether PFC enhances irinotecan sensitivity in a mouse subcutaneous tumor model. The mice were divided into four groups and administered the therapeutic agent intraperitoneally. The changes in tumor volume are shown in Figure 7A. The rate of increase was evaluated using the volume on the first day of administration as the baseline. Since this study focused on the extent to which PFC enhances irinotecan sensitivity, we compared two groups: b) the irinotecan-only group and c) the phenylfurocoumarin derivative + irinotecan-only group. From day 12 onwards, the tumor growth rate was significantly smaller in c) the irinotecan-only group. Furthermore, although the mice's body weight gradually decreased, the decrease remained below 20% in all groups (Figure 7B). Regarding tumor weight, there was no significant difference between the b) irinotecan-administered group and the c) phenylfurocoumarin derivative + irinotecan-administered group. However, the tumors tended to be smaller in the c) group (207.8 ± 70.4 mg) and the c) group (159.7 ± 37.9 mg) (Figure 7C).

[0135] 4. Discussion As shown in the above tests, compound 14 (PFC) showed inhibition of substrate efflux in flow cytometry and enhanced sensitivity to anticancer drugs in cytotoxicity tests. Furthermore, considering the results of ATPase assay and RT-qPCR, it was thought that PFC did not alter ABCG2 expression but acted directly on the substrate binding site of ABCG2, exhibiting an inhibitory effect. In addition, in a subcutaneous tumor model of nude mice, PFC enhanced sensitivity to irinotecan without exhibiting significant side effects.< / em>

Claims

1. An ABC transporter function inhibitor comprising a phenylfurocoumarin derivative represented by the following general formula (I) or a salt thereof: 【Chemistry 1】 [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 1 to 3. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

2. A pharmaceutical product for enhancing the antitumor effect of an anticancer agent, comprising a phenylfurocoumarin derivative represented by the following general formula (I) or a salt thereof: 【Chemistry 2】 [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 1 to 3. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

3. Pharmaceuticals for antitumor treatment comprising phenylfurocoumarin derivatives represented by the following general formula (I) or salts thereof, and anticancer agents: 【Transformation 3】 [In the formula, l represents an integer from 1 to 3. m represents an integer from 0 to 2. n represents an integer from 1 to 3. R represents an alkyl group, alkoxy group, or halogen atom. If n is 2 or greater, multiple Rs may be the same or different.]

4. basis 【Chemistry 4】 [In the formula, n and R are the same as above.] However, 【Transformation 5】 [In the formula, n'' represents an integer between 0 and 2. R is the same as above.] The functional inhibitor described in claim 1 or The pharmaceutical product according to claim 2 or claim 3.

5. Phenylfurocoumarin derivatives or salts thereof represented by the following general formula (I'): 【Transformation 6】 [In the formula, l' represents an integer from 1 to 3. m' represents an integer from 0 to 2. n' represents an integer from 1 to 3. R' represents an alkyl group, alkoxy group, or halogen atom. If n' is 2 or more, the multiple R's may be the same or different. However, 【Transformation 7】 [Excluding...]

6. basis 【Transformation 8】 [In the formula, n' and R' are the same as above.] However, 【Chemistry 9】 [In the formula, n''' represents an integer between 0 and 2. R' is the same as above.] The phenylfurocoumarin derivative or salt thereof according to claim 5.

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