JP5936213B2 - PDT effect enhancer - Google Patents

Description

  The present invention relates to a light beam comprising a compound used in combination with a photosensitizer, 5-aminolevulinic acid (5-ALA) or a derivative thereof, or a salt thereof (hereinafter, these may be collectively referred to as “ALAs”). The present invention relates to an effect enhancer for mechanical therapy (PDT).

  Cancer (malignant tumor) is the largest cause of death in Japan, and it is said that one out of two Japanese people will be affected. In other developed countries, cancer is ranked among the top causes of death. Therefore, the development of effective treatments for cancer is a long-cherished desire for the people of Japan and the world. However, because cancer is a patient's own cells, it is not easy to develop effective therapeutic agents and treatment methods that can effectively treat cancer without side effects, and there are still sufficient therapeutic agents and treatment methods. Is not obtained.

  In recent years, photodynamic therapy (hereinafter also referred to as “PDT”), which has been widely used, has recently been easy to treat, has little bioinvasiveness, and can preserve organs. , It is attracting attention as a new cancer treatment method considering QOL. In PDT, administered porphyrin-related compounds are directly accumulated in tumor tissues and new blood vessels, and then singlet oxygen generated by excitation of light such as laser light causes the cells to degenerate and become necrotic. It is something that demonstrates.

  Photosensitizers approved in Japan for cancer treatment include hematoporphyrin derivative porfimer sodium (trade name: Photofrin) and talaporfin sodium derived from plant chlorophyll (trade name: resaphyrin) . Porfimer sodium is known to cause sunlight hypersensitivity because it has a slow metabolism and tends to accumulate in the vicinity of epidermal tissue.

  On the other hand, 5-ALA is a kind of natural amino acid that is widely contained in animals, plants, and fungi and is contained in the living body, and is metabolized in cells as a common precursor of chlorophyll and heme. 5-ALA itself is not photosensitive, and in PDT, administered ALAs are metabolized to proporphyrin IX (PpIX) by a series of enzymes in the heme biosynthetic pathway, and PpIX is directly It is said that singlet oxygen generated by the specific accumulation in tumor tissues and new blood vessels, which are excited by light such as laser light, causes the cells to degenerate and become necrotic, thereby producing a therapeutic effect. Studies on photodynamic therapy (ALA-PDT) using a kind are being conducted. Further, as an action enhancer in PDT, a cancer cell necrosis action enhancer, which is a photodynamic therapy agent, composed of a vasodilator anesthetic has been reported (for example, see Patent Document 1).

  However, it is also known that there are so-called ALA-PDT resistant cells with low ALA-PDT efficiency depending on the cancer cell type and malignancy. For example, in cells where ABCG2, which is one of the ABC transporter families using porphyrins such as PpIX as a substrate in cancer cells, is highly expressed, PpIX is excreted into the cytoplasm, and PpIX does not accumulate sufficiently. ALA-PDT It has been confirmed that the effect may be low (see Non-Patent Document 1). The present inventors have previously used an ABCG2 inhibitor to block ABCG2 itself, or by using an anticancer agent which is a substrate for ABCG2 transporter and 5-aminolevulinic acid in combination. We have devised methods to enhance the anticancer effects of drugs.

  However, high expression of ABCG2 is not necessarily observed in all ALA-PDT resistant cells, and the cause of ALA-PDT resistance cannot be attributed to ABCG2. Some cancer cells exhibiting ALA-PDT resistance, on the contrary, have low expression of ABCG2, and naturally these cells are not effective by the above-described ABCG2 inhibitor or ABCG2 blocking method, It was practically the limit of treatment with PDT.

Table 2007/086395

Tamura, A. et al, (2007) Drug Metabolism and Pharmacokinetics 22, No. 6, 428-440

  The subject of this invention is providing the enhancer which can enhance a PDT effect with respect to a target cell, especially the ALA-PDT resistant cancer cell with low expression of ABCG2 in performing PDT.

  The present inventors have demonstrated that the effect of 18 kinds of compounds on ALA-PDT is enhanced by MKN-45 of human gastric cancer cells that have been confirmed to be 5-ALA-PDT resistant cancer cells with low expression of ABCG2. When examined using cells, in ALA-PDT, N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5- It has been found that when dichlorobenzaldehyde, 5-chlorosalicylaldehyde, or 3-chloro-2-hydroxybenzaldehyde is used in combination with ALAs, the effect of PDT is remarkably enhanced, and the enhancer of the present invention Confirmed that it has no effect on normal cells, and completed the present invention.

  That is, the present invention provides the following formula (I), which is used in combination with [1] a photosensitizer or aminolevulinic acid (ALA).

[Wherein, R ¹ represents —CHO or a group that can be converted to —CHO, and R ³ and R ⁵ each represent H or an electron-withdrawing group (provided that at least one is an electron-withdrawing group). , R ² , R ⁴ , and R ⁶ each represent H or —OH], relates to an effect enhancer for photodynamic therapy (PDT).

In the present invention, [2] the group that can be converted to —CHO in the formula (I) is —CH═NR ⁷ , —CH═NOR ⁸ , or —CH (OR ⁹ ) OR ¹⁰ , and R ⁷ Is a substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched carbon group An alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms, a substituted or unsubstituted carbon number 3-9 heterocyclic group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted naphthyl group, R ⁸ is hydrogen, methanesulfonyl group, trifluoromethanesulfonyl group, a benzenesulfonyl group, or Parato Indicates Ensuruhoniru group, R ^9, R ¹⁰ are each a substituted or unsubstituted straight or branched chain alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, A substituted or unsubstituted linear or branched alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched carbon number 2 -6 alkynyl group, substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, or R ⁹ and R ¹⁰ may constitute one diol molecule, in which case R ⁹ and R ¹⁰ Is an enhancer according to the above [1], which represents a substituted or unsubstituted alkanediol group having 2 to 10 carbon atoms, or a substituted or unsubstituted catechol group, and [3] a compound represented by the formula (I), N-3 ', 5'- Chloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichlorobenzaldehyde, 5-chloro-2-hydroxybenzaldehyde (5-chlorosalicylaldehyde), And 3-chloro-2-hydroxybenzaldehyde (3-chlorosalicylaldehyde), wherein the enhancer according to the above [1] or [4] PDT is intended for cancer. The enhancer according to any one of [1] to [3] above, or [5] the enhancer according to [4] above, wherein the cancer is composed of ABCG-2 low-expressing cells. About.

Further, the present invention provides a compound of the above formula (I) in combination with [6] photosensitizer or aminolevulinic acid (ALA), wherein R ¹ represents a group that can be converted to —CHO or —CHO, R ³ and R ⁵ each represent H or an electron-withdrawing group (however, at least one is an electron-withdrawing group), and R ² , R ⁴ and R ⁶ each represent H or —OH. A method for enhancing the effect of photodynamic therapy (PDT), comprising administering a compound represented by formula (I) to a subject, or [7] the above formula (I) [wherein R ⁷ is substituted or unsubstituted. A linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted straight chain or branched chain alkenyl having 2 to 6 carbon atoms Group, substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms, substituted or unsubstituted 3 to 9 carbon atoms ring group, a substituted or unsubstituted phenyl group or a show substituted or unsubstituted naphthyl group,; R ⁸ is hydrogen, methanesulfonyl group, trifluoromethanesulfonyl group, a benzenesulfonyl group or a p-toluenesulfonyl, It is shown; R ^9, R ¹⁰ are each a substituted or unsubstituted straight or branched chain alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted A substituted straight or branched alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted straight chain or branched chain having 2 to 6 carbon atoms An alkynyl group, a substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, or R ⁹ and R ¹⁰ may constitute one diol molecule, in which case R ⁹ and R ¹⁰ are substituted Alternatively, it represents an unsubstituted alkanediol group having 2 to 10 carbon atoms, or a substituted or unsubstituted catechol group. ] The group which can be converted into -CHO in -CH = NR < ⁷ >, -CH = NOR < ⁸ >, or -CH (OR < ⁹ >) OR < ¹⁰ >, The method of said [6] description characterized by the above-mentioned, [8 The compound represented by the above formula (I) is N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichloro. The method according to [6] above, which is selected from benzaldehyde, 5-chlorosalicylaldehyde, and 3-chloro-2-hydroxybenzaldehyde, and [9] PDT is intended for cancer. The method according to [6] above, or [10] the method according to [9] above, wherein the cancer is composed of ABCG-2 low-expressing cells.

Furthermore, the present invention relates to the above formula (I) [wherein R ¹ ] for enhancing the effect of photodynamic therapy (PDT) in combination with [11] photosensitizer or aminolevulinic acid (ALA). Represents a group that can be converted to -CHO or -CHO, R ³ and R ⁵ each represent H or an electron-withdrawing group (provided that at least one is an electron-withdrawing group), and R ² and R ⁴ , R ⁶ each represents H or —OH. ] [12] The above formula (I) [wherein R ⁷ is a substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted carbon number. 3-6 cyclic alkyl groups, substituted or unsubstituted linear or branched alkenyl groups having 2 to 6 carbon atoms, substituted or unsubstituted cyclic alkenyl groups having 3 to 6 carbon atoms, substituted or unsubstituted A linear or branched alkynyl group having 2 to 6 carbon atoms, a substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted naphthyl group; R ⁸ is hydrogen, methanesulfonyl group, trifluoromethanesulfonyl group, a benzenesulfonyl group, or a p-toluenesulfonyl group; R ^9, R ¹⁰ are each substituted or unsubstituted straight or branched carbon An alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted carbon atom A cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms, a substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, or R ⁹ , R ¹⁰ may constitute one diol molecule, in which case R ⁹ and R ¹⁰ represent a substituted or unsubstituted alkanediol group having 2 to 10 carbon atoms, or a substituted or unsubstituted catechol group. Show. ] The group which can be converted into -CHO is -CH = NR < ⁷ >, -CH = NOR < ⁸ >, or -CH (OR < ⁹ >) OR < ¹⁰ >, [13] The compound represented by the above formula (I) is N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichloro. The compound according to [11] above and [14] PDT, which are selected from benzaldehyde, 5-chlorosalicylaldehyde, and 3-chloro-2-hydroxybenzaldehyde, are intended for cancer. The compound according to any one of [11] to [13] above, or [15] the cancer is composed of ABCG-2 low-expressing cells. Relates to compounds.

Furthermore, the present invention provides the above formula (I) [wherein, for preparing a photodynamic therapy (PDT) effect enhancer, which is used in combination with a photosensitizer or aminolevulinic acid (ALA). R ¹ represents —CHO or a group that can be converted to —CHO, R ³ and R ⁵ each represent H or an electron-withdrawing group (provided that at least one is an electron-withdrawing group), R ² , R ⁴ and R ⁶ each represent H or —OH. Or a compound represented by the following formula (I): wherein R ⁷ is a substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted A cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, substituted or non-substituted A substituted linear or branched alkynyl group having 2 to 6 carbon atoms, a substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted naphthyl group ; R ⁸ is hydrogen, methanesulfonyl group, trifluoromethanesulfonyl group, a benzenesulfonyl group or p-toluenesulfonyl group; R ^9, R ¹⁰ are each a substituted or unsubstituted straight or branched An alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted A cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms, a substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms, or , R ⁹ and R ¹⁰ may constitute one diol molecule, in which case R ⁹ and R ¹⁰ are substituted or unsubstituted alkanediol groups having 2 to 10 carbon atoms, or substituted or unsubstituted catechol. Indicates a group. The group according to [16] above, wherein the group that can be converted to —CHO is —CH═NR ⁷ , —CH═NOR ⁸ , or —CH (OR ⁹ ) OR ^10; The compound represented by the above formula (I) is N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichloro. The use according to [16] above, wherein [19] PDT is selected from cancer, wherein the use is selected from benzaldehyde, 5-chlorosalicylaldehyde, and 3-chloro-2-hydroxybenzaldehyde. The use according to any one of [16] to [18] above, or [20] the use according to [19] above, wherein the cancer is composed of ABCG-2 low-expressing cells. Related.

  According to the present invention, the dose of a photosensitizer and the like in PDT can be reduced, and even when the target cell of PDT is an ALA-PDT resistant cell, the therapeutic effect of PDT can be enhanced. Can be reduced.

The schematic diagram of the evaluation method by an isobologram is shown. When two drug agents A and B are used in combination, when the drug concentration showing 50% cell growth inhibition is plotted on a graph, there is a synergistic effect when these points are on the lower concentration side of the curve (synergistic) Can be determined. N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-prepared to a concentration of 0,0.41,1.23,3.70,11.1,33.3, and 100 μM It is a figure which shows the result at the time of adding 5-chloro-nitroaniline (TX-816) and ALA prepared so that a density | concentration may be 0, 50, 100, and 200 micromol and performing 5-ALA-PDT. . It is a figure which shows the result of having examined the 5-ALA-PDT enhancement effect of TX-816 by the isobologram. It was confirmed that the 5-ALA-PDT enhancing effect of TX-816 belongs to the category of synergistic effects. It is a figure which shows the graph which plotted the cell growth inhibition rate (inhibition ratio: IR) in each density | concentration of TX-816 on the semivariable graph. The IC ₅₀ (50% inhibitory concentration) value was calculated to be 36.5 μM. It is a figure which shows each structure of (a) TX-816, (b) 3, 5- dichloro salicylaldehyde (DCSA), (c) 2-chloro- 4-nitroaniline (CNA). It is a figure which shows the comparison of IR about the test compound (compound number: 3, 6, 7, 8, 11, 12) represented by Table 3. FIG. It is a figure which shows IR comparison about the test compound (reference number: 7-2, 7-3, 7-4, 7-5, 7-6, 7-7) represented by Table 5. FIG. It is a figure which shows the comparison of IR of DCSA and 3-fluorosalicylaldehyde (3-FSA). It is a figure which shows accumulation | storage of intracellular PpIX to MKN-45 cell in 50 micromol 5-ALA single processing (a) and simultaneous processing (b) of 50 micromol 5-ALA and 100 micromol DCSA.

  The PDT effect enhancer of the present invention is not particularly limited as long as it contains a compound represented by the above formula (I) used in combination with a photosensitizer or aminolevulinic acid (ALA), and the PDT of the present invention. There is no particular limitation on the method for enhancing the effect of the present invention as long as it is a method of administering the compound represented by the above formula (I) to a subject in combination with a photosensitizer or aminolevulinic acid (ALA). As a compound that is used in combination with a photosensitizer or aminolevulinic acid (ALA) to enhance the effect of photodynamic therapy (PDT), as long as it is a compound represented by the above formula (I), As the use of the present invention, the use of a compound represented by the above formula (I) for preparing a photodynamic therapy (PDT) effect enhancer in combination with a photosensitizer or aminolevulinic acid (ALA). Especially if limited However, the above-mentioned PDT is a method in which treatment is performed by irradiating a target cell in a lesion site in which a compound that reacts with light is accumulated by administration of a photosensitizer or ALA, and ALA-PDT. Administers ALAs that do not themselves have a photosensitizing action, specifically accumulates PpIX induced through the chromogenic biosynthetic pathway in the target cell, and PpIX is exposed to surrounding oxygen molecules by light irradiation. And the singlet oxygen produced as a result of this exerts a cell killing effect due to its strong oxidizing power. The wavelength of light for exciting the PpIX is 400 nm to 700 nm, preferably 625 nm. ˜642 nm can be mentioned, and 635 nm is particularly preferable.

In the above formula (I), R ¹ represents —CHO or a group that can be converted to —CHO, and R ³ and R ⁵ each represent H or an electron-withdrawing group (provided that at least one is an electron-withdrawing group). R ² , R ⁴ , and R ⁶ each represent H or —OH.

The group that can be converted to —CHO in the present invention is a group that is converted to —CHO by hydrolysis, specifically, an imine represented by —CH═NR ⁷ , an oxime represented by —CH═NOR ⁸ ; or ^-CH (oR 9) can be mentioned acetal represented by ^{oR 10.}

Examples of the electron withdrawing group in R ³ and R ⁵ include fluorine, chlorine, bromine, trifluoromethyl group, nitro group, sulfonyl group, carbonyl group, carboxyl group, and cyano group, and fluorine, chlorine, bromine. And a nitro group are preferable, and fluorine or chlorine is more preferable.

R ⁷ includes a substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear group, or Branched alkenyl group having 2 to 6 carbon atoms, substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, substituted or unsubstituted straight chain or branched chain alkynyl group having 2 to 6 carbon atoms, substituted or Examples thereof include an unsubstituted heterocyclic group having 3 to 9 carbon atoms, a substituted or unsubstituted phenyl group, and a substituted or unsubstituted naphthyl group. 6 alkyl groups, substituted or unsubstituted phenyl groups, or substituted or unsubstituted naphthyl groups are preferred, and substituted or unsubstituted phenyl groups are more preferred.

Examples of R ⁸ include hydrogen, a methanesulfonyl group, a trifluoromethanesulfonyl group, a benzenesulfonyl group, and a paratoluenesulfonyl group.

R ⁹ and R ¹⁰ are each a substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms, substituted or unsubstituted. A linear or branched alkenyl group having 2 to 6 carbon atoms, a substituted or unsubstituted cyclic alkenyl group having 3 to 6 carbon atoms, a substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms Group, substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms,
Alternatively, R ⁹ and R ¹⁰ may constitute one diol molecule, in which case R ⁹ and R ¹⁰ are substituted or unsubstituted alkanediol groups having 2 to 10 carbon atoms, or substituted or unsubstituted. Of catechol groups.

In the above R ⁷ , R ⁹ and R ¹⁰ , each substituted or unsubstituted linear or branched alkyl group having 1 to 6 carbon atoms includes methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl. Group, sec-butyl group, tert-butyl group, cyclopropylmethyl group, pentyl group, isopentyl group, neopentyl group, cyclobutylmethyl group, hexyl group, methylpentyl group, ethylbutyl group, 3,3-dimethyl-butyl- Examples thereof include 2-yl group, cyclopentylmethyl group, 2 ′, 2′-dimethylcyclopropylmethyl group, 1-methylcyclopropylmethyl group, and halogen substituents such as fluorine, chlorine, bromine and iodine.

Specific examples of the substituted or unsubstituted cyclic alkyl group having 3 to 6 carbon atoms in R ⁷ , R ⁹ and R ¹⁰ include a cyclopropyl group, a cyclobutyl group, a 1-methylcyclopropyl group, and a cyclopentyl group. Group, 2 ′, 2′-dimethylcyclopropyl group, 1-methylcyclobutyl group, cyclohexyl group, 1-methylcyclopentyl group, and halogen substituents thereof such as fluorine, chlorine, bromine and iodine, A cyclohexyl group is preferred.

In the above R ⁷ , R ⁹ and R ¹⁰ , each substituted or unsubstituted linear or branched alkenyl group having 2 to 6 carbon atoms includes a vinyl group, an allyl group, a crotonyl group, a butenyl group, and a crotononitrile group. , A pentenyl group, a 2-oxo-3-pentenyl group, a hexenyl group, a cyclopentenylmethyl group, and halogen substituents thereof such as fluorine, chlorine, bromine and iodine, and an allyl group is preferable.

In the above R ⁷ , R ⁹ and R ¹⁰ , each substituted or unsubstituted linear or branched cyclic alkenyl group having 3 to 6 carbon atoms includes a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, and a cyclohexenyl group. , 2-oxo-cyclohexenyl groups, and halogen substituents thereof such as fluorine, chlorine, bromine, iodine, etc., and cyclohexenyl groups are preferred.

In the above R ⁷ , R ⁹ and R ¹⁰ , each substituted or unsubstituted linear or branched alkynyl group having 2 to 6 carbon atoms includes ethynyl group, propargyl group, butynyl group, pentynyl group, hexynyl group, and These halogen substitution products such as fluorine, chlorine, bromine and iodine can be mentioned, and propargyl group is preferred.

In the above R ⁷ , R ⁹ and R ¹⁰ , each substituted or unsubstituted heterocyclic group having 3 to 9 carbon atoms includes pyrrolidinyl group, N-methylpyrrolidinyl group, N-ethylpyrrolidinyl group, N- Acetylpyrrolidinyl group, N-tert-butoxycarbonylpyrrolidinyl group, tetrahydrofuranyl group, 2-oxotetrahydrofuranyl group, 2,5-dioxotetrahydrofuranyl group, 3,4-dihydroxy-5-hydroxymethyltetrahydrofuranyl group Group, tetrahydrothiophenyl group, pyrrolyl group, furyl group, thiophenyl group, methyl-3-yl group of 2-thiophenecarboxylate, methyl-3-yl group of 4-methyl-2-thiophenecarboxylate, piperidinyl group, N-methyl Piperidinyl group, N-ethylpiperidinyl group, N-isopropylpiperidinyl group 4-pyrrolidinylpiperidinyl group, 2,2,6,6-tetramethylpiperidinyl group, tetrahydropyranyl group, tetrahydropyranyl group, 2-oxotetrahydropyranyl group, 2,5-dioxotetrahydro Pyranyl group, 3-acetylamino-4,5-dihydroxy-6-hydroxymethyltetrahydropyranyl group, 3,4,5-trihydroxy-6-hydroxymethyltetrahydropyranyl group, tetrahydrothiopyranyl group, pyridinyl group , Methylpyridinyl group, ethylpyridinyl group, methoxypyridinyl group, ethoxypyridinyl group, methoxycarbonylpyridyl group, nitropyridinyl group, cyanopyridinyl group, 3-nitro-4-methylpyridinyl group, 2-nitro-5-methylpyridinyl group, imidazolyl group N-methylimidazolyl group Methyl imidazolyl group, cyano imidazolyl group, nitro imidazolyl group, pyrazoyl group, N-methyl pyrazoyl group, N-phenyl pyrazoyl group, methyl pyrazoyl group, tert-butyl pyrazoyl group, phenyl pyrazoyl group, thiazolyl group, Nitrothiazolyl, methylthiazolyl, dimethylthiazolyl, methoxythiazolyl, benzothiazolyl, nitrobenzothiazolyl, methylbenzothiazolyl, dimethylbenzothiazolyl, methoxybenzothiazolyl, cyano Imidazolyl group, pyrazinyl group, methylpyrazinyl group, indolyl group, methylindolyl group, benzoimidazolyl group, nitrobenzoimidazolyl group, quinolyl group, isoquinolyl group, chromenyl group, isochromenyl group, and their fluorine, chlorine, bromine, iodine Halogen-substituted derivatives can be mentioned etc., pyridinyl group, chloropyridinyl group, Nitoropirijiniru group, chloro nitro pyridinylalkyl group.

In the above R ⁷ , the substituted or unsubstituted phenyl group includes phenyl group, tolyl group, dimethylphenyl group, trimethylphenyl group, methoxyphenyl group, dimethoxyphenyl group, trimethoxyphenyl group, ethylphenyl group, diethylphenyl group, Triethylphenyl group, ethoxyphenyl group, diethoxyphenyl group, triethoxyphenyl group, ethynylphenyl group, nitrophenyl group, dinitrophenyl group, trinitrophenyl group, 3-methoxy-2-methylphenyl group, methylnitrophenyl group, N, N-dimethylaminophenyl group, propylphenyl group, isopropylphenyl group, isopropoxyphenyl group, 2,3-methylenedioxyphenyl group, 3,4-methylenedioxyphenyl group, butylphenyl group, sec-butylphenol Nyl group, tert-butylphenyl group, 2-methyl-6-ethylphenyl group, 2-methyl-5-isopropylphenyl group, 2-tert-butyl-6-methylphenyl group, biphenyl group, indanyl group, and these Examples include halogen substituents such as fluorine, chlorine, bromine and iodine, such as 2-chloro-4-nitrophenyl group, 2-nitro-4-chlorophenyl group, 2,6-dichloro-4-nitrophenyl group, 2 , 6-Nitro-4-chlorophenyl group, 2,4,6-trichlorophenyl group, 2-chloro-4-cyanophenyl group, 2-cyano-4-chlorophenyl group, 2,6-dichloro-4-cyanophenyl group 2,6-cyano-4-chlorophenyl group is preferable, and 2-chloro-4-nitrophenyl group is more preferable.

In R ⁷ , the substituted or unsubstituted naphthyl group includes a naphthyl group, a methylnaphthyl group, a dimethylnaphthyl group, a trimethylnaphthyl group, a methoxynaphthyl group, a dimethoxynaphthyl group, a trimethoxynaphthyl group, an ethylnaphthyl group, and a diethylnaphthyl group. , Triethylnaphthyl group, ethoxynaphthyl group, diethoxynaphthyl group, triethoxynaphthyl group, nitronaphthyl group, dinitronaphthyl group, trinitronaphthyl group, methylnitronaphthyl group, N, N-dimethylaminonaphthyl group, tetrahydroxynaphthyl group And halogen substituents such as fluorine, chlorine, bromine and iodine, and nitronaphthyl group, chloronaphthyl group and chloronitronaphthyl group are preferable.

In the above R ⁹ and R ¹⁰ , when R ⁹ and R ¹⁰ constitute one diol molecule, the substituted or unsubstituted alkanediol group having 2 to 10 carbon atoms may be an ethylenediol group or 1,2-propanediol. Group, 1,3-propanediol group, 1,2-butanediol group, 1,3-butanediol group, 1,4-butanediol group, 2,3-butanediol group, 1,2-dihydroxy-2- Methylpropane group, 2-methyl-1,3-propanediol group, 2-methyl-1,4-butanediol group, 1,2-pentanediol group, 1,3-pentanediol group, 1,4-pentanediol Group, 1,5-pentanediol group, 2,3-pentanediol group, 2,4-pentanediol group, 1,2-hexanediol group, 1,3-hexanediol group 1,4-hexanediol group, 1,5-hexanediol group, 1,6-hexanediol group, 2,3-hexanediol group, 2,4-hexanediol group, 2,5-hexanediol group, 3, 4-hexanediol group, 1,5-dihydroxy-2-methylpentane group, 1,2-heptanediol group, 1,3-heptanediol group, 1,4-heptanediol group, 1,5-heptanediol group, 1,6-heptanediol group, 1,7-heptanediol group, 2,3-heptanediol group, 2,4-heptanediol group, 2,5-heptanediol group, 2,6-heptanediol group, 3, 4-heptanediol group, 3,5-heptanediol group, 1,2-octanediol group, 1,3-octanediol group, 1,4-octanediol group, 1, -Octanediol group, 1,6-octanediol group, 1,7-octanediol group, 1,8-octanediol group, 2,3-octanediol group, 2,4-octanediol group, 2,5-octane Diol group, 2,6-octanediol group, 2,7-octanediol group, 3,4-octanediol group, 3,5-octanediol group, 3,6-octanediol group, and fluorine, chlorine thereof, And halogen-substituted products such as bromine and iodine, such as ethylenediol group, 1,2-propanediol group, 1,3-propanediol group, 1,2-butanediol group, 1,3-butanediol group, 2,3-butanediol group, 1,2-pentanediol group, 1,3-pentanediol group, 2,3-pentanediol group, 2,4-pentanediol Group, 1,2-hexanediol group, 1,3-hexanediol group, 2,3-hexanediol group, 2,4-hexanediol group, 3,4-hexanediol group, ethylenediol group, 1 1,2-propanediol group, 1,3-propanediol group, 1,3-butanediol group, 2,4-pentanediol 2,4-hexanediol group are more preferable.

In the above R ⁹ and R ¹⁰ , when R ⁹ and R ¹⁰ constitute one diol molecule, the substituted or unsubstituted catechol group includes catechol group, methyl catechol group, ethyl catechol group, vinyl catechol group, propyl catechol group Group, allyl catechol group, butyl catechol group, methoxy catechol group, ethoxy catechol group, propoxy catechol group, butoxy catechol group, fluoro catechol group, chloro catechol group, bromo catechol group, acetyl catechol group, nitro catechol group, cyano catechol group, α, β-dihydroxynaphthyl group and halogen-substituted products thereof such as fluorine, chlorine, bromine, iodine and the like can be mentioned, and catechol group, methyl catechol group, methoxy catechol group, ethoxy catechol group, propoxyca A techol group and an α, β-dihydroxynaphthyl group are preferred, and a methoxycatechol group is more preferred.

In the above formula (I), R ¹ is —CH═NR ⁷ , R ² is OH, R ³ and R ⁵ are chlorine, R ⁴ and R ⁶ are hydrogen, and R ⁷ is 2- N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, which is chloro-4-nitrophenyl, R ¹ is —CHO, R ² is OH, R 3,5-dichlorosalicylaldehyde in which ³ and R ⁵ are chlorine, and R ⁴ and R ⁶ are hydrogen, R ¹ is —CHO, R ² , R ⁴ and R ⁶ are hydrogen, and R ³ And 3,5-dichlorobenzaldehyde, wherein R ⁵ is chlorine,
R ¹ is —CHO, R ² is OH, R ³ , R ⁴ and R ⁶ are hydrogen, and R ⁵ is chlorine, or R ¹ is —CHO, R 3-chloro-2-hydroxybenzaldehyde, wherein ² is OH, R ³ is chlorine, and R ⁴ , R ⁵ and R ⁶ are hydrogen,
Particularly preferred is 3-fluoro-2-hydroxybenzaldehyde, wherein R ¹ is —CHO, R ² is OH, R ³ is fluorine, and R ⁴ , R ⁵ and R ⁶ are hydrogen. it can.

Of the compounds represented by formula (I), when R ¹ is —CH═NR ⁷ which is a group convertible to —CHO, these are represented by formula (I) wherein R ¹ is —CHO. It can be obtained by a dehydration condensation reaction between the compound and R ⁷ NH ₂ . Specifically, the compound of formula (I) in which R ¹ is —CHO and R ⁷ NH ₂ are dissolved in an organic solvent such as dichloromethane, and a dehydrating agent is added to obtain the target compound. it can. At this time, sodium sulfate, magnesium sulfate, neutral alumina, basic alumina, or the like can be used as the dehydrating agent. When the target compound is not obtained or difficult to obtain in the above reaction, benzene or toluene is used as a reaction solvent, and a non-volatile acid such as benzene sulfonic acid, toluene sulfonic acid, camphor sulfonic acid or the like is used as a catalyst. The target compound can be obtained by using a reaction apparatus.

Of the compounds represented by formula (I), when R ¹ is —CH═NOR ⁸ which is a group convertible to —CHO, these are represented by formula (I) wherein R ¹ is —CHO. By dissolving the compound and hydroxylamine hydrochloride in an organic solvent such as dichloromethane and adding an organic base such as triethylamine, a target compound in which R ⁸ is hydrogen can be obtained. After this compound is isolated, it is dissolved again in an organic solvent such as dichloromethane, and methanesulfonyl chloride, trifluoromethanesulfonyl chloride, benzenesulfonyl chloride, or paratoluenesulfonyl chloride is added in the presence of an organic base such as triethylamine. Thus, a target compound in which R ⁸ is a methanesulfonyl group, a trifluoromethanesulfonyl group, a benzenesulfonyl group, or a paratoluenesulfonyl group can be obtained.

Among the compounds represented by the formula (I), when R ¹ is —CH (OR ⁹ ) OR ¹⁰ which is a group convertible to —CHO, these are the formulas (I) in which R ¹ is —CHO. It can be obtained by a dehydration reaction between the represented compound and R ⁹ OH or R ¹⁰ OH. Specifically, a compound represented by the formula (I) in which R ¹ is —CHO and R ⁹ OH and R ¹⁰ OH are dissolved in benzene or toluene, and benzenesulfonic acid, toluenesulfonic acid, camphorsulfonic acid, etc. The target compound can be obtained by using a Dean-Stark reactor using a non-volatile acid as a catalyst.

Examples of the enhancing action in the present invention include an additive action and a synergistic action, and a synergistic action is preferable. For example, when two kinds of drugs are used in combination, the synergistic action includes two kinds of specific actions. This is an effect that exhibits an effect (synergistic effect) that exceeds the effect (additive effect) of the combined action of the drugs. The interaction between the two drugs can be determined based on an isobologram evaluation proposed by Steel et al. (1979) et al. That is, for example, when two agents of A agent and B agent are used in combination, IC ₅₀ , IC ₄₀ , IC ₃₀ , IC ₂₀ , IC ₁₀ of A agent alone are a5, a4, a3, a2, a1 respectively. Similarly, IC ₅₀ , IC ₄₀ , IC ₃₀ , IC ₂₀ , IC ₁₀ with B agent alone are set to b5, b4, b3, b2, b1, respectively, and then (0, a5) is plotted on the Y axis on the graph (B5, 0) is plotted on the X axis, and (b5-b1, a1), (b5-b2, a2), (b5-b3, a3), (b5-b4, a4), (b1 , A5-a1), (b2, a5-a2), (b3, a5-a3), (b4, a5-a4) are plotted on the same graph to draw a curve connecting the points, Using the two drugs B together, the drug concentration showing 50% cell growth inhibition is shown in the graph above. When these points are on the lower concentration side of the curve, it is judged that there is a synergistic effect (synergistic), and when they are within the curve, it is judged that there is an additive effect (additive), By determining that there is an antagonistic effect when one drug counteracts the action of another agent when it is on the high concentration side, the concentration that produces a synergistic effect can be determined. A schematic diagram is shown in FIG.

  Examples of the administration target of the enhancer of the present invention and the compound represented by formula (I) include mammals, specifically humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, Rabbits, sheep, goats, cats, and dogs can be exemplified, and humans can be preferably exemplified.

  The target cell in the administration subject is not particularly limited as long as PDT can be applied, and examples thereof include cancer cells, warts, dysplasias, bacterial / fungal infection site cells, and virus-infected cells. Cancer cells are preferred, PDT resistant cancer cells are more preferred, ALA-PDT resistant cancer cells are more preferred, PEPT1-expressing / ABCG2 low-expressing ALA-PDT resistant cancer cells are more preferred, and used in combination with an ABCG-2 inhibitor. However, PEPT1-expressing / ABCG-2 low-expressing ALA-PDT resistant cancer cells in which no PDT enhancing action is observed are particularly preferred.

  The above cancers include malignant melanoma, skin cancer, lung cancer, tracheal and bronchial cancer, oral epithelial cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, colon cancer, liver and Cancers / tumors and muscles with malignant epithelial cells such as intrahepatic bile duct cancer, kidney cancer, pancreatic cancer, prostate cancer, breast cancer, cervical cancer, uterine cancer, ovarian cancer, and brain tumor Tumors, osteosarcomas, Ewing sarcomas, etc., cancers / tumors with malignant muscles and bones that constitute the supporting tissues, and cancers derived from hematopoietic cells, such as leukemia, malignant lymphoma, myeloma, Burkitt lymphoma A tumor etc. can be mentioned.

  The enhancer of the present invention can enhance the effect of PDT in both in vitro and in vivo systems. As a method for use in an in vitro system, (a) the target cancer cell derived from the species to be administered is added to the enhancer of the present invention and the photosensitizer or ALAs (hereinafter referred to as “photosensitizer”) of the present invention. And a method including a step of performing PDT after the addition of the sensitizer, etc., and the step (a) includes (a1) the enhancement of the present invention in a medium containing target cancer cells. 5 minutes to 25 hours, more preferably 10 minutes to 15 hours, still more preferably 2 hours to 7 hours, particularly preferably 3 to 6 hours after adding and mixing the agent and mixing with the photosensitizer and the like. Examples of the step of culturing and then removing the medium containing the enhancer, the photosensitizer and the like of the present invention, and then newly adding the medium to perform PDT are exemplified.

  The amount of the enhancer of the present invention used in the above in vitro system is not particularly limited as long as the enhancer effect of the present invention is obtained. As the final concentration in the medium for culturing ALA-PDT sensitive target cells, for example, ALA is 25 μM. ˜125 μM can be mentioned, and 25 μM to 50 μM is preferable, and ALA is preferably 300 μM or more for ALA-PDT low-sensitivity target cells. DCSA can include 10 μM to 100 μM, and more preferably 30 μM to 100 μM.

  The administration route when the enhancer of the present invention or the compound represented by formula (I) is used in an in vivo system includes intravenous administration, intramuscular injection, intraarterial administration, etc .; oral administration; transdermal administration; Examples include mucosal administration; transrectal administration; transluminal administration; local administration to the brain, etc., among which intravenous administration, intramuscular injection, intraarterial administration, etc .; oral administration; It can be illustrated. The administration route of the enhancer of the present invention or the compound represented by formula (I) and the administration route of the photosensitizer in the present invention may be the same or different. Further, the enhancer of the present invention, the compound represented by the formula (I), the photosensitizer and the like may be administered to the subject simultaneously, or any of them may be administered first, but it is more excellent. From the viewpoint of obtaining the enhancing effect of the present invention, a mode in which the enhancing agent of the present invention or the compound represented by the formula (I) is administered first and the photosensitizer and the like are administered later can be preferably exemplified. .

  The amount of the enhancer of the present invention and the compound represented by the formula (I) used in the in vivo system is not particularly limited as long as the enhancer effect of the present invention is obtained, but it is once per adult in terms of DCSA of the present invention. Preferably, 1 mg to 10000 mg, more preferably 3 mg to 3000 mg, and even more preferably 10 mg to 1000 mg per PDT can be exemplified.

  The enhancer of the present invention and the compound represented by formula (I) can contain other optional components that enhance the PDT effect as necessary, as long as the enhance effect of the present invention is obtained. Other ingredients such as excipients can be added. The dosage form when the enhancer of the present invention or the compound represented by formula (I) is used in an in vivo system includes injections, instillations, intravesical injections, tablets, capsules, fine granules, syrups , Sprouting agents, suppositories and the like. These can be formulated according to a conventional method using a solvent, a dispersion medium, a bulking agent, an excipient and the like as appropriate.

  The mechanism of potentiation of the PDT effect brought about by the structure of the enhancer of the present invention or the compound represented by the formula (I) is unknown, but from the cell killing effect by PDT exceeding the effect presumed from intracellular PpIX accumulation. Then, one of the effect enhancing actions is an increasing action of intracellular PpIX accumulation.

  The photosensitizer in the present invention absorbs visible light and emits fluorescence, and specifically accumulates in tumor tissue and new blood vessels to generate singlet oxygen. As these photosensitizers, any photosensitizer used in PDT can be used, and examples thereof include tetrapyrrole compounds. Specifically, PpIX, porfimer sodium ( Photofrin), talaporfin sodium (resaphyrin), benzoporphyrin, foscan, chlorin, uroporphyrin I, uroporphyrin III, heptacarboxyl porphyrin I, heptacarboxyl porphyrin III, hexacarboxyl porphyrin I, hexacarboxyl porphyrin III, pentacarboxyl porphyrin I, pentacarboxyl porphyrin III, coproporphyrin I, coproporphyrin III, isocoproporphyrin, harderoporphyrin, isoharderoporphyrin, hematoporphyrin, mesoporphyrin, Ethioporphyrin, pyroporphyrin, deuteroporphyrin IX, pentoporphyrin, ATXs-10, tetra (m-hydroxyphenyl) chlorine, tin (IV) chlorin e6, pheophorbide, pyropheophorbide, bacteriochlorin, phthalocyanine, phenothiazine, ethyl Examples include ethylpurine tin and pullpurinimide.

Examples of the ALAs include compounds represented by the general formula (II) R ² R ¹ NCH ₂ COCH ₂ CH ₂ COR ³ or salts thereof. In the above formula (II), R ¹ and R ² can be exemplified. Are each independently a hydrogen atom, an alkyl group having 1 to 24 carbon atoms, an acyl group having 1 to 12 carbon atoms, an alkoxycarbonyl group having 2 to 13 carbon atoms, an aryl group having 6 to 16 carbon atoms, or 7 to 22 carbon atoms. R ³ is a hydroxy group, an alkoxy group having 1 to 24 carbon atoms, an acyloxy group having 1 to 12 carbon atoms, an alkoxycarbonyloxy group having 2 to 13 carbon atoms, or an aryloxy group having 6 to 16 carbon atoms 5-ALA or a derivative thereof, or a salt thereof, which is an aralkyloxy group having 7 to 22 carbon atoms or an amino group, is preferable.

  As a C1-C24 alkyl group in Formula (II), a C1-C24 linear or branched alkyl group can be mentioned, A C1-C18 alkyl group is preferable, and especially carbon number 1-6 alkyl groups are preferred. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, t-butyl group, n-pentyl group, n- A hexyl group etc. can be mentioned.

  As a C1-C12 acyl group in Formula (II), a C1-C12 linear or branched alkanoyl group, a C3-C12 linear or branched alkenylcarbonyl group, C5-C12 12 monocyclic or polycyclic aroyl groups, or monocyclic or polycyclic aryloxycarbonyl groups having 5 to 12 carbon atoms, and particularly preferably alkanoyl groups having 1 to 6 carbon atoms. Can include formyl, acetyl, propionyl, butyryl, pentanoyl and the like. The carbon number in the acyl group having 1 to 12 carbon atoms includes carbonyl carbon.

  The alkoxycarbonyl group having 2 to 13 carbon atoms in the formula (II) is preferably an alkoxycarbonyl group having 2 to 7 carbon atoms, specifically, a methoxycarbonyl group, an ethoxycarbonyl group, an n-propoxycarbonyl group, an isopropoxy group. Examples include carbonyl group, butoxycarbonyl group, pentyloxycarbonyl group, hexyloxycarbonyl group, heptyloxycarbonyl group, octyloxycarbonyl group, nonyloxycarbonyl group, decyloxycarbonyl group, undecyloxycarbonyl group, dodecyloxycarbonyl group, etc. be able to. The carbon number in the alkoxycarbonyl group having 2 to 13 carbon atoms includes carbonyl carbon.

  Examples of the aryl group having 6 to 16 carbon atoms in the formula (II) include a monocyclic or polycyclic aryl group, and specifically include a phenyl group, a naphthyl group, an anthryl group, a phenanthryl group, and a pyrenyl group. Etc.

  Examples of the aralkyl group having 7 to 22 carbon atoms in the formula (II) include a group consisting of the aryl group having 6 to 16 carbon atoms and the alkyl group having 1 to 6 carbon atoms. , A benzyl group, a phenethyl group, and the like.

  As a C1-C24 alkoxy group in Formula (II), a C1-C24 linear or branched alkoxy group can be mentioned, A C1-C16 alkoxy group is preferable, and especially carbon number 1-12 alkoxy groups are preferred. Specific examples include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, a pentyloxy group, a hexyloxy group, an octyloxy group, a decyloxy group, and a dodecyloxy group.

  As a C1-C12 acyloxy group in Formula (II), a C1-C12 linear or branched alkanoyloxy group can be mentioned, A C1-C6 alkanoyloxy group is preferable. Specific examples include an acetoxy group, a propionyloxy group, a butyryloxy group, and a pentanoyloxy group.

  The alkoxycarbonyloxy group having 2 to 13 carbon atoms in the formula (II) is preferably an alkoxycarbonyloxy group having 2 to 7 carbon atoms, specifically, a methoxycarbonyloxy group, an ethoxycarbonyloxy group, or n-propoxycarbonyloxy group. Group, isopropoxycarbonyloxy group, butoxycarbonyloxy group, pentyloxycarbonyloxy group, hexyloxycarbonyloxy group and the like. The carbon number in the alkoxycarbonyloxy group having 2 to 13 carbon atoms includes carbonyl carbon.

  Examples of the aryloxy group having 6 to 16 carbon atoms in the formula (II) include monocyclic or polycyclic aryloxy groups, and specifically include a phenoxy group, a naphthyloxy group, an anthryloxy group, A phenanthryloxy group, a pyrenyloxy group, etc. can be mentioned. As the aralkyloxy group having 7 to 22 carbon atoms, those having the aralkyl group are preferable, and specific examples include a benzyloxy group and a phenethyloxy group.

In formula (II), R ¹ and R ² are preferably hydrogen atoms. R ³ is preferably a hydroxy group, an alkoxy group or an aralkyloxy group, more preferably a hydroxy group or an alkoxy group having 1 to 12 carbon atoms, and particularly preferably a methoxy group or a hexyloxy group.

In the formula (II), a compound in which R ¹ and R ² are hydrogen atoms and R ³ is a hydroxy group is 5-ALA, and can be particularly preferably mentioned. As preferable examples of 5-ALA derivatives other than 5-ALA, 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester, 5-ALA pentyl ester, 5-ALA hexyl ester 5-ALA esters such as 5-ALA methyl ester or 5-ALA hexyl ester can be particularly preferably mentioned. It is disclosed, for example, in Japanese Patent Application Laid-Open No. 11-501914 that an ester of 5-ALA exhibits a physiological effect similar to that of 5-ALA.

  Examples of pharmacologically acceptable salts of 5-ALA or 5-ALA derivatives include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Examples of acid addition salts include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, and other inorganic acid salts, formate, acetate, propionate, toluenesulfonic acid Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Mention may be made of organic acid addition salts. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc. Examples of ammonium salts include ammonium salts and alkylammonium salts such as tetramethylammonium salts. Examples of the organic amine salt include triethylamine salts, piperidine salts, morpholine salts, toluidine salts and the like.

  5-ALA or 5-ALA derivatives can be produced by any method of chemical synthesis, production by microorganisms, and production by enzymes. For example, an acyl group in an amino group of a 5-ALA derivative, an ester group in a carboxyl group, or the like can be produced by acylation of an amino group, esterification of a carboxyl group, or the like by a conventional method of chemical synthesis. Further, when it is desired to obtain a salt of 5-ALA or a 5-ALA derivative, if the compound represented by the general formula (II) is obtained in the form of a salt, it may be purified as it is, or obtained in a free form. In such a case, the salt may be dissolved or suspended in a suitable organic solvent, and an acid or base may be added to form a salt by an ordinary method. 5-ALA or 5-ALA derivatives can also be present in the form of adducts with water or various solvents.

  The dose of the photosensitizer and the like in the present invention is not particularly limited as long as the enhancing effect of the enhancer of the present invention and the compound represented by the formula (I) is obtained. For example, the photosensitizer and the like are ALAs. In the case of the above, the oral dose is 1 mg to 1000 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg, still more preferably 15 mg to 25 mg per kg body weight in terms of 5-ALA. When used in combination with an agent, 10 to 90%, preferably 20 to 80%, more preferably 30 to 70%, and still more preferably 40 to 60% of the amount recommended for normal PDT. In addition, when using ALAs in the form of a solution, it is preferable to prepare the solution so that the aqueous solution does not become alkaline in order to prevent decomposition of the ALAs. When it becomes alkaline, decomposition of the active ingredient can be prevented by removing oxygen.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Example 1]
[Search for new PDT effect enhancer 1]
PDT (5-ALA-PDT) using 18 kinds of test compounds and 5-ALA hydrochloride for 18 kinds of test compounds provided by Prof. Hitoshi Hori and Associate Professor Yoshihiro Utsu of Tokushima University ) To examine the effect of enhancing the effect of PDT.

As cancer cells, MKN-45 cells, which are human gastric cancer cell lines, were used. Such MKN-45 cells were prepared in RPMI-1640 medium (Nissui) supplemented with 10% (v / v) inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 50 μg / mL kanamycin (Meiji Seika Co., Ltd.). The cells were cultured and maintained in a 5% CO ₂ incubator at 37 ° C. in 10% FBS-RPMI. The cells were subjected to experiments as floating cells in 0.25% trypsin-2 mM EDTA solution. The above-mentioned MKN-45 cells express ALA uptake transporter (PEPT1) but have low expression of ABCG2 transporter and do not show enhanced PDT effect when used in combination with an ABCG-2 inhibitor. This has already been confirmed by the inventors.

The MKN-45 cells were seeded at 5 × 10 ³ cells / well in a 96-well plate and cultured overnight at 37 ° C. As the medium, RPMI-1640 medium supplemented with 10% FBS (manufactured by HyClone Laboratories, South Logan, UT, USA) was used. After culture, the medium was removed, and 189 μL of 10% FBS-RPMI was newly added to the well. Each test compound prepared to be dissolved in DMSO to 2 mM and 20 mM was serially diluted with DMSO to 1 μL / well. Added and mixed with mixer (final concentrations in media 10 μM and 100 μM). Then, 10 μL of 5-ALA hydrochloride prepared to 2 mM by dissolving in distilled water for injection was added and mixed with a mixer (final concentration 100 μM), and then cultured at 37 ° C. for 4 hours in a CO ₂ incubator. .

  After culturing, the medium containing each test compound and 5-ALA hydrochloride was removed, 200 μL of 10% FBS-RPMI was newly added, and a 96-well plate PDT irradiation apparatus was used at a wavelength of 628.1 nm for 5 minutes. Irradiation and PDT were performed. As a control group, a well not added with ALA (ALA (-)) and / or a well not added with a test compound was provided, and PDT was performed in the same manner.

For the measurement of cell killing effect, Cell-Counting Kit-8 (manufactured by Dojindo Laboratories) is used to measure the concentration of MTT formazan produced using tetrazolium salt WST-8 that produces water-soluble formazan as a chromogenic substrate. Performed by the MTT improved method. Specifically, after 72 hours of 5-ALA-PDT treatment, the medium was removed, washed with 200 μL of PBS, and 200 μL of Cell-Counting Kit-8 solution diluted 40-fold was dispensed into each well, and a CO ₂ incubator was further added. Incubated at 37 ° C for 1 hour. About the produced | generated MTT formazan density | concentration, the light absorbency in 490 nm was measured using the immunoleader (made by Nihon Intermed), and IR was computed using the following formula | equation. The results are shown in Table 1 below.

[Equation 1]
IR (%) = (1-T / C) × 100
(However, T is the average absorbance of each well to which 5-ALA hydrochloride and each test compound were added, and C is the average absorbance of the wells in the control group.)

(result)
As is apparent from Table 1, when 10 μM of compound TX-816 was added without adding 5-ALA hydrochloride and PDT was performed, only 3% IR was shown for MKN-45 cells. When 100 μM 5-ALA hydrochloride was added and 10 μM compound TX-816 was added and PDT was performed, IR was close to 80%, and the most effective among the 18 test compounds. The 5-ALA-PDT enhancing action was shown. In the control in which 100 μM 5-ALA hydrochloride was added and PDT was performed without adding the test compound, the IR was 33%.

[Example 2]
[Examination of the effect of TX-816]
Regarding the TX-816, the ALA-PDT effect enhancing action was examined in more detail. 1 μL / well of TX-816 (20 mM for a final concentration of 100 μM) prepared to have a final concentration of 0, 0.41, 1.23, 3.70, 11.1, 33.3, and 100 μM Add 10 μL of ALA (4 mM for a final concentration of 200 μM) prepared by dissolving in distilled water for injection to a final concentration of 0, 50, 100, 200 μM and mix for 5 hours after mixing with a mixer. Then, 5-ALA-PDT was performed in the same procedure as in Example 1, and then the MTT improvement method was performed. The IR at each concentration of TX-816 was plotted on a single variable graph to determine the IC ₅₀ (50% inhibitory concentration) value. The results are shown in FIGS.

(result)
As is clear from FIG. 2, TX-816 was confirmed to enhance the ALA-PDT effect in a concentration-dependent manner, and confirmed to have the most concentration-dependent PDT action enhancing effect when 5-ALA hydrochloride was 50 μM. It was done.

  As is clear from FIG. 3, as a result of determining the combined effect by the isobologram method, it was confirmed that the ALA-PDT enhancing effect of TX-816 is a synergistic effect.

As can be seen from FIG. 4, the IC ₅₀ value for the growth inhibitory effect of TX-816 on MKN-45 cells after 72 hours of contact is calculated to be 36.5 μM, and a synergistic effect is obtained at a low toxicity concentration even after prolonged contact. It was confirmed that it can be demonstrated.

(Discussion)
Although MKN-45 cells are ALA-PDT resistant cells, it has been confirmed that ABCG2 expression is low and an ALA uptake transporter (PEPT1) is expressed. The event that 5-ALA is not taken up by the cells is unlikely. In addition, we examined the expression of genes encoding metabolic enzymes that work stepwise in the process of ALA incorporated into cells being metabolized into porphyrins. ) Therefore, it can be said that TX-816 can synergistically enhance the effect of PDT in ALA-PDT resistant cells when used in combination with ALAs.

[Example 3]
[Examination of the effects of DCSA and CAN]
As shown in FIG. 5, TX-816 has N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline in which dichlorophenol and chloronitrobenzene are bound as a Schiff base (FIG. 5 ( a)). Since TX-816 has low water solubility, it was dissolved in DMSO and used in the study of the present application, but decomposes into DCSA (see FIG. 5 (b)) and CNA (see FIG. 5 (c)) in DMSO. This has been revealed by the inventors' analysis. Therefore, the active body of the PDT action effect-enhancing agent of the present invention may be either DCSA or CNA. Compared with DCSA, CAN, TX-816, the ALA-PDT effect on MKN-45 cells The potentiating action was examined. DCSA, CNA, and TX-816 prepared to have concentrations of 11.1, 33.3, and 100 μM were added at 1 μL / well, respectively, and dissolved in DMSO to obtain final concentrations of 0, 31.25, 62. Further, 10 μL of 5-ALA hydrochloride prepared to 5, 125, 250, 500, 1000 μM was added, mixed with a mixer, cultured for 5 hours, and then subjected to 5-ALA-PDT in the same manner as in Example 1. Then, the MTT improvement method was performed, and IR was calculated. The results are shown in Table 2 below.

(result)
As is clear from Table 2 above, DCSA shows a potentiating effect similar to that of TX-816, while CNA does not show any enhancing action, and DCSA is a compound having an ALA-PDT effect enhancing action. confirmed.

[Example 4]
[Search for new PDT effect enhancer 2]
The 18 compounds shown in Table 3 below, which are related compounds and derivatives of DCSA, were picked up as subjects for further search for compounds having a PDT effect enhancing action. Among them, 6 compounds (compound numbers: 3, 6, 7, 8, 11, 12) were compared with DCSA (compound number: 1) as a test compound for enhancing the ALA-PDT effect in MKN-45 cells. And examined. Each test compound prepared so that the final concentration is 0, 3.125, 6.25, 12.5, 25, 50, 100 μM is added at 1 μL / well, and the final concentration is adjusted to 0, 50 μM. Further, 10 μL of 5-ALA hydrochloride was added and mixed with a mixer, followed by culturing for 5 hours. 5-ALA-PDT was performed in the same procedure as in Example 1, then MTT improvement was performed, and IR was calculated. . The results are shown in FIG.

(result)
Among the DCSA-related compounds, among the compounds shown in Table 3 above, compounds 6 (3,5-dichlorobenzaldehyde), 7 (5-chlorosalicylaldehyde), and 8 (3-chloro-2-hydroxybenzaldehyde) In the compounds having an aldehyde group such as), a potentiating action comparable to that of DCSA was observed (see FIG. 6). On the other hand, the compound 3 in which the aldehyde group is substituted with a carboxyl group has no activity, and the compound 12 in which the aldehyde group is substituted with an amide group or the compound 11 having no aldehyde group has low activity, so the aldehyde group is active. It was confirmed to be important for expression. Therefore, further improvement in activity can be expected by introducing a substituent that enhances the electrophilicity of the aldehyde group into benzaldehyde.
It was confirmed that the aldehyde group is important for activity expression.

[Comparative Example 1]
(Enhancement effect in normal cells)
Using the MKN-45 cells as cancer cells and human umbilical cord endothelial cells HUVEC cells (C2517A; manufactured by Lonza, Walkersville, MD, USA) as normal cells, respectively, ALA-PDT sensitivity was compared.

MKN-45 cells and HUVEC cells were seeded at 1 × 10 ⁴ cells / well in 96-well plates and cultured overnight at 37 ° C. As an exclusive medium for each cell, RPMI-1640 medium (10% FBS-RPMI; manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 10% FBS (manufactured by HyClone Laboratories, South Logan, UT, USA) is used for MKN-45 cells. For HUVEC cells, endothelial cell medium kit-2 (EGM-2 BulletKit; CC-3162, Lonza) was used.

After culturing, each of the dedicated media is removed, and for each cell, 189 μL of each of the dedicated growth media is added to the well and then dissolved in DMSO to prepare DCSA prepared to a final concentration of 30 μM or 100 μM. / Well added, ALA dissolved in distilled water for injection and serially diluted is further added and mixed with a mixer, 5-ALA-PDT is performed in the same procedure as in Example 1, then MTT improvement is performed, IC ₅₀ was calculated. The results are shown in Table 4 below.

(result)
As shown in Table 4 above, since HUVEC cells have low 5-ALA-PDT sensitivity, the effect of DCSA on normal cells was clearly different from the effect on cancer cells.

[Comparative Example 2]
[Search for new PDT effect enhancer 3]
As the DCSA-related compounds, the test compounds (reference numbers: 7-2, 7-3, 7-4, 7-5, 7-6, 7-7) shown in Table 5 below are used as DCSA (reference numbers). : The ALA-PDT effect enhancement action compared with 7-1) was examined. 1 μL / well of each compound dissolved in DMSO and adjusted to a final concentration of 0, 0.41, 1.23, 3.70, 11.1, 33.3, and 100 μM in the medium was added. Add 10 μL of ALA dissolved in distilled water for injection to a final concentration of 0, 50 μM, mix with a mixer, incubate for 5 hours, and follow the same procedure as in Example 1 to 5-ALA. -PDT was performed, then MTT improvement was performed, and IR was calculated. The results are shown in FIG.

(result)
The above compounds include salicylic acid and acetylsalicylic acid (aspirin) having anti-inflammatory, antipyretic, analgesic and antiplatelet actions. As is clear from FIG. 7, 3-hydroxybenzaldehyde (HBA), benzaldehyde (BA), and salicylaldehyde (SA) did not show an effect enhancing effect even if they had an aldehyde group. It has been clarified that the positional relationship between the aldehyde group and the introduced substituent such as a hydroxyl group or a chlorine atom is important in enhancing the ALA-PDT effect. In addition, it is speculated that salicylic acid and acetylsalicylic acid have no effect-enhancing action and have an action mechanism different from the prostaglandin (PG) synthesis inhibitory action in salicylic acid non-steroidal anti-inflammatory drugs.

[Example 5]
[Search for new PDT effect enhancer 4]
The ALA-PDT effect enhancing action was examined using 3-fluorosalicylaldehyde (3-FSA) into which a fluorine atom was introduced instead of a chlorine atom in 3-chloro-2-hydroxy-benzaldehyde. Add 1 μL / well of 6 mM (final concentration 30 μM) DCSA, 6 mM (final concentration 30 μM) 3-FSA, 20 mM (final concentration 100 μM) 3-FSA and dissolve in distilled water for injection. 10 μL of ALA prepared to 7.8, 15.6, 31, 63, 125, 250 μM was further added, mixed with a mixer, cultured for 5 hours, and 5-ALA was obtained in the same manner as in Example 1. performed-PDT, then subjected to MTT improved method to calculate the cell inhibition rate and _{IC 50.} The results are shown in FIG.

(result)
As is clear from FIG. 8, 100 μM activity was equivalent to 30 μM DCSA. The activity of 3-FSA was estimated to be about one-third that of DCSA.

[Example 6]
[Investigation of intracellular PpIX accumulation]
For the purpose of examining the mechanism of DCSA's enhancement of the ALA-PDT effect, MKN-45 cells were used using an LLS-405VISLED light source and a SEC2000-VIS / NIR spectrometer SLIT200 fluorescence measurement device (manufactured by BSA). The effect of DCSA on intracellular PpIX accumulation was analyzed. 5 × 10 ⁶ MKN-45 cells were seeded in a 15 cm dish and cultured until subconfluent. When the cells were fully grown, 19 mL of warm, fresh 10% FBS-supplemented RPMI-1460 medium was added. After DCSA dissolved in DMSO was added to the medium to a final concentration of 30 μM or 100 μM (DMSO final concentration was 0.5%), 1 mL of 1 mM or 2 mM 5-ALA (final concentrations of 50 μM and 100 μM) was added. . After culturing for 5 hours, the cells were collected by trypsin-EDTA, suspended in 10 mL of RPMI-1460 medium supplemented with 10% FBS, and the number of cells was counted. After centrifugation, the cells were washed twice with PBS, and the cells were lysed with 2% Triton X-100. After centrifugation at 15,000 rpm for 15 minutes, the supernatant was collected, and the fluorescence intensity was measured with a SEC2000-VIS / NIR spectrometer. The results are shown in FIG.

(result)
As shown in FIG. 9, the intracellular PpIX was about 5.5 times higher when 50 μM 5-ALA and 100 μM DCSA were treated simultaneously (b) than when 50 μM 5-ALA alone was treated (a). Also increased. One of the effect enhancing actions of the enhancer of the present invention is presumed to be an effect of increasing the intracellular PpIX accumulation of the enhancer of the present invention itself.

  The enhancer of the present invention is useful in the medical field using PDT.

Claims (8)

A photodynamic therapy (PDT) effect enhancer comprising a compound represented by the following formula (I), which is used in combination with a photosensitizer or aminolevulinic acid (ALA).
[Wherein R ¹ represents —CHO or —CH═NR ⁷ (where R ⁷ represents a substituted or unsubstituted phenyl group) ,
R ³ and R ⁵ each represent H or an electron-withdrawing group selected from fluorine, chlorine, bromine and nitro groups (provided that at least one is an electron-withdrawing group);
R ² , R ⁴ and R ⁶ each represent H or —OH. ] The compound represented by the formula (I) is N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichlorobenzaldehyde. The enhancer according to claim 1, wherein the enhancer is selected from: 5-chlorosalicylaldehyde, and 3-chloro-2-hydroxybenzaldehyde. The potentiator according to claim 1 or 2 , wherein PDT targets cancer. The enhancer according to claim 3 , wherein the cancer is composed of cells with low expression of ABCG-2. Use of a compound represented by the following formula (I) for preparing a photodynamic therapy (PDT) effect enhancer in combination with a photosensitizer or aminolevulinic acid (ALA).
[Wherein R ¹ represents —CHO or —CH═NR ⁷ (where R ⁷ represents a substituted or unsubstituted phenyl group) ,
R ³ and R ⁵ each represent H or an electron-withdrawing group selected from fluorine, chlorine, bromine and nitro groups (provided that at least one is an electron-withdrawing group);
R ² , R ⁴ and R ⁶ each represent H or —OH. ] The compound represented by the formula (I) is N-3 ′, 5′-dichloro-2′-hydroxybenzylidene-2-chloro-4-nitroaniline, 3,5-dichlorosalicylaldehyde, 3,5-dichlorobenzaldehyde. 6. Use according to claim 5 , characterized in that it is selected from: 5-chlorosalicylaldehyde and 3-chloro-2-hydroxybenzaldehyde. The use according to claim 5 or 6 , wherein PDT targets cancer. The use according to claim 7 , wherein the cancer is composed of cells with low expression of ABCG-2.