WO2018101434A1 - Glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof, pharmaceutical composition, method for destroying target, and method for producing glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof - Google Patents

Abstract

The present invention addresses the problem of providing a glycosylated chlorin e6 derivative which has excellent tumor cell-killing properties (phototoxic property) and growth-inhibiting effect on tumor tissues and which is used for photodynamic therapy. The glycosylated chlorin e6 derivative according to the present invention is applicable for use in photodynamic diagnosis as well. The present invention pertains to a glycosylated chlorin e6 derivative represented by general formula (1) or a pharmaceutically acceptable salt thereof.

Description

Glycosylated chlorin e6 derivative, or pharmaceutically acceptable salt thereof, pharmaceutical composition, method of destroying target, and method for producing glycosylated chlorin e6 derivative, or pharmaceutically acceptable salt thereof

The present invention is a glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof, that can be suitably used in photodynamic therapy (PDT) and / or photodynamic diagnosis (PDD). Relating to salt. The present invention also relates to a pharmaceutical composition, a method for destroying a target, and a method for producing a glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.

Photodynamic therapy (PDT) involves administering a photosensitive substance to a patient having a target diseased tissue (hereinafter also referred to as “target tissue”), and the target tissue (cancer tissue, tumor tissue, skin lesion, and This is a treatment method that selectively destroys only a target tissue by irradiating light having an appropriate wavelength for exciting the photosensitive substance after the photosensitive substance is accumulated in the neovascularization. In this case, photo-excited and activated photosensitizers directly or indirectly transfer energy to nearby molecular oxygen, and the generation of singlet oxygen by this is the main mechanism of target tissue destruction. It is considered.

As a photosensitive substance used in PDT, a hematoporphyrin derivative (for example, Photofrin (registered trademark)) is well known and has already been put into practical use. However, hematoporphyrin derivatives are known to cause temporary photosensitivity as a side effect when administered to the human body. In addition, the selectivity of hematoporphyrin derivatives for cancer tissues is not sufficient, and accumulation in normal tissues is also observed. Therefore, the patient who received the drug is dark for a long time until it is excreted outside the body so that normal cells are not destroyed by the photosensitizing action of hematoporphyrin derivative (porfimer sodium) accumulated in normal tissues. It is necessary to stay in place. However, it has been reported that photosensitivity remains for more than 4 weeks because porfimer sodium has a slow elimination rate from normal tissues. In addition, since the maximum absorption wavelength of the porphyrin derivative is about 630 nm and the molar extinction coefficient is low, the therapeutic effect of PDT (in other words, the target tissue) is limited to surface cancers of about 5 to 10 nm.

For such compounds, phthalocyanine compounds having absorption in a longer wavelength region (650 nm to 800 nm), chlorin compounds, and the like have been proposed as second-generation drugs. For example, talaporfin sodium (hereinafter also referred to as “TS” in the present specification, Rezaphyrin (registered trademark)) in which aspartic acid is bound to a chlorin-based compound has already been put into practical use, and its clinical use amount is gradually increased. Yes. Although such drugs have cleared problems such as metabolic property and longer absorption wavelength, there are problems such as insufficient tumoricidal properties and tumor tissue growth inhibitory effects.

As a drug for PDT as described above, Patent Document 1 proposes a compound obtained by binding chlorin e6, which is a chlorin compound, and folic acid. Patent Document 2 proposes a compound in which a chlorin compound and galactosamine are bound to detect a galectin biomarker. Further, Non-Patent Document 1 synthesizes a compound in which a sugar is bound to a chlorin compound labeled with 124 iodine, and proposes to PET (Positron Emission Tomography) imaging and photodynamic therapy.

Special table 2011-518890 gazette KR2009047872A

As described above, porphyrin compounds and chlorin compounds have also been developed and researched, but there is room for improvement in terms of tumoricidal properties and tumor tissue growth inhibitory effects.
Accordingly, an object of the present invention is to provide a glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof that has excellent tumoricidal properties (phototoxicity) and excellent tumor tissue growth inhibitory effect. .
Another object of the present invention is to provide a pharmaceutical composition, a method for destroying a target, and a method for producing a glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.

As a result of studying various porphyrin derivatives and chlorin derivatives, the present inventors have become a photosensitive substance useful as a photodynamic therapeutic drug, ensuring safety to the living body and exhibiting high phototoxicity in a small amount. The present inventors have found a novel glycosylated chlorin e6 derivative and a method for producing the same, and have reached the present invention.

That is, the present invention is as follows.
[1] A glycosylated chlorin e6 derivative represented by the following general formula (1) or a pharmaceutically acceptable salt thereof.
[2] In the general formula (1) described later, R ¹ , R ² and R ³ are each independently an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms, [1 Or a pharmaceutically acceptable salt thereof.
[3] The glycosylated chlorin e6 derivative according to [1] or [2], wherein R ¹ , R ² and R ³ in the general formula (1) described later are methyl groups, or a pharmaceutically acceptable salt thereof Salt.
[4] In the general formula (1) described later, —X— is —X ³ —O—, and the glycosyl according to any one of [1] to [3] represented by the general formula (2) described later Chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[5] In the general formula (2) described later, X ³ is a group bonded to an anomeric carbon atom of R or a group bonded to a carbon atom adjacent to the anomeric carbon atom. A glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[6] Glycosylated chlorin according to [4] or [5], wherein —X ³ — is —S—X ⁴ — in the general formula (2) described later, and represented by the general formula (3) described later e6 derivative, or a pharmaceutically acceptable salt thereof.
[7] The glycosylated chlorin e6 derivative according to [6] or a pharmaceutical product thereof according to [6], wherein in the general formula (3) described below, X ⁴ is a linear or branched alkylene group having 1 to 16 carbon atoms Acceptable salt.
[8] As described in [6] or [7], in general formula (3) described later, X ⁴ is an alkylene group represented by — (CH ₂ ) _n —, and n is an integer of 1 to 16. Glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[9] Any one of [6] to [8], wherein in the general formula (3) described later, X ⁴ is an alkylene group represented by — (CH ₂ ) _n —, and n is an integer of 3 to 10. Or a pharmaceutically acceptable salt thereof.
[10] The saccharide is any one of [1] to [9], wherein the saccharide is a monosaccharide, an oligosaccharide, a polysaccharide, a monosaccharide containing an amino group, an oligosaccharide containing an amino group, or a polysaccharide containing an amino group. The glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[11] The glycosylated chlorin e6 derivative according to any one of [1] to [10], wherein the saccharide is a monosaccharide and S is bonded to the anomeric carbon atom of R, or a pharmaceutically acceptable salt thereof salt.
[12] The glycosylated chlorin e6 derivative or the pharmaceutically acceptable salt thereof according to any one of [1] to [11], wherein the sugar is glucose, galactose or mannose.
[13] The glycosylated chlorin e6 derivative according to any one of [1] to [12] represented by the following general formula (4), (5) or (6), or a pharmaceutically acceptable salt thereof.
[14] A chlorin e6 derivative or a pharmaceutically acceptable salt thereof according to any one of [1] to [13], which is used for photodynamic treatment of a tumor, skin disease, eye disease or age-related macular degeneration. A pharmaceutical composition comprising as an active ingredient.
[15] The glycosyl according to any one of [1] to [13], wherein the target is selected from the group consisting of a virus, a microorganism and any of these cells, an infected cell, a tumor cell, a tumorous tissue, and a neovascularization. Irradiating the target with light having a wavelength that is absorbed by the chlorin e6 derivative or a pharmaceutically acceptable salt thereof after contacting the chlorinated e6 derivative or a pharmaceutically acceptable salt thereof. Including a method of destroying a target.
[16] A pharmaceutical composition comprising the glycosylated chlorin e6 derivative according to any one of [1] to [13] or a pharmaceutically acceptable salt thereof as an active ingredient.
[17] The pharmaceutical composition according to [16] for treatment, diagnosis or detection of a tumor, skin disease, eye disease or age-related macular degeneration.
[18] The glycosylated chlorin e6 derivative according to any one of [1] to [13], or a pharmaceutically acceptable salt thereof, comprising a step of binding chlorin e6 and a sugar via a linking group Manufacturing method.

The glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof (hereinafter also simply referred to as “the present chlorin derivative”) according to an embodiment of the present invention has very low toxicity in the dark, In vitro and in vivo, the target virus, bacteria, or these infected cells, tumor cells, or After contacting with a tumorous tissue, irradiation with light having a wavelength that is absorbed by the present chlorin derivative or the like can be applied to destroy the target. Therefore, the present chlorin derivative and the like can be used as a medicament containing the present chlorin derivative or the like as an active ingredient, particularly as a photodynamic therapeutic agent for tumors or skin diseases, or a photodynamic diagnostic agent.

It is a figure for demonstrating an example of the manufacturing method of the glycosylated chlorin e6 derivative which concerns on embodiment of this invention. It is a figure for demonstrating an example of the manufacturing method of the glycosylated chlorin e6 derivative which concerns on embodiment of this invention. It is a graph showing the intracellular uptake | capture performance evaluation result of the glycosylated chlorin e6 derivative based on embodiment of this invention using an esophageal cancer cell line and an immortalized esophageal normal epithelial cell line. It is a graph showing the evaluation result of the antitumor effect of the glycosylated chlorin e6 derivative which concerns on embodiment of this invention in photodynamic therapy.

[Glycosylated chlorin e6 derivative and its production method]
Hereinafter, among the present chlorin derivatives and the like, in particular, a glycosylated chlorin e6 derivative may be described as an example, but the description is pharmaceutically acceptable for the glycosylated chlorin e6 derivative unless otherwise specified. The same applies to salt.

In the general formula (1), X ¹ and X ² are each independently a group represented by H (hydrogen atom) or R—X— * (* represents a bonding position), and X ¹ and At least one of X ² is a group represented by R—X— *. Synthesis is easier viewpoint, i.e., from the viewpoint of having superior productivity, it is preferable that either one of X ¹ and X ² is a group represented by R-X- *, X ¹ is More preferably, it is a group represented by R—X— *, and X ² is H (hydrogen atom).

Here, R represents a sugar residue (hereinafter referred to as “sugar residue”). The sugar residue means a residue obtained by removing one hydroxyl group bonded to a carbon atom of the sugar, and a residue obtained by removing a hemiacetal (anomeric) hydroxyl group of a sugar is preferable.
X is a divalent group bonded to any one of the carbon atoms constituting R, and R is C (carbon atom), N (nitrogen atom), O (oxygen atom), H (hydrogen atom), And a linear or branched divalent group consisting of at least one atom selected from the group consisting of S and sulfur (sulfur atom). X is, for example, —S—, —O—, —NR _x — (R _x is a hydrogen atom or a hydrocarbon group which may have a hetero atom), a carbonyl group, an alkylene group, an alkenylene group, and And a combination thereof, preferably containing O (oxygen atom) and / or S (sulfur atom), and selected from the group consisting of —S—, —O—, and an alkylene group A group in which the above is combined is more preferable, and a group in which —S—, —O—, and an alkylene group are combined is more preferable.

The sugar of R is not particularly limited. For example, aldpentose (ribose, arabinose, xylose, lyxose, etc.), aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose, etc.) ), Aldoheptose, ketopentose (such as ribulose and xylulose), ketohexose (such as psicose, fructose, sorbose, and tagatose), ketoheptose (such as cedoheptulose and coliose), and derivatives thereof having an amino group, etc. Monosaccharides;
Oligosaccharides such as sucrose, maltose, lactose, maltotriose, raffinose and maltotetraose, and derivatives thereof having an amino group;
And polysaccharides such as starch, amylose and glycogen, and derivatives thereof having an amino group; Among these, monosaccharides are preferable, hexose or hexosamine is more preferable, hexose is more preferable, and glucose is particularly preferable.
The monosaccharide may be D-form or L-form, but D-form is preferred.

In the present specification, oligosaccharide means a compound containing 2 to 9 monosaccharide units, and polysaccharide means a compound containing 10 or more monosaccharide units (JD ROBERTS & M. C. CASERIO (1964) BASIC PRINCIPLES OF ORGANIC CHEMISTRY.WA Benjamin.Inc. (Quoted more). The monosaccharides that are glycosidically linked may be the same or different. The glycosidic bond between monosaccharides may be an α-bond or a β-bond.

Specific examples of hexose include glucose, galactose, mannose, allose, altrose, gulose, idose, and talose. Among these, glucose is most preferable. This is because the phototoxicity of glucose is excellent.

Specific examples of hexosamine include glucosamine, galactosamine, mannosamine, daunosamine, and perosamine. Of these, glucosamine is most preferable. This is because the phototoxicity of glucosamine is excellent.

In formula (1), R ¹ , R ² and R ³ are each independently H (hydrogen atom), an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms, and R ¹ , R ² and R ³ are each an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms.
Here, examples of the acetoxyalkyl having 1 to 6 carbon atoms include acetoxymethyl, acetoxyethyl, acetoxypropyl, and acetoxybutyl. The hydrocarbon having 1 to 6 carbon atoms is a straight chain having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, and cyclohexyl. Examples include chain, branched chain, or cyclic alkyl. Among them, R ¹ , R ² and R ³ are each independently an acetoxyalkyl group having 1 to 6 carbon atoms or 1 carbon atom in that a glycosylated chlorin e6 derivative having a better effect of the present invention can be obtained. It is preferably a hydrocarbon group of ˜6. It is because the uptake | capture property with respect to a cancer cell improves.

R ¹ , R ² and R ³ are each independently preferably a hydrocarbon group having 1 to 3 carbon atoms, more preferably a methyl group, from the viewpoint of water solubility.

In the group represented by R—X— * (* represents the bonding position), the divalent group (linking group) X preferably contains O (oxygen atom), and O and S (sulfur atoms) ) Is more preferable.
Among them, the group represented by R—X— * includes a group represented by R—X ³ —O— * in that a glycosidated chlorin e6 derivative having a better effect of the present invention can be obtained. preferable. Here, X ³ is a linear or branched divalent group consisting of at least one selected from the group consisting of C, N, O, H, and S, and constitutes R It is bonded to any one of the carbon atoms. No particular limitation is imposed on the divalent group X ^3, the same form as the divalent group X already described.
That is, the glycosylated chlorin e6 derivative is preferably represented by the following formula (2). In the formula (2), the form of the sugar residue R is as already described as R in the formula (1).

The group represented by R—X ³ —O— * is a group represented by R—L—S—X ⁴ —O— * in that a glycosidated chlorin e6 derivative having an excellent effect of the present invention can be obtained. More preferred are the groups Here, L represents a single bond or a divalent group. Although it does not restrict | limit especially as a bivalent coupling group of L, For example, it is as having already demonstrated as a bivalent group of X.
Among them, it is preferable that L in the group represented by RLS—X ⁴ —O— * is a single bond in that a glycosidated chlorin e6 derivative having a superior effect of the present invention can be obtained. . That is, the group represented by R—X— * is a group represented by R—S—X ⁴ —O— *, in other words, the sugar residue R is directly linked to —S—X ⁴ —O—. The groups are preferred. Here, the direct linkage refers to, for example, a structure (—C—S—X ⁴ —O—) in which C (carbon atom) at the anomeric position of a sugar and —S—X ⁴ —O— are linked.

In addition, S (sulfur atom) in the group represented by R—S—X ⁴ —O— * is a linking group bonded to the anomeric carbon atom of R (carbon atom at position 1), or the anomeric position from the viewpoint of synthesis. A linking group bonded to a carbon atom adjacent to the carbon atom (2-position carbon atom) is preferable, and a linking group bonded to the anomeric carbon atom (1-position carbon atom) of R is more preferable.

X ⁴ is bonded to O (oxygen atom) and S (sulfur atom). X ⁴ is a linear or branched divalent group having C (carbon atom) and H (hydrogen atom). X ⁴ is not particularly limited, and examples thereof include an alkylene group, an oxyalkylene group, and an alkyleneoxy group. Among these, a linear or branched alkylene group having 1 to 16 carbon atoms may be used. A linear alkylene group represented by — (CH ₂ ) n— is more preferable. n is preferably an integer of 1 to 16, more preferably n is an integer of 2 to 13, and further preferably n is an integer of 3 to 10. This is because the synthesis is easy and the water solubility of the compound is increased.

In the formula (3), the form of the sugar residue R is as already described as R in the formula (1).

Among these, the glycosylated chlorin e6 derivative is preferably represented by the following formula (4), (5), or (6) from the viewpoint of having particularly excellent effects of the present invention. In the following formulas (4) to (6), n represents an integer of 3 to 10, respectively.

In addition, one kind of glycosylated chlorin e6 derivative may be used alone, or two or more kinds may be used in combination.

Pharmaceutically acceptable salts include alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as magnesium and calcium salts), ammonium salts, mono-, di- or tri -Lower (alkyl or hydroxyalkyl) ammonium salts (eg ethanolammonium salt, diethanolammonium salt, triethanolammonium salt, tromethamine salt), hydrochloride, hydrobromide, hydroiodide, nitrate, phosphate, Sulfate, formate, acetate, citrate, oxalate, fumarate, maleate, succinate, malate, tartrate, trichloroacetate, trifluoroacetate, methanesulfonate, Benzene sulfonate, p-toluene sulfonate, mesitylene sulfonate and naphthalene Sulfonic acid salts.
The salt may be an anhydride or a solvate, and examples of the solvate include hydrate, methanol solvate, ethanol solvate, propanol solvate, and 2-propanol solvate.

The present chlorin derivative having the above-described configuration does not show cytotoxicity in the dark, but shows strong cytotoxicity under light irradiation. And it is estimated that the absorption in a long wavelength is large compared with a porphyrin derivative (absorption maximum wavelength 650nm), and the compound has high cell affinity and / or cell permeability by the coupling | bonding of saccharide | sugar.

Next, a method for producing the present chlorin derivative and the like will be described. The method for producing a chlorin derivative or the like according to an embodiment of the present invention includes a step of linking chlorin e6 and a sugar via a linking group (glycosidation). More specifically, it includes a step of glycosylation by binding a 3-position double bond of chlorin e6 alkyl ester and a sugar via a linking group. The procedure for binding the linking group may be to link the sugar and the linking group and then link to chlorin e6. Alternatively, the linking group and chlorin e6 may be combined and then linked to the saccharide, depending on the purpose. The manufacturing method may be selected.

In the following, a manufacturing method in which a sugar and a linking group are first bonded and then linked to chlorin e6 will be described in detail with reference to FIG.
First, the thiol sugar (R-SH) has a functional group that can be linked to the thiol sugar, such as a leaving group (E) such as a halogen, a tosyl group, and a mesyl group, and a hydroxyl group (-OH). A linking group (EX ⁴ —OH) is introduced.

The sugar residue R of the thiol sugar (R-SH) used here is preferably protected with a protecting group such as an acyl group. Protecting groups include acetyl and aliphatic acyl groups such as pivaloyl; aromatic acyl groups such as benzoyl group; and aralkyl groups such as benzyl group. Of these protecting groups, an acetyl group is particularly preferred.

Examples of the linking group (EX ⁴ —OH) linked to the thiol sugar (R—SH) include chloroalcohol, bromoalcohol, iodoalcohol, tosylalcohol, and mesylalcohol.

The solvent used in this reaction is not particularly limited as long as the reaction proceeds. For example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride Halogenated hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, And ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide; water; and mixtures thereof. A particularly preferred solvent for the above reaction is chloroform or dichloromethane.

When a base is used in this reaction, for example, basic salts such as sodium carbonate, potassium carbonate, and cesium carbonate; inorganic bases such as sodium hydroxide and potassium hydroxide; pyridine, lutidine, etc. Aromatic amines such as: triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine, and N-methylmorpholine Tertiary amines; Sodium hydrides and alkali metal hydrides such as potassium hydride; Metal amides such as sodium amide, lithium diisopropylamide and lithium hexamethyldisilazide; Sodium methoxide and sodium ethoxy Do And metal alkoxides such as potassium tert- butoxide; is selected from the like.
The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractional distillation, crystallization, recrystallization, and chromatography.

Next, a thiol sugar linking group conjugate (RS—X ⁴ —OH) is introduced into chlorin e6 trimethyl ester.

First, chlorin e6 trimethyl ester hydrogen halide adduct is obtained by adding hydrogen halide to the double bond at the 3-position in the porphyrin ring 1-24 number method with respect to chlorin e6 trimethyl ester. Examples of the hydrogen halide used include hydrogen chloride, hydrogen bromide, and hydrogen iodide. As the hydrogen halide, hydrogen bromide is preferable from the viewpoint of the reactivity and stability of the chlorin e6 trimethyl ester hydrogen halide adduct.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds, but formic acid, acetic acid, propionic acid and the like can be mentioned.

The resulting chlorin e6 trimethyl ester hydrogen halide adduct is allowed to act on a thiol sugar linking group conjugate (RS—X ⁴ —OH) to give a thiol sugar linking group conjugate (RS—X ⁴ —OH). ) To a chlorin e6 trimethyl ester hydrohalide to give a sugar-linked chlorin e6 trimethyl ester. The thiol sugar linking group conjugate (RS—X ⁴ —OH) added in the above reaction is preferably added in an amount of 3 equivalents or more with respect to the chlorin e6 trimethyl ester hydrogen halide adduct. This is because the yield of sugar-linked chlorin e6 trimethyl ester is improved.

When a base is used in this reaction, for example, basic salts such as sodium carbonate, potassium carbonate, and cesium carbonate; inorganic salts such as sodium hydroxide and potassium hydroxide; pyridine, and Aromatic amines such as lutidine; triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine, and N-methylmorpholine Tertiary amines such as sodium hydride and alkali metal hydrides such as potassium hydride; metal amides such as sodium amide, lithium diisopropylamide and lithium hexamethyldisilazide; sodium methoxide, Sodium etoki De, and it is selected from metal alkoxides such as potassium tert- butoxide.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds. For example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride Halogenated hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, And ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide, or a mixture of two or more of these. Particularly preferred solvents for the above reaction are dichloromethane, chloroform and mixtures thereof from the viewpoint of reactivity.

The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractional distillation, crystallization, recrystallization, and chromatography.

Next, a production method in which a linking group and chlorin e6 are bonded and then linked to a saccharide will be described in detail with reference to FIG.

First, the chlorin e6 trimethyl ester has a functional group capable of being linked to a sugar, for example, a linking group (E) having a leaving group (E) such as a halogen, a tosyl group, and a mesyl group, and a hydroxyl group (—OH). -X ⁴ -OH).

In the chlorin e6 trimethyl ester, a chlorin e6 trimethyl ester hydrogen halide adduct is obtained by adding a hydrogen halide to the double bond at the 3-position in the porphyrin ring 1-24 number method. Examples of the hydrogen halide used include hydrogen chloride, hydrogen bromide, and hydrogen iodide. As the hydrogen halide, hydrogen bromide is preferable from the viewpoint of the reactivity and stability of the chlorin e6 trimethyl ester hydrogen halide adduct.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds, and formic acid, acetic acid, propionic acid and the like can be mentioned.

To the resulting chlorin e6 trimethyl ester hydrogen halide adduct, is reacted with a linking group ^(E-X 4 -OH), a linking group ^(E-X 4 -OH) the chlorin e6 trimethyl ester hydrogen halide adduct Combine to give the linking group-bound chlorin e6 trimethyl ester.
Examples of the linking group (EX ⁴ —OH) include chloroalcohol, bromoalcohol, iodoalcohol, tosylalcohol, and mesylalcohol. The linking group (EX ⁴ -OH) added in the above reaction is preferably added in an amount of 10 equivalents or more with respect to the chlorin e6 trimethyl ester hydrogen halide adduct. This is because the yield of the linking group-bound chlorin e6 trimethyl ester is improved.

When a base is used in this reaction, for example, basic salts such as sodium carbonate, potassium carbonate, and cesium carbonate; inorganic bases such as sodium hydroxide and potassium hydroxide; pyridine, lutidine, etc. Aromatic amines such as: triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine, and N-methylmorpholine Tertiary amines; Sodium hydrides and alkali metal hydrides such as potassium hydride; Metal amides such as sodium amide, lithium diisopropylamide and lithium hexamethyldisilazide; Sodium methoxide and sodium ethoxy Do And it is selected from metal alkoxides such as potassium tert- butoxide.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds. For example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride Halogenated hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, And ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide; and mixtures of two or more of these. Particularly preferred solvents for the above reaction are dichloromethane, chloroform and mixtures thereof from the viewpoint of reactivity.

The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractional distillation, crystallization, recrystallization, and chromatography.
Next, a thiol sugar (R-SH) is introduced into the linking group-bound chlorin e6 trimethyl ester.

As the thiol sugar (R-SH) used here, it is preferable to use a sugar in which a hydroxyl group in the sugar is protected with a protecting group such as an acyl group. Examples of the protecting group include an acetyl group and an aliphatic acyl group such as a pivaloyl group; an aromatic acyl group such as a benzoyl group; an aralkyl group such as a benzyl group; Of these protecting groups, an acetyl group is particularly preferred.

The solvent used in this reaction is not particularly limited as long as the reaction proceeds. For example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride Halogenated hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, Examples thereof include ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide; water; a mixture of two or more of these. Particularly preferred solvents for the above reaction are chloroform and dichloromethane.

When a base is used in this reaction, for example, basic salts such as sodium carbonate, potassium carbonate and cesium carbonate; inorganic bases such as sodium hydroxide and potassium hydroxide; pyridine, lutidine and the like Aromatic amines; triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylpyrrolidine, N-methylmorpholine, etc. Tertiary amines; alkali metal hydrides such as sodium hydride and potassium hydride; metal amides such as sodium amide, lithium diisopropylamide and lithium hexamethyldisilazide; sodium methoxide, sodium ethoxide , And metal alkoxides such as potassium tert- butoxide; is selected from the like.
The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractional distillation, crystallization, recrystallization, and chromatography.

In the above production method, when a sugar residue R in which a hydroxyl group is blocked with a protecting group is used, the protecting group is then eliminated and removed by alkali treatment or the like. When the protecting group is an acyl group, it may be hydrolyzed by adding an alkaline solution. For example, alkali metals such as sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide, and potassium tert-butoxide in dichloromethane, methanol, ethanol, tetrahydrofuran, or a mixed solvent thereof The protecting group is removed by treating the protected compound with an alkali such as an alkoxide. When the protecting group is an aralkyl group, it can be removed by hydrogenation using a palladium catalyst. Deprotecting the hydroxyl group of the sugar residue R further promotes the transfer of the present chlorin derivative and the like into the cell, and is superior in cytotoxicity.

[Use]
This chlorin derivative, etc. does not show cytotoxicity in the dark, but makes use of the fact that it exhibits strong cytotoxicity under irradiation with light, so that it is brought into contact with the target biological material in vitro or in vivo in the dark. It can be used for the purpose of destroying the target by irradiating with light having an absorption wavelength such as the present chlorin derivative after incorporation into the cell.

Here, examples of the target include a target selected from the group consisting of viruses, microorganisms and their infected cells, tumor cells, tumorous tissues, and neovascularization. Therefore, it can be used for tumor destruction.

Therefore, the present chlorin derivative and the like can be used as a therapeutic agent for cancer classified as a malignant tumor. Examples of the malignant tumor include an epithelial malignant tumor and a malignant tumor having a non-epithelial tissue classified as a sarcoma as a development base. The present chlorin derivative is particularly useful for the treatment of solid tumors in which tumor cells grow massively and solidly, and surface cancers that reach light, among tumor cells.
Specific examples include esophageal cancer, lung cancer, gastric cancer, cervical cancer, uterine cancer such as uterine cancer, skin cancer, prostate cancer, and kidney cancer. Skin cancer includes primary (squamous cell carcinoma, basal cell carcinoma, and epidermis cancer), as well as skin metastasis of visceral cancer.

In addition, since the present chlorin derivative has an affinity for benign tumors among tumor cells, photodynamics of skin diseases such as actinic keratosis, severe acne, and skin psoriasis can be obtained by local administration. It can also be used as a therapeutic drug and as a photodynamic therapeutic drug for eye diseases such as age-related macular degeneration.

Furthermore, by utilizing the tumor accumulation property of the glycosylated chlorin e6 derivative of the present invention, it can be used for diagnosis of cancer in combination with PET or the like. Furthermore, the glycosylated chlorin derivative of the present invention can also be used for tumor detection by utilizing its tumor accumulation property and affinity for benign tumors.

[How to destroy a target]
As already explained, the present chlorin derivative and the like can be applied to the method for destroying the above target. The method for destroying a target according to an embodiment of the present invention includes a step of irradiating the target with light having a wavelength absorbed by the chlorin derivative or the like after contacting the target with the chlorin derivative or the like.
In addition, the said method can be implemented in a human individual and a non-human individual. That is, the method for destroying the target can be carried out in a human individual, and can be carried out in a form other than that in a human individual.

[Pharmaceutical composition]
A pharmaceutical composition according to an embodiment of the present invention is for photodynamic treatment of tumors, skin diseases, eye diseases or age-related macular degeneration, and contains the glycosylated chlorin e6 derivative as an active ingredient. In particular, it has a better effect for photodynamic therapy of tumors.

As described above, the glycosylated chlorin e6 derivative, which is an active ingredient, has excellent water solubility, excellent cell permeability, and high phototoxicity. Therefore, it is a drug for photodynamic treatment of tumors (particularly solid cancer). Can be suitably used.

The pharmaceutical composition can be administered by catheter, intravenous or intramuscular injection, and can be administered by other parenteral routes.
Moreover, it may be a creamy pharmaceutical composition and can be administered transdermally. In addition, it can be locally injected directly into the tumor tissue deep inside the body.

In addition, as long as the present pharmaceutical composition contains the present chlorin derivative and the like, it may contain other components as necessary. Examples of other components include excipients.

Excipients include, for example, lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, and sodium chloride as solids, Examples thereof include glycerin, peanut oil, polyvinyl pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

In addition to excipients, this pharmaceutical composition can be used as necessary, including bases, surfactants, preservatives, emulsifiers, colorants, flavoring agents, fragrances, stabilizers, preservatives, antioxidants, An agent, an antibacterial agent, a solubilizing agent, a suspending agent, a binder, and a disintegrating agent may be contained.

The dosage form of the pharmaceutical composition is not particularly limited, and examples thereof include tablets, powders, granules, capsules, troches, syrups, emulsions, soft gelatin capsules, gels, pastes, injectable preparations, creams and gels. , Lotions, patches and the like.

The carrier used in the present pharmaceutical composition is appropriately selected according to the type of preparation. When it is prepared as an injectable preparation, it can be formulated into a sterile aqueous solution or dispersion containing the present chlorin derivative or the like or a sterile lyophilized preparation containing the present chlorin derivative or the like. As the liquid carrier, for example, water, physiological saline, ethanol, hydrous ethanol, glycerol, propylene glycol, vegetable oil and the like are preferable.

In the present pharmaceutical composition, together with the present chlorin derivative, etc., diluents such as lactose, sucrose, dicalcium phosphate and carboxymethylcellulose; lubricants such as magnesium stearate, calcium stearate and talc; starch, glucose, molasses , Polyvinyl pyrrolidone, cellulose, and derivatives thereof, and the like.

The content of the active ingredient per preparation in the present embodiment can be appropriately determined according to the subject to be treated and the usage, but for example, the amount of glycosylated chlorin e6 derivative can be 1 to 2000 mg. -1000 mg is preferable, and 10-500 mg is more preferable.

The dose of the present chlorin derivative and the like varies depending on the subject to be treated and the purpose, but in general, the amount of the present chlorin derivative and the like is preferably 0.1 to 30 mg / kg for tumor diagnosis or detection and tumor treatment. The standard is 0.2 to 20 mg / kg.
Since the present chlorin derivative, which is an active ingredient, has high cytotoxicity, it can be expected to obtain an effect equal to or higher than that of conventional products (photofurin and resafirin). This means that the time required for metabolism and excretion is short, and the convenience of using photodynamic therapy is enhanced.

To treat the tumor, after administering the present chlorin derivative or the like, the region to be treated is irradiated with light containing the absorption band of the compound. Specifically, singlet oxygen can be generated by irradiation with light having a wavelength of 500 nm or more, and the desired cytotoxicity can be exhibited, but irradiation with light having a high ratio of light having the maximum absorption wavelength is preferable.

As the irradiation source, an LED (Light Emitting Diode), a laser, a halogen lamp, or the like is used. As the laser, a dye laser, a semiconductor laser, an argon laser, or the like may be used as long as it can obtain light having a wavelength necessary for excitation.

Hereinafter, the present invention will be described in detail based on examples and comparative examples. However, the present invention is not limited by these. The compound manufactured based on the Example and the comparative example evaluated by the following method.

[Evaluation method of phototoxicity]
IC ₅₀ (Half maximum (50%) inhibitory concentration) was used for the phototoxicity evaluation, and the measurement was performed according to the following procedure.
MKN45 (obtained from JCRB cell bank and subcultured for 6 months) and MKN28 (obtained from Immunobiological Laboratories and subcultured for 6 months), which are human gastric cancer-derived cells, were each 96. 5 × 10 ³ cells / well were seeded in a predetermined number of wells in a well plate (RPMI 1640 medium containing 10% FBS, 100 μL) and cultured at 37 ° C. for 24 hours in the presence of 5% CO ₂ . Next, 100 μL of the same medium solution containing the drug at different concentrations is added to each well, each well is adjusted to a predetermined drug concentration, cultured for 4 hours, and then 100 μL of phosphate buffered saline (PBS). Washed once with Buffered Saline), 100 μL of PBS was added again. Then, light was irradiated for 8 minutes and 40 seconds (16 J / cm ² ) using a 660 nm LED light source (30.8 mW / cm ² , LEDR-660DL, OptoCode). After irradiation, PBS was removed, and 100 μL of RPMI 1640 medium containing 2% FBS was added and cultured for 24 hours.

After culturing, operation was performed according to the protocol of Cell Counting kit-8 (Dojindo Laboratories: Viable Cell Counting Kit), and the drug was added by measuring the absorbance at 450 nm with a microplate reader (SPECTRA MAX340, manufactured by Molecular Devices). The cell viability as a relative value with respect to the case where it did not was calculated. The measurement was carried out at n = 8, except for the maximum and minimum values obtained in 8 times, the average value was the IC ₅₀ value, and those having an IC ₅₀ of less than 0.01 μmol / L were A, 0.01 μmol. / L or more and less than 0.1 μmol / L is B, 0.1 μmol / L or more and less than 1 μmol / L is C, 1 μmol / L or more and less than 10 μmol / L is D, 10 μmol / L or more is E It was evaluated.

[Method for evaluating tumor growth inhibition rate]
Nude mice (4-6 mice in each group, 4-5 weeks old female, 20 ± 2 g body weight, obtained from BioLASCO) on the right buttock, 10% fetal bovine serum (Gibco) at 37 ° C. under 5% CO ₂ 2 × 10 ⁷ human colon cancer cell lines HT29 grown in Dulbecco's modified Eagle's medium containing 1 mmol / l sodium pyruvate (Gibco BRL) supplemented with 1% antibiotics (manufactured by BRL) (0 days) The tumor size was measured every 2 days. Tumor volume was calculated by 1/2 (4π / 3) (L / 2) (W / 2) H (L: length of tumor, W: width, H: height). When the tumor volume reached 50 to 100 mm3 (10th day), 0.1 ml of physiological saline (control group) was added from the lateral tail vein of nude mice, and the drug concentration was 1 in physiological saline or 20% PEG aqueous solution. 0.1 ml of drug solution dissolved at 25 mM was administered. At 4 or 24 hours after administration, the tumor was irradiated with a diode laser (100 mW / cm ² , CrystaLaser CL660) with a spot of 660 nm (15 Jcm ⁻² ) and a diameter of 2 cm.
The tumor size is measured every 2 days, and the tumor growth inhibition rate (TGI%) is calculated from the tumor volume on the 24th day according to the following formula. When the TGI% is less than 65%, D, 65% or more and 70 Less than% was evaluated as C, 70% or more and less than 75% was evaluated as B, and 75% or more was evaluated as A.

TGI% = ((Tumor volume in control group) − (Tumor volume in treatment group)) / (Tumor volume in control group) × 100%

[Example 1]
Production and evaluation of 1- (3-hydroxy-propanethio) -β-D-glucose-linked chlorin e6 trimethyl ester (1-1) Synthesis of 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate Nitrogen atmosphere Then, 1-thio-β-D-glucose tetraacetate (2.73 g, 7.50 mmol) was dissolved in chloroform (5.0 ml), and triethylamine (2.08 ml, 15.00 mmol) was added. The resulting solution was cooled to 0 ° C. and stirred while 3-bromo-1-propanol (0.85 ml, 9.75 mmol) was slowly added dropwise. After completion of the dropwise addition, the temperature of the solution was raised to 25 ° C. and stirred for 3 hours. Water (50 ml) and chloroform (30 ml) were added to the stirred solution, and a liquid separation operation was performed to separate the chloroform layer. The obtained chloroform layer was washed with saturated brine (50 ml) and dehydrated with anhydrous sodium sulfate.
Next, the obtained chloroform solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and hexane. The eluent was concentrated under reduced pressure to obtain 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate (yield 2.68 g, yield 85%).

The obtained compound is J.P. Org. Chem. Based on 2013, 78, 5196-5204, it was confirmed by 1H-NMR (400 MHz, deuterated chloroform solvent).

(1-2) Synthesis of Chlorine e6 Trimethyl Ester Under a nitrogen atmosphere, chlorin e6 (11.93 g, 20.00 mmol) was dissolved in a mixed solvent of dehydrated dichloromethane (500 ml) and dehydrated methanol (250 ml). Trimethylsilyldiazomethane (2.0 mol / l hexane solution, 34.0 ml) was added dropwise to the resulting solution, and the mixture was stirred at 25 ° C. for 2 hours. The solvent was distilled off from the obtained reaction solution under reduced pressure, and the resulting residue was recrystallized from dichloromethane / hexane to obtain chlorin e6 trimethyl ester (yield 11.64 g, yield 91%).

The obtained compound was confirmed by ¹ H-NMR (400 MHz, deuterated chloroform solvent) based on Photochemistry and Photobiology, 2007, 83, 1006-1015.

(1-3) Synthesis of 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester Chlorine e6 trimethyl ester synthesized in the above (1-2) (0.95 g, 1.48 mmol) ) Was dissolved in 25% hydrogen bromide-acetic acid solution (18.0 ml) under a nitrogen atmosphere and stirred at 30 ° C. for 2 hours. The hydrogen bromide-acetic acid solution was distilled off under reduced pressure from the resulting reaction solution to dry the reaction solution. The obtained residue was dissolved in dehydrated dichloromethane (50 ml), potassium carbonate (2.05 g, 14.80 mmol) was added, and the mixture was stirred at 30 ° C. for 30 minutes. 1- (3-Hydroxy-propanethio) -β-D-glucose tetraacetate (1.88 g, 4.45 mmol) synthesized in (1-1) above was dissolved in dehydrated dichloromethane (50 ml) in the resulting solution. And then stirred at 30 ° C. for 5 hours. Water (100 ml) and dichloromethane (200 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (200 ml) and saturated brine (200 ml), and dried over anhydrous sodium sulfate. The obtained dichloromethane solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluent was concentrated under reduced pressure to obtain 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 0.42 g, yield 27%).

In the resulting compound, the peak derived from the 3-position double bond of chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent), and 1- (3-hydroxy-propanethio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed that the molecular weights coincided with each other by analysis by an ESI + MS (Electrospray Ionization Mass Spectrometry) method.
MS: m / z ([M + H] +); calcd. _{1061.45 for C 54 H 68 N 4} O 16 S, found. 1061.41.

(1-1 ′) Synthesis of 3-bromo-1-propanol-linked chlorin e6 trimethyl ester Chlorine e6 trimethyl ester (0.96 g, 1.50 mmol) synthesized in (1-2) above was treated with a 25% odor in a nitrogen atmosphere. Dissolved in a hydrogen fluoride-acetic acid solution (18.0 ml) and stirred at 30 ° C. for 2 hours. The hydrogen bromide-acetic acid solution was distilled off under reduced pressure from the resulting reaction solution to dry the reaction solution. The obtained residue was dissolved in dehydrated dichloromethane (50 ml), potassium carbonate (2.07 g, 15.00 mmol) was added, and the mixture was stirred at 30 ° C. for 30 min. To the obtained solution, 3-bromo-1-propanol (2.60 ml, 30.00 mmol) was added dropwise and stirred at 30 ° C. for 5 hours. Water (100 ml) and dichloromethane (200 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (200 ml) and saturated brine (200 ml), and dehydrated with anhydrous sodium sulfate. The obtained dichloromethane solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluent was concentrated under reduced pressure to obtain 3-bromo-1-propanol-linked chlorin e6 trimethyl ester (yield: 0.91 g, yield: 78%).

In the obtained compound, the peak derived from the 3-position double bond of chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent), and 3-bromo-1-propanol was bonded. confirmed. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

MS: m / z ([M + H] +); calcd. _{777.29 for C 40 H 49 BrN 4} O 7, found. 777.29.

Synthesis of (1-2 ′) 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester Under nitrogen atmosphere, 1-thio-β-D-glucose tetraacetate (131 mg, 0. 36 mmol) was dissolved in chloroform (1.0 ml) and triethylamine (90 μl, 0.65 mmol) was added. The resulting solution was cooled to 0 ° C. and stirred while 3-bromo-1-propanol linked chlorin e6 trimethyl ester (86 mg, 0.11 mmol) was slowly added dropwise. After completion of dropping, the temperature of the solution was raised to 20 ° C. and stirred for 14 hours. Water (10 ml) and chloroform (10 ml) were added to the stirred solution, and a liquid separation operation was performed to separate the chloroform layer. The obtained chloroform layer was washed with saturated brine (10 ml) and dehydrated with anhydrous sodium sulfate. The obtained chloroform solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluent was concentrated under reduced pressure to give 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester (yield 52 mg, 45%).
It was confirmed that 1-thio-β-D-glucose tetraacetate was bound to the obtained compound by ¹ H-NMR (400 MHz, deuterated chloroform solvent). Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

MS: m / z ([M + H] +); calcd. _{1061.45 for C 54 H 68 N 4} O 16 S, found. 1061.45.

(1-4) Synthesis of 1- (3-hydroxy-propanethio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (3-hydroxy-synthesized in (1-3) and (1-2 ′) above Propanethio) -β-D-glucose tetraacetate linked chlorin e6 (107 mg, 0.10 mmol) was dissolved in dehydrated methanol (8.0 ml) under a nitrogen atmosphere, and sodium methoxide (54 mg, 1.00 mmol) was added to add 25 Stir at 30 ° C. for 30 minutes. Acetic acid (58 μl, 1.00 mmol) was added to the resulting reaction solution to stop the reaction. The solvent was distilled off from the resulting solution under reduced pressure, and the residue was loaded onto a PLC glass plate (silica gel 60 F254, manufactured by Merck & Co., Inc.) and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, filled in reverse phase silica gel chromatography (Sep-Pak C18, manufactured by Waters), the salt was eluted with ion-exchanged water, and then eluted with methanol. The obtained methanol solution was concentrated under reduced pressure to obtain 1- (3-hydroxy-propanethio) -β-D-glucose linked chlorin e6 trimethyl ester (yield 64 mg, yield 72%).
In the obtained compound, the peak derived from the acetyl group of 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester disappeared in ¹ H-NMR (400 MHz, deuterated chloroform solvent). Confirmed that. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

MS: m / z ([M + H] +); calcd. _{893.40 for C 46 H 60 N 4} O 12 S, found. 893.45.

(1-5) Evaluation of 1- (3-hydroxy-propanethio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (3-hydroxy-propanethio) -β-D- obtained by the above production method Glucose-linked chlorin e6 trimethyl ester was evaluated based on a phototoxicity evaluation method and a tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 2]
Production and evaluation of 1- (6-hydroxy-hexanethio) -β-D-glucose-linked chlorin e6 trimethyl ester (2-1) Synthesis of 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate Nitrogen atmosphere Then, 1-thio-β-D-glucose tetraacetate (1.37 g, 3.75 mmol) was dissolved in chloroform (2.5 ml), and triethylamine (1.04 ml, 7.50 mmol) was added. The resulting solution was cooled to 0 ° C. and stirred while 6-bromo-1-hexanol (0.66 ml, 4.88 mmol) was slowly added dropwise. After completion of the dropwise addition, the temperature of the solution was raised to 25 ° C. and stirred for 3 hours. Water (25 ml) and chloroform (15 ml) were added to the stirred solution, and a liquid separation operation was performed to separate the chloroform layer. The obtained chloroform layer was washed with saturated brine (25 ml) and dehydrated with anhydrous sodium sulfate. The obtained chloroform solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and hexane. The eluent was concentrated under reduced pressure to obtain 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate (yield 1.39 g, yield 80%).

Structural identification is described in J. Org. Org. Chem. Based on 2013, 78, 5196-5204, ¹ H-NMR (400 MHz, deuterated chloroform solvent) was used.

¹ H-NMR (CDCl3, 400 MHz) δ 5.22 (t, J = 9.4 Hz, 1H, H-3), 5.08 (t, J = 10.0 Hz, 1H, H-4), 5.04 (T, J = 10.0 Hz, 1H, H−2) 4.48 (d, J = 10.8 Hz, 1H, H−1), 4.25 (dd, J = 11.2 Hz, J = 2. 3Hz, 1H, H-6), 4.14 (dd, J = 11.2Hz, J = 2.3Hz, 1H, H-6), 3.70-3.72 (m, 1H, H-5) , 3.6-3.65 (m, 2H, CH ₂ OH), 2.64-2.72 (m, 2H, SCH ₂ ), 2.06, 2.05, 2.03, 2.01 ( s, 12H, 4 × OCOCH ₃ ), 1.52-1.65 (m, 4H, —CH ₂ —), 1.35—1.42 (m, 4H, —CH ₂ —)

(2-2) Synthesis of 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester Chlorine e6 trimethyl ester (664 mg, 1.04 mmol) synthesized in Example 1 was placed under a nitrogen atmosphere. , 25% hydrogen bromide-acetic acid solution (12.0 ml) and stirred at 30 ° C. for 2 hours. The hydrogen bromide-acetic acid solution was distilled off under reduced pressure from the resulting reaction solution to dry the reaction solution. The obtained residue was dissolved in dehydrated dichloromethane (35 ml), potassium carbonate (1437 mg, 10.40 mmol) was added, and the mixture was stirred at 30 ° C. for 30 min. 1- (6-Hydroxy-hexanethio) -β-D-glucose tetraacetate (1450 mg, 3.12 mmol) synthesized in (1-1) above was dissolved in dehydrated dichloromethane (35 ml) and added dropwise to the resulting solution. And stirred at 30 ° C. for 5 hours. Water (70 ml) and dichloromethane (140 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (140 ml) and saturated brine (140 ml), and dried over anhydrous sodium sulfate. The obtained dichloromethane solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluent was concentrated under reduced pressure to obtain 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester (yield 145 mg, yield 13%).

In the resulting compound, the peak derived from the 3-position double bond of chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent), and 1- (6-hydroxy-hexanethio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

(2-3) Synthesis of 1- (6-hydroxy-hexanethio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (6-hydroxy-hexanethio) -β-D- synthesized in (2-2) above Glucose tetraacetate-linked chlorin e6 trimethyl ester (134 mg, 0.12 mmol) was dissolved in dehydrated methanol (10.0 ml) under a nitrogen atmosphere, sodium methoxide (65 mg, 1.21 mmol) was added, and the mixture was stirred at 25 ° C. for 30 minutes. did. Acetic acid (69 μl, 1.21 mmol) was added to the resulting solution to stop the reaction. The solvent was distilled off from the resulting solution under reduced pressure, and the residue was loaded onto a PLC glass plate (silica gel 60 F254, manufactured by Merck & Co., Inc.) and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, filled in reverse phase silica gel chromatography (Sep-Pak C18, manufactured by Waters), the salt was eluted with ion-exchanged water, and then eluted with methanol. The obtained methanol solution was concentrated under reduced pressure to obtain 1- (6-hydroxy-hexanethio) -β-D-glucose linked chlorin e6 trimethyl ester (yield 75 mg, 67%).

In the resulting compound, the peak derived from the acetyl group of 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent). I confirmed. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

(2-4) Evaluation of 1- (6-hydroxy-hexanethio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (6-hydroxy-hexanethio) -β-D- obtained by the above production method Glucose-linked chlorin e6 trimethyl ester was evaluated based on a phototoxicity evaluation method and a tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 3]
Production and evaluation of 1- (10-hydroxy-decanethio) -β-D-glucose-linked chlorin e6 trimethyl ester (3-1) Synthesis of 1- (10-hydroxy-decanthio) -β-D-glucose tetraacetate Nitrogen atmosphere Then, 1-thio-β-D-glucose tetraacetate (1.37 g, 3.75 mmol) was dissolved in chloroform (2.5 ml), and triethylamine (1.04 ml, 7.50 mmol) was added. The resulting solution was cooled to 0 ° C. and stirred while 10-bromo-1-decanol (1.00 ml, 4.88 mmol) was slowly added dropwise. After completion of the dropwise addition, the temperature of the solution was raised to 25 ° C. and stirred for 3 hours. Water (25 ml) and chloroform (15 ml) were added to the stirred solution, and a liquid separation operation was performed to separate the chloroform layer. The obtained chloroform layer was washed with saturated brine (25 ml) and dehydrated with anhydrous sodium sulfate. The obtained chloroform solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and hexane. The eluent was concentrated under reduced pressure to obtain 1- (10-hydroxy-decanthio) -β-D-glucose tetraacetate (yield 1.60 g, yield 82%).

Structural identification is described in J. Org. Org. Chem. Based on 2013, 78, 5196-5204, ¹ H-NMR (400 MHz, deuterated chloroform solvent) was used.

¹ H-NMR (CDCl3, 400 MHz) δ 5.22 (t, J = 9.4 Hz, 1H, H-3), 5.07 (t, J = 10.0 Hz, 1H, H-4), 5.03 (T, J = 10.0 Hz, 1H, H−2) 4.49 (d, J = 10.8 Hz, 1H, H−1), 4.25 (dd, J = 11.2 Hz, J = 2. 3Hz, 1H, H-6), 4.13 (dd, J = 11.2Hz, J = 2.3Hz, 1H, H-6), 3.69-3.72 (m, 1H, H-5) , 3.61-3.64 (m, 2H, CH ₂ OH), 2.60-2.72 (m, 2H, SCH ₂ ), 2.07, 2.05, 2.02, 2.01 ( s, 12H, 4 × OCOCH ₃ ), 1.52-1.65 (m, 4H, —CH ₂ —), 1.25-1.42 (m, 12H, —CH ₂ —)

(3-2) Synthesis of 1- (10-hydroxy-decanothio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester Chlorine e6 trimethyl ester (715 mg, 1.12 mmol) synthesized in Example 1 was placed under a nitrogen atmosphere. , 25% hydrogen bromide-acetic acid solution (13.0 ml), and stirred at 30 ° C. for 2 hours. The hydrogen bromide-acetic acid solution was distilled off under reduced pressure from the resulting reaction solution to dry the reaction solution. The obtained residue was dissolved in dehydrated dichloromethane (40 ml), potassium carbonate (1549 mg, 11.20 mmol) was added, and the mixture was stirred at 30 ° C. for 30 min. To the obtained solution, 1- (10-hydroxy-decanthio) -β-D-glucose tetraacetate (1750 mg, 3.36 mmol) synthesized in (3-1) above was dissolved in dehydrated dichloromethane (40 ml) and added dropwise. And stirred at 30 ° C. for 5 hours. Water (80 ml) and dichloromethane (160 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (160 ml) and saturated brine (160 ml), and dried over anhydrous sodium sulfate. The obtained dichloromethane solution was suction filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluent was concentrated under reduced pressure to obtain 1- (10-hydroxy-decanthio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 285 mg, yield 22%).

In the resulting compound, the peak derived from the 3-position double bond of chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent), and 1- (10-hydroxy-decanothio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

(3-3) Synthesis of 1- (10-hydroxy-decanethio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (10-hydroxy-decanethio) -β-D- synthesized in (3-2) above Glucose tetraacetate-linked chlorin e6 trimethyl ester (268 mg, 0.23 mmol) was dissolved in dehydrated methanol (20.0 ml) under nitrogen atmosphere, sodium methoxide (125 mg, 2.31 mmol) was added, and the mixture was stirred at 20 ° C. for 1 hour. did. Acetic acid (132 μl, 2.31 mmol) was added to the resulting solution to stop the reaction. The solvent was distilled off from the resulting solution under reduced pressure, and the residue was loaded onto a PLC glass plate (silica gel 60 F254, manufactured by Merck & Co., Inc.) and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, filled in reverse phase silica gel chromatography (Sep-Pak C18, manufactured by Waters), the salt was eluted with ion-exchanged water, and then eluted with methanol. The obtained methanol solution was concentrated under reduced pressure to obtain 1- (10-hydroxy-decanthio) -β-D-glucose linked chlorin e6 trimethyl ester (yield 149 mg, yield 65%).

In the obtained compound, the peak derived from the acetyl group of 1- (10-hydroxy-decanthio) -β-D-glucose tetraacetate linked chlorin e6 trimethyl ester disappeared in ¹ H-NMR (400 MHz, deuterated chloroform solvent). I confirmed. Moreover, it was confirmed by the analysis by ESI + MS method that the molecular weights coincided.

(3-4) Evaluation of 1- (10-hydroxy-decanothio) -β-D-glucose-linked chlorin e6 trimethyl ester 1- (10-hydroxy-decanothio) -β-D- obtained by the above production method Glucose-linked chlorin e6 trimethyl ester was evaluated based on a phototoxicity evaluation method and a tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 4]
Production and evaluation of 1- (3-hydroxy-propanethio) -β-D-galactose-linked chlorin e6 trimethyl ester (4-1) Synthesis of 1-thio-β-D-galactose tetraacetate 2,3,4, 6-Tetra-O-acetyl-D-galactopyranose (348 mg, 1.0 mmol) was dissolved in dichloromethane (5.0 ml), tetrabromomethane (663 mg, 2.0 mmol) and triphenylphosphine (525 mg, 2.0 mmol). ) And stirred at 25 ° C. for 4 hours. A mixed solution of carbon disulfide (114 mg, 1.5 mmol) and sodium sulfide nonahydrate (480 mg, 2.0 mmol) dissolved in dimethylformamide (2.0 ml) was added to the resulting reaction solution, and 25 Stir at 5 ° C. for 5 minutes. Water (30 ml) and dichloromethane (50 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (30 ml) and dehydrated with anhydrous sodium sulfate. The obtained dichloromethane layer was suction filtered and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and hexane. The eluent was concentrated under reduced pressure to obtain 1-thio-β-D-galactose tetraacetate (yield 291 mg, yield 80%).

Structural identification is described by Beilstein J. et al. Org. Chem. Based on 2013, 9, 974-982, ¹ H-NMR (400 MHz, deuterated chloroform solvent) was used.

(4-2) Synthesis and evaluation of 1- (3-hydroxy-propanethio) -β-D-galactose-linked chlorin e6 trimethyl ester (4-1) instead of 1-thio-β-D-glucose tetraacetate 1-thio-β-D-galactose tetraacetate synthesized in the same manner as in Example 1 was used to produce 1- (3-hydroxy-propanethio) -β-D-galactose-linked chlorin e6 Trimethyl ester was obtained.

The 1- (3-hydroxy-propanethio) -β-D-galactose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 5]
Production and evaluation of 1- (6-hydroxy-hexanethio) -β-D-galactose-linked chlorin e6 trimethyl ester 1- synthesized in (4-1) instead of 1-thio-β-D-glucose tetraacetate The same production method as in Example 2 was performed using thio-β-D-galactose tetraacetate to obtain 1- (6-hydroxy-hexanethio) -β-D-galactose-linked chlorin e6 trimethyl ester. .

The 1- (6-hydroxy-hexanethio) -β-D-galactose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 6]
Production and evaluation of 1- (10-hydroxy-decanthio) -β-D-galactose-linked chlorin e6 trimethyl ester 1- synthesized in (4-1) instead of 1-thio-β-D-glucose tetraacetate The same production method as in Example 3 was carried out using thio-β-D-galactose tetraacetate to obtain 1- (10-hydroxy-decanthio) -β-D-galactose-linked chlorin e6 trimethyl ester. .

The 1- (10-hydroxy-decanthio) -β-D-galactose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 7]
Production and evaluation of 1- (3-hydroxy-propanethio) -β-D-mannose-linked chlorin e6 trimethyl ester (7-1) Synthesis of 1-thio-β-D-mannose tetraacetate 2,3,4, 6-Tetra-O-acetyl-D-mannopyranose (348 mg, 1.0 mmol) is dissolved in dichloromethane (5.0 ml), tetrabromomethane (663 mg, 2.0 mmol) and triphenylphosphine (525 mg, 2. 0 mmol) and stirred at 25 ° C. for 4 hours. A mixed solution of carbon disulfide (114 mg, 1.5 mmol) and sodium sulfide nonahydrate (480 mg, 2.0 mmol) dissolved in dimethylformamide (2.0 ml) was added to the resulting reaction solution, and 25 Stir at 5 ° C. for 5 minutes. Water (30 ml) and dichloromethane (50 ml) were added to the resulting reaction solution to carry out a liquid separation operation, and a dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (30 ml) and dehydrated with anhydrous sodium sulfate. The obtained dichloromethane layer was suction filtered and the solvent was distilled off under reduced pressure. The obtained crude product was packed in a silica gel column (High Flash column, manufactured by Yamazen Co., Ltd.) and eluted with a mixed solvent of ethyl acetate and hexane. The eluent was concentrated under reduced pressure to obtain 1-thio-β-D-mannose tetraacetate (yield 255 mg, yield 70%).

Structural identification is described by Beilstein J. et al. Org. Chem. Based on 2013, 9, 974-982, ¹ H-NMR (400 MHz, deuterated chloroform solvent) was used.

(7-2) Synthesis and evaluation of 1- (3-hydroxy-propanethio) -β-D-mannose-linked chlorin e6 trimethyl ester (7-1) instead of 1-thio-β-D-glucose tetraacetate 1-thio-β-D-mannose tetraacetate synthesized in the same manner as in Example 1 was used to produce 1- (3-hydroxy-propanethio) -β-D-mannose linkage. Chlorine e6 trimethyl ester was obtained.

The 1- (3-hydroxy-propanethio) -β-D-mannose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 8]
Production and evaluation of 1- (6-hydroxy-hexanethio) -β-D-mannose-linked chlorin e6 trimethyl ester 1-thio- synthesized in (4-1) instead of 1-thio-β-D-glucose tetraacetate The same production method as in Example 2 was carried out using β-D-mannose tetraacetate to obtain 1- (6-hydroxy-hexanethio) -β-D-mannose-linked chlorin e6 trimethyl ester. .

The 1- (6-hydroxy-hexanethio) -β-D-mannose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Example 9]
Production and evaluation of 1- (10-hydroxy-decanthio) -β-D-mannose linked chlorin e6 trimethyl ester 1-thio- synthesized in (4-1) instead of 1-thio-β-D-glucose tetraacetate The same production method as in Example 3 was carried out using β-D-mannose tetraacetate to obtain 1- (10-hydroxy-decanthio) -β-D-mannose-linked chlorin e6 trimethyl ester. .

1- (10-Hydroxy-decanthio) -β-D-mannose-linked chlorin e6 trimethyl ester obtained based on the above production method was evaluated based on a phototoxicity evaluation method and a tumor growth inhibition rate evaluation method. Carried out. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Comparative Example 1]
Evaluation of talaporfin sodium Using commercially available talaporfin sodium (mono-L-aspartyl chlorin e6, manufactured by Medko Biosciences), evaluation was performed based on the phototoxicity evaluation method and tumor growth inhibition rate evaluation method. did. The results are shown in Table 1, Table 2 and Table 3. Further, it was confirmed that all nude mice were alive without weight loss within the evaluation period of the tumor growth inhibition rate, and no adverse effects on the nude mice due to drug administration and laser irradiation were observed.

[Results of phototoxicity evaluation]
The evaluation results of phototoxicity are shown in Tables 1 and 2 below. It should be noted that, in the table, the term "IC50" refers to the _{"IC 50".}

[Evaluation results of tumor growth inhibition rate]

From Tables 1 and 2, 1- (3-hydroxy-propanethio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 1), 1- (6-hydroxy-hexanethio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 2), 1- (10-hydroxy-decanthio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 3), 1- (3-hydroxy-propanethio) -β-D-galactose Linked chlorin e6 trimethyl ester (Example 4), 1- (6-hydroxy-hexanethio) -β-D-galactose Linked chlorin e6 trimethyl ester (Example 5), 1- (10-hydroxy-decanethio) -β-D -Galactose-linked chlorin e6 trimethyl ester (Example 6), 1- (3-hydroxy Propanethio) -β-D-mannose linked chlorin e6 trimethyl ester (Example 7), 1- (6-hydroxy-hexanethio) -β-D-mannose linked chlorin e6 trimethyl ester (Example 8) and 1- (10- hydroxy - Dekanchio) _{IC 50} of-beta-D-mannose linked chlorin e6 trimethyl ester (example 9) were both found to be smaller than the _{IC 50} of talaporfin sodium (Comparative example 1).

From Table 3, 1- (3-hydroxy-propanethio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 1), 1- (6-hydroxy-hexanethio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 2), 1- (10-hydroxy-decanthio) -β-D-glucose linked chlorin e6 trimethyl ester (Example 3), 1- (3-hydroxy-propanethio) -β-D-galactose linked chlorin e6 Trimethyl ester (Example 4), 1- (6-hydroxy-hexanethio) -β-D-galactose linked chlorin e6 trimethyl ester (Example 5), 1- (10-hydroxy-decanthio) -β-D-galactose linked Chlorine e6 trimethyl ester (Example 6), 1- (3-hydroxy-propa Thio) -β-D-mannose linked chlorin e6 trimethyl ester (Example 7), 1- (6-hydroxy-hexanethio) -β-D-mannose linked chlorin e6 trimethyl ester (Example 8) and 1- (10- It was found that the tumor growth inhibition rate of hydroxy-decanothio) -β-D-mannose-linked chlorin e6 trimethyl ester (Example 9) was higher than that of talaporfin sodium (Comparative Example 1).

From the above Examples and Comparative Examples, 1- (3-hydroxy-propanethio) -β-D-glucose linked chlorin e6 trimethyl ester, 1- (6-hydroxy-hexanethio) -β-D-glucose linked chlorin e6 trimethyl ester 1- (10-hydroxy-decanothio) -β-D-glucose linked chlorin e6 trimethyl ester, 1- (3-hydroxy-propanethio) -β-D-galactose linked chlorin e6 trimethyl ester, 1- (6-hydroxy- Hexanethio) -β-D-galactose-linked chlorin e6 trimethyl ester, 1- (10-hydroxy-decanthio) -β-D-galactose-linked chlorin e6 trimethyl ester, 1- (3-hydroxy-propanethio) -β-D-mannose Linked chlorin e6 trimethyl ester Ter, 1- (6-hydroxy-hexanethio) -β-D-mannose linked chlorin e6 trimethyl ester and 1- (10-hydroxy-decanthio) -β-D-mannose linked chlorin e6 trimethyl ester compared to talaporfin sodium It has excellent phototoxicity and excellent tumor growth inhibitory effect.

[Evaluation of phototoxicity with different cell lines]
Esophageal cancer cell line: KYSE30 (No. 11D028; ECACC), OE21 (No. 11D028; ECACC), gastric cancer cell line: MKN45 (No. 0254; Japan Cancer Research Bank), and colon cancer cell line: HT29 (No. HTB-38 (ATCC) was used and cultured under the following conditions.

KYSE30: RPMI 1640
OE21: RPMI 1640 and half mixture of Ham's F12 MKN45: RPMI 1640
HT29: McCoy's 5A
All cultures contained 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, 0.25 mg / ml amphotericin B, and were cultured under the conditions of 5% CO ₂ and 37 ° C.

Using the above cells, the 1- (3-hydroxy-propanethio) -β-D-glucose-linked chlorin e6 trimethyl ester of Example 1 (in Table 3) was prepared in the same manner as the “phototoxicity evaluation method” described above. IC ₅₀ (50% cancer cell killing concentration) was determined for TS (comparative example) and TS (comparative example), and evaluated according to the same criteria as “evaluation method for phototoxicity”. The results are shown in Table 4.

From the results shown in Table 4, it was found that the compound of Example 1 had excellent phototoxicity and had the effects of the present invention.
On the other hand, the TS of the comparative example did not have the effect of the present invention regardless of the type of cancer cell line.

[Evaluation of incorporation into cancer cell lines]
As a cell line, a human gastric cancer cell line: MKN45 (No. 0254; Japan Cancer Research Bank) and a colon cancer cell line: HT29 (No. HTB-38; ATCC) were used.

Cell culture conditions and equipment used are as follows.

Culture conditions:
As the culture solution, RPMI 1640 was used for MKN45, and McCoy's 5A was used for HT29. All cultures contained 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, 0.25 mg / ml amphotericin B, and were cultured under the conditions of 5% CO ₂ and 37 ° C.

Experimental equipment used:
FACSCanto II (BD Biosciences) was used for Flow cytometry.

(Test method)
Using a 6 cm culture plate, 2 × 10 ⁵ cells / well of the above cancer cell lines were cultured under the above conditions for 28 hours.
Next, the culture solution was removed, (1) only the culture solution (control), (2) a culture solution containing 5 μM of the glycosylated chlorin e6 derivative of Example 1, and (3) 5 μM Talaporfin Sodium (TS; (Registered trademark, Meiji Seika Pharma) was replaced with the culture solution, and further cultured for 4 hours.
After culturing for 4 hours, the culture solution was removed, washed three times with phosphate-buffered saline (PBS), and cells were collected from the culture plate using TrypLE-Express (Invitrogen).
Next, using the FACSCanto II, excitation was performed at 405 nm, and fluorescence emission at 680 nm was measured. At least 10,000 events were measured for each sample. The measurement was carried out by measuring the mean ammonia area (MAA) that evaluates TS and the vicinity of 650 nm, which is the fluorescence wavelength band of the compound of Example 1, and the results are shown in Table 5.

As shown in Table 5, when MKN45 was used as the cell line, the MAA of TS (Comparative Example) was 235, the MAA of the compound of Example 1 was 17051, and the MAA of the compound of Example 1 was the MAA of TS. It was about 70 times higher.
When HT29 is used as a cell line, the MAA of TS (Comparative Example) is 97, the MAA of the compound of Example 1 is 18669, and the MAA of the compound of Example 1 is about 190 times higher than TS. was.

From the above, it was found that the compound of Example 1 has about 70 to 190 times higher uptake into cells in vitro compared to TS (Comparative Example) which is clinically applied. From this, it can be inferred that, when this chlorin derivative or the like is used, strong tumor fluorescence is obtained, and therefore, when applied to photodynamic diagnosis (PDD), a superior sensitivity can be obtained. In addition, the present chlorin derivative and the like have better uptake performance into cancer cell lines compared to TS, and from this, the present chlorin derivative and the like have a cell killing effect by PDT superior to TS. It was found that

[Intracellular uptake evaluation using esophageal cancer cell line and immortalized esophageal normal epithelial cell line]
The esophageal cancer cell line: OE21 (No. 11D028; ECACC) and the immortalized esophageal epithelial cell line: Het-1A (No. 3527836; ATCC) were used to evaluate the intracellular uptake performance.

Cell culture conditions are as follows.
OE21: Mixture of half volume of RPMI 1640 and Ham's F12 Het-1A: BEGM Bullet kit was used as a culture solution. All culture solutions were 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, 25 mg / ml Of amphotericin B and cultured under conditions of 5% CO ₂ concentration and 37 ° C.

(Test method)
Cells of 5 × 10 ³ cells / well were cultured for 24 hours using a 96-well plate. Thereafter, each medium was replaced with a culture solution containing 1 μM Talaporfin Sodium (TS; Rezaphyrin (registered trademark), Meiji Seika Pharma) and a culture solution containing 1 μM of the compound of Example 1, and cultured for 4 hours. Next, after removing the culture medium by suction, the cells were washed with phosphate-buffered saline (PBS), each well was filled with 100 μl of PBS, and excited using a spectrometer (Gemini EM, Molecular Device) with light at a wavelength of 405 nm. Then, the intensity of fluorescence emission from the cells at a wavelength of 650 nm was measured. The fluorescence intensity of each specimen was a value divided by a blank. The obtained results were analyzed with SoftMAX pro software. Results were analyzed from 8 independent experiments. The results are shown in FIG. In addition, the vertical axis | shaft of the graph of FIG. 3 shows fluorescence emission intensity, and it represents that the compound is taken in in a cell, so that this is large.

From the results shown in the graph of FIG. 3, in Het-1A, which is normal esophageal epithelium, there was no difference in the intracellular uptake performance of the compound of TS (Comparative Example) and Example 1. On the other hand, in OE21 which is an esophageal cancer cell line, the intracellular uptake performance of the compound of Example 1 was significantly enhanced as compared with TS (Comparative Example) (Bonferroni test P = 0.0002).

[Evaluation of antitumor effect in photodynamic therapy]
The mouse colon cancer cell line: CT26 (No. CRL-2638; ATCC) was used as the cell line. Cell culture conditions, animals used, and methods for preparing mouse subcutaneous tumor models are as follows.

Culture conditions:
DMEM (Dulbecco's Modified Eagle Medium) contained 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, 0.25 mg / ml amphotericin B, and cultured at 5% CO ₂ concentration at 37 ° C. .

Animals used:
The mice were 8-10 weeks old, female, approximately 20 g body weight mice (BALB / c CrSlc). Mice were raised for 2 weeks and adapted to the environment.

Creation of mouse subcutaneous tumor model:
The hair was removed in a circular shape of about 10 mm around the back of the right thigh root of the mouse, and 1 × 10 ⁶ CT26 were inoculated subcutaneously using a 27G needle. Seven days after inoculation, a subcutaneous tumor model with an average tumor size of 100 mm ³ (major axis (mm) × minor axis (mm) × minor axis (mm)) was prepared.

(Test method)
The prepared mouse subcutaneous tumor model was divided into three groups (untreated control group, treatment group with the compound of Example 1, Talaporfin Sodium (TS (comparative example); Rezaphyrin (registered trademark), Meiji Seika Pharma) treatment group) average tumor Ten animals were allocated so that the sizes were uniform. The compound of Example 1 or TS at a concentration of 1.56 μmol / Kg was intravenously administered from the mouse tail vein, and 30 minutes later, a 664 nm red semiconductor laser (KOYO-PDL664, OK Fiber Technology) was used, for a total of 100 J / cm ² (150 mW / cm ² ) single irradiation was performed. Tumor size after treatment was measured using electronic calipers every 3 days. The measurement results were compared between two groups using Welch's t-test. The results are shown in the graph of FIG.

As shown in the graph of FIG. 4, the group to which the compound of Example 1 was administered showed significantly tumor suppression compared to the control, and the mortality rate of the mice was 0% (n = 10). Furthermore, the complete disappearance rate of the tumor was 40% in 4 of 10 cases. On the other hand, in the group administered with TS, all cases died within a few days after treatment.
From the above results, when the compound of Example 1 was used, even when irradiated with light 30 minutes after administration, the mortality rate of the mice was 0% and showed a high antitumor effect. This indicates that the compound of Example 1 is more easily transferred out of the body and is safer even when irradiated with light at an early stage after administration. On the other hand, when TS was irradiated with light 30 minutes after administration, all mice died, indicating that safety was poor when irradiated with light early after administration.

[Single intravenous toxicity study using mice]
The compound of Example 1 was intravenously administered to mice (Crl: CD1 (ICR), 5 males and 5 females / dose group) at doses of 60, 125, and 250 mg / kg, and the observed toxicity changes were confirmed. .
In order to confirm the difference in the toxicity expression due to the lighting conditions, two breeding conditions were set in this test under normal lighting (light conditions) and dark conditions. The administration liquid volume was 10 ml / kg. A pharmacological physiological saline containing 10% Cremophor + 5% ethanol was used as the administration liquid medium.

Results of administration: In the 250 mg / kg group raised in light conditions, 3 males, 1 female died on the 2nd day, and 1 female died on the 3rd day. In the 250 mg / kg group kept in the dark, 2 males died on the 2nd and 5th days, and 3 females died on the 2nd to 4th days. In some of these animals, decreased locomotor activity, slow breathing, and decreased body temperature were observed. There was no difference between the light and dark conditions in the status of death.

General state observation in surviving cases: In 2 males and 3 females in the 250 mg / kg group bred under light conditions, swelling of the tail was observed after the second day, and discoloration of the auricles was observed from the ninth day. The swelling of the tail disappeared and recovered on the 12th day, but the discoloration of the auricle did not recover until the time of dissection, and a part of the auricle was lost in 2 males. In one male of the 250 mg / kg group fed in the dark condition, the color change of the pinna was observed from the 14th day, but it was localized at the tip of the pinna, and the symptom was prominently observed in the bright condition. did.

Body weight measurement: Weight loss was observed on the 4th or 8th day in the 125 and 250 mg / kg groups for both bright and dark conditions. Both were temporary and showed a tendency to recover. There was no difference between light and dark conditions in weight fluctuation.

Necropsy results: Only the discoloration of the auricle, the defect, and the crust of the tail, which were recognized as general symptoms in the 250 mg / kg group, were observed.

From the above results, it was concluded that the minimum lethal dose in the single intravenous administration of the compound of Example 1 was 250 mg / kg for both male and female in the light and dark breeding conditions. Moreover, under the light condition breeding, swelling of the tail and discoloration of the auricle were observed after administration of the 250 mg / kg group, and a difference depending on the lighting conditions was observed.
From the above results, it was presumed that the compound of Example 1 could obtain sufficient effects of the present invention at a dose at which safety was recognized.

[Pharmacokinetic study of the compound of Example 1]
¹⁴ C-labeled compound of Example 1 was intravenously administered to rats, and the following results were obtained (dose: 5 mg / kg).
Plasma radioactivity concentration 23.30 μg eq. 5 minutes after administration, which is the first measurement time. of S Example 1 showed compound / ml and then decreased rapidly. Six to eight hours after administration, after a re-elevation of plasma radioactivity concentration was observed, it disappeared at the elimination half-life (t _1/2 ) of 42.65 h. The plasma radioactivity concentration (C ₀ ) immediately after administration was 51.52 μg eq. / Ml, AUC _0-last is 30.42 μg eq h / ml, AUC _0-∞ is 31.89 μg eq. -H / ml. The results of the compound of Example 1 and TS are shown in Table 6.

From the results shown in Table 6, it is presumed that the compound of Example 1 tends to disappear from plasma (disappears quickly) compared to TS.

88.9% of the administered radioactivity was excreted in the bile up to 4 hours after administration, and 94.9% was excreted by 48 hours. 4.4% and 0.8% of the administered radioactivity was excreted in the urine and feces up to 48 hours after administration, respectively. The survival rate in the body 48 hours after administration was 0.8%, and the total recovery rate was 100.9%. From the above results, it was suggested that the main excretion route of the compound of Example 1 was fecal excretion via bile. The results are shown in Table 7.

From the results shown in Table 7, it is presumed that the compound of Example 1 is more likely to be discharged, as compared to TS, the discharge amount after 0-24 hours and 48 hours exceeded TS. The

Claims (18)

1. A glycosylated chlorin e6 derivative represented by the following general formula (1), or a pharmaceutically acceptable salt thereof.
   

   

   
   In the formula:
   X ¹ and X ² are each independently a group represented by H or R—X— *, * represents a bonding position, and at least one of X ¹ and X ² is represented by R—X— * R is a sugar residue, and X is a linear or branched 2 consisting of at least one atom selected from the group consisting of C, N, O, H, and S. And X is bonded to any one of the carbon atoms constituting R;
   R ¹ , R ² and R ³ are each independently H, an acetoxyalkyl group having 1 to 6 carbon atoms, or a hydrocarbon group having 1 to 6 carbon atoms, and at least ^one of R ¹ , R ² and R ³ One is an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms.
2. 2. The general formula (1), wherein R ¹ , R ² and R ³ are each independently an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms. Glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
3. The glycosylated chlorin e6 derivative according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, in the general formula (1), R ¹ , R ² and R ³ are methyl groups.
4. The glycosylated chlorin e6 derivative according to any one of claims 1 to 3, wherein in the general formula (1), -X- is -X ³ -O-, and is represented by the following general formula (2): Or a pharmaceutically acceptable salt thereof.
   

   

   
   In the formula:
   R is a sugar residue;
   X ³ is a linear or branched divalent group consisting of at least one selected from the group consisting of C, N, O, H, and S, and a carbon atom constituting R Combined with any one.
5. The glycosylated chlorin according to claim 4, wherein, in the general formula (2), X ³ is a group bonded to an anomeric carbon atom of R or a group bonded to a carbon atom adjacent to the anomeric carbon atom. e6 derivative, or a pharmaceutically acceptable salt thereof.
6. 6. The glycosylated chlorin e6 derivative according to claim 4 or 5, wherein —X ³ — is —S—X ⁴ — and represented by the following general formula (3), or a pharmaceutical product thereof: Acceptable salt.
   

   

   
   In the formula:
   X ⁴ is a linear or branched divalent group having C and H.
7. The glycosylated chlorin e6 derivative according to claim 6, or a pharmaceutically acceptable salt thereof, wherein, in the general formula (3), X ⁴ is a linear or branched alkylene group having 1 to 16 carbon atoms. Salt.
8. The glycosylated chlorin e6 derivative according to claim 6 or 7, wherein in the general formula (3), X ⁴ is an alkylene group represented by-(CH ₂ ) _n- , and n is an integer of 1 to 16, Or a pharmaceutically acceptable salt thereof.
9. The glycosyl according to any one of claims 6 to 8, wherein in the general formula (3), X ⁴ is an alkylene group represented by-(CH ₂ ) _n- , and n is an integer of 3 to 10. Chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
10. The sugar according to any one of claims 1 to 9, wherein the sugar is a monosaccharide, an oligosaccharide, a polysaccharide, a monosaccharide containing an amino group, an oligosaccharide containing an amino group, or a polysaccharide containing an amino group. Glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
11. The glycosylated chlorin e6 derivative according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein the sugar is a monosaccharide and S is bonded to an anomeric carbon atom of R.
12. The glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the sugar is glucose, galactose or mannose.
13. The glycosylated chlorin e6 derivative according to any one of claims 1 to 12, represented by the following general formula (4), (5) or (6), or a pharmaceutically acceptable salt thereof.
    

    

    
    

    

    
    

    

    
    In the formulas (4) to (6), n is an integer of 3 to 10.
14. The glycosylated chlorin e6 derivative according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof is effective for photodynamic treatment of tumor, skin disease, eye disease or age-related macular degeneration. A pharmaceutical composition comprising as an ingredient.
15. A glycosylated chlorin e6 according to any one of claims 1 to 13 to a target selected from the group consisting of viruses, microorganisms and infected cells of any of these, tumor cells, tumorous tissue, and neovascularization. Irradiating the target with light having a wavelength that is absorbed by the glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof after contacting the derivative or a pharmaceutically acceptable salt thereof. A method of destroying said target.
16. A pharmaceutical composition comprising the glycosylated chlorin e6 derivative according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof as an active ingredient.
17. The pharmaceutical composition according to claim 16, for treating, diagnosing or detecting a tumor, skin disease, eye disease or age-related macular degeneration.
18. The method for producing a glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, comprising a step of binding chlorin e6 and a sugar via a linking group.

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