JP7003057B2 - A method for destroying a glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition or a target, and a method for producing a glycosylated chlorin e6 derivative or a pharmaceutically acceptable salt thereof. - Google Patents

Description

The present invention is a glycosylated chlorin e6 derivative, or pharmaceutically acceptable thereof, which can be suitably used in photodynamic therapy (PDT) and / or photodynamic diagnosis (PDD). Regarding salt. The invention also relates to pharmaceutical compositions, methods of destroying targets, and methods of producing glycosylated chlorin e6 derivatives, or pharmaceutically acceptable salts thereof.

In photodynamic therapy (PDT), a photosensitizer is administered 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 skin lesion) is administered. , New blood vessels, etc.), and then by irradiating light with an appropriate wavelength to excite the photosensitizer, it is a therapeutic method that selectively destroys only the target tissue. In this case, the photosensitizer activated by photoexcitation directly or indirectly transfers energy to the neighboring 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 for PDT, a hematoporphyrin derivative (for example, Photofurin (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 the hematoporphyrin derivative for cancer tissues is not sufficient, and the accumulation in normal tissues is also observed. Therefore, the treated patients are darkened for a long time until they are excreted from the body so that the photosensitizing action of the hematoporphyrin derivative (porphymer sodium) accumulated in normal tissues does not destroy normal cells. It is necessary to stay in place. However, it has been reported that photosensitivity remains for more than 4 weeks due to the slow rate of excretion of porphymer sodium from normal tissues. In addition, since the maximum absorption wavelength of the porphyrin derivative is about 630 nm and the molar absorption coefficient is low, the therapeutic effect of PDT (in other words, the target tissue) is limited to surface cancer of about 5 to 10 nm.

With respect to 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 the clinical use amount is gradually increased. There is. Although such a drug has solved the problems of metabolicity and lengthening of the absorption wavelength, it has a problem of insufficient tumor-killing cell-like effect and growth-suppressing effect on tumor tissue.

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

Japanese Patent Publication No. 2011-518890 KR2009048772A

Ravindra K. Pandey et al., Journal of Medical Chemistry 2009, 52, 445-455

As described above, porphyrin-based compounds and chlorin-based compounds are also being developed and studied, but there is room for improvement in terms of tumor-killing cell-killing and tumor tissue growth-suppressing effects.
Therefore, it is an object of the present invention to provide a glycosylated chlorin e6 derivative having excellent tumor-killing cell properties (phototoxicity) and an excellent tumor tissue growth inhibitory effect, or a pharmaceutically acceptable salt thereof. ..
It is also an object of the present invention 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 obtained a photosensitizer that is useful as a photodynamic therapeutic agent, secures safety to a living body, and exhibits high phototoxicity in a small amount. , A novel glycosylated chlorin e6 derivative and a method for producing the same have been found, and the present invention has been reached.

That is, the present invention is as follows.
[1] A glycosylated chlorin e6 derivative represented by the general formula (1) described later, or a pharmaceutically acceptable salt thereof.
[2] In the general formula (1) described later, R ¹ , R ² and R ³ are independently acetoxyalkyl groups having 1 to 6 carbon atoms or hydrocarbon groups having 1 to 6 carbon atoms, respectively [1]. ] The glycosylated chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[3] In the general formula (1) described later, the glycosylated chlorin e6 derivative according to [1] or [2], wherein R ¹ , R ² and R ³ are methyl groups, or pharmaceutically acceptable thereof. Salt.
[4] The glycosylation according to any one of [1] to [3], wherein -X- is -X ³ -O- in the general formula (1) described later, and is represented by the general formula (2) described later. Chlorin e6 derivative, or a pharmaceutically acceptable salt thereof.
[5] Described in [4], 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. Glycosylated chlorin e6 derivative of, or a pharmaceutically acceptable salt thereof.
[6] The glycosylated chlorin according to [4] or [5], which is represented by the general formula (3) described later, in which -X ^3- is -S-X ^4- in the general formula (2) described later. An e6 derivative, or a pharmaceutically acceptable salt thereof.
[7] The glycosylated chlorin e6 derivative according to [6], or a pharmacy thereof, wherein X4 is a linear or branched alkylene group having 1 to 16 carbon atoms in the general formula ( ³ ) described later. Acceptable salt.
[8] The general formula (3) described later, according to [6] or [7], wherein 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] In the general formula (3) described later, any one of [6] to [8], wherein X ⁴ is an alkylene group represented by − (CH ₂ ) _n − and n is an integer of 3 to 10. The glycosylated chlorin e6 derivative according to the above, or a pharmaceutically acceptable salt thereof.
[10] The sugar is any of [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. The above-mentioned 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 sugar is a monosaccharide and S is bonded to an anomeric carbon atom of R, or a pharmaceutically acceptable thereof. salt.
[12] The glycosylated chlorin e6 derivative according to any one of [1] to [11], or a pharmaceutically acceptable salt thereof, 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] The chlorin e6 derivative according to any one of [1] to [13], or a pharmaceutically acceptable salt thereof, for photodynamic therapy of tumor, skin disease, eye disease or age-related macular degeneration. A pharmaceutical composition containing as an active ingredient.
[15] The glycosyl according to any one of [1] to [13], which is a target selected from the group consisting of viruses, microorganisms and infected cells of any of these, tumor cells, neoplastic tissues, and new blood vessels. After contacting the chlorin e6 derivative or a pharmaceutically acceptable salt thereof, a step of irradiating the target with light having a wavelength absorbed by the chlorin e6 derivative or the pharmaceutically acceptable salt thereof. How to destroy a target, including.
[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 the treatment, diagnosis or detection of tumor, skin disease, eye disease or age-related macular degeneration.
[18] The glycosylated chlorin e6 derivative according to any one of [1] to [13], which comprises a step of binding chlorin e6 and a sugar via a linking group, or a pharmaceutically acceptable salt thereof. Manufacturing method.

The glycosylated chlorin e6 derivative according to the embodiment of the present invention or a pharmaceutically acceptable salt thereof (hereinafter, also simply referred to as “the present chlorin derivative or the like”) has very low toxicity in a dark place and is a living body. Targeted viruses, bacteria, or infected cells, tumor cells, or in vitro or in vivo because they are safe against chlorinating and have excellent tumor-killing cell-killing and tumor tissue growth-suppressing effects by light irradiation. It can be applied to the application of destroying the target by irradiating the chlorin derivative or the like with light having a wavelength that is absorbed after contact with the tumor-like tissue. Therefore, the chlorin derivative and the like can be used as a drug containing the chlorin derivative and the like as an active ingredient, particularly as a photodynamic therapeutic agent for tumors or skin diseases, or as 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 which shows the intracellular uptake performance evaluation result of the glycosylated chlorin e6 derivative which concerns on embodiment of this invention using an esophageal cancer cell line and an immortalized esophageal normal epithelial cell line. It is a graph which shows 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]
In the following, the glycosylated chlorin e6 derivative may be described as an example among the present chlorin derivatives and the like, but the description thereof is pharmaceutically acceptable for the glycosylated chlorin e6 derivative unless otherwise specified. It also applies to salt.

In the general formula (1), X ¹ and X ² are independently represented by H (hydrogen atom) or RX- * (* represents a bond position), and X ¹ and X 2 are represented. At least one of X ² is a group represented by RX- *. From the viewpoint of easier synthesis, that is, from the viewpoint of having higher productivity, it is preferable that either X ¹ or X ² is a group represented by RX- *, and X ¹ is preferable. It is more preferable that the group is represented by RX- * and X ² is H (hydrogen atom).

Here, R represents a sugar residue (hereinafter referred to as "sugar residue"). The sugar residue represents a residue excluding one hydroxyl group bonded to a carbon atom of the sugar, and a residue excluding a hemiacetal (anomeric) hydroxyl group of the 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 (sulfur atom). Examples of X include -S-, -O-, -NR _x- (R _x is a hydrocarbon group which may have a hydrogen atom or a heteroatom), a carbonyl group, an alkylene group, an alkenylene group, and , A group combining these, preferably containing O (oxygen atom) and / or S (sulfur atom), and two 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 further preferable.

The sugar of R is not particularly limited, and is, for example, aldopentose (ribose, arabinose, xylulose, and lyxose, etc.), aldohexose (allose, altorose, glucose, mannose, growth, idose, galactose, and tarose, etc.). ), Aldoheptos, ketopentose (ribulose, xylulose, etc.), ketohexose (psicose, fructose, sorbose, and tagatose, etc.), ketoheptos (sedoheptulose, corioth, etc.), and derivatives thereof having an amino group, etc. Monosaccharides;
Oligosaccharides such as sucrose, maltose, lactose, maltotriose, raffinose, and maltotetraose, as well as derivatives having an amino group;
Polysaccharides such as starch, amylose, and glycogen, and derivatives thereof having an amino group; and the like can be mentioned. Of these, monosaccharides are preferred, hexoses or hexosamines are more preferred, hexoses are even more preferred, and glucose is particularly preferred.
The monosaccharide may be D-form or L-form, but D-form is preferable.

In the present specification, the oligosaccharide means a compound containing 2 to 9 monosaccharide units, and the polysaccharide means a compound containing 10 or more monosaccharide units (JD ROBERTS & M. C.CASERIO (1964). BASIC PRINCIPLES OF ORGANIC CHEMISTRY. WA Benjamin. Inc. (JD Roberts & MC Caserio Oki Michinori (Translation) (1969). Roberts Organic Chemistry Co., Ltd. Tokyo Chemicals Dojin) More quoted). The glycosidic-bonded monosaccharides may be the same or different. Further, the glycosidic bond between monosaccharides may be an α-bond or a β-bond.

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

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

In formula (1), R ¹ , R ² and R ³ are independently H (hydrogen atom), an acetoxyalkyl group having 1 to 6 carbon atoms or a hydrocarbon group having 1 to 6 carbon atoms, respectively, and R ¹ , R ² and R ³ are at least one 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. Hydrocarbons having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl, and cyclohexyl having 1 to 6 carbon atoms. Chain-like, branched chain-like or cyclic alkyl can be mentioned. Among them, R ¹ , R ² and R ³ independently have an acetoxyalkyl group having 1 to 6 carbon atoms or an acetoxyalkyl group having 1 carbon atom in that a glycosylated chlorin e6 derivative having a more excellent effect of the present invention can be obtained. It is preferably a hydrocarbon group of ~ 6. This is because the uptake property for cancer cells is improved.

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

In the group represented by RX- * (* represents a bond position), the divalent group (linking group) X preferably contains O (oxygen atom), and O and S (sulfur atom). ) Is more preferable.
Among them, the group represented by RX3- * is a group represented by RX3 ^- O- * in that a glycosidated chlorin e6 derivative having a more excellent 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. The divalent group of X ³ is not particularly limited, but has the same form as the divalent group of X described above.
That is, the glycosidated chlorin e6 derivative is preferably represented by the following formula (2). The form of the sugar residue R in the formula (2) is as described above as R in the formula (1).

The group represented by R-X ³ -O- * is represented by R-L-S-X ⁴ -O- * in that a glycosidated chlorin e6 derivative having a further excellent effect of the present invention can be obtained. The groups to be added are more preferred. Here, L represents a single bond or a divalent group. The divalent group of L is not particularly limited, but is as described above as, for example, the divalent group of X.
Among them, it is preferable that L of the group represented by RL-S-X4 ^- O- * is a single bond in that a glycosidated chlorin e6 derivative having a more excellent effect of the present invention can be obtained. .. That is, the group represented by RX- * is a group represented by R-SX ⁴ -O- *, in other words, the sugar residue R is directly linked to -SX ⁴ -O-. A group is preferred. Here, the direct link refers to 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.

Further, S (sulfur atom) in the group represented by R-S-X ⁴ -O- * is a linking group bonded to an anomer-position carbon atom (1-position carbon atom) of R from the viewpoint of synthesis, or an anomer-position. It is preferably a linking group bonded to a carbon atom (2-position carbon atom) adjacent to the carbon atom, and more preferably a linking group bonded to an anomaly-position carbon atom (1-position carbon atom) of R.

X ⁴ is bonded to O (oxygen atom) and S (sulfur atom). Further, ^X4 is a linear or branched divalent group having C (carbon atom) and H (hydrogen atom). The X ⁴ is not particularly limited, and examples thereof include an alkylene group, an oxyalkylene group, and an alkyleneoxy group. Among them, a linear or branched alkylene group having 1 to 16 carbon atoms may be used. It is more preferably a linear alkylene group represented by − (CH ₂ ) n−. n is preferably an integer of 1 to 16, more preferably n is an integer of 2 to 13, and even more preferably n is an integer of 3 to 10. This is because it is easy to synthesize and the water solubility of the compound is increased.

The form of the sugar residue R in the formula (3) is as described above as R in the formula (1).

Among them, the glycosylated chlorin e6 derivative is preferably represented by the following formula (4), (5), or (6) in that it has a particularly excellent effect of the present invention. In the following equations (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 (eg, sodium and potassium salts, etc.), alkaline earth metal salts (eg, magnesium and calcium salts, etc.), ammonium salts, mono, di, or tri. -Lower (alkyl or hydroxyalkyl) ammonium salts (eg ethanolammonium salt, diethanolammonium salt, triethanolammonium salt, tromethamine salt), hydrochlorides, hydrobromide, hydride iodide, nitrates, phosphates, Sulfate, formate, acetate, citrate, oxalate, fumarate, maleate, succinate, malate, tartrate, trichloroacetate, trifluoroacetate, methanesulfonate, Examples thereof include benzene sulfonate, p-toluene sulfonate, mesitylene sulfonate, naphthalene sulfonate and the like.
The salt may be an anhydride or a solvate, and examples of the solvate include hydrates, methanol hydrates, ethanol hydrates, propanol hydrates, and 2-propanol hydrates.

The chlorin derivative and the like having the above-mentioned structure do not show cytotoxicity in the dark, but show strong cytotoxicity under light irradiation. It is presumed that the absorption at a long wavelength is larger than that of the porphyrin derivative (absorption maximum wavelength of 650 nm), and that the compound has high cell affinity and / or cell permeability due to the ligation of sugars.

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

In the following, first, a production method in which sugar and a linking group are bound and then linked to chlorin e6 will be described in detail with reference to FIG. 1.
First, the thiol sugar (R-SH) has a leaving group (E) such as a functional group that can be linked to the thiol sugar, for example, a halogen, a tosyl group, and a mesyl group, and has a hydroxyl group (-OH). A linking group (EX ⁴ -OH) is introduced.

It is preferable that the hydroxyl group of the sugar residue R contained in the thiol sugar (R-SH) used here is protected by a protecting group such as an acyl group. Examples of the protecting group include an aliphatic acyl group such as acetyl and pivaloyl; an aromatic acyl group such as a benzoyl group; and an aralkyl group such as a benzyl group. Of these protecting groups, the acetyl group is particularly preferred.

Examples of the linking group (EX ⁴ -OH) to be linked to the thiol sugar (R-SH) include chloroalcohol, bromoalcohol, iodine alcohol, tosyl alcohol, and mesylate alcohol.

The solvent used in this reaction is not particularly limited as long as the reaction proceeds, but for example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride. Halogenated hydrocarbons such as; aliphatic hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, etc. Examples include ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide; water; and mixtures thereof. A particularly preferable 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, rutidin, etc. Aromatic amines; such as triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidin, N-methylpyrrolidin, and N-methylmorpholin. Tertiary amines; alkali metal hydrides such as sodium hydride and potassium hydride; metal amides such as sodium amide, lithium diisopropylamide, and lithium hexamethyldisilazide; sodium methoxydo, sodium ethoxy And metal amides such as potassium tert-butoxide; etc.
The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractionation, crystallization, recrystallization, and chromatography.

Next, a thiol sugar linking group conjugate (RSX ⁴ -OH) is introduced into the chlorin e6 trimethyl ester.

First, a hydrogen halide is added to the double bond at the 3-position of the porphyrin ring in the 1 to 24 numbering method with respect to the chlorin e6 trimethyl ester to obtain a chlorin e6 trimethyl ester hydrogen halide adduct. Examples of the hydrogen halide used include hydrogen chloride, hydrogen bromide, hydrogen iodide and the like. 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 examples thereof include formic acid, acetic acid, and propionic acid.

A thiol sugar linking group conjugate (RSX ⁴ -OH) was allowed to act on the obtained chlorin e6 trimethyl ester halogenated hydrogen adduct to form a thiol sugar linking group conjugate (RSX ⁴ -OH). ) Is bonded to a chlorin e6 trimethyl ester halide hydrogen adduct to obtain a sugar-linked chlorin e6 trimethyl ester. The thiol sugar linking group conjugate (RSX ⁴ -OH) added in the above reaction is preferably added in an amount of 3 or more with respect to the chlorin e6 trimethyl ester hydrogen halide adduct. This is because the yield of the sugar-linked chlorin e6 trimethyl ester is improved.

When bases are 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, and Aromatic amines such as rutidin; triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidin, N-methylpyrrolidin, and N-methylmorpholin 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 methoxydo, etc. It is selected from sodium ethoxydo and metal amides such as potassium tert-butoxide.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds, and for example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride. Halogenated hydrocarbons such as; aliphatic hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, etc. Examples include ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide, or mixtures of two or more thereof. Particularly preferred solvents for the above reactions 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, fractionation, crystallization, recrystallization, and chromatography.

Next, a production method in which the linking group and chlorin e6 are bound and then linked to sugar will be described in detail with reference to FIG. 2.

First, a linking group (E) having a leaving group (E) such as a functional group capable of linking with a sugar, for example, a halogen, a tosyl group, and a mesyl group, and a hydroxyl group (-OH) with respect to the chlorin e6trimethyl ester. -X ⁴ -OH) is introduced.

In the chlorin e6 trimethyl ester, a hydrogen halide is added to the double bond at the 3-position in the 1 to 24 numbering method of the porphyrin ring to obtain a chlorin e6 trimethyl ester hydrogen halide adduct. Examples of the hydrogen halide used include hydrogen chloride, hydrogen bromide, hydrogen iodide and the like. 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 examples thereof include formic acid, acetic acid, and propionic acid.

A linking group (EX ⁴ -OH) was allowed to act on the obtained chlorin e6 trimethyl ester hydrogen halide adduct to convert the linking group (EX ⁴ -OH) into a chlorin e6 trimethyl ester hydrogen halide adduct. Bonding gives a linking group-bonded chlorin e6 trimethyl ester.
Examples of the linking group (EX ⁴ -OH) include chloroalcohol, bromoalcohol, iodine alcohol, tosyl alcohol, mesyl alcohol and the like. The linking group (EX ⁴ -OH) added in the above reaction is preferably added in an amount of 10 or more with respect to the chlorin e6 trimethyl ester hydrogen halide adduct. This is because the yield of the linking group-bonded 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, rutidin, etc. Aromatic amines; such as triethylamine, tripropylamine, tributylamine, cyclohexyldimethylamine, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylpiperidin, N-methylpyrrolidin, and N-methylmorpholin. Tertiary amines; alkali metal hydrides such as sodium hydride and potassium hydride; metal amides such as sodium amide, lithium diisopropylamide, and lithium hexamethyldisilazide; sodium methoxydo, sodium ethoxy It is selected from metal amides such as potassium tert-butoxide and the like.
The solvent used in this reaction is not particularly limited as long as the reaction proceeds, and for example, aromatic amines such as pyridine, lutidine, and quinoline; dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride. Halogenated hydrocarbons such as; aliphatic hydrocarbons such as hexane, pentane, and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene; diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, etc. Examples include ethers such as dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide and N, N-dimethylacetamide; mixtures of two or more of these. Particularly preferred solvents for the above reactions 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, fractionation, crystallization, recrystallization, and chromatography.
Next, a thiol sugar (R-SH) is introduced into the linking group-bonded chlorin e6 trimethyl ester.

As the thiol sugar (R-SH) used here, it is preferable to use one in which the 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; and the like. Of these protecting groups, the acetyl group is particularly preferred.

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

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-methylpiperidin, N-methylpyrrolidin, N-methylmorpholin and the like. Tertiary amines; alkali metal hydrides such as sodium hydride and potassium hydride; metal amides such as sodium amide, lithium diisopropylamide, and lithium hexamethyldisilazide; sodium methoxydo, sodium ethoxydo , And metal amides such as potassium tert-butoxide; etc.
The final product obtained by this reaction can be isolated and purified from the reaction mixture by known means such as concentration, solvent extraction, fractionation, crystallization, recrystallization, and chromatography.

In the above production method, when the sugar residue R whose hydroxyl group is blocked by the protecting group is used, then the protecting group is 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, in dichloromethane, methanol, ethanol, tetrahydrofuran, or a mixed solvent thereof, alkali metals such as sodium methoxyd, sodium ethoxyoxide, sodium-t-butoxide, potassium methoxyd, potassium ethoxyoxide, and potassium-t-butoxide, and the like. Protective groups are removed by treating the compound protected with an alkali such as alkoxide. If the protecting group is an aralkyl group, it can be removed by hydrogenation using a palladium catalyst. Deprotection of the hydroxyl group of the sugar residue R further promotes the transfer of the chlorin derivative and the like into the cell, and is more excellent in cytotoxicity.

[Use]
This chlorin derivative and the like do not show cytotoxicity in the dark, but by utilizing the fact that they show strong cytotoxicity under light irradiation, they are brought into contact with the target biological material in vitro or in vivo in the dark. After being taken up into cells, it can be used for destroying targets by irradiating it with light having an absorption wavelength such as this chlorin derivative.

Here, the target includes a target selected from the group consisting of viruses, microorganisms and their infected cells, tumor cells, tumor-like tissues, and new blood vessels, and particularly has an affinity for tumor cells and tumor cells. It can be used for tumor destruction because it is easily accumulated.

Therefore, the chlorin derivative and the like can be used as a therapeutic agent for cancer classified as a malignant tumor. Examples of malignant tumors include epithelial malignancies and malignant tumors originating from non-epithelial tissues such as those classified as sarcoma. Among the tumor cells, the chlorin derivative and the like are particularly useful for the treatment of solid carcinoma (solid carcinoma) in which the tumor cells proliferate in a mass and solid manner, and superficial carcinoma to which light reaches.
Specific examples thereof include uterine cancer such as esophageal cancer, lung cancer, gastric cancer, cervical cancer and endometrial cancer, skin cancer, prostate cancer, and kidney cancer. Skin cancer includes primary (squamous cell carcinoma, basal cell carcinoma, and epidermal accessory machine carcinoma) as well as skin metastases of visceral cancer.

In addition, since this chlorin derivative and the like have an affinity for benign tumors among tumor cells, photodynamics of skin diseases such as actinic keratosis, severe acne, and skin psoriasis by local administration. It can be used as a therapeutic agent for the purpose and also as a photodynamic therapeutic agent for eye diseases such as age-related yellow spot degeneration.

Furthermore, by utilizing the tumor accumulation property of the glycosylated chlorin e6 derivative of the present invention, it can be used for the 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 and affinity for benign tumors.

[How to destroy the target]
As described above, the chlorin derivative and the like can be applied to the above-mentioned method for destroying a target. The method for destroying a target according to an embodiment of the present invention includes a step of contacting the target with the chlorin derivative or the like and then irradiating the target with light having a wavelength absorbed by the chlorin derivative or the like.
The above method can be carried out on human individuals and non-human individuals. That is, the method for destroying the target can be carried out in a human individual, and can also be carried out in a form other than the implementation in a human individual.

[Pharmaceutical composition]
The pharmaceutical composition according to the embodiment of the present invention is for photodynamic therapy of tumor, skin disease, eye disease or age-related macular degeneration, and contains the above-mentioned glycosylated chlorin e6 derivative as an active ingredient. Among them, it has a better effect for photodynamic therapy of tumors.

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

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

Further, the pharmaceutical composition may contain other components as needed, as long as it contains the chlorin derivative and the like. Other components include, for example, excipients.

Examples of the excipient include lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride and the like as solids, and liquid substances. Examples thereof include glycerin, peanut oil, polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, water and the like.

In addition to excipients, this pharmaceutical composition contains bases, surfactants, preservatives, emulsifiers, colorants, odorants, fragrances, stabilizers, preservatives, antioxidants, and abundant materials as needed. It may contain an agent, an antibacterial agent, a solubilizing agent, a suspending agent, a binder, a disintegrant, and the like.

The dosage form of the present pharmaceutical composition is not particularly limited, and is, for example, tablets, powders, granules, capsules, troches, syrups, emulsions, soft gelatin capsules, gels, pastes, injectable formulations, creams and gels. , Lotions, patches, and the like.

The carrier used in the present pharmaceutical composition is appropriately selected according to the type of the pharmaceutical product. When prepared as an injectable preparation, it can be formulated in the form of a sterile aqueous solution or dispersion containing the chlorin derivative or the like or a sterile lyophilizer containing the 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 pharmaceutical composition, along with the chlorin derivative and the like, diluents such as lactose, sucrose, calcium ferric phosphate and carboxymethyl cellulose; lubricants such as magnesium stearate, calcium stearate and talc; starch, glucose and sugar honey. , Polyvinylpyrrolidone, cellulose, and derivatives thereof; and the like;

The content of the active ingredient per preparation in the present embodiment can be appropriately adjusted depending on the subject to be treated and the usage, and for example, the amount of the glycosylated chlorin e6 derivative can be 1 to 2000 mg. It is preferably up to 1000 mg, more preferably 10 to 500 mg.

The dose of the chlorin derivative and the like varies depending on the subject and purpose to be treated, but in general, the amount of the chlorin derivative and the like is preferably 0.1 to 30 mg / kg for tumor diagnosis or detection and tumor treatment. As a guide, 0.2 to 20 mg / kg.
Since this chlorin derivative, which is an active ingredient, has high cytotoxicity, it can be expected that the dose is smaller than that of conventional products (photofurin and rezaphyrin) to obtain the same or higher effect. This means that the time required for metabolism and excretion can be shortened, which enhances the convenience of utilizing photodynamic therapy.

For the treatment of tumor, after administration of this chlorin derivative or the like, the site to be treated is irradiated with a light beam containing an absorption band of the relevant compound. Specifically, singlet oxygen can be generated by irradiation with a light beam of 500 nm or more to exhibit the desired cytotoxicity, but it is preferable to irradiate a light beam having a high proportion of light having a maximum absorption wavelength.

As an irradiation source, an LED (Light Emitting Diode), a laser, a halogen lamp, or the like is used. The laser may be a dye laser, a semiconductor laser, an argon laser, or the like, as long as it can obtain a light beam having a wavelength required 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 compounds prepared based on Examples and Comparative Examples were evaluated by the following methods.

[Evaluation method of phototoxicity]
IC ₅₀ (Half maximal (50%) inhibitory concentration) was used for the phototoxicity evaluation, and the measurement was performed by the following procedure.
96 of MKN45 (obtained from JCRB cell bank and subcultured for 6 months) and MKN28 (obtained from Immuno-Biological Laboratory Co., Ltd. and subcultured for 6 months), which are cells derived from human gastric cancer, respectively. 5 × 10 ³ cells / well seeded (RPMI1640 medium containing 10% FBS, 100 μL) in a predetermined number of wells on a well plate 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 a different concentration is added to each well, the well is adjusted to the predetermined drug concentration, and after culturing for 4 hours, 100 μL of phosphate buffered saline (PBS: Phosphate-). It was washed once with Buffered Saline) and 100 μL of PBS was added again. Then, the light was irradiated for 8 minutes and 40 seconds using a 660 nm LED light source (30.8 mW / cm ² , LEDR-660DL, OptoCode) (16 J / cm ² ). After irradiation, PBS was removed, 100 μL of RPMI1640 medium containing 2% FBS was added, and the cells were cultured for 24 hours.

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

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

TGI% = ((tumor volume of control group)-(tumor volume of treatment group)) / (tumor volume of control group) x 100%

[Example 1]
Production and Evaluation of 1- (3-Hydroxy-Propanthio) -β-D-Glucose Linked Chloroform e6trimethyl Ester (1-1) Synthesis of 1- (3-Hydroxy-Propanthio) -β-D-Glucose Tetraacetate Nitrogen Atmosphere Below, 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 obtained solution was cooled to 0 ° C., and 3-bromo-1-propanol (0.85 ml, 9.75 mmol) was slowly added dropwise and stirred. After completion of the dropping, the temperature of the solution was raised to 25 ° C. and the mixture was stirred for 3 hours. Water (50 ml) and chloroform (30 ml) were added to the stirred solution to perform a liquid separation operation, and the chloroform layer was separated. 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 Corporation) and eluted with a mixed solvent of ethyl acetate and hexane. The eluate was concentrated under reduced pressure to give 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate (yield 2.68 g, 85% yield).

The obtained compound was J.I. Org. Chem. It was confirmed by 1H-NMR (400 MHz, deuterated chloroform solvent) based on 2013, 78, 5196-5204.

(1-2) Synthesis of chlorin 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 obtained 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 obtained residue was recrystallized from dichloromethane / hexane to obtain a 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 The chlorin e6 trimethyl ester synthesized in (1-2) above (0.95 g, 1.48 mmol) ) Was dissolved in a 25% hydrogen bromide-acetic acid solution (18.0 ml) under a nitrogen atmosphere, and the mixture was stirred at 30 ° C. for 2 hours. The hydrogen bromide-acetic acid solution was distilled off under reduced pressure from the obtained reaction solution, and the reaction solution was dried. 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. In the obtained solution, 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). Was added dropwise, and the mixture was stirred at 30 ° C. for 5 hours. Water (100 ml) and dichloromethane (200 ml) were added to the obtained reaction solution to carry out a liquid separation operation, and the dichloromethane layer was separated. The resulting 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 Corporation) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluate was concentrated under reduced pressure to give 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 0.42 g, yield 27%).

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 1- (3-hydroxy-propanethio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed that the molecular weights were the same by the analysis by the 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 The chlorin e6 trimethyl ester (0.96 g, 1.50 mmol) synthesized in (1-2) above has a 25% odor under a nitrogen atmosphere. It was dissolved in a hydrogen chlorinated-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 obtained reaction solution, and the reaction solution was dried. 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 minutes. 3-Bromo-1-propanol (2.60 ml, 30.00 mmol) was added dropwise to the obtained solution, and the mixture was stirred at 30 ° C. for 5 hours. Water (100 ml) and dichloromethane (200 ml) were added to the obtained reaction solution to carry out a liquid separation operation, and the 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 Corporation) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluate was concentrated under reduced pressure to give 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 bound. confirmed. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

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

(1-2') Synthesis of 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester Under a 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 obtained solution was cooled to 0 ° C., and 3-bromo-1-propanol-linked chlorin e6trimethyl ester (86 mg, 0.11 mmol) was slowly added dropwise and stirred. After completion of the dropping, the temperature of the solution was raised to 20 ° C. and the mixture was stirred for 14 hours. Water (10 ml) and chloroform (10 ml) were added to the stirred solution to perform a liquid separation operation, and the chloroform layer was separated. 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 Corporation) and eluted with a mixed solvent of ethyl acetate and dichloromethane. By concentrating the eluent under reduced pressure, 1- (3-hydroxy-propanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 52 mg, yield 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 that the molecular weights were the same by the analysis by the ESI + MS method.

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 e6trimethyl ester 1- (3-hydroxy-) synthesized in (1-3) and (1-2') above. Propanthio) -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 25. The mixture was stirred at ° C for 30 minutes. Acetic acid (58 μl, 1.00 mmol) was added to the obtained reaction solution to terminate the reaction. The solvent was distilled off under reduced pressure from the obtained solution, the residue was filled in a PLC glass plate (silica gel 60 F254, manufactured by Merck Group, Inc.), and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, charged in a reverse phase silica gel chromatograph (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 e6trimethyl 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 by ¹ H-NMR (400 MHz, deuterated chloroform solvent). I confirmed that. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

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-Propanthio) -β-D-Glucose Linked Chlorin e6 trimethyl Ester 1- (3-Hydroxy-Propanthio) -β-D- obtained by the above production method The glucose-linked chlorin e6 trimethyl ester was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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 chloroform e6trimethyl ester (2-1) Synthesis of 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate Nitrogen atmosphere Below, 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 obtained solution was cooled to 0 ° C., and 6-bromo-1-hexanol (0.66 ml, 4.88 mmol) was slowly added dropwise and stirred. After completion of the dropping, the temperature of the solution was raised to 25 ° C. and the mixture was stirred for 3 hours. Water (25 ml) and chloroform (15 ml) were added to the stirred solution to perform a liquid separation operation, and the chloroform layer was separated. 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 Corporation) and eluted with a mixed solvent of ethyl acetate and hexane. The eluate was concentrated under reduced pressure to give 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate (yield 1.39 g, yield 80%).

The structure identification was performed by J. Org. Chem. It was carried out by ¹ H-NMR (400 MHz, deuterated chloroform solvent) based on 2013, 78, 5196-5204.

^{1 1} H-NMR (CDCl 3,400 MHz) δ5.22 (t, J = 9.4 Hz, 1H, H-3), 5.08 (t, J = 10.0 Hz, 1H, H-4), 5.04 (T, J = 10.0Hz, 1H, H-2) 4.48 (d, J = 10.8Hz, 1H, H-1), 4.25 (dd, J = 11.2Hz, 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) , 3.63-3.65 (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 The chlorin e6 trimethyl ester (664 mg, 1.04 mmol) synthesized in Example 1 was added under a nitrogen atmosphere. , Dissolved in 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 obtained reaction solution, and the reaction solution was dried. 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 minutes. In the obtained solution, 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. Then, the mixture was stirred at 30 ° C. for 5 hours. Water (70 ml) and dichloromethane (140 ml) were added to the obtained reaction solution to carry out a liquid separation operation, and the dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (140 ml) and saturated brine (140 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 Corporation) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluate was concentrated under reduced pressure to give 1- (6-hydroxy-hexanethio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 145 mg, yield 13%).

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 1- (6-hydroxy-hexanethio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

(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) is dissolved in dehydrated methanol (10.0 ml) under a nitrogen atmosphere, sodium methoxide (65 mg, 1.21 mmol) is added, and the mixture is stirred at 25 ° C. for 30 minutes. did. Acetic acid (69 μl, 1.21 mmol) was added to the obtained solution to terminate the reaction. The solvent was distilled off under reduced pressure from the obtained solution, the residue was filled in a PLC glass plate (silica gel 60 F254, manufactured by Merck Group, Inc.), and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, charged in a reverse phase silica gel chromatograph (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, yield 67%).

In the obtained 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 that it was there. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

(2-4) Evaluation of 1- (6-Hydroxy-hexanethio) -β-D-glucose-linked chlorin e6trimethyl ester 1- (6-hydroxy-hexanethio) -β-D- obtained by the above production method The glucose-linked chlorin e6 trimethyl ester was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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-Decantio) -β-D-Glucose Linked Chlorin e6 Trimethyl Ester (3-1) Synthesis of 1- (10-Hydroxy-decantio) -β-D-Glucose Tetraacetate Nitrogen Atmosphere Below, 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 obtained solution was cooled to 0 ° C., and 10-bromo-1-decanol (1.00 ml, 4.88 mmol) was slowly added dropwise and stirred. After completion of the dropping, the temperature of the solution was raised to 25 ° C. and the mixture was stirred for 3 hours. Water (25 ml) and chloroform (15 ml) were added to the stirred solution to perform a liquid separation operation, and the chloroform layer was separated. 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 Corporation) and eluted with a mixed solvent of ethyl acetate and hexane. The eluate was concentrated under reduced pressure to give 1- (10-hydroxy-decantio) -β-D-glucose tetraacetate (yield 1.60 g, 82% yield).

The structure identification was performed by J. Org. Chem. It was carried out by ¹ H-NMR (400 MHz, deuterated chloroform solvent) based on 2013, 78, 5196-5204.

^{1 1} H-NMR (CDCl 3,400 MHz) δ5.22 (t, J = 9.4 Hz, 1H, H-3), 5.07 (t, J = 10.0 Hz, 1H, H-4), 5.03 (T, J = 10.0Hz, 1H, H-2) 4.49 (d, J = 10.8Hz, 1H, H-1), 4.25 (dd, J = 11.2Hz, 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-decantio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester The chlorin e6 trimethyl ester (715 mg, 1.12 mmol) synthesized in Example 1 was added under a nitrogen atmosphere. , Dissolved in 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 obtained reaction solution, and the reaction solution was dried. 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 minutes. In the obtained solution, 1- (10-hydroxy-decantio) -β-D-glucose tetraacetate (1750 mg, 3.36 mmol) synthesized in (3-1) above was dissolved in dehydrated dichloromethane (40 ml) and added dropwise. Then, the mixture was stirred at 30 ° C. for 5 hours. Water (80 ml) and dichloromethane (160 ml) were added to the obtained reaction solution to carry out a liquid separation operation, and the dichloromethane layer was separated. The obtained dichloromethane layer was washed with water (160 ml) and saturated brine (160 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 Corporation) and eluted with a mixed solvent of ethyl acetate and dichloromethane. The eluate was concentrated under reduced pressure to give 1- (10-hydroxy-decantio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester (yield 285 mg, 22% yield).

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 1- (10-hydroxy-decantio) -β-D- It was confirmed that glucose tetraacetate was bound. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

(3-3) Synthesis of 1- (10-hydroxy-decantio) -β-D-glucose-linked chlorin e6trimethyl ester 1- (10-hydroxy-decantio) -β-D- synthesized in (3-2) above Glucose tetraacetate-linked chlorin e6trimethyl ester (268 mg, 0.23 mmol) is dissolved in dehydrated methanol (20.0 ml) under a nitrogen atmosphere, sodium methoxide (125 mg, 2.31 mmol) is added, and the mixture is stirred at 20 ° C. for 1 hour. did. Acetic acid (132 μl, 2.31 mmol) was added to the obtained solution to terminate the reaction. The solvent was distilled off under reduced pressure from the obtained solution, the residue was filled in a PLC glass plate (silica gel 60 F254, manufactured by Merck Group, Inc.), and eluted with a mixed solvent of dichloromethane and methanol. The eluent was concentrated under reduced pressure, charged in a reverse phase silica gel chromatograph (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-decantio) -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-decantio) -β-D-glucose tetraacetate-linked chlorin e6 trimethyl ester disappeared by ¹ H-NMR (400 MHz, deuterated chloroform solvent). I confirmed that it was there. Moreover, it was confirmed that the molecular weights were the same by the analysis by the ESI + MS method.

(3-4) Evaluation of 1- (10-Hydroxy-decantio) -β-D-glucose-linked chlorin e6trimethyl ester 1- (10-hydroxy-decantio) -β-D- obtained by the above production method The glucose-linked chlorin e6 trimethyl ester was evaluated based on the phototoxicity evaluation method and the tumor growth inhibition rate evaluation method. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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). ) Was added and stirred at 25 ° C. for 4 hours. To the obtained reaction solution, a mixed solution of carbon disulfide (114 mg, 1.5 mmol) dissolved in dimethylformamide (2.0 ml) and sodium sulfide nine hydrate (480 mg, 2.0 mmol) was added, and 25 The mixture was stirred at ° C for 5 minutes. Water (30 ml) and dichloromethane (50 ml) were added to the obtained reaction solution, and a liquid separation operation was performed to separate the dichloromethane layer. 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 The obtained crude product was packed in a silica gel column (high flash column, manufactured by Yamazen Corporation) and eluted with a mixed solvent of ethyl acetate and hexane. The eluate was concentrated under reduced pressure to give 1-thio-β-D-galactose tetraacetate (yield 291 mg, yield 80%).

Structural identification was performed by Belstein 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 Instead of 1-thio-β-D-glucose tetraacetate (4-1) Using the 1-thio-β-D-galactose tetraacetate synthesized in 1 above, the same production method as in Example 1 was carried out, and 1- (3-hydroxy-propanethio) -β-D-galactose linked chlorin e6 was carried out. A 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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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 carried out 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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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-decantio) -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-decantio) -β-D-galactose-linked chlorin e6 trimethyl ester. ..

The 1- (10-hydroxy-decantio) -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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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 e6trimethyl 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) was dissolved in dichloromethane (5.0 ml), tetrabromomethane (663 mg, 2.0 mmol) and triphenylphosphine (525 mg, 2. 0 mmol) was added and the mixture was stirred at 25 ° C. for 4 hours. To the obtained reaction solution, a mixed solution of carbon disulfide (114 mg, 1.5 mmol) dissolved in dimethylformamide (2.0 ml) and sodium sulfide nine hydrate (480 mg, 2.0 mmol) was added, and 25 The mixture was stirred at ° C for 5 minutes. Water (30 ml) and dichloromethane (50 ml) were added to the obtained reaction solution, and a liquid separation operation was performed to separate the dichloromethane layer. 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 The obtained crude product was packed in a silica gel column (high flash column, manufactured by Yamazen Corporation) and eluted with a mixed solvent of ethyl acetate and hexane. The eluate was concentrated under reduced pressure to give 1-thio-β-D-mannose tetraacetate (yield 255 mg, 70% yield).

Structural identification was performed by Belstein J. et al. Org. Chem. It was carried out by ¹ H-NMR (400 MHz, deuterated chloroform solvent) based on 2013, 9, 974-982.

(7-2) Synthesis and evaluation of 1- (3-hydroxy-propanethio) -β-D-mannose linked chlorin e6 trimethyl ester Instead of 1-thio-β-D-glucose tetraacetate (7-1) Using the 1-thio-β-D-mannosetetraacetate synthesized in 1 above, the same production method as in Example 1 was carried out, and 1- (3-hydroxy-propanethio) -β-D-mannose linkage was carried out. Chlorin 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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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-Hexanthio) -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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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-Decantio) -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-decantio) -β-D-mannose linked chlorin e6 trimethyl ester. ..

The 1- (10-hydroxy-decantio) -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. Was carried out. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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 Evaluation was carried out using commercially available talaporfin sodium (mono-L-aspartylchlorin e6, manufactured by Medkoo Biosciences) based on the above-mentioned phototoxicity evaluation method and tumor growth suppression rate evaluation method. did. The results are shown in Table 1, Table 2 and Table 3. In addition, it was confirmed that all nude mice survived without weight loss during 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.

[Evaluation results of phototoxicity]
The evaluation results of phototoxicity are shown in Tables 1 and 2 below. In the table, " _IC50 " means "IC50".

[Evaluation result 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 e6trimethylester (Example 2), 1- (10-hydroxy-decantio) -β-D-glucose linked chlorin e6trimethylester (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-decantio) -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 The IC _50s of -D-mannose linked chlorin e6 trimethyl ester (Example 8) and 1- (10-hydroxy-decantio) -β-D-mannose linked chlorin e6 trimethyl ester (Example 9) are both talaporfin sodium. It was found to be smaller than the IC ₅₀ of (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-decantio) -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 linkage Chlorin e6 trimethyl ester (Example 5), 1- (10-hydroxy-decantio) -β-D-galactose linkage 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- The tumor growth inhibitory rates of mannose-linked chlorin e6 trimethyl ester (Example 8) and 1- (10-hydroxy-decantio) -β-D-mannose-linked chlorin e6 trimethyl ester (Example 9) are both talaporfin sodium (Example 9). It was found that it was larger than the tumor growth inhibition rate of 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-decantio) -β-D-glucose linked chlorin e6 trimethyl ester, 1- (3-hydroxy-propanethio) -β-D-galactose linked chlorin e6 trimethyl ester, 1- (6-hydroxy- Hexanthio) -β-D-galactose linked chlorin e6 trimethyl ester, 1- (10-hydroxy-decantio) -β-D-galactose linked chlorin e6 trimethyl ester, 1- (3-hydroxy-propanethio) -β-D-mannose Linked chlorin e6 trimethyl ester, 1- (6-hydroxy-hexanethio) -β-D-mannose Linked chlorin e6 trimethyl ester and 1- (10-hydroxy-decantio) -β-D-mannose linked chlorin e6 trimethyl ester It has excellent phototoxicity and excellent tumor growth inhibitory effect as compared with porfin sodium.

[Evaluation of phototoxicity by changing the cell line used]
Esophageal cancer cell line: KYSE30 (No. 11D028; ECACC), OE21 (No. 11D028; ECACC), Gastric cancer cell line: MKN45 (No. 0254; Japanese Cancer Research Bank), and colorectal cancer cell line: HT29 (No. HTB-38; ATCC) was used and cultured under the following conditions.

KYSE30: RPMI 1640
OE21: Mixture of RPMI 1640 and Ham's F12 in half MKN45: RPMI 1640
HT29: McCoy's 5A
All cultures contained 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, and 0.25 mg / ml amphotericin B and were cultured under 5% CO ₂ concentration, 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 carried out in the same manner as in the "phototoxicity evaluation method" described above. "Example 1") and TS (comparative example) were determined to have an _IC50 (50% cancer cell killing concentration) and evaluated according to the same criteria as in the "phototoxicity evaluation method". 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 effect 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 uptake performance into cancer cell lines]
As cell lines, a human gastric cancer cell line: MKN45 (No. 0254; Japanese Cancer Research Bank) and a colorectal cancer cell line: HT29 (No. HTB-38; ATCC) were used.

The conditions for cell culture and the equipment used are as follows.

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

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

(Test method)
The cancer cell lines of 2 × 10 ⁵ cell / well were cultured for 28 hours under the above conditions using a 6 cm culture plate.
Next, the culture broth was removed, and (1) the culture broth only (control), (2) the culture broth containing the glycosylated chlorin e6 derivative of Example 1 of 5 μM, and (3) 5 μM Talaporfin Sodium (TS; Rezaphyrin). It was replaced with a culture solution containing (registered trademark), Meiji Seika Pharma) and further cultured for 4 hours.
After culturing for 4 hours, the culture broth was removed, washed 3 times with phosphate-buffered saline (PBS), and cells were collected from the culture plate using TripLE-Express (Invitrogen).
Next, using the above FACSCantoII, excitation was performed at 405 nm, and fluorescence emission at 680 nm was measured. At least 10,000 events were measured for each sample. For the measurement, TS and Mean Amcyan area (MAA) for evaluating the vicinity of 650 nm, which is the fluorescence wavelength band of the compound of Example 1, were measured, 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 than.
When HT29 was used as the cell line, the MAA of TS (Comparative Example) was 97, the MAA of the compound of Example 1 was 18669, and the MAA of the compound of Example 1 was about 190 times higher than that of TS. was.

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

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

The conditions for cell culture are as follows.
OE21: Half of RPMI 1640 and Ham's F12 mixed solution Het-1A: BEGM Bullet kit was used as the culture medium. All culture solutions were 10% fetal bovine serum, 100 U / ml penicillin and streptomycin, 25 mg / ml. Amphotelicin B was contained and cultured under the conditions of 5% CO ₂ concentration and 37 ° C.

(Test method)
5 × 10 ³ cell / well cells were cultured for 24 hours using a 96-well plate. Then, the cells were replaced with a culture solution containing 1 μM of Talaporfin Sodium (TS; Rezaphyrin®, 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 phosphorate-buffered saline (PBS), 100 μl of PBS was added to each well, and the spectrometer (Gemini EM, Molecular Device) was used to excite with light having 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 sample was the value divided by the blank. The obtained results were analyzed by SoftMAX pro software. Results were analyzed from 8 independent experiments. The results are shown in FIG. The vertical axis of the graph in FIG. 3 indicates the fluorescence emission intensity, and the larger the value, the more the compound is incorporated into the cell.

From the results shown in the graph of FIG. 3, there was no difference in the intracellular uptake performance of TS (Comparative Example) and the compound of Example 1 in Het-1A, which is a normal esophageal epithelium. 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]
As the cell line, a mouse colorectal cancer cell line: CT26 (No. CRL-2638; ATCC) was used. The conditions for cell culture, the animals used, and the method for preparing a mouse subcutaneous tumor model are as follows.

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

Animals used:
As mice, 8 to 10 week old females, about 20 g body weight mice (BALB / c CrSlc) were used. Mice were bred for 2 weeks and adapted to the environment.

Preparation of mouse subcutaneous tumor model:
Hair was removed in a circle of about 10 mm centered on the dorsal side of the right femur of the mouse, and 1 × 10 ⁶ CT26s were inoculated subcutaneously using a 27 G needle. Seven days after inoculation, a subcutaneous tumor model having 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 3 groups (untreated control group, treatment group with the compound of Example 1, Talaporfin Sodium (TS (comparative example); Rezaphyrin®, Meiji Seika Pharma) treatment group). 10 animals were allotted so that the sizes would be even. The compound of Example 1 or TS having a concentration of 1.56 μmol / Kg was intravenously administered from the tail vein of a mouse, and 30 minutes later, a 664 nm red semiconductor laser (KOYO-PDL664, OK fiber technology) was used, for a total of 100 J / cm. A single irradiation of ² (150 mW / cm ² ) was performed. Tumor size after treatment was measured every 3 days using an electronic caliper. The measurement results were compared between the 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 significant tumor suppression as compared with the control, and the mouse mortality rate was 0% (n = 10). Furthermore, the complete tumor disappearance rate was 40% in 4 of 10 cases. On the other hand, in the TS-administered group, all cases died within a few days after the treatment.
From the above results, when the compound of Example 1 was used, the mortality rate of the mice was 0% and a high antitumor effect was shown even when irradiated with light rays 30 minutes after the administration. This indicates that the compound of Example 1 is more easily transferred to the outside of the body and is safer even when irradiated with light at an early stage after administration. On the other hand, TS was shown to be inferior in safety when irradiated with light rays 30 minutes after administration, because all mice died when irradiated with light rays 30 minutes after administration.

[Single intravenous dose toxicity test 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 in a single intravenous dose, and the toxic changes that appeared were confirmed. ..
In order to confirm the difference in toxicity development depending on the lighting conditions, two breeding conditions were set in this test, under normal lighting (light conditions) and under dark conditions. The dose was 10 ml / kg. A 10% Cremophor + 5% ethanol-containing Japanese Pharmacopoeia saline solution was used as the administration solution medium.

Results of administration: In the 250 mg / kg group reared under light conditions, 3 males and 1 female died on the 2nd day and 1 female died on the 3rd day. Also, in the 250 mg / kg group reared under dark conditions, 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, bradypnea, and decreased body temperature were noted. There was no difference in the incidence of death between light and dark conditions.

Observation of general condition in surviving cases: In 2 males and 3 females in the 250 mg / kg group kept under light conditions, swelling of the tail was observed after the 2nd day and discoloration of the auricle was observed from the 9th 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 autopsy, and a part of the auricle was deleted in 2 males. Discoloration of the auricle was also observed in one male in the 250 mg / kg group reared under dark conditions from the 14th day, but it was localized at the tip of the auricle, and the symptom was markedly expressed in the reared under light conditions. did.

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

Autopsy results: Only discoloration and defects of the auricle and crusting of the tail were observed as general symptoms in the 250 mg / kg group.

From the above results, it was concluded that the minimum lethal dose of the compound of Example 1 in a single intravenous administration was 250 mg / kg for both males and females under both the light breeding condition and the dark breeding condition under the conditions of this test. In addition, in captivity under light conditions, swelling of the tail and discoloration of the auricle were observed after administration of the 250 mg / kg group, and differences were observed depending on the lighting conditions.
From the above results, it was speculated that the compound of Example 1 could sufficiently obtain the effect of the present invention at a dose at which safety was recognized.

[Pharmacokinetic test of the compound of Example 1]
¹⁴ C-labeled compound of Example 1 was intravenously administered to rats with the following results (dose: 5 mg / kg).
Plasma radioactivity concentration 23.30 μg eq. 5 minutes after administration, which is the first measurement time. After showing of S Example 1 compound / ml, it decreased rapidly. At 6 to 8 hours after administration, a re-increase in plasma radioactivity concentration was observed, and then disappeared with a elimination half-life (t _1/2 ) 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. -It was 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 is more likely to disappear from plasma (faster disappearance) than TS.

88.9% of the administered radioactivity was excreted in the bile up to 4 hours after administration, and 94.9% by 48 hours after administration. Up to 48 hours after administration, 4.4% and 0.8% of the administered radioactivity were excreted in urine and feces, respectively. The survival rate in the body at 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 compounds of Example 1 are more easily discharged because the emissions after 0 to 24 hours and 48 hours both exceed TS as compared with TS. To.

Claims (5)

A glycosylated chlorin e6 derivative represented by the following general formula (4), (5) or (6), or a pharmaceutically acceptable salt thereof.

In equations (4) to (6), n is an integer of 3 to 10. A pharmaceutical composition for photodynamic therapy of tumor, skin disease, eye disease or age-related macular degeneration, which comprises the glycosylated chlorin e6 derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition comprising the glycosylated chlorin e6 derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 3 , for the treatment, diagnosis or detection of tumor, skin disease, eye disease or age-related macular degeneration. The method for producing a glycosylated chlorin e6 derivative according to claim 1 , or a pharmaceutically acceptable salt thereof, which comprises a step of binding chlorin e6 and a sugar via a linking group.

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