JPH08268917A - Antitumor agent having high transition to cancer tissue - Google Patents

Abstract

PURPOSE: To obtain an antitumor agent capable of safely taking an antitumor agent in low-density lipoprotein(LDL) in in vivo without using an isolated material of LDL or or a solubilizing agent by binding a steroidal compound to a water-soluble compound and binding the steroidal compound to an antitumor agent. CONSTITUTION: This antitumor agent is obtained by binding (A) a steroidal compound of formula I [A is an amino or OH; B is methyl, carboxyl, etc.; D is H or OH; E is B; G is D; F is a (substituted)3-9C (branched) alkyl, with the proviso that when F has no functional group, at least one of B, D, E and F is A or carboxyl], formula II or formula III to (B) a water-soluble compound such as phosphoric acid, sulfuric acid, succinic acid, maleic acid or methylmalonic acid by ester bond of one OH group of the component A with an acidic functional group or carboxyl of the component B and binding the component A to (C) a carcinostatic agent by urethane bond, acid amino bond or ester bond of OH, carboxyl and amino of the component A with a functional group of the component C.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to an antitumor agent having a high transferability to cancer tissues.

[0002]

It is well known that anticancer compounds such as melphalan, methotrexate, 5-fluorouracil, mitomycin C, cytarabine and doxorbin are used as anticancer drugs. However,
When such a carcinostatic drug is systemically administered, it is generally distributed to normal organs other than cancer cells, and therefore it exhibits toxicity even in the normal organs in which it is distributed.

Therefore, if the selective transfer of the anticancer drug to the cancer tissue is enhanced, not only the drug effect may be increased but also the toxicity to normal organs may be reduced.

In this connection, many cancers already have LDL via the low density lipoprotein (LDL) receptor.
It has been reported to have the property of taking up more than normal cells (YK Ho, et al., Blood, 52 (1
978) 1099. ), An attempt to utilize LDL as a carrier for an anticancer drug by utilizing this fact has already been examined by several research institutions. Most of them are based on a method using LDL isolated from blood (see “Lipoproteins as carrierrs of pharmacological age” edited by JM Shaw).
nts "1991, Marcel Dekker New York). In other words, isolated LDL can be incorporated with a lipophilic anticancer drug or LDL.
By attaching a carcinostatic drug to the surface of the
It has been reported that DL is loaded with an anticancer drug and is injected intravenously. However, in order to industrialize such a method, a large amount of human LDL must be stably supplied, and further, aseptic formulation including a step of loading an anticancer drug on LDL and a method for removing pyrodiene. Contains many difficult problems to be solved.

[0005] On the other hand, it has been reported that intravenous injection of a highly lipophilic compound is taken up by lipoprotein in blood (Tokui et al., “Pharmacokinetics”, 6 supleme).
nt (1991) 75). However, such highly lipophilic compounds are generally poorly water-soluble, and a solubilizing agent such as a surfactant is required to make such a compound an injection.

[0006]

Under the background of the prior art described in the preceding paragraph, the present invention provides an anticancer drug without using isolated LDL and without using a solubilizing agent of which toxicity is a concern. It is intended to develop and provide a method for incorporating LDL into LDL in vivo.

[0007]

Means for Solving the Problems As a result of earnest research to achieve the object described in the preceding paragraph, the present inventor has found that a water-soluble group (a water-soluble residue of a water-soluble compound is added to a hydroxyl group of a steroid compound carrying an anticancer drug It was found that a carcinostatic agent which was made water-soluble by introducing) becomes lipophilic by removing the water-soluble group in the body after administration,
The present invention has been completed based on such knowledge.

That is, the present invention provides the following general formula (I):
The steroid compound represented by (II) or (III) is phosphoric acid, sulfuric acid, succinic acid, maleic acid, methylmalonic acid,
A water-soluble compound selected from fumaric acid, citric acid, tartaric acid, malic acid, lysine, arginine, and β-sulfopyruvic acid is bonded to each other through an ester bond between one hydroxyl group of the former and an acidic functional group or carboxyl group of the latter. And a cancer tissue characterized in that the steroid compound is bound to a carcinostatic agent by a urethane bond, an acid amide bond or an ester bond between the former carboxyl group, amino group or hydroxyl group and the latter functional group. The present invention relates to a highly anticancer drug.

[0009]

Embedded image

However, in the above general formulas (I) to (III), A represents an amino group or a hydroxyl group, B represents a methyl group, a carboxyl group or a hydroxymethyl group, and D represents a hydrogen atom or a hydroxyl group. , E represents a methyl group, a carboxyl group or a hydroxymethyl group,
G represents a hydrogen atom or a hydroxyl group, and F is a linear or branched alkyl group having 3 to 9 carbon atoms, which may have one functional group selected from a hydroxyl group, an amino group and a carboxyl group. Represents a group. However, when F has no functional group, at least one of B, D, E and G represents a hydroxyl group, an amino group or a carboxyl group as a whole.

When the steroid compound has only a hydroxyl group as a functional group and the anticancer drug is bound via an ester bond, the anticancer drug has 2 hydroxyl groups.
The primary hydroxyl group and the hydroxyl group to which the water-soluble compound binds is 1
It is a primary hydroxyl group. Further, when the steroid compound has an amino group or a carboxyl group as a functional group, it has only one of them, and the amino group or the carboxyl group is always bonded to the functional group of the anticancer drug. .
Further, when the carcinostatic drug is ester-bonded to the carboxyl group of the steroid compound, the carbohydryl group of the carcinostatic drug is a secondary hydroxyl group, and the hydroxy group of the steroid compound to which the water-soluble compound is bonded is 1 It is a primary hydroxyl group.

Further, the functional group of the anticancer drug has an amino group or a hydroxyl group corresponding to that the bond with the steroid compound is a urethane bond, an acid amide bond and an ester bond.
An amino group or a carboxyl group and a hydroxyl group or a carboxyl group.

The present invention will be described in detail below.

The steroid compound has the general formula (I):
A compound represented by (II) or (III),
Specific examples include lithocholic acid and 3-hydroxy-5-
Cholen-24-oic acid, colane-3,24-diol, 5-cholen-3,24-diol, 3-amino-24-hydroxy-5-cholene, deoxycholic acid,
Chenodeoxycholic acid, ursodeoxycholic acid, 19
-Hydroxycholesterol, 3-hydroxy-5-cholestene-19-euic acid, 18-hydroxycholesterol, 3-hydroxy-5-cholestene-18
-Oic acid, 24-amino-3-hydroxycholane, 26-hydroxy-7-dehydrocholesterol and the like can be mentioned.

The structures of these compounds are shown more specifically in Table 1 below.

[0016]

[Table 1]

In particular, F is a linear or branched alkyl group having 3 to 9 carbon atoms which may have one of functional groups selected from a hydroxyl group, an amino group and a carboxyl group,
For example, an alkyl group bonded to the 17-position such as cholesterol analogs and bile acids can be used as it is, or an alkyl group obtained by treating it enzymatically or organically synthetically can be used. Furthermore, if a steroid compound that can be alkylated at the 17-position of the steroid ring such as dehydroepiandrosterone is used as a raw material, it is possible to introduce a desired alkyl group that does not exist naturally as a so-called steroid compound, so that the conditions already described are satisfied. The alkyl group is not particularly limited as long as it is an alkyl group.

The steroid compound having no alkyl group as F is a so-called steroid hormone itself such as sex hormone or adrenocortical hormone,
It is often a compound that can be metabolized and converted into them in vivo. It is speculated that such a compound would be unsuitable as a chemical modifier for pharmaceuticals because it exerts its medicinal effect in a trace amount. Therefore, an alkyl group having 2 or less carbon atoms is not preferable. When the number of carbon atoms is 10 or more, synthesis and the like become difficult, which is not preferable.

Of these steroid compounds, lithocholic acid and 3-hydroxy-5-cholen-24- are particularly preferable because they are easily available, chemically stable, and have low toxicity. Examples thereof include euic acid, colane-3,24-diol, 5-cholene-3,24-diol and the like.

These steroid compounds are produced by chemically or enzymatically or fermenting naturally occurring compounds as described below, or commercially available products are purchased, or after the commercially available products are purchased. It can be obtained by a method such as chemical or enzymatic conversion or conversion by fermentation. For example, lithocholic acid, 3-hydroxy-5-cholene-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, 18-hydroxycholesterol, 19-hydroxycholesterol, etc. are commercially available, or
Since it is a natural product, it can be produced chemically or enzymatically or by fermentation. 24-Amino-3-hydroxychorane, cholane-3,24-diol and 5-cholene-3,24-diol are respectively lithocholic acid amide, lithocholic acid and 3-hydroxy-5-cholene-.
It can be synthesized by a conventional method using 24-oic acid as a raw material and a reducing agent such as lithium aluminum hydride. 3-Hydroxy-5-cholestene-18-euic acid and 3-hydroxy-5-cholestene-19-
Euic acid is obtained by chemically or enzymatically oxidizing 18-hydroxycholesterol and 19-hydroxycholesterol, respectively. 3-
Amino-24-hydroxy-5-cholene can be obtained by a general method for converting the hydroxyl group of 3-hydroxy-5-cholene-24-oic acid into an amino group. In addition,
Hydroxy-7-dehydrocholesterol can be obtained by a method such as hydroxylating 7-dehydrocholesterol with an enzyme such as C ₂₆ -hydroxylase.

As mentioned above, the water-soluble compound is selected from phosphoric acid, sulfuric acid, succinic acid, maleic acid, methylmalonic acid, fumaric acid, citric acid, tartaric acid, malic acid, lysine, arginine and β-sulfopyruvic acid. Be done.

Examples of the anticancer drug include the above-mentioned melphalan, methotrexate, 5-fluorouracil, mitomycin C, cytarabine and doxorbin.

The order of binding the anticancer drug and the water-soluble compound to the steroid compound depends on the kinds of the anticancer drug and the water-soluble compound selected. If it can be composed,
It goes without saying that whichever comes first.

The ester bond between the steroid compound and the water-soluble compound having a hydroxyl group can be formed by a general method in organic synthesis reaction. For example, in order to obtain a phosphoric acid ester, the hydroxyl group is phosphorylated using a pentavalent phosphorylating agent such as phosphorus oxychloride, or a trivalent phosphorus such as di-tert-butyldiethylphosphoramidite is used. There is a method in which a derivative and a compound having the hydroxyl group are condensed and then oxidized to induce a pentavalent phosphate ester. Further, in order to obtain a sulfate ester, a method of mixing the compound having the hydroxyl group with concentrated sulfuric acid and heating if necessary, or a method of sulfating the hydroxyl group using a sulfating agent such as dimethylsulfate, etc. may be used. is there. Furthermore, in order to form an ester bond with a water-soluble carboxylic acid, a desired acid anhydride or acid chloride is used, or a compound having the hydroxyl group is mixed with a desired carboxylic acid in the presence or absence of a catalyst as appropriate. There is a method of adding a dehydration condensing agent such as dicyclohexylcarbodiimide in the presence.

When the anticancer drug is bound to the hydroxyl group of the steroid compound via an ester bond, the hydroxyl group is a secondary hydroxyl group. When the anticancer drug binds to the hydroxyl group of the steroid compound via a urethane bond, the type of the hydroxyl group does not matter. However, in any case, the steroid compound has neither an amino group nor a carboxyl group.

When the anticancer drug binds to the amino group or the carboxyl group of the steroid compound, the amino group or the carboxyl group possessed by the steroid compound is either one of them or not having two or more of them. .

When the anticancer drug binds to the carboxyl group of the steroid compound via an ester bond, the hydroxyl group of the anticancer drug is a secondary hydroxyl group, and the hydroxyl group of the steroid compound to which the water-soluble compound binds is a primary hydroxyl group. It is a hydroxyl group.

The ester bond between the anticancer drug and the steroid compound is formed, for example, by dissolving the two in an appropriate solvent and using a dehydration condensing agent such as dicyclohexylcarbodiimide in the presence or absence of a catalyst as appropriate. It can be formed by a general method such as a method of converting the compound having a carboxyl group into an acid chloride or the like and mixing the compound having a hydroxyl group in an anhydrous organic solvent.

The urethane bond between the anticancer drug and the steroid compound is obtained by, for example, imidazolylformylating or chloroformylating the hydroxyl group with carbonyldiimidazole or phosgene, and the amine and the amine, It can be formed by a general method such as mixing in an anhydrous organic solvent.

The amide bond between the anticancer drug and the steroid compound can be obtained by, for example, mixing the acid chloride, acid anhydride or active ester of the carboxylic acid with the amine in an anhydrous organic solvent, or It can be formed by a general method such as a method of dissolving a carboxylic acid and the amine in a suitable solvent, and forming them by using a dehydration condensing agent such as dicyclohexylcarbodiimide in the presence or absence of a catalyst as appropriate. .

The derivative of the anticancer drug thus obtained is, of course, also an anticancer compound and becomes an anticancer drug. This anticancer agent is water-soluble, but when administered by intravenous injection, it becomes fat-soluble by eliminating the water-soluble compound in the body. The lipid-soluble steroid compound carrying the anticancer drug is taken up by the cancer tissue more and released in the cancer tissue. Thus, more anticancer drug will be distributed in the cancer tissue.

[0032]

EXAMPLES The present invention will be further described below with reference to examples.
The structures of the compounds used in the examples are shown in FIGS.
B and FIG. 1C.

Example 1 Synthesis of 5-cholene-3,24-diol (Compound 1) Tetrahydrofuran (hereinafter abbreviated as THF. 50 m
l) was cooled with ice, and lithium aluminum hydride (1.1
g) was added slowly. THF (3
3-hydroxy-5-cholen-2 dissolved in
4-Oic acid (4.8 g) was gradually added dropwise, and after the addition was completed, stirring was continued at room temperature. After 0.5 hours, thin layer chromatography (chloroform: acetone = 10:
After confirming the disappearance of the raw materials in 1), the reaction solution was ice-cooled, acetone (10 ml) was gradually added dropwise, and chloroform (15 ml) was added.
0 ml) was added. 5N hydrochloric acid was gradually added dropwise to the suspension to adjust the pH to 3. The organic layer was concentrated under reduced pressure, and the obtained white solid residue was recrystallized from a mixed solution of methanol and THF to obtain 4.1 g of compound 1 as white crystals.

NMR (in DMSO-d ₆ ): δpp
m 5.26 (1H, broad d), 4.58 (1H, br
load), 4.30 (1H, broad), 3.34 (2H,
m), 3.25 (1H, m), 2.16-2.04 (2H, m), 1.
97-1.88 (2H, m), 1.82-1.74 (2H, m), 1.68
-1.65 (1H, m), 1.56-1.20 (11H, m), 1.16
-0.85 (13H, m), 0.65 (3H, s).

Example 2: 3- (Hydroxy) -24-
Synthesis of (triphenylmethyloxy) -3--5-cholene (Compound 2) Compound 1 (7.19 g) was dissolved in pyridine (200 ml), and triphenylmethyl chloride (5.8 g) was added under ice cooling. Dissolved. The resulting solution was left in the cold room (4 ° C.) for 3 days and then at room temperature for another 4 days. The reaction mixture was concentrated under reduced pressure and water was added to solidify. The white precipitate obtained was collected by filtration and washed with water. After air drying, it was dissolved in a mixed solution of chloroform and THF, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue is subjected to silica gel column chromatography (chloroform: acetone =
Purification at 100: 8) yielded 10.5 g of compound as an amorphous.

NMR (in DMSO-d ₆ ): δpp
m 7.38-7.23 (15H, m), 5.26 (1H, bro
dd, J = 5Hz), 4.57 (1H, d, J = 4.5H)
z), 3.26 (1H, m), 2.94 (2H, m), 2.12 (2
H, m), 1.98-1.86 (2H, m), 1.77-1.72 (2
H, m). 1.68-1.65 (1H, m), 1.62-1.31 (11
H, m), 1.22-0.85 (13H, m), 0.62 (3H,
s).

Example 3: 32- (5-Fluorouracil-1-yl-carbonyloxy) -24-hydroxy-
Synthesis of 5-cholene (Compound 3) Compound 2 (5.3 g) was dissolved in THF (40 ml), triethylamine (4 g) was added, and the mixture was ice-cooled. In this solution, triphosgene (0.88 g) in THF (10
ml) solution was added slowly. After the addition was complete, the reaction mixture was stirred at room temperature for 0.5 hours. In this, 5-fluorouracil (1.7 g) and triethylamine (1.4 ml)
Was added and the mixture was further stirred for 2 hours. The white precipitate in the reaction solution was filtered off, and the filtrate was evaporated under reduced pressure. The obtained residue was partitioned with chloroform and water, the organic layer was dried over anhydrous sodium sulfate and heated to 85% in an oil bath at 90 ° C.
Acetic acid (70 ml) was added and the mixture was stirred for 2 hours. After cooling, the solvent was concentrated under reduced pressure, and when the white solid began to precipitate, the reduced pressure was released. The obtained slurry-like suspension was added dropwise to an aqueous solution of sodium hydrogen carbonate (1000 ml), and the white powder deposited was collected by filtration and dissolved in a small amount of THF, in which diethyl ether and n- were added. Hexane was added and the precipitated white powder was collected by filtration. The obtained white powder was recrystallized from a mixed solution of THF and acetonitrile to obtain 2.9 g of compound 3 as white crystals.

NMR (in DMSO-d ₆ ): δpp
m 11.99 (1H, d, J = 5Hz), 8.25 (1H, d,
J = 7Hz), 5.40 (1H, m), 4.61 (1H, m),
4.31 (1H, broad), 3.34 (2H, m), 2.40-
0.89 (31H, m), 0.66 (3H, s).

Example 4: 3- (5-Fluorouracil-
1-yl-carbonyloxy) -24-hydroxy-5
-Synthesis of cholen-24-phosphate monotriethylammonium salt (Compound 4) THF (1 ml) containing pyridine (280 mg) was ice-cooled, and phosphorus oxychloride (153 mg) was added dropwise thereto. Compound 3 (100 mg) in THF (2
ml) solution was added dropwise. After disappearance of the raw materials was confirmed by thin layer chromatography, water (0.5 ml) was added under ice cooling. The mixture was added dropwise to distilled water (200 ml) to obtain a solution, which was adjusted to pH 3 with 5N hydrochloric acid, and the white powder precipitated was collected by filtration and washed with water. The obtained white powder was dissolved in a mixed liquid of chloroform and THF, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in N, N-dimethylformamide (2 ml) containing an excess of triethylamine (1 ml), diethyl ether was added, and the precipitated white crystalline powder was collected by filtration. The white crystals obtained were dried to obtain 80 mg of compound 4.

NMR (in DMSO-d ₆ ): δpp
m 8.21 (1H, d, J = 7.5Hz), 5.39 (1H,
m), 4.60 (1H, m), 3.64 (2H, broad
m), 2.94 (6H, q), 2.46-0.90 (43H, m),
0.67 (3H, s).

Example 5: 3- (5-Fluorouracil-
1-yl-carbonyloxy) -24-hydroxy-5
-Synthesis of cholen-24-hemisuccinate (Compound 5) Compound 3 (1.55 g) and triethylamine (1.
0 g) was dissolved in THF (30 ml). Succinic anhydride (0.4 g) was added and dissolved in this solution. This solution was left at room temperature for 15 hours. The disappearance of Compound 3 was confirmed by thin layer chromatography, and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in a mixed solution of THF and chloroform and partitioned with an aqueous citric acid solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. A small amount of ethanol was added to the residue to dissolve it, and n-hexane was added with heating and the mixture was allowed to cool. The precipitated white crystals were dried under reduced pressure to obtain 1.2 g of compound 5.

NMR (in DMSO-d ₆ ): δpp
m 12.04 (2H, broad), 8.24 (1H, d, J =
7Hz), 5.40 (1H, m), 4.62 (1H, m), 3.97
(2H, m), 3.32 (4H, s), 2.48-0.90 (31
H, m), 0.67 (3H, s).

Example 6: 3- (5-Fluorouracil-
1-ylcarbonyloxy) -24-hydroxy-5-
Synthesis of cholen-24-L-lysine ester dihydrochloride (Compound 6) Compound 3 (0.52 g) and N- (tert-butyloxycarbonyl) -L-lysine (0.45 g) were added to methylene chloride (4 ml) and It was dissolved in a mixed solvent with pyridine (2 ml), N, N'-dicyclohexylcarbodiimide (0.3 g) was added thereto, and the mixture was stirred for 2 hours. The precipitated white crystals were filtered off, and the filtrate was concentrated under reduced pressure. Diethyl ether was added to the obtained residue, and the insoluble material was filtered off. Chloroform was added to the filtrate and it was washed with an aqueous citric acid solution.
The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Diethyl ether was added to the obtained residue, and n
-When hexane was added, a white powder precipitated. This powder was collected by filtration, and a 4N hydrogen chloride solution in dioxane (10 ml) was added and dissolved. The mixture was stirred at room temperature for 1 hour, and the obtained white powder was collected by filtration. This powder was washed with diethyl ether and dried to obtain 0.33 g of compound 6.

NMR (in DMSO-d ₆ ) δ ppm
12.00 (1H, d, J = 4Hz), 8.55 (3H, bro
ad), 8.25 (1H, d, J = 7Hz), 7.98 (3H,
Broad), 5.40 (1H, m), 4.61 (1H, m),
4.14 (2H, m), 2.75 (2H, m), 2.46-0.88 (3
8H, m), 0.67 (3H, s).

Example 7: Synthesis of cholan-3,24-diol (Compound 7) THF (50 ml) was ice-cooled, and lithium aluminum hydride (3.0 g) was gradually added. Lithocholic acid (10.0 g) dissolved in THF (300 ml) was gradually added dropwise to the obtained suspension, and after completion of the addition, stirring was continued at room temperature. After 0.5 hours, the disappearance of the raw materials was confirmed by thin layer chromatography (chloroform: acetone = 10: 1), the reaction solution was ice-cooled, acetone (10 ml) was gradually added dropwise, and chloroform (150 ml) was further added. Was added. 5N hydrochloric acid was gradually added dropwise to the suspension to adjust the pH to 3. The organic layer was washed with an aqueous sodium sulfate solution and then dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the obtained white solid residue was recrystallized from a mixed solution of methanol and THF to obtain 9.1 g of compound 7 as white crystals.

NMR (in DMSO-d ₆ ): δpp
m 4.43 (1H, broad), 4.30 (1H, broad
d), 3.4-3.3 (3H, m), 1.94-0.87 (34H,
m), 0.62 (3H, s).

Example 8: 3- (Hydroxy) -24-
Synthesis of (triphenylmethyloxy) -chorane (Compound 8) Compound 7 (9.0 g) was dissolved in pyridine (100 ml), and triphenylmethyl chloride (8.3 g) was added under ice cooling to dissolve it. The resulting solution was left at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, the obtained residue was dissolved in THF, and this was added dropwise to water (900 ml) to obtain a yellowish white viscous substance, which was added to a mixed solution of THF and chloroform. Was dissolved in water, washed with water, and dried over anhydrous sodium sulfate. After evaporating the solvent, the obtained residue was subjected to silica gel column chromatography (chloroform: acetone = 1.
It refine | purified in 00: 8) and obtained 13.6 g of compound 8 as an amorphous substance.

NMR (in DMSO-d ₆ ): δpp
m 7.38-7.19 (15H, m), 4.44 (1H, d, J =
4.5Hz), 3.36 (1H, m), 2.93 (2H, m),
1.92-0.85 (34H, m), 0.58 (3H, s).

Example 9: Synthesis of 24- (tert-butyldimethylsilyloxy) -3- (hydroxy) -cholane (Compound 9) Compound 7 (2.94 g), 4- (N, N-dimethylamino)- Pyridine (100 mg) and triethylamine (1.1 g) were dissolved in a mixed solution of THF (25 ml) and N, N-dimethylformamide (10 ml). This container was ice-cooled, tert-butyldimethylchlorosilane (1.6 g) was added, and the mixture was stirred. After 2 hours, a large amount of water was added to the reaction solution and the precipitated white solid was collected by filtration. The obtained solid was dissolved in chloroform, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform: methanol = 80: 1) to obtain 2.8 g of compound 9 as an amorphous substance.

NMR (in CDCl ₃ ): δ ppm
3.63 (1H, m), 3.58 (2H, t), 1.98 (1H,
m), 1.90-1.73 (4H, m), 1.67 (1H, m), 1.
63-1.49 (4H, m), 1.46-1.31 (9H, m), 1.31
-1.19 (3H, m), 1.18-0.99 (6H, m), 0.99-
0.93 (7H, m), 0.91 (9H, s), 0.65 (3H,
s), 0.06 (6H, s).

Example 10: 24- (tert-Butyldimethylsilyloxy) -3- [2- (dansylamino)
Ethylaminocarbonyloxy] -cholan (Compound 1
Synthesis of 0) Compound 9 (6.3 g) was dissolved in THF (20 ml),
Carbonyldiimidazole (2.5 g) was added thereto and heated in an oil bath (55 to 60 ° C.) for 0.5 hours. After confirming the disappearance of compound 9 by thin layer chromatography, after cooling, monodansyl ethylenediamine (4.7 g) was added and the mixture was stirred at room temperature. The insoluble material was filtered off, and the filtrate was evaporated under pressure. The residue was purified by silica gel column chromatography (chloroform: methanol = 80: 1).
0 g of compound 10 was obtained as an amorphous.

NMR (in CDCl ₃ ): δ ppm
8.55 (1H, d), 8.25 (2H, t), 7.58 (1H,
t), 7.52 (1H, t), 7.19 (1H, d), 5.25 (1
H, broad), 4.81 (1H, broad), 4.53
(1H, m), 3.57 (2H, t), 3.21 (2H, bro
ad), 3.02 (2H, q), 2.90 (6H, s), 2.0-
0.90 (43H, m), 0.65 (3H, d), 0.04 (6H,
s).

Example 11: Synthesis of 3- [2- (dansylamino) ethylaminocarbonyloxy] -24- (hydroxy) -cholane (Compound 11) Compound 10 (3.1 g) was dissolved in acetic acid (30 ml). Then, distilled water (15 ml) was added, and the mixture was allowed to stand at room temperature for 15 hours.
The disappearance of Compound 10 was confirmed by thin layer chromatography, and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in chloroform and then washed with an aqueous sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue is subjected to silica gel column chromatography (chloroform: methanol = 50: 1).
Was purified by the above procedure to obtain 1.8 g of Compound 11 as an amorphous substance.

NMR (in CDCl ₃ ): δ ppm
8.55 (1H, d), 8.25 (2H, t), 7.58 (1H,
t), 7.52 (1H, t), 7.19 (1H, d), 5.23 (1
H, broad), 4.81 (1H, broad), 4.53
(1H, m), 3.62 (2H, t), 3.21 (2H, bro
ad), 3.02 (2H, q), 2.90 (6H, s), 2.00-
1.97 (1H, m), 1.88-1.71 (4H, m), 1.67-1.
61 (2H, m), 1.52 (1H, m), 1.5-0.92 (34
H, m), 0.65 (3H, s). NMR (in DMSO-d ₆ ): δppm 8.46 (1
H, d, J = 9Hz), 8.26 (1H, d, J = 7.5H
z), 7.96 (1H, t), 7.61 (2H, m), 7.26 (1
H, d, J = 7Hz), 6.88 (1H, t), 4.37 (1
H, m), 4.30 (1H, t), 3.34 (2H, m), 2.96
(2H, m), 2.83 (6H, s), 2.79 (2H, m),
1.93-0.87 (34H, m), 0.61 (3H, s).

Example 12: Synthesis of 3- [2- (dansylamino) ethylaminocarbonyloxy] -24- (hydroxy) -chorane-24-phosphate ester monotriethylammonium salt (Compound 12) Pyridine (700 mg) THF containing (2 ml) was cooled with ice,
Phosphorus oxychloride (320 mg) was added dropwise thereto. A solution of compound 11 (180 mg) in THF (4 ml) was added dropwise to this solution. After disappearance of the raw materials was confirmed by thin layer chromatography, water (0.5 ml) was added under ice cooling. This mixture was added dropwise to distilled water (200 ml) to give a solution of 5
N hydrochloric acid was added to adjust the pH to 3, and the precipitated white powder was collected by filtration.
Washed with water. The fluorescent yellow-green powder obtained was mixed with chloroform and T.
After dissolving in a mixed solution of HF and drying with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was dissolved in N, N-dimethylformamide (5 ml) containing an excess of triethylamine (1.5 ml), diethyl ether was added, and the precipitated fluorescent yellow-green crystalline powder was collected by filtration. The obtained crystals were dried to obtain 170 mg of compound 12.

NMR (in DMSO-d ₆ ): δpp
m 8.46 (1H, d, J = 8.5Hz), 8.26 (1H,
d, J = 8.5Hz), 8.09 (1H, d, J = 7.5H)
z), 7.96 (1H, broad), 7.60 (2H, m),
7.25 (1H, d, J = 7.5Hz), 6.89 (1H, br
od), 4.36 (1H, m), 3.64 (2H, m), 2.96
-2.78 (16H, m), 1.93-0.85 (43H, m), 0.
61 (3H, s).

Example 13: Synthesis of 3- (5-fluorouracil-1-yl-carbonyloxy) -24-hydroxychorane (Compound 13) THF (100 ml) was ice-cooled and triphosgene (2.1) was added.
g) was added and dissolved. Triethylamine (5 ml) was gradually added thereto. Compound 8 in this mixture
(7.6 g) was added and dissolved. This reaction solution is 40 ℃
After heating for 15 minutes, the disappearance of the raw materials was confirmed by silica gel thin layer chromatography, and the reaction solution was concentrated under reduced pressure. To the obtained residue, a solution of 5FU (3 g) and triethylamine (2 ml) in N, N-dimethylformamide (20 ml) was added and mixed, and the mixture was mixed in a water bath at 60 ° C.
Heated for 5 hours. The reaction solution was added to distilled water (800 ml) with stirring, and a white precipitate obtained was collected by filtration. This precipitate was dissolved in chloroform, washed with water and dried over anhydrous sodium sulfate. After evaporating the solvent, a white amorphous substance was obtained.

The obtained amorphous substance was dissolved in THF (10 ml), 90% acetic acid (40 ml) was added, and the mixture was heated in a 70 ° C. oil bath for 45 minutes. This solution is distilled water (900 ml)
The white powder was added dropwise to the mixture, which was collected by filtration and washed with water. The obtained powder was dissolved in chloroform containing THF, washed with water, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The obtained residue was solidified with diethyl ether, and the precipitated white powder was recrystallized from a mixed solution of THF-acetonitrile to obtain 2.8 g of compound 13 as white powdery crystals.

NMR (in DMSO-d ₆ ): δpp
m 11.99 (1H, s), 8.25 (1H, d, J = 7H
z), 4.79 (1H, m), 4.32 (1H, t), 3.34 (2
H, m), 1.99-0.98 (m), 0.92 (3H, s), 0.88
(3H, d, J = 6.5Hz), 0.63 (3H, s).

Example 14: Synthesis of 3- (5-fluorouracil-1-yl-carbonyloxy) -24-hydroxycholane-24-phosphate monotriethylammonium salt (Compound 14) Pyridine (420 mg) in THF ( 1.5 ml) was ice-cooled, and phosphorus oxychloride (230 mg) was added dropwise thereto.
Compound 13 (150 mg) in THF (2 ml) in this solution
The solution was added dropwise. The disappearance of the raw materials was confirmed by silica gel thin layer chromatography, and distilled water (0.5 ml) was added under ice cooling. This mixed solution was added dropwise to distilled water (200 ml), and the resulting solution was adjusted to pH 3 with 5N hydrochloric acid. The white powder deposited was collected by filtration and washed with water. The obtained white powder was dissolved in chloroform, this chloroform solution was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in N, N-dimethylformamide (2 ml) containing triethylamine (0.5 ml), diethyl ether was added, and the precipitated white crystalline powder was collected by filtration (Compound 14, 152 mg).

NMR (in DMSO-d ₆ , phos
phase): δppm 11.98 (1N, s), 8.25 (1
H, d, J = 7 Hz), 4.79 (1 H, m), 3.77 (2
H, m), 2.00-1.91 (2H, m), 1.84-1.73 (4
H, m), 1.64-1.57 (2H, m), 1.56-1.34 (H, m)
m), 1.25-1.17 (H, m), 1.12-1.00 (H, m),
0.92 (3H, s), 0.89 (3H, d), 0.63 (3H,
s). NMR (in DMSO-d ₆ , Et ₃ N sal
t): δppm 8.25 (1H, d, J = 7Hz), 4.78
(1H, m), 3.65 (2H, m), 2.93 (6H, m),
1.98-0.99 (m), 1.13 (9H, s), 0.91 (3H,
s), 0.89 (3H, d, J = 6.5Hz), 0.63 (3
H, s).

Example 15: Synthesis of 3- (5-fluorouracil-1-yl-carbonyloxy) -24-hydroxycholan-24-hemisuccinate (Compound 15) Compound 13 (1.55 g) and triethylamine (1. 0 g) was dissolved in THF (30 ml). Succinic anhydride (0.4 g) was added to and dissolved in this solution as a solid. This solution was left at room temperature for 15 hours.
Starting compound 1 by silica gel thin-layer chromatography
After confirming disappearance of 3, the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a mixture of THF-chloroform and partitioned with an aqueous citric acid solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. A small amount of ethanol was added to the residue to dissolve it, and n-hexane was added with heating and the mixture was allowed to cool. The white crystals obtained were filtered and dried under reduced pressure.
5 g of compound 15 was obtained as white crystals.

NMR (in DMSO-d ₆ ): δpp
m 12.04 (1H, broad), 12.00 (1H, broad
ad), 8.24 (1H, d, J = 7.5Hz), 4.79 (1
H, m), 3.97 (2H, m), 3.32 (4H, s), 2.47
(4H, m), 1.99-0.85 (m) 0.63 (3H, s).

Example 16: 3- (5-Fluorouracil-1-yl-carbonyloxy) -24-hydroxycholan-24-L-lysine ester dihydrochloride (Compound 1
Synthesis of 6) Compound 13 (0.52 g) and α, ε-di-N-tert
-Butyloxycarbonyl-L-lysine (0.45g)
And were dissolved in a mixed solvent of methylene chloride (4 ml) and pyridine (2 ml), N, N-dicyclohexylcarbodiimide (0.3 g) was added thereto, and the mixture was stirred for 2 hours. The precipitated white crystals were filtered off, and the filtrate was concentrated under reduced pressure.
Diethyl ether was added to the obtained residue, and the insoluble material was filtered off. Chloroform was added to the filtrate and it was washed with an aqueous citric acid solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. When diethyl ether was added to the obtained residue and n-hexane was added, a white powder was deposited. This powder was collected by filtration, and 4N hydrogen chloride (dioxane solution, 10 ml) was added and dissolved. The mixture was stirred at room temperature for 1 hour, and the obtained white powder was collected by filtration. This powder was washed with diethyl ether and dried to give 0.41 g of compound 1
6 was obtained as a white powder.

NMR (in DMSO-d ₆ ): δpp
m 12.00 (1H, d, J = 4.5Hz), 8.49 (3H,
Broad), 8.24 (1H, d, J = 7Hz), 7.91
(3H, broad), 4.79 (1H, m), 4.14 (2
H, t), 3.99 (1H, m), 2.75 (2H, m), 1.95
-1.02 (m), 0.92 (3H, s), 0.90 (3H, d, J
= 6.5 Hz), 0.64 (3 H, s).

Reference Example 1: Synthesis of 12- (5-fluorouracil-1-yl-carbonyloxy) -1-stearyl alcohol-1-phosphate monosodium salt (Comparative Compound A) THF (containing pyridine (280 mg) ( 1 ml) on ice,
Phosphorus oxychloride (153 mg) was added dropwise thereto. To this solution, a solution of separately synthesized 12- (5-fluorouracil-1-yl-carbonyloxy) -1-stearyl alcohol (100 mg) in THF (2 ml) was added dropwise.
After disappearance of the raw materials was confirmed by thin layer chromatography, water (0.5 ml) was added under ice cooling. This mixed solution was added to distilled water (2
(00 ml) and 5N hydrochloric acid was added to the resulting solution to adjust the pH to 3 to obtain a white viscous substance. This was dissolved in a mixed liquid of chloroform and THF, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a mixed solution of diethyl ether: methanol = 3: 1, and a diluted solution of sodium methylate in methanol was added dropwise thereto. A white powdery solid was deposited, which was collected by filtration and washed with diethyl ether to obtain 75 mg of comparative compound A.

NMR (in D ₂ O): δppm 8.03
(1H, s), 5.05 (1H, m), 3.74 (2H, m),
1.72 (2H, m), 1.58 (2H, m), 1.38-1.24 (2
6H, m), 0.87 (3H, broad).

Evaluation Example 1: Carcinogenicity of the compound of the present invention in tumor-bearing mice (adenocarcinoma 755) (a) Evaluation method BDF ₁ male mice were passaged subcutaneously on the ventral side (11 days).
Adenocarcinoma 755 solid tumor cells were transplanted subcutaneously to the ventral region of 6-week-old BDF ₁ male mice (5 × 10 ⁵
cells / 0.05 ml), and on day 13, each test compound was intravenously administered once. The dose of the 5FU derivative was 10 mg / kg (76.9 μmol / kg) as 5FU, and the dose of the dansyl derivative was 20 μmol / kg.
After a certain time from the administration, the mouse was anesthetized with diethyl ether, blood was collected from the abdominal aorta of the abdomen, and treated with heparin.
The tumor and thigh muscle were resected respectively. Immediately centrifuge the collected blood (3,000 rpm, 15 min, 4 min.
The obtained plasma was frozen. The excised tissues were frozen after measuring their wet weight. All biological samples were stored frozen until just before the quantitative analysis described below.

5FU in the biological sample was treated with 5-chlorouracil as an internal standard substance, deproteinized homogenized tissue or plasma, and then extracted with ethyl acetate to obtain a normal phase high-speed liquid. It was separated and quantified by chromatography (HPLC) (detection was by ultraviolet absorption). Similarly, the dansyl derivative in the biological sample was similarly subjected to deproteinization using a fluorescent substance as an internal standard substance, and then separated and quantified by reverse phase HPLC (detection was by fluorescence).

(B) Evaluation Result The evaluation result will be described with reference to the drawings.

FIG. 2 shows the time-dependent changes in the concentration of 5FU detected in the tumor after 5FU, compound 4 or comparative compound A was intravenously administered at the above dose.
U, black circles indicate the results of Compound 4 and triangles indicate the results of Comparative Compound A administration group. The area under the tumor concentration curve (AUC) of the compound 4 administration group was twice that of the 5FU administration group. On the other hand, the intratumoral 5FU concentration of the comparative compound A administration group was 5FU.
It did not exceed that of the treatment group.

FIG. 3 shows plasma (FIG. 3A), tumor (FIG. 3B) and muscle (FIG. 3) after intravenous administration of comparative compound A (dansyl-L-alanine) or compound 12 at the above doses.
Compound 12 (open circle), compound 11 detected in C)
The changes over time in the concentrations of (black circle) and comparative compound A (triangle) are shown. Administered compound 12 was rapidly converted to compound 11 and maintained a higher intratumoral concentration as compared to dansyl-L-alanine of comparative compound A for a long time (intratumoral AUC was 4.1 times that of dansyl-L-alanine). , The average residence time is 2.5 times). On the other hand, when comparing the normal intramuscular concentration, no significant difference was observed between the compound of the present invention and Comparative Compound A.

(C) Discussion A major structural difference between the compound 4 of the present invention and the comparative compound A lies in the lipid to which 5FU binds. That is, 5 FU is bound to the steroid derivative of the compound 4 and 5 FU is bound to the linear lipid of the comparative compound A, respectively. This difference in lipids probably influences the intratumoral transfer of 5FU.

The water-soluble compounds (Compound 4 and Compound 12) according to the present invention are poorly water-soluble compounds (Compound 12) in the body.
In the case of, the compound was rapidly converted to compound 11) and distributed in the cancer tissue. The distribution amount and the residence time after the distribution are the same as those of the comparative control compound dansyl-L-
It was higher than when alanine was administered (Fig. 3B). As one of the factors, the difference in plasma concentration of both compounds (Fig. 3
The effect by A) is presumed, and this suggests the possibility of the difference in the distribution of LDL in the blood initially assumed by the present inventor. Moreover, since the distribution of Compound 11 in normal muscle tissue was not enhanced as much as that in cancer tissue (FIG. 3C), it is considered that the expression of toxicity to normal tissue by this compound is not increased. The above results show that the compound of the present invention has relatively high cancer transferability, which makes it possible to provide a carcinostatic agent having high selective toxicity to cancer.

[0075]

INDUSTRIAL APPLICABILITY According to the present invention, a carcinostatic agent having a high selective migration to a cancer tissue can be easily provided.

[Brief description of drawings]

FIG. 1A shows the structures of the compounds treated in the examples.

FIG. 1B shows the structures of the compounds treated in the examples.

FIG. 1C shows the structures of the compounds treated in the examples.

FIG. 2 shows results in Evaluation Example 1.

FIG. 3A shows results in Evaluation Example 1.

FIG. 3B shows results in Evaluation Example 1.

FIG. 3C shows results in Evaluation Example 1.

Claims (2)

[Claims] 1. A compound represented by the following general formula (I), (II) or (III):
) Is a water-soluble compound selected from phosphoric acid, sulfuric acid, succinic acid, maleic acid, methylmalonic acid, fumaric acid, citric acid, tartaric acid, malic acid, lysine, arginine and β-sulfopyruvic acid. , The former one hydroxyl group and the latter acidic functional group or a carboxyl group by an ester bond, and the steroid compound is a carcinostatic agent, the former carboxyl group, amino group or hydroxyl group and the latter functional group A carcinostatic agent having a high migration property to a cancer tissue, which is bound by a urethane bond, an acid amide bond or an ester bond. Embedded image However, in the above general formulas (I) to (III), A is
Represents an amino group or a hydroxyl group, B represents a methyl group, a carboxyl group or a hydroxymethyl group, D represents a hydrogen atom or a hydroxyl group, E represents a methyl group, a carboxyl group or a hydroxymethyl group, and G represents Represents a hydrogen atom or a hydroxyl group, and F represents a linear or branched alkyl group having 3 to 9 carbon atoms, which may have one of functional groups selected from a hydroxyl group, an amino group and a carboxyl group. . However, when F does not have a functional group, B, D, E
At least one of G and G represents a hydroxyl group, an amino group or a carboxyl group as a whole. When the steroid compound has only a hydroxyl group as a functional group and the anticancer drug is bound via an ester bond, the anticancer drug is bound to a secondary hydroxyl group and is a water-soluble compound. The hydroxyl group to which is bonded is a primary hydroxyl group. Further, when the steroid compound has an amino group or a carboxyl group as a functional group, it has only one of them, and the amino group or the carboxyl group is always bonded to the functional group of the anticancer drug. . Further, when the carcinostatic drug is ester-bonded to the carboxyl group of the steroid compound, the carbohydryl group of the carcinostatic drug is a secondary hydroxyl group, and the hydroxy group of the steroid compound to which the water-soluble compound is bonded is 1 It is a primary hydroxyl group. In addition, the functional group of the anticancer drug is an amino group or a hydroxyl group, an amino group or a carboxyl group, and a hydroxyl group or a carboxyl group corresponding to the bond with the steroid compound being a urethane bond, an acid amide bond and an ester bond. is there. 2. The steroid compound is lithocholic acid, 3-
Hydroxy-5-cholene-24-oic acid, colane-3,24-diol, or 5-cholene-3,24
-A carcinostatic agent according to claim 1, which is a diol.