MX2008003047A - Antitumor agent. - Google Patents

Abstract

A sugar-cholestanol compound having a sufficient antitumor activity is provided by a simple synthesis. Disclosed is an antitumor agent containing a cholestanol compound represented by the formula (1) below or a cyclodextrin clathrate thereof as an active ingredient. (1).

Description

ANTITUMOR AGENT TECHNICAL FIELD OF THE INVENTION The present invention relates to a drug containing a sugar-cholestanol compound as an active ingredient.

TECHNICAL BACKGROUND A sugar-cholestanol compound, in which GIcNAc-Gal-, GIcNAc-Gal-Glc-, Fue-Gal-, Gal-Glc- or Gal- binds to cholestanol which has a carbon-carbon double bond saturated in the B ring of cholesterol, exhibits excellent antitumor activity (see patent documents 1 , 2 and 3). The majority of said sugar-cholestanol compounds consisting of at least two portions of sugar chain exhibit anti-tumor activities. However, these compounds require a complicated procedure for synthesis and a higher production cost since the number of sugar chain portions increases. Meanwhile, a sugar-cholestanol compound that has galactose as a single serving of sugar has been reported so far, but its activity is still unsatisfactory. In addition, these sugar-cholestanol compounds share the same problem as they are generally insoluble in water. Therefore, there has been a growing demand for a new sugar-cholestanol compound having a small number of short sugar chain portions, which has sufficient anticancer activity, and which is excellent in solubility. A cholestanol compound having N-acetyl-D-glucosamine (GIcNAc) as a sugar chain portion is a known compound (see document which is not patent 1), but a report of its bioactivity has not yet been made. [Patent Document 1] JP-A-11-60592 [Patent Document 2] JP-A-2000-191685 [Patent Document 3] WO 2005/007172 Pamphlet [document that is not patent 1] Studies on steroids. Part CXXXXV. Synthesis of conjugated cholesterol and cholestanols. (19J9), 27 (8), 1926-31.

BRIEF DESCRIPTION OF THE INVENTION PROBLEMS TO BE RESOLVED BY THE INVENTION An object of the present invention is to provide a sugar-cholestanol compound which can be easily synthesized, and which exhibits satisfactory antitumor activity.

Means to solve problems In a sugar-cholestanol, no antitumor activity is recognized about either its portion of sugar chain or portion of cholestanol at all, so the entire structure of sugar-cholestanol is essential for the expression of antitumor activity. But it has been extremely difficult to deduce the number or type of sugar chain portions of the correlated structure. Under such circumstance, the inventors of the present have found that a cholestanol compound represented by the formula (1) described below having GIcNAc as a single sugar portion, exhibits a strong inhibitory activity against growth of cancer cells. The inventors of the present invention have also found that the cholestanol compound, when in the form of a cyclodextrin inclusion complex or a liposomal preparation, can overcome the disadvantages attributable to insolubility, and that this compound is much more effective with respect to the above mentioned activity. Accordingly, the present invention provides the following. 1) An anti-cancer agent containing, as an active ingredient, a cholestanol compound represented by the following formula (1): or a cyclodextrin inclusion complex containing the compound. 2) a liposomal formulation containing the cholestanol compound. 3) A cyclodextrin inclusion complex containing the cholestanol compound. 4) The use of the cholestanol compound or a cyclodextrin inclusion complex containing the compound to produce an anticancer agent. 5) A method for preventing or treating cancer, characterized in that it comprises the administration of the cholestanol compound or a cyclodextrin inclusion complex containing the compound.

EFFECTS OF THE INVENTION The cholestanol compound of the present invention exhibits excellent anti-tumor effects, and can be easily synthesized in comparison with the case of a conventional sugar-cholestanol compound. Therefore, the cholestanol compound of the present invention is useful as a drug to prevent or treat cancer. A cyclodextrin inclusion complex or liposomal formulation containing the cholestanol compound exhibits excellent solubility, and is remarkably effective because the compound exhibits anticancer activity.

DETAILED DESCRIPTION OF THE INVENTION The cholestanol compound of the present invention, which is a known compound (see document which is not patent 1), can be produced, for example, by the following method.

Specifically, the cholestanol compound of the present invention can be produced from 4-methoxyphenyl-3,4,6-tri-0-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside through four steps (see the production example described below). The sugar chain portion of the cholestanol compound of the present invention is preferably GIcNACß-. Therefore, the cholestanol compound of the present invention is preferably 5-a-cholestan-3β-l-2-acetamido-2-deoxy-p-D-glucopyranoside (la). The cholestanol compound of the present invention can be easily included in a cyclodextrin compound or a derivative thereof to form a complex. Cyclodextrin is not necessarily adapted to all insoluble compounds, since, conceivably, the formation of a cyclodextrin inclusion complex is affected by the size of a host molecule to be included, the van der Waals interaction between the host molecule and cyclodextrin, or the link of hydrogen between the host molecule and hydroxyl groups of cyclodextrin. However, cyclodextrin can form a good inclusion complex with the cholestanol compound of the present invention. Examples of the cyclodextrin used to form the cyclodextrin inclusion complex of the present invention include cyclodextrin compounds such as α-cyclodextrin, β-cyclodextrin and β-cyclodextrin, and cyclodextrin derivatives such as metill-β-cyclodextrin, 2-hydroxypropyl -β-cyclodextrin, monoacetyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. Of these, 2-hydroxypropyl-β-cyclodextrin is preferred to obtain improved solubility. The cyclodextrin inclusion complex can be prepared, for example, by the following procedure: an appropriate aqueous solution (e.g., 20 to 40%) of a cyclodextrin compound or a derivative thereof is prepared, and the The cholestanol compound of the present invention is added to the aqueous solution, followed by stirring the resulting mixture. No particular limitation is imposed on the amount of the cholestanol compound to be added, provided that the cholestanol compound can form an inclusion complex with cyclodextrin. The amount is generally 1 to 50% by mass, preferably from about 10 to about 30% by mass. The cyclodextrin inclusion complex thus formed is readily soluble in water, and therefore exhibits its effects efficiently in vivo. The inclusion complex of cyclodextrin is also advantageous since it can be reliably evaluated in an in vitro test system. A liposomal formulation prepared from the cholestanol compound of the present invention can be efficiently delivered to a site where the effects of the compound are to be presented. The liposomal formulation can also be reliably evaluated in an in vitro test system. The liposomal formulation preferably contains the cholestanol compound of the present invention, a membrane component, and an aliphatic or aromatic amine. The amount of the cholestanol compound contained in the liposomal formulation is 0.3 to 2.0 moles, preferably 0.8 to 1.5 moles, based on 1 mole of the membrane component. Examples of the membrane component include phospholipids.

The phospholipids that are preferably used include naturally occurring phospholipids and synthesized phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidic acid; mixtures of these phospholipids and naturally occurring processed phospholipids such as aqueous lecithin. Most preferably, 1a- dipalmitoylphosphatidylcholine (DPPC), which is a compound of phosphatidylcholine. The aliphatic or aromatic amine is generally added to positively charge the surface of a lipid membrane. Examples of the amine to be added include aliphatic amines such as stearylamine and oleylamine.; and aromatic amines such as fluorenoethylamine. Particularly, stearylamine is preferably used. The amount of the amine contained in the liposomal formulation is 0.04 to 0.15 moles, preferably 0.1 to 0.15 moles, based on 1 mole of the membrane component (phospholipid). If necessary, the liposomal formulation may contain, in addition to the aforementioned components, a membrane structure stabilizer such as cholesterol, a fatty acid or diacetyl phosphate. The aqueous solution used to disperse the membrane component is preferably water, saline, a pH regulator. an aqueous solution of a sugar, or a mixture thereof. The pH regulator to be used is preferably an organic or inorganic pH regulator having pH-regulating action in the vicinity of the hydrogen ion concentration of body fluids. For example, a phosphate pH regulator can be used. No particular limitation is imposed on the method for preparing the liposomal formulation, and the formulation can be prepared by a generally used method. For example, the liposomal formulation is can be prepared by the method described in JP-A-57-82310, JP-A-60-12127, JP-A-60-58915, JP-A-1-117824, JP-A-1-167218, JP- A-4-29925 or JP-A-9-87168, the method described in Methods of Biochemical Analysis (1988) 33, p 337 or the method described in "Líposome" (Nankodo). Next, a method for preparing a liposomal formulation from the cholestanol compound of the present invention will be described. First, an organic solvent and water are added to, and mixed with, the compound of cholestanol of the present invention, any component of aforesaid membrane, and an aliphatic or aromatic amine, and subsequently the organic solvent is completely removed by means of a rotary evaporator or similar apparatus, followed by water removal. The mixing ratios of the membrane component, the alkylamine and the cholestanol compound can be, for example, 52: 8: 20 (per mole) However, as long as the mixing ratios do not deviate significantly from them, no particular problems will arise.

When the mixing ratio of the cholestanol compound is low, if necessary, a membrane structure stabilizer such as cholesterol can be added. However, when the mixing ratio of the cholestanol compound is high, the addition of said membrane structure stabilizer is not necessarily required. No particular limitation is imposed on the organic solvent that It must be used, provided it is a volatile organic solvent that is soluble in water. Examples of the organic solvent that can be used include chloroform, chloromethane, benzene and hexane. When considering the solubility, an organic solvent having relatively high polarity (e.g., ethanol or methanol) can be suitably added to said water-insoluble solvent, and the organic solvent mixture thus prepared can be used. No particular limitation is imposed on the mixing ratios of the organic solvent and water mixture, provided that a uniform solvent mixture is obtained. In the case where the water is added for the preparation of the liposomal formulation, the removal of water is generally carried out by freeze drying. However, the removal of water is not necessarily done through freeze drying, and can be done through drying in a desiccator of reduced pressure. After removing the water, the aforementioned aqueous solution for dispersion is added, followed by stirring by means of, for example, a swirling mixer, to thereby form the liposomal formulation. Liposomes having a uniform particle size can be prepared, for example, by ultrasonic treatment, extrusion treatment by means of a porous membrane filter, treatment by means of a high pressure injection emulsifier, or combination of said treatments. Smaller liposome particles can be prepared, for example, by performing ultrasonic treatment for a long time period. The particle size of the liposomes is preferably 40 nm to 300 nm. The cholestanol compound of the present invention can be used in form, in addition to the aforementioned liposomal formulation, of a variety of stabilizers, and the shape of the stabilizer varies in response to a dosage form to be used. As described below in Examples, compound cholestanol of the present invention, an inclusion complex of cyclodextrin containing compound cholestanol, and a liposomal formulation containing the compound of cholestanol (hereinafter these may be referred collectively as "the cholestanol compound, etc.") have a much stronger cell proliferation inhibiting effect, as compared to the case of a cholestanol compound having a sugar chain portion formed of GIcNAc-Gal- or Gal-. Thus, a formulation containing, as an active ingredient, the cholestanol compound of the present invention is useful as a drug to prevent or treat cancer. The dosage form of the anti-cancer agent of the present invention can be determined appropriately when considering the treatment site or the therapeutic purpose. Provided that an additive preventing the stability of the liposomal formulation form is not used, the anti-cancer agent may be in any dosage form of, for example, a peroral product, an injection, a suppository, an ointment, and a patch, each of which can be produced through a conventional formulation method known to those skilled in the art. A peroral solid product (e.g., a tablet, a coated tablet, a granule, a powder or a capsule) can be prepared by adding, to the cholestanol compound, etc., of the present invention, an excipient and, if necessary, an additive such as a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, or a deodorant, followed by customary processing. The additive to be used may be an additive that is generally used in the art. Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, liquid glucose, liquid starch, liquid gelatin, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrating agent include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricant include purified talc, salts of stearic acid, borax, and polyethylene glycol. Examples of the flavoring agent include sucrose, orange peel, citric acid, and tartaric acid. A peroral liquid product (e.g., a peroral solution, a syrup, or an elixir) can be prepared by adding, to the cholestanol compound, etc., of the present invention, a flavoring agent, a pH regulator, a stabilizer, a deodorant, or the like, followed by the usual processing. The flavoring agent to be used for this preparation can be any of the aforementioned. Examples of the pH regulator include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic and gelatin. An injection (e.g., a subcutaneous injection, an intramuscular injection or an intravenous injection) can be prepared by adding to the cholestanol compound, etc., of the present invention, a pH adjusting agent, a pH regulator, a stabilizer , an isotonizing agent, a local anesthetic agent, or the like, followed by the usual processing. Examples of the pH adjusting agent and the pH regulator used for this preparation include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the local anesthetic agent include procaine hydrochloride and lidocaine hydrochloride. Examples of the isotonizing agent include sodium chloride and glucose. A suppository can be prepared by adding, to the cholestanol compound, etc., of the present invention, a drug preparation vehicle known in the art, such as polyethylene glycol, lanolin, cocoa butter, or fatty acid triglyceride and, if necessary, a surface active agent such as Tween (registered trademark), followed by customary processing. An ointment is prepared by mixing, through a technique customary, the cholestanol compound, etc., of the present invention, if necessary, with a generally used additive such as a base, a stabilizer, a humectant or a preservative. Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, and propyl p-hydroxybenzoate. A patch can be prepared by applying, to a generally used support, the aforementioned ointment and a cream, a gel, a paste, or the like. Examples of suitable supports include woven fabrics and non-woven fabrics formed of cotton, short fiber, and fibers and chemical films and foam sheets formed of soft vinyl chloride, polyethylene and polyurethane. The dose of the cholestanol compound, etc., of the present invention varies depending on the symptom, body weight, age, sex, etc., of a patient who needs it. The daily dose of the cholestanol compound, etc., for an adult is typically from about 0.01 to about 200 mg / kg, preferably from 0.1 to 50 mg / kg, as reduced to the cholestanol compound (1). Preferably, the daily dose is administered once a day, or in a divided manner (2 to 4 times a day). The present invention will be described below in more detail by way of examples.

EXAMPLES EXAMPLE OF PRODUCTION 1 Production of GIcNAc-cholestanol (1) Production of ethyl-3, 4,6-tri-0-acetyl-2-deoxy-2-phthalimido-1-thio-PD-glucopyranoside (compound (2)) In an argon atmosphere, a solution of 4-methoxyphenyl-3,4,6-tri- 0-acetyl-2-deoxy-2-phthalimido-pD-glucopyranosid (2 g, 4.19 mmol) and ethanethiol (0.40 ml, 5.45 mmol) in sodium chloride. dry methylene (30 ml) was cooled with ice, and boron trifluoride-diethyl ether complex (3.19 ml, 25.14 mmol) was added dropwise to the solution, followed by stirring at room temperature overnight. A saturated aqueous solution of sodium bicarbonate was added to the resulting reaction mixture under cooling with ice, and the resulting organic layer was dried over sodium sulfate. The solvent was removed by evaporation under reduced pressure, and the residue was recrystallized from ethyl acetate-hexane, to thereby give 1.62 g of the title compound. Form: white powder; 1 H-NMR (CDCl 3) d: 1.22 (3 H, t, J = 7.3 Hz), 1.86 (3 H, s), 2.04 (3 H, s), 2.11 (3 H, s), 2.61-2.76 (2 H, m), 3.90 (1 H, ddd, J = 2.4Hz, 4.9Hz, 10.3Hz), 4.18 (1 H, dd, J = 2.2Hz, 12.4Hz), 4.31 (1 H, dd, J = 2.2Hz, 12.4Hz) , 4.39 (1 H, dd, J = 10.3 Hz, 10.3 Hz), 5.18 (1 H, dd, J = 9.2 Hz, 10.3 Hz), 5.49 (1 H, d, J = 10.3 Hz), 5. 83 (1 H, dd, J = 9.2Hz, 10.3Hz), 7.74 (2H, dd, J = 2.4Hz, 5.4Hz), 7.86 (2H, dd, J = 2.4Hz, 5.4Hz). (2) Production of 5-a-cholestan-3β-yl-2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D-qlucopyranoside (compound (3)) In an argon atmosphere, Dry ethylene chloride (4.6 ml) was added to a mixture of compound (2) obtained in (1) above (400 mg, 0.834 mmol), 5-a-cholestan-3ß-ol (389 mg, 1.0 mmol), and Activated molecular sieves 4A (1.88 g), followed by stirring at room temperature for one hour. Subsequently, methyl trifluoromethanesulfonate (0.28 ml, 2.48 mmol) was added to the resulting mixture, followed by stirring overnight. Triethylamine (1 ml) was added to the resulting reaction mixture, followed by stirring for 30 min. Subsequently, the resulting mixture was subjected to celite filtration, and the filtrate was washed with methylene chloride. The solvent was removed by evaporation of a mixture of the filtrate and the washing liquid, and the residue was purified by means of column chromatography on silica gel (hexa no: ethyl acetate = 3: 1 (by volume), the same will be applied later), to thereby give 510 mg of the title compound. Form: white powder; 1 H-NMR (CDCl 3) d: 0.51-2.10 (55H, m), 3.48-3.56 (1 H, m), 3.85 (1 H, ddd, J = 2.4Hz, 4.9Hz, 10.3Hz), 4.15 (1 H , dd, J = 2.2Hz, 12.2Hz), 4.26-4.34 (3H, m), 5.15 (1H, dd, J = 8.9Hz, 10.0Hz), 5.46 (1H, d, J = 8.4Hz), 5.77 (1 H, dd, J = 8.9Hz, 10.8Hz), 7.74 (2H, dd, J = 2.4Hz, 5.4Hz), 7.86 (2H, dd, J = 2.4Hz, 5.4Hz). (3) Production of 5-a-cholestan-3β-yl-2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-qlucopyranoside (compound (4)) Ethylenediamine (5.0 ml ) was added to a suspension of the compound (3) obtained in (2) above (510 mg, 0.633 mmol) in 1-butanol (5.0 ml), and the resulting mixture was stirred at 120 ° C overnight. The solvent was removed from the reaction mixture by evaporation under reduced pressure, and the residue was mixed with pyridine (5 ml), acetic anhydride (5 ml), and 4-dimethylaminopyridine (catalytic amount), followed by stirring during the night. The solvent was removed from the reaction mixture by evaporation under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was washed with 1 M aqueous solution of hydrochloric acid, a saturated aqueous solution of sodium bicarbonate, and a saturated aqueous saline solution, and then the resulting organic layer was dried over sodium sulfate. The solvent was removed by evaporation under reduced pressure, and the residue was purified by column chromatography on silica gel (hexane: ethyl acetate = 1: 4), to thereby give 250 mg of the title compound. Form: white powder; 1 H-NMR (CDCl 3) d: 0.55-2.07 (58H, m), 3.52-3.72 (3H, m), 4.10 (1H, dd, J = 2.2Hz, 11.3Hz), 4.26 (1H, dd, J = 2.2 Hz, 12.2Hz), 4.86 (1 H, d, J = 8.4Hz), 5.03 (1 H, dd, J = 9.7Hz, 9.7Hz), 5. 35-5.43 (2H, m). (4) Production of 5-a-cholestan-3ß-yl-2-acetamido-2-deoxy-β-D-chloropyranoside (compound (Ia)) A solution of sodium methoxide-28% methanol (0.07 ml, 0. 363 mmole) was added to a solution of compound (4) obtained in (3) above (250 mg, 0.348 mmol) in a mixture of methanol (5.0 ml) and methylene chloride (5.0 ml), followed by stirring at room temperature overnight. The resulting reaction mixture was neutralized with Amberlyst 15, and then the reaction mixture was subjected to filtration. The solvent was removed from the filtrate through evaporation under reduced pressure, and the residue was suspended in methanol. The insoluble materials obtained through filtration were washed with methanol, and then dried, to thereby give 159 mg of the title compound. Form: 1H-NMR white powder (DMSO-d6 + D20) d: 0.62-1.94 (49H, m), 2.99-3.05 (2H, m), 3.22-3.50 (4H, m), 3.66 (1H, d, J = 11.6Hz), 4.40 (1H, d, J = 7.6Hz), 7.64 (1H, d, J = 8 .4Hz).

EXAMPLE 1 Production of liposomal formulation and cyclodextrin inclusion complex A solution of 20 μmol / ml each of GIcNAc-cholestanol (compound (la)) and cholestanol (dissolved in chloroform / methanol = 5/1 (v / v)) was used as a starting material. (1) Preparation of liposomal formulation 1a-dipalmitoylphosphatidylcolin, stearylamine, and sugar-cholestanol were mixed in proportions of 52/8/20 (per mole) to obtain a total amount of 700 μl, and subsequently a solvent organic (chloroform / methanol = 2/1 (V / V) (300 μl) and distilled water (1 ml) were added to and mixed with the resulting mixture, then the organic solvent was completely removed by means of a rotary evaporator. , and the resulting product was subjected to freeze drying to completely remove the water.The freeze-dried product was dissolved in PBS (1 ml), followed by ultrasonic treatment (15 W, 15 minutes), to form liposomes that they had a uniform particle size of about 40 to about 300 nm The liposomes thus formed were used in Example 2. (2) Preparation of cyclodextrin inclusion complex A 20% aqueous solution of hydroxypropyl-β-cyclodextrin was prepared, and GIcNAc-cholestanol was added to the solution, followed by stirring / mixing, to thereby form a cyclodextrin inclusion complex. with GIcNAc-cholestanol. The formation of a good inclusion complex is confirmed by observing that GIcNAc-cholestanol (ie, an insoluble compound) is solubilized by stirring.

EXAMPLE 2 Inhibitory effect of cell proliferation of GIcNAc-cholestanol A line of cultured cancer cells (HT-29 (human)) was inoculated into a 96-well plate (1 x 10 4 cells / 100 μl / well), and subsequently the liposomal formulation containing GIcNAc-cholestanol compound or inclusion complex The cyclodextrin prepared in Example 1 was added to the 96-well plate, followed by incubation at 37 ° C for three days. Subsequently, the MTT test was performed, and the number of cells was determined. The percent inhibition of proliferation was obtained by using the formula described below. The results are shown in table 1. As a result, GIcNAc-cholestanol showed an inhibitory effect of strong cell proliferation.

Percent inhibition of cell proliferation (percent CPI) (%) = (1-DO of treated cells / OD of untreated cells) x 100 The OD of treated cells and the OD of untreated cells were measured at 450 nm and 650 nm, respectively (in accordance with the MTT test).

TABLE 1 Cell inhibiting effect of sugar-cholestanol 1) GIcNAcGalChol: GIcNAcßl, 3Galß-cholestanol GIcNAc Chol: GIcNAcß-cholestanol GalChol: Galß-cholestanol Chol: cholestanol 2) Concentration required to achieve 50% of CPI

Claims (5)

NOVELTY OF THE INVENTION CLAIMS

1. An anti-cancer agent containing, as an active ingredient, a cholestanol compound represented by the following formula (1): or a cyclodextrin inclusion complex containing the compound.

2. A liposomal formulation containing a cholestanol compound according to claim 1. 3.- A cyclodextrin inclusion complex containing a cholestanol compound according to claim 1. 4.- The inclusion inclusion complex with claim 3, further characterized in that the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin. 5. The use of a cholestanol compound represented by the following formula (1): or a cyclodextrin inclusion complex containing the compound for the manufacture of a medicament useful for preventing or treating cancer.