JPS6132319B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the following general formula [] [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different and represent hydrogen or an acetyl group, R ⁵ and R ⁶ represent hydrogen or a methyl group, and X is

【Formula】Also teeth

Represents [formula]. However, R ⁵ and R ⁶ are not the same. ] Cholesteryl pyranoside derivative, 20(s)-cholesteryl pyranoside derivative, 27-nor-25-ketocholesteryl pyranoside derivative, or 20(s)-27-nor-25-ketocholesteryl pyranoside The present invention relates to derivatives and pharmaceuticals having immunosuppressive and anti-inflammatory effects thereof. Among the compounds represented by the above general formula [], examples of compounds that are preferably used include 25-hydroxycholesteryl-β-D-glucopyranoside derivatives and 20(s)-25-hydroxycholesteryl β-
It is a D-glucopyranoside derivative. All of these compounds are new substances, and the present invention is based on the discovery that these compounds have immunosuppressive and anti-inflammatory effects. Compounds having a steroid skeleton have various biological activities, and many of them are used as medicines. Among these, cholesterol is a very important compound that exists widely and in sufficient quantities in living organisms, and is being elucidated to play an important role in the structure and function of biological membranes. In addition, various steroid hormones and bile acids derived from cholesterol are also important substances for living organisms. Recently, there have been many very interesting reports regarding the biological activities of compounds in which steroids are oxidized at specific positions. 7-ketocholesterol and 7-hydroxycholesterol have immunosuppressive effects, and 7-β-hydroxycholesterol, 7-β-hydroxycampesterol, and ergosterol peroxide have a concentration that does not affect normal cells, which inhibits the proliferation of immune cells. There is a statement that an inhibitory effect was observed (J.Chem.
Research(S), 1977, 217; ibid, 1977, 218;
ibid, 1977, 219; ibid, 1979, 84-85). Furthermore, in research on the cholesterol biosynthesis mechanism, it was discovered that cholesterol metabolites inhibit cholesterol biosynthesis through a feedback mechanism. Furthermore, since it is known that cholesterol biosynthesis in various tissues is involved in cell division, the application of these cholesterol metabolites to cancer therapy is expected. However, the metabolic intermediates of cholesterol themselves are very easily metabolized, so their effects in vivo cannot be expected. In order to improve this point, attempts have been made to esterify the hydroxyl group at the 3-position, but satisfactory results have not yet been obtained (Science Vol. 201, No. 4355, 498-
501 (1978)). Various compounds having a steroid skeleton are used as medicines, but they generally have strong side effects. For example, corticosteroid hormone preparations are widely used as immunosuppressants and anti-inflammatory agents, but their use is severely limited due to the side effects specific to corticosteroid preparations, such as moon-like facial appearance, irritability, euphoria, and hypertension. There is a desire to reduce the side effects. The present inventors have discovered that sterol glycosides and their derivatives have vascular reinforcing and hemostatic effects. At this time, it has been discovered that there are two phases in the expression of physiological activities of these sterol glycosides.
That is, the drug exhibits strong physiological activity several hours after administration, and then the activity decreases, but strong activity reappears several tens of hours later. This fact can be interpreted to mean that sterol glycosides are metabolized in vivo, metabolites accumulate, and become active again after several tens of hours. The present inventors paid attention to the above-mentioned facts and worked diligently to find a compound that retains high physiological activity, does not have the side effects inherent to adrenal steroid hormones, is chemically and physically stable, and is resistant to catabolism in the body. We conducted research and found that cholesterol oxidized at the 25th position as aglycone, its 20s isomer, and steryl pyranosides containing 27-nor-25-ketocholesterol have strong immunosuppressive and anti-inflammatory effects. The present invention has been completed by discovering that the compound has the following properties, has low toxicity, and is not susceptible to metabolism. Compounds having immunosuppressive and anti-inflammatory effects of the present invention include cholesteryl pyranoside derivatives, 20(s)-cholesteryl pyranoside derivatives, and 27-nor-25-ketocholesteryl pyranoside derivatives represented by the general formula []. or 20(s)-27-nor-25-ketocholesteryl pyranoside derivatives. Eight types of compounds shown in Table 1 are exemplified as compounds represented by the general formula []. These compounds were synthesized according to the routes described below.

【table】 Compounds represented by formula []a or []b, steryl-2', 3', 4', 6'-tetra-O-acetyl-β-D-glucopyranosides (5), (6), (7), (8 ) are the so-called 3-hydroxysterols (1), (2), (3), (4) represented by the corresponding formula []a or []b, respectively, which are reacted with acetobromoglucoside.
Produced by Konigs-Knorr reaction. 3-Hydroxysterols represented by the formula []a or []b are known compounds that can be used in various ways, for example (JCS chem: Comm, 1974, 15; G.
P.2415676.Biochemistry, 1969, 8, 671.Helv.
chem.Acta, 1974, 57, 764.ibid, 1974, 57,
771.), but as a raw material for the present invention, Helv.chem.Acta, 1974, 57, 771
Manufactured according to the method. The reaction is carried out by dissolving the compound of formula []a or []b in an organic solvent (e.g. methylene chloride, chloroform, ethylene dichloride, ether or a mixture thereof), adding freshly prepared silver oxide AgO, a dehydrating agent (e.g. hydrogen This is easily carried out by dropping a solution of bromoacetoglucoside in an organic solvent (the above-mentioned solvent) under stirring in the presence of a small amount of iodine, preferably some iodine, and preferably in the absence of light. Compound steryl-2′ of []a or []b,
3', 4', 6'-tetra-O-acetyl-β-D-glucopyranosides (5), (6), (7), and (8) can be obtained. Compound steryl-β-D-glucopyranosides (9) represented by formula []a or []b,
(10), (11), and (12) are the corresponding expressions []a or []
Steryl-2', 3', 4', 6'-tetra-O-acetyl-β-D-glucopyranosides represented by b
Produced by hydrolyzing (5), (6), (7), and (8). The reaction is carried out by dissolving the compound of formula []a or []b in an organic solvent (e.g. methanol, ethanol, dioxane or a mixture thereof) and adding an alkali (e.g. sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate). The compound steryl-β-D- of the formula []a or []b can be easily added as a solid or by adding it dissolved in the above solvent or water, preferably by stirring at room temperature.
Glucosides (9), (10), (11), and (12) can be obtained. Compounds (5) and (6) represented by formula [a] are:
Compounds (7) and (8) respectively represented by the corresponding formula []b
is produced by the Grignard reaction by reacting with methylmagnesium halide. In the reaction, the compound of formula []b is dissolved in an organic solvent (e.g. ether,
Compound (5) of formula []a, ( 6) You can get it. Compounds of general formulas []a and []b exhibit immunosuppressive effects at very low concentrations and also exhibit anti-inflammatory effects. The pharmacologically effective amounts of the compounds of general formulas []a and []b are shown below. Effect on delayed-type hypersensitivity reaction in mice (inhibitory effect). Experimental method a. Animals used: SPF-ICR, male, 14 weeks old. b. Sheep red blood cells (SRBC): Less than 1 month after blood collection from the Japan Biomaterials Center. c Control drug: cortisone acetate (Tokyo Kasei) d Preparation method: In vitro for each drug (15 mm x
105mm) with polyoxyl stearate 40, and 0.25mg/Kg in 0.5% CMC solution, 1
mg/Kg, adjusted to 4 mg/Kg. The dose was 0.2 ml per mouse. e DTH: SRBC 1.5×10 ⁵ cells/mouse/
The animals were immunized intravenously with 0.25 ml, and each drug was orally administered once at the same time. 0.2 ml of 0.5% CMC solution of polyoxyl stearate 40 was administered to the control. 4 days after sensitization, intracutaneously into the left foot pad of the mouse.
Inoculate SRBC1×10 ⁸ cells/40ml, induce a reaction, and measure the thickness of the left and right footpads 24 hours later.
The difference in thickness between the left and right sides was taken as the reaction value. The results are shown in Table 2.

[Table] As described above, the compound of the present invention exhibited a strong immunosuppressive effect at low doses. EXAMPLES The production of the compounds of the present invention will be described below with reference to Examples, but the present invention is not limited thereto. Example 1 (27-nor-25-ketocholesteryl) 2', 3',
Production of 4',6'-tetra-O-acetyl-β-D-glucopyranoside (7) 27-nor-25-ketocholesterol (3) 3.0
Dissolve g in 100 ml of dry methylene chloride and 100 ml of dry ethylene dichloride, and add 15.0 g of freshly prepared silver oxide, 2.0 g of calcium hydride, and 0.1 g of iodine to this solution while shielding from light. Then, while stirring at room temperature, a solution of 15 g of bromoacetyl glucoside dissolved in 50 ml of dry methylene chloride was added dropwise over a period of 1 hour. When about half the amount is dropped, 5.0 g of silver oxide and 0.1 g of iodine are further added to the reaction solution. Stir at room temperature for 10 hours and then filter. Add 1.0g of activated carbon to the liquid and add 10
Stir for a minute and then strain. The solvent is distilled off from the solution under reduced pressure in a nitrogen stream. The residue was separated by silica gel column chromatography, and 6.0 g of the crude title compound was obtained from the fraction eluted with a mixed solvent of benzene and ether (1:1). Recrystallization from ethanol gives the title compound 4.9 as colorless needle-shaped crystals with a melting point of 156.4°C.
get g. Yield is 87.1%. Physical properties 1 Melting point 156.4℃ 2 Infrared absorption spectrum ν ^KBr _nax cm ^-1 1755
，
1715 3 Mass spectrometry m/e: 716 (M ⁺ ) 4 Light intensity [α] ²⁴ _D : -27.3° (Chloroform:
C=0.8781) 5 Elemental analysis value [C ₄₀ H ₆₀ O ₁₁ ] C% H% Calculated value 67.02 8.44 Actual value 66.93 8.69 Example 2 (25-hydroxycholesteryl)-2', 3',
Production of 4',6'-tetra-O-acetyl-β-D-glucopyranoside (5) (27-nor-25-ketocholesteryl)-2',
Ether obtained by dehydrating 3.1 g of 3', 4', 6'-tetra-O-acetyl-β-D-glucopyranoside (7)
Dissolve in 200 ml and 50 ml of dehydrated tetrahydrofuran. Under a nitrogen stream at room temperature, a solution prepared by diluting 5 ml of an ether solution of methylmagnesium iodide (containing about 2 moles per 1 mol) with 30 ml of ether is added dropwise. Stir the reaction at room temperature for 1.5 hours. A saturated aqueous solution of sodium sulfate is then added to destroy the excess Grignard reagent. After adding anhydrous sodium sulfate, filter and wash the solid with 100 ml of chloroform. The organic solvent portions were combined and the solvent was distilled off under reduced pressure in a nitrogen stream to obtain 2.5 g of a viscous oily substance.
Purified by silica gel column chromatography,
Further recrystallization from acetone and n-hexane yields 1.7 g of the title compound as colorless needle-like crystals. Yield is 47.3%. Physical properties 1 Melting point 185.8℃ 2 Infrared absorption spectrum ν ^KBr _nax cm ^-1 3515,
1760 3 Mass spectrometry m/e: 732 (M ⁺ ) 4 Light intensity [α] ²⁵ _D : -25.5° (Chloroform:
C=0.5245) 5 Elemental analysis value [ _{C41H64O11 ]} C% H% Calculated value 67.19 8.80 Actual value 67.15 9.12 Example 3 25- _{Hydroxycholesteryl} -β-D-glucopyranoside (9) (25-Hydroxycholesteryl) −2′, 3′,
Dissolve 775 mg of 4',6'-tetra-O-acetyl-β-D-glucopyranoside (5) in 5 ml of dioxane and 50 ml of methanol, add a solution of 700 mg of anhydrous potassium carbonate in 4 ml of water, and stir at room temperature for 1.5 hours. do. The reaction solution was poured into ice water 2, and the resulting precipitate was removed to obtain 550 mg of colorless crude crystals. Recrystallization from dioxane gives 520 mg of the title compound as a colorless crystalline powder. Yield is 86.6%. Physical properties 1 Melting point 274.0°C (color decomposition) 2 Infrared absorption spectrum ν ^KBr _nax cm ^-1 3390 3 Mass spectrometry m/e: 545 (M ⁺ -18) 4 Light intensity [α] ²⁵ _D : -48.3° (pyridine: C=
1.0100) 5 Nuclear magnetic resonance spectrum δ (C ₅ D ₅ N): 0.67
(s, 3H) 0.95 (s, 3H) 0.97 (d, 3H)
1.42 (s, 6H) 5.35 (m, 2H) 6 Elemental analysis value [C ₃₃ H ₅₆ O ₇ ] C% H% Calculated value 70.17 9.99 Actual value 69.95 10.18 Example 4 20(s)-25-Hydroxycholesteryl-
2', 3', 4', 6'-tetra-O-acetyl-β-
Production of D-glucopyranoside (6) 1.5 g of 20(s)-25-hydroxycholesterol (2) was dissolved in 100 ml of dry methylene chloride and 100 ml of dry ether, and 3.0 g of freshly prepared silver oxide and 3.0 g of freshly prepared silver oxide were added to this solution in the dark. Add 500 mg of calcium hydride and 100 mg of iodine. Then, while stirring at room temperature, a solution of 12.0 g of bromoacetyl glucoside dissolved in 40 ml of dry methylene chloride was added dropwise over 30 minutes. When about half the amount is dropped, 3.0 g of silver oxide is further added to the reaction solution. Stir overnight at room temperature and then strain.
Add 500 mg of activated carbon to the liquid, stir for 10 minutes, and strain. The solvent is distilled off under reduced pressure in a nitrogen stream, and the resulting oily substance is purified by silica gel column chromatography to obtain 1.47 g of crystals. Yield is 53.8%. Physical properties 1 Melting point 165.0℃ 2 Infrared absorption spectrum ν ^KBr _nax cm ^-1 3500
1760 3 Mass spectrometry m/e: 732 (M ⁺ ) 4 Nuclear magnetic resonance spectrum δ (CDCl ₈ ): 0.69
(s, 3H) 0.82 (d, 3H, J=7Hz), 0.99
(s, 3H), 1.21 (s, 6H) 1.95, 1.98, 2.01,
2.03 [12H (s, 3H x 3)] 5.30 (m, 1H) 5 Light intensity [α] ²⁴ _D -36.1° (chloroform, C
=0.555%) 6 Elemental analysis value [C ₄₁ H ₆₄ O ₁₁ ] C% H% Calculated value 67.19 8.80 Actual value 67.18 8.84 Example 5 20(s)-25-hydroxycholesteryl-β
-Production of D-darcopyranoside (10) 20(s)-25-hydroxycholesteryl-
2', 3', 4', 6'-tetra-O-acetyl-β-D
- Dissolve 750 mg of glucopyranoside (6) in 6 ml of dioxane and 40 ml of methanol, add a solution of 624 mg of anhydrous potassium carbonate in 4 ml of water, and stir at room temperature for 30 minutes. The reaction solution was poured into ice water, and the resulting precipitate was removed and thoroughly washed with water to obtain 450 mg of crude crystals. Recrystallization from dioxane yields 390 mg of the title compound as a colorless crystalline powder. Yield is 67.1%. Physical properties 1 Melting point 255℃ (color decomposition) 2 Infrared absorption spectrum ν ^KBr _nax cm ^-1 3400 3 Mass spectrometry 546 (M ⁺ -18) 4 Nuclear magnetic resonance spectrum δ (C ₅ D ₅ N): 0.67
(s, 3H) 0.85-1.00 (broad, 6H), 1.42
(s, 6H), 5.45 (m, 1H) 5 Light intensity [α] ²⁵ _D -55.1° (pyridine, C=
0.558%) 6 Elemental analysis value [C ₃₃ H ₅₆ O ₇ ] C% H% Calculated value 70.17 9.99 Actual value 70.10 10.22 Example 6 25-Hydroxycholesteryl-2', 3', 4',
Production of 6'-tetra-O-acetyl-β-D-glucopyranoside (5) 25-hydroxycholesterol (1) 4.0g
was dissolved in 200 ml of dry methylene chloride, and 8.0 g of freshly prepared silver oxide, 2.0 g of calcium hydride, and 200 mg of iodine were added to this solution while shielded from light. Then, while stirring at room temperature, a solution of 15 g of bromoacetyl glucoside dissolved in 100 ml of dry methylene chloride was added dropwise over 30 minutes. When about half the amount is dropped, 4.0 g of silver oxide is further added to the reaction solution. Thereafter, in the same manner as in Example 4, 3.1 g of the title compound was obtained. Yield is 42.6%. The physical properties were the same as in Example 2.

Claims (1)

[Claims] First-order general formula [] [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different and represent hydrogen or an acetyl group, R ⁵ and R ⁶ represent hydrogen or a methyl group, and X is [Formula] or [Formula] represent However, R ⁵ and R ⁶ are not the same. ] A compound represented by 2nd order general formula [] [In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different and represent hydrogen or an acetyl group, R ⁵ and R ⁶ represent hydrogen or a methyl group, and X is [Formula] or [Formula] represent However, R ⁵ and R ⁶ are not the same. ] An immunosuppressant whose main ingredient is a compound represented by 3. The immunosuppressant according to claim 2, wherein the immunosuppressant is an anti-inflammatory agent.