JPS6250478B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel α,α-trehalose-6,
This invention relates to a method for producing 6′-fatty acid diester. α,α-trehalose is a type of so-called disaccharide composed of two molecules of glucose, and is a compound widely distributed in nature. Various derivatives of this compound have also been synthesized and their pharmacological activities are being investigated. For example, it has been reported that mixtures of various diesters with different fatty acid bonding positions have antitumor effects. The present inventors have demonstrated that α,α-trehalose 6,
Various studies were conducted on 6'-fatty acid diesters, and the known α,α-trehalose-6,
It has been concluded that the method for producing 6'-fatty acid diesters is not a satisfactory method for producing the substance advantageously. That is, conventional α,α-trehalose-6,
For example, 6'-fatty acid diester (); (hereinafter simply referred to as "trehalose fatty acid diester") was produced by the following method (Carbohydrate
Research, 44 , 308 (1975)). (In the formula, R′CO represents an acyl group, and TMS represents a trimethylsilyl group.) However, this method has a low yield in each step, resulting in a poor overall yield, from (′) to (′). It is difficult to selectively convert to
Since it uses a TMS-forming agent, it has disadvantages such as being economically disadvantageous, and it is difficult to adopt it as an industrial production method aimed at mass synthesis. Therefore, the present inventors conducted intensive studies on a method for producing trehalose fatty acid diester that can be used industrially, and found that α,α-trehalose (2,3,2′,3′-tetra-O- benzyl-α,α-trehalose () and then reacted with fatty acid () or its active derivative to form a novel intermediate 2,3,2′,3′-tetra-O-benzyl-α,α- The inventors discovered that trehalose fatty acid diester (2) can be advantageously obtained by obtaining trehalose-6,6'-fatty acid diester (2) and further reducing and debenzylating it using a reduction catalyst, and completed the present invention. The present invention is shown by the following reaction formula. (In the formula, RCO represents a fatty acid residue having 30 or less carbon atoms, and Bn represents a benzyl group.) In the method of the present invention, 2,3,2',3'-tetra-O-benzyl represented by the formula () ―α、α
-Trehalose is produced by reacting benzaldehyde with trehalose represented by the formula () using a conventional method to produce 4,6,4',6'-di-O-benzylidene-
It can be obtained by converting α,α-trehalose into a 2,3,2',3'-tetra-O-benzyl form with benzyl chloride, and then reacting with an acid. In addition, the reaction between the compound () and the fatty acid () or its active derivative follows the general esterification reaction conditions, usually in the presence of a deoxidizing agent such as pyridine or triethylamine, and for 1 mol of the compound (),
This is preferably carried out by reacting fatty acids () or active derivatives thereof in a proportion of 1.8 to 2.4 mol. This reaction is preferably carried out at ~30° C. for 1 to 24 hours under ice cooling in the presence or absence of a solvent. Examples of the solvent used here include methylene chloride, chloroform, benzene, toluene, ether, tetrahydrofuran, and dioxane. The reductive debenzyl reaction of the compound () thus obtained is carried out by catalytic reduction using preferably 0.5 to 10 moles of a reduction catalyst per 1 mole of the compound () in an appropriate solvent. Examples of the reduction catalyst include palladium black, palladium-carbon, and Raney nickel. Also,
Examples of the solvent used here include alcohols such as methanol, ethanol, and isopropanol; halides such as chloroform and methylene chloride; and mixed solvents thereof. The manufacturing method of the present invention comprises the formula (), This method takes advantage of the fact that the hydroxyl groups of α,α-trehalose, represented by , have different reactivity toward esterification. That is, in the present invention, the compound ( ) is symmetrical, and the reactivity of each hydroxyl group is 6,
Since the order is 6'≫2, 2'>3, 3'>4, 4', 2,
If the esterification reaction is carried out by protecting the hydroxyl groups at the 2' and 3,3' positions, the 4,4' positions, which have the lowest reactivity, will not be esterified, and only the 6,6' positions will be regioselectively esterified. This is based on the knowledge that only the desired 6,6'-diester can be obtained by removing the protecting groups at the 2, 3, 2', and 3' positions. Therefore, according to the method of the present invention, α,α-trehalose-6,
6'-fatty acid diesters can be easily obtained in high yield and purity. Next, the pharmacological action of trehalose fatty acid diester () obtained by the method of the present invention will be shown. (1) Cell-killing effect of trehalose diester derivatives: To examine the cell-killing effect of trehalose diester derivatives, Lyukemia L-5178Y cells were used and incubated at 10 ⁵ in RPMI1640 medium containing 10% fetal bovine serum.
The sample was added to this medium at a predetermined concentration. After culturing in a 5% CO ₂ incubator at 37°C for 48 hours, the number of viable cells was counted under a microscope, the proliferation rate was determined by comparing it with the control group, and the resulting straight line was plotted on log probability paper. The IC ₅₀ value was determined. Sample concentrations are 25, 50, 100, 200 and 400μ
g/ml. The results are shown in Table 1.

【table】

[Table] (2) Activation effect on reticuloendothelial system function: The experimental method was based on the so-called carbon clearance method to measure the activation effect on the phagocytic ability of reticuloendothelial cells. A predetermined amount of the test compound suspended in sterile physiological saline was intraperitoneally administered to 1 group of 10 ddY male mice, 4 weeks old and weighing 20 to 25 g. Zymosan was administered in the same manner as the standard drug, and sterile physiological saline alone was administered in the same manner to the control group. 24 hours after administration of the test compound, 0.1 ml/10 g of Pelican ink (C ₁₁ /1431a) prepared at a concentration of 1.6 mg/0.1 ml of carbon was injected into the tail vein of the mouse, and immediately pipetted into one orbital venous plexus. Using
0.02 ml of blood was collected and added to 2.5 ml of 0.1% sodium carbonate solution for hemolysis. After exactly 4 minutes, 0.02 ml of blood was collected from the other orbital venous plexus and subjected to the same procedure, and the absorbance at a wavelength of 600 nm was measured. /Z)
were calculated and shown in Table 2. Phagocytic index K value: K=log C ₁ -log C ₂ /T ₂ -T ₁ However, C ₁ is the absorbance immediately after carbon ink administration, C ₂ is after 4 minutes, T ₂ - T ₁ = 4 (min). Relative activity (T/Z): Relative activity (T/Z) to zymosan administration group
%) =K _T -K _C /K _Z -K _C ×100% where K _T is the average K value of the test compound administration group, K _Z is zymosan, and K _C is the average K value of the control group.

[Table] (3) Anticancer effect of trehalose fatty acid diester: Six 5-week-old std-ddY male mice were used in each group, and 5 x 10 ⁶ Sarcoma 180 cancer cells were intraperitoneally implanted into each mouse. did. 30% after 24 hours
Test compound suspended in propylene glycol
It was administered intraperitoneally once a day for 5 consecutive days at a dose of 150 mg/Kg. Ascites was collected on the 7th day after cancer cell transplantation.
Ascites volume and cell ratio were determined. Tumor growth rate (T/C
(%) was calculated from the following formula using TPCV (total cell volume), which is the product of ascites volume and cell ratio, to determine the anticancer activity of the test compound. The results are shown in Table 3. T/C (%) = TPCV of treated group/TPCV of control group
×100

[Table] As described above, trehalose fatty acid diester has anticancer effects and reticuloendothelial system function activation effects. The toxicity of the trehalose fatty acid diester produced by the method of the present invention is such that similar substances such as sucrose fatty acid esters are designated as food additives and are widely used as harmless surfactants in foods, medicines, cosmetics, etc. As is clear from the above, it is a safe compound with almost no toxicity. In addition, when this is used as a pharmaceutical, for example, an immunostimulant, various dosage forms may be used, such as tablets, capsules, powders, etc., depending on the mode of administration such as oral or parenteral administration.
Orally administered preparations such as granules and liquid preparations; parenteral administration preparations such as subcutaneous, intramuscular or intravenous injections, preparations for mixing with circular fluid, or suppositories. The above formulation can be performed by a method known per se. That is, trehalose fatty acid diester is combined with excipients such as starch, lactose, and mannitol; binders such as sodium carboxymethyl cellulose and hydroxypropyl cellulose; disintegrants such as crystalline cellulose and calcium carboxymethyl cellulose; lubricants such as talc and magnesium stearate. Tablets, capsules, powders, or granules can be produced by appropriately combining and formulating fluidity improvers such as light anhydrous silicic acid.
In addition, solutions and injections can be produced by dissolving trehalose fatty acid diester in vegetable oil etc. to make an oil-based injection, or by dissolving or suspending it in water etc. using a conventional method to make syrups etc. can. Furthermore, for making suppositories, commonly used bases,
For example, it can be produced by dispersing it in cacao butter, synthetic oil, etc. by a conventional method and then solidifying it. The dosage of trehalose fatty acid diester according to the method of the present invention varies depending on the severity of the disease, but it is usually 0.1 to 0.1 when administered orally to adults.
2000mg/Kg, 0.05-1000mg/for parenteral administration
It is preferable to administer Kg once a day or in divided doses. Next, the present invention will be explained with reference to Examples and Reference Examples. Example 1 2,3,2',3'-tetra-O-benzyl-α,
Add 20 ml of pyridine and 20 ml of dichloromethane to 2.0 g of α-trehalose, dissolve, and stir at 0°C under ice cooling to 1.8 g of stearoyl chloride.
was added dropwise. After stirring for 30 minutes, the mixture was further stirred at room temperature for 1 hour, and the reaction mixture was poured into ice water and extracted with chloroform. Chloroform was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 2,3,2',3'-tetra-O-benzyl-6,6'-di-O-stearoyl-α,α - Obtained 2.9g of trehalose. Colorless waxy substance. Yield 83
%. [α] ²⁵ _D +58.8° (C=1.0, chloroform) IRν ^KBr _nax cm ^-1 1740 NMR (60MHz, heavy chloroform) δppm; 0.88 ^t (6H, J=5Hz), 1.23 ^m (60H), 2.0−
2.5 ^m (4H), 3.3−4.3 ^m (12H), 5.14 ^d (2H,
J=3.5Hz), 4.66 ^s (4H), 4.86 ^d (4H), 7.0
-7.4 _n (20H) Example 2 2,3,2',3'-tetra-O-benzyl-6,
1.24 g of 6'-di-O-stearoyl-α,α-trehalose was dissolved in 20 ml of chloroform, 0.5 g of palladium black was added as a catalyst, and hydrogen gas was introduced for catalytic reduction for 1 hour. The reaction solution was filtered to remove the catalyst, and the solution was concentrated to dryness under reduced pressure. The residue was recrystallized from isopropanol to give 6,6'-
0.55 g of colorless needle crystals of di-O-stearoyl-α,α-trehalose were obtained. Yield 63%. Melting point 157-160℃ [α] ²⁰ _D ; +80.8゜ (C=1.0, chloroform) IRν ^KBr _nax cm ^-1 ; 1735 (>C=O) NMR (100MHz, d ₅ -pyridine) δppm; 0.88 ^t ( 6H, J=6.5Hz) 1.1-1.8 ^m (60H) 2.2-2.4 ^t (4H, J=7Hz) 4.0-2.5 ^m (4H) 4.5-5.2 ^m (8H) 5.84 ^d (2H, J=3.5Hz) Implementation The reaction was carried out according to Example 1 to obtain the compounds shown in Table 4.

[Table] The debenzylation reaction was carried out in the same manner as in Example 2.
The compounds of the present invention shown in the table were obtained.

[Table] Reference Example 1 Tablet One tablet with the following ingredients and amounts was manufactured by a conventional method. Trehalose fatty acid diester (behenate)
100mg D-mannitol 150mg Crystalline cellulose 50mg Starch 28mg Carboxymethyl cellulose calcium 16mg Talc 4mg Magnesium stearate 2mg Total amount 350mg Reference example 2 Capsules The following ingredients were prepared using a conventional method. Produce a quantity of granules,
This was filled into one No. 3 capsule. Trehalose fatty acid diester (palmitate) 25 mg Crystalline cellulose 17 mg Light anhydrous silicic acid 7 mg Magnesium stearate 1 mg Reference Example 3 Injection One injection was manufactured from the following ingredients and quantities by a conventional method. Trehalose fatty acid diester (stearate) 25mg Add olive oil to make total volume 1ml. Reference Example 4 Suppositories After melting and stirring the following ingredients and amounts using the usual method,
The mixture was molded and solidified to produce one suppository. Trehalose fatty acid diester (myristate) 100mg Cocoa butter 1100mg Total amount 1200mg

Claims (1)

[Claims] 1 2,3,2',3'-tetra-O-benzyl-
By reacting α,α-trehalose with a fatty acid represented by the following formula (), RCOOH () (in the formula, RCO represents a fatty acid residue having 30 or less carbon atoms) or an active derivative thereof, the formula (), (In the formula, RCO has the same meaning as above and Bn represents a benzyl group) 2,3,2',3'-tetra-O-benzyl-α,α-trehalose-6,6' - formula (), characterized in that the fatty acid diester is removed and then the benzyl group is removed; (In the formula, RCO is the same as above.) A method for producing α,α-trehalose-6,6′-fatty acid diester represented by: