JPS6360749B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to C-benzyl-β-D-xylopyranoside, and more specifically, it has the property of changing the quality and quantity of complex carbohydrates (proteoglycans) present on the surface of cell membranes, and has anticancer effects. , C-benzyl-, which is expected to have anti-arteriosclerotic effects, anti-thrombotic effects, etc.
It relates to β-D-xylopyranoside.

Conventionally, O-β-D-xylopyranoside compounds have been used to modify the properties of certain cell membrane surfaces by changing the amount of so-called proteoglycans, which exist on the surface of cell membranes or between cells and are important constituents of living tissues. [Journal of Biochemistry (J.
Biochem.), 74 , 1069-1073 (1973)].

Taking cancer cells as an example, this property shows that O-β-D
- Xylopyranoside compounds change the properties of proteoglycans on the surface of cancer cells, reducing their amount and rendering cancer cells naked, thereby increasing the body's immunity to cancer cells, thereby preventing carcinogenesis. It is fully expected to enhance the therapeutic effect of cancer cell immunity. However, O-β-D-xylopyranoside compounds are easily hydrolyzed by enzymes, and for example, when administered into the human body for the purpose of anticancer effects, most of them are degraded before they exhibit their effects. It becomes useless. Therefore, the present inventors discovered a C-β-D-xylopyranoside compound that is resistant to enzymatic hydrolysis and can change the quality and quantity of proteoglycans on the cell surface, and completed the present invention. It has arrived.

An object of the present invention is to provide novel C-β-D-xylopyranoside compounds.

The present invention, namely, the following formula (): [In the formula, R represents a benzyl group. ] C-benzyl-β-D-xylopyranoside is provided. The compound represented by formula () is a new compound.

The compound of the present invention represented by formula () can be synthesized according to the reaction route shown below.

[In the above routes and formulas, Ac represents acetyl (CH ₃ CO), X represents a halogen atom such as a bromine atom, and R has the above-mentioned meaning. ] That is, D-xylose () was prepared by the method of Hudson et al.
S.Hudson), J.M.
Johnson), Journal of the American Chemical Society (J.Am.Chem.
Soc., 37 , 2748 (1915)] to obtain tetraacetate (), which was processed by the method of Holland et al. [CVHolland, D. Holton (D.
Horton), G.S.G.
Compound () is obtained by treatment with aluminum chloride. At this time, if () is treated with aluminum chloride for a short time, the β-isomer of () will be obtained, but if treated for a long time, the thermodynamically more stable α-isomer will be obtained. () is also () in the presence of zinc chloride,
It can also be obtained by treatment with acetyl chloride [supra, J.Am.Chem.Soc., 37 , 2748
(1915)].

Compound () is then treated with excess Grignard reagent and then acetylated to obtain compound (). At this time, α-form and β-form are generated. α−
Separation of the β-isomer and the β-isomer can be performed by applying techniques such as chromatography and recrystallization. The β-isomer () thus obtained can be treated with a catalytic amount of lithium hydroxide in methanol to obtain the target compound () of the present invention.

The C-benzyl-β- of the present invention thus obtained
D-xylopyranoside is a good initiator for chondroitin sulfate biosynthesis, as shown in the Test Example below and FIG. 2. Moreover, the glycosaminoglycans synthesized using C-benzyl-β-D-xylopyranoside of the present invention as an initiator do not bind protein components compared to normal proteoglycans (molecular weight 2.5×10 ⁶ or more). Moreover, the molecular weight is extremely low (molecular weight 2.0×10 ⁴ - 3.0×10 ⁴ )
Therefore, it is difficult to remain in the tissue, and in a tissue culture system, it is released into the medium, and in an animal body, it escapes from the tissue and is released into the bloodstream. This shows that by administering C-benzyl-β-D-xylopyranoside of the present invention to a living body, the amount of proteoglycans on the surface of cell membranes constituting tissues can be reduced, and the C-benzyl-β-D-xylopyranoside of the present invention can be reduced.
This results in the release of benzyl-β-D-xylopyranoside-initiated low molecular weight glycosaminoglycans (chondroitin sulfate) into the bloodstream. Taking cancer cells as an example, the amount of proteoglycans on the surface of cancer cells becomes extremely small.
Cancer cells become naked, so to speak, and this results in increased immunity by immune cells. Therefore, the compounds of the present invention are fully expected to be useful in the prevention and treatment of cancer.

In addition, glycosaminoglycan (chondroitin sulfate) released into the bloodstream has the same effect on the body as chondroitin sulfate that is specially administered from outside the body, causing lipid deposition on blood vessel walls and various causes of arteriosclerosis. It is expected that it will be useful in the prevention and treatment of diseases. Furthermore, C-benzyl-β- of the present invention
D-xylopyranoside is less susceptible to hydrolysis by acids and enzymes than conventional O-β-D-xylopyranoside compounds, and there is no risk that the compound of the present invention will be degraded before reaching the so-called target organ, so it is safe for living organisms. The administered compound acts effectively, and in this respect it has an advantage over conventional O-β-D-xylopyranoside compounds.

Hereinafter, the present invention will be explained in more detail with reference to Examples and Test Examples.

Example Tri-O-acetyl-
An ether solution (500 ml) containing 50 g of α-D-xylosyl chloride () was added dropwise over 30 minutes. After dripping,
The reaction mixture was refluxed for 2 hours. After cooling the reaction mixture to room temperature, it was slowly poured into ice-water 1. After making this acidic with acetic acid, the organic layer was separated. The aqueous layer was concentrated under reduced pressure and dried under vacuum to obtain a white solid. After crushing this finely, it was placed in the eggplant flask (No. 2). In this, anhydrous sodium acetate
Add 60g and 700ml of acetic anhydride, and heat the reaction mixture to 100℃.
The mixture was stirred for 2 hours. After stirring, acetic anhydride was distilled off under reduced pressure, followed by extraction with ethyl acetate, washing with water, and drying to obtain syrup. This syrup was subjected to silica gel column chromatography to obtain tri-O
-Acetyl-C-benzyl-β-D-xylopyranoside (22g) was obtained.

A mixture of 22 g of tri-O-acetyl-C-benzyl-β-D-xylopyranoside prepared above, 100 mg of lithium hydroxide and 100 ml of dry methanol was stirred at room temperature for 1 hour. After the reaction mixture was concentrated under reduced pressure, the residue was subjected to silica gel column chromatography to obtain 10 g of the target C-benzyl-β-D-xylopyranoside (C-β-D-xylopyranosylmethylbenzene). Yield: 30%.

[α] ²⁰ _D = -56.3° (c = 1, H ₂ O).

IR (cm ^-1 ): 3430, 1600cm ^-1 .

'HNMR (D ₂ O), δppm: 2.3 to 4.3 (m, 8H),
7.4 (m, 5H).

mp: 105-107℃.

Test Example Epiphyseal cartilage was collected from a 12-day-old chick embryo in Tyrode's medium while cooling on ice, and excess tissue was removed. 5ml of BGJb for 150mg of cartilage equivalent to 5 animals
[Completely synthetic medium, GIBCO (Grand Island)
[prepared according to the recipe of Biological Company] was added thereto, and pre-incubation was performed at 37°C. After replacing the medium, 1 ml of BGJb was newly added, 5 μCi of Na ₂ ³⁵ SO ₄ was added, and the mixture was incubated at 37° C. for 3 hours. Furthermore, the medium was replaced with 1 ml of fresh medium (chase medium) containing no isotope, and after incubation at 37°C for 1 hour, the medium and tissue were separated. In order to examine the effect of xyloside compounds on the synthesis of glycosaminoglycans, xyloside compounds were added to the preculture and culture medium at a constant concentration.

After culturing, labeled medium (labeled medium,
Medium _containing ^Na235SO4 ) and chase medium (chase
medium) and 0.5M Tris-HCl buffer (PH
Pronase-P was added in 8.0) and the mixture was digested at 50°C for 16 hours. The digestion reaction solution was purified using Bio-Gel P-2 (Bio-gel) using 0.2M ammonium formate solution as the eluent.
Gel filtration was performed using a column (1.5 x 14 cm) packed with Gel (trade name, manufactured by Bio-Rad), the Vo fraction was collected, and then lyophilized to obtain crude glycosaminoglycan.

On the other hand, ice-cold 4M guanidine hydrochloride was added to the tissue separated from the medium in the above procedure, and after being left overnight at -20°C, the tissue was homogenized. After leaving the obtained homogenate at room temperature overnight,
Centrifugation was performed at 8500 rpm to obtain a supernatant. Three times the amount of water was added to this supernatant, and three times the amount of 95% ethanol (containing 1.3% potassium acetate) was added to obtain a precipitate. After repeating this operation two more times,
The resulting precipitates were combined and dried in a desiccator. The obtained precipitate was dissolved in 0.02M Tris-HCl buffer (PH8.0) and digested with pronase in the same manner as in the case of the medium described above to obtain crude glycosaminoglycan.

As xyloside compounds, conventional O-paranitrophenyl-β-D-xylopyranoside and C-benzyl-β-D-xylopyranoside of the present invention were used, and the total amount of [ ³⁵ S] glycosaminoglycan synthesized in each case was determined. ( ³⁵ S uptake amount) (×...
×), the amount released into the culture medium (●---●), and the amount remaining in the tissue (〇-〇) are illustrated in Figures 1 and 2, respectively.

In the figure, the vertical axis represents the amount of [ ³⁵ S] chondroitin sulfate ( ³⁵ S cpm × 10 ^-4 / μmol uronic acid),
The horizontal axis represents the concentration (mM) of each xylopyranoside compound in the medium.

Looking at Figure 1, O-paranitrophenyl-β
-D-xylopyranoside concentration from 0.05mM
The total amount of ^35S incorporated into glycosaminoglycans increased as the concentration increased to 1.0mM, and at a concentration of 1mM,
It showed 40800 counts per minute (cpm), which was 2.35 times that of the control. Also, at that time, 95% of the control was released into the medium, while the [ ³⁵ S] glycosaminoglycan of the tissue was
It decreased to 11%. This has been confirmed by previous reports [J.
Biochem., 74 , 1069-1073 (1973)],
O-paranitrophenyl-β-D-xylopyranoside has been shown to be highly effective as an initiator for chondroitin sulfate synthesis.

On the other hand, the C-benzyl-β-D-
As for xylopyranoside, as shown in Figure 2, as in the case of the conventional compound described above, as the concentration increases, the total amount of ³⁵ S incorporated into glycosaminoglycan increases and is released into the medium. While the amount increased, the amount retained in tissues decreased. This indicates that C-benzyl-β-D-xylopyranoside of the present invention is a good initiator for chondroitin sulfate synthesis, although it requires a higher concentration than O-β-D-xylopyranoside compounds. There is.

[Brief explanation of drawings]

Figures 1 and 2 show O-paranitrophenyl-β-D-xylopyranoside and C-
It is a graph showing the influence of benzyl-β-D-xylopyranoside (the compound of the present invention) on chondroitin sulfate synthesis. The vertical axis represents the amount of [ ³⁵ S]chondroitin sulfate ( ³⁵ S cpm×10 ⁻⁴ /μmol·uronic acid), and the horizontal axis represents the concentration (mM) of each xylopyranoside compound in the medium. ×……×: Total amount of [ ³⁵ S] chondroitin sulfate uptake, ●――●: Amount of [ ³⁵ S] chondroitin sulfate in the medium, 〇――〇: Amount of [ ³⁵ S] chondroitin sulfate in the tissue.

Claims (1)

[Claims] Primary formula: [In the formula, R represents a benzyl group. ] C-benzyl-β-D-xylopyranoside shown.