JPS6363553B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a novel N-D-xylopyranoside compound, 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. The present invention relates to N-D-xylopyranoside compounds that are expected to have an arteriosclerosis inhibiting effect, a blood clot inhibiting effect, and the like.

Conventionally, O-β-D-xylopyranoside compounds exist on the surface of cell membranes or between cells, and become important constituents of biological tissues, changing the amount of so-called proteoglycans and greatly altering the properties of certain cell membrane surfaces. [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 an N-D-xylopyranoside compound that is resistant to hydrolysis by enzymes and can change the quality and quantity of proteoglycans on the cell surface, and completed the present invention. .

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

The present invention, namely, the following formula (): N-paracarbamoyl phenyl-D-
It provides xylopyranosylamine.

The compound represented by formula () is a new compound.

The compound of the present invention represented by formula () can be obtained by reacting D-xylose with p-aminobenzamide in the presence of an acid.

The thus obtained N-paracarbamoylphenyl-D-xylopyranosylamine of the present invention serves as a good initiator for chondroitin sulfate biosynthesis, as shown in Test Examples and FIG. 2 below. Moreover, the N-paracarbamoyl phenyl-
Glycosaminoglycans synthesized using D-xylopyranosylamine as an initiator do not bind protein components and have an extremely low molecular weight (molecular weight of 2.5 x 10 ⁶ or more) compared to normal proteoglycans (molecular weight of 2.5 2.0×10 ⁴ to 3.0×10 ⁴ ), it is difficult to remain in tissues, and in tissue culture systems, it is released into the medium, and in animal bodies, it leaves the tissues and is released into the bloodstream. This shows that by administering the N-paracarbamoylphenyl-D-xylopyranosylamine of the present invention to a living body, the amount of proteoglycans on the surface of cell membranes constituting tissues can be reduced, This results in the release of carbamoylphenyl-D-xylopyranosylamine-initiated low molecular weight glycosaminoglycans (chondroitin sulfate) into the bloodstream. Taking cancer cells as an example, the amount of proteoglycan on the surface of cancer cells becomes extremely small, and the cancer cells become naked, so to speak, resulting 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, the N-paracarbamoylphenyl-D-xylopyranosylamine of the present invention is less susceptible to hydrolysis by acids and enzymes than conventional O-β-D-xylopyranoside compounds, and the compound of the present invention can be used as a so-called target. There is no risk of decomposition before reaching the organs, and the substance administered to the living body will have an effective effect, which is an advantage that conventional O-β-D-xylopyranoside compounds do not have. ing.

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

Example 15 g (0.10 mol) of D-xylose and 14.05 g (0.10 mol) of p-aminobenzamide were added to 30 ml of water, and 6 ml of acetic acid was added to this liquid over 5 minutes with stirring.
When the stirring was continued after the dropwise addition was completed, crystals precipitated and the entire liquid solidified. Next, the mixture was left at room temperature for 1 hour and filtered through a glass filter. After washing with a small amount of cold water, the resulting white solid was dissolved in ethyl acetate and the solution was then dried over anhydrous magnesium sulfate. The solvent was distilled off to obtain 20 g of white needle-like crystals of N-paracarbamoylphenyl-D-xylopyranosylamine.

mp: 177~178℃ IR (KBr:cm ^-1 ): 3500, 3400, 3340, 3280,
1650, 1615, 1055.

NMR (δ, ppm): 2.8-4.2 (m, 5H), 4.8 (m,
1H), 6.97 (d, 2H, J=8.6Hz), 7.77 (d,
2H, J=8.6Hz).

Nitrogen analysis value ( _{as C12H16O5N2} ): Actual _value 10.44%, theoretical _value 10.45%.

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.
The mixture was subjected to gel filtration using a column (1.5 x 14 cm) packed with Gel (trade name, manufactured by Bio-Rad), and the Vo fraction was collected, followed by freeze-drying 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 the tissue was left at -20°C overnight and 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 0.2M 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 the xyloside compound, O-paranitrophenyl β-D-xylopyranoside of the conventional example and N-paracarbamoylphenyl-D-xylopyranosylamine of the present invention were used, and the total synthesis amount of [ ³⁵ S] glycosaminoglycan was determined. The effect on ( ³⁵ S uptake amount) is illustrated in Figures 1 and 2.

Looking at Figure 1, we see that O-paranitrophenyl β
-D-xylopyranoside concentration from 0.05mM
The total amount of ³⁵ S incorporated into glycosaminoglycans increased as the concentration increased to 1.0 mM.
showed 40,800 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
Tissue [ ^35S ] glycosaminoglycans control
This decreased to 11%. Similar to previous reports [J.Biochem., 74 , 1069-1073 (1973)], this indicates that O-paranitrophenyl β-D-xylopyranoside is extremely effective as an initiator for chondroitin sulfate synthesis. It shows that it is valid.

On the other hand, for N-paracarbamoylphenyl-D-xylopyranosylamine, which is a compound of the present invention, as shown in ^FIG . The total uptake into glycosaminoglycans increased and therefore the amount released into the medium increased, while the amount retained in the tissues decreased. This means that although the N-paracarbamoylphenyl-D-xylopyranosylamine of the present invention requires higher concentrations compared to O-β-D-xylopyranoside compounds,
It has been shown to be a good initiator for chondroitin sulfate synthesis.

[Brief explanation of drawings]

Figures 1 and 2 show the chondroitin sulfate synthesis of O-paranitrophenyl β-D-xylopyranoside (conventional example) and N-paracarbamoylphenyl-D-xylopyranosylamine (inventive compound), respectively. It is a graph showing the influence. The vertical axis represents the total uptake of [ ³⁵ S] chondroitin sulfate ( ³⁵ Scpm×10 ⁻⁴ /μmol·uronic acid), and the horizontal axis represents the concentration (mM) of each xylopyranoside compound in the medium.

Claims (1)

[Claims] Primary formula: N-paracarbamoyl phenyl-D-
Xylopyranosylamine.