JPS637555B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel derivatives of N-acetylneuraminic acid, particularly derivatives having anticancer activity with high cell selectivity, and a method for producing the same. N-acetylneuraminic acid is known to exist in the animal kingdom and on the cell surface of some bacteria as sialocomplexes (glycoproteins, glycolipids, oligosaccharides and polysaccharides). In recent years, this compound has gained medical and pharmaceutical importance in regulating nerve function, promoting cell differentiation, treating cancer markers, inflammation, immune diseases, viral infections, etc., and as a hormone receptor. It has been attracting attention as a unique localized active molecule. However, the role played by N-acetylneuraminic acid in the sialo complex is currently unclear. In recent years, there have been reports that it may be one of the cancer cell markers. This N-acetylneuraminic acid has been studied by many natural product organic chemists, and there have already been some reports on simple derivatives. However, these did not show significant physiological activity. Under these circumstances, the applicant has been conducting a series of studies to obtain N-acetylneuraminic acid derivatives with few side effects and cell selectivity, and as part of this research, a new derivative with cancer activity has been developed. I got it. That is, the main object of the present invention is to provide a novel derivative of N-acetylneuraminic acid, which is an anticancer agent having a cancer-reducing effect. Another object of the present invention is to provide a method for producing the above derivatives. These and other objects of the invention will become apparent from the detailed description below. The novel N-acetylneuraminic acid derivative of the present invention has the general formula []: However, R ¹ represents a hydrogen atom or an acetyl group, R ² represents a hydrogen atom or a methyl group,
Z is

【formula】

[expression] or

[Formula] is represented by . These novel derivatives of the present invention exhibit cell selectivity and are expected to serve as anticancer agents with strong cancer-removing effects. The novel derivative of the present invention comprises a chlorinated derivative of N-acetylneuraminic acid and a predetermined compound:

【formula】

[expression] or

It can be produced by reacting with [Formula]. Here, the chlorinated derivative of N-acetylneuraminic acid as a starting material has the following formula: However, Ac represents an acetyl group, which is a useful intermediate in the production of N-acetylneuraminic acid derivatives, and the production method is described in detail in JP-A-58-992. . The catalyst for this reaction is Ag ₂ CO ₃ ,
_Ag2O , Hg(CN) ₂ , _HgBr2, etc. can be used, with the preferred _catalyst being _Ag2CO3 . The reaction temperature is not particularly critical, but for economic reasons and ease of operation it is generally carried out at room temperature. A reaction time of about 10 to 72 hours is sufficient. Furthermore, as reaction solvents, CH ₃ CN, C ₆ H ₆ ,
Examples include CH ₂ Cl ₂ , CH ₃ NO ₂ and the like, and among them, CH ₃ CN is particularly preferred. Deacetylation reduces the product of the above reaction to about -20 to 0
The reaction solution is stirred with an alkali metal alcoholate in a solvent such as methanol for about 20 minutes at a temperature of ℃, the reaction solution is neutralized with Dowex 50 x 8 (H ⁺ ), and the following treatment is carried out according to a conventional method. In this way, the H-form of the above derivative can be obtained. The compound of the present invention obtained according to the above method can be purified by conventionally known methods such as silica gel chromatography. Further, the above compounds [] ₁ to [] ₃ can be obtained according to various methods, one example of which will be described in detail in the following example. As already mentioned above, the N-acetylneuraminic acid derivative of the present invention has an excellent cell selection effect. The cancer-reducing effect can be confirmed by the following method. ^3H -thymidine uptake action of K562 cells in adult chronic myeloid leukemia: K562 cells have ^3H -thymidine uptake action without activation, and when drugs or N-acetylneuraminic acid derivatives are added to this system, , the uptake effect is suppressed. The applicant judged this effect to be one of the cancer-removing effects and investigated its effect. That is,
The K562 cells used were those that had been subcultured in the applicant's laboratory. The culture medium used was RPMI with 10% fetal bovine serum added, and
5×10 ⁴ cells (200 μ) were seeded per well in a microplate. Subsequently, an N-acetylneuraminic acid derivative and a control drug, adriacin and mitomycin, were added in triplicate, and culture was carried out on day 0. On the 5th day of culture, 10μ of 50μCi/ml of ³ H-thymidine was added, and 6 hours later, the cells were collected using a cell harvester and measured using a liquid scintillation counter. Regarding the compound represented by the general formula [],
At concentrations of 10 ^-6 M and 10 ^-8 M, it showed a marked inhibitory effect compared to mitomycin and adriacin. Especially 10 ^-8 M
The inhibitory effect of concentration was remarkable. It had no effect on normal cells. From the above results, the compound of the present invention has selectivity between cancer cells and normal cells, and also suppresses ^3H -thymidine uptake, which is a characteristic of cancer cells, and therefore normalizes the properties of cancer cells. It was determined that the cancer had been removed. Therefore, it is expected to be useful in clinical applications as an ideal anticancer agent. The present invention will be explained below using examples. These examples are merely illustrative of the invention and therefore, of course, are not intended to limit it. Example 1 Methyl 5-(acetylamino)-2,3,5-
Trideoxy-4,7,8,9-tetra-O-
Acetyl-2-(5-fluoro-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidin-1-yl)-D-glycero-α-D-galacto-nonulosonate 1 2,4-bis- Trimethylsilyloxy-5
-Synthesis of fluoropyrimidine [] ₁ 5-Fluorouracil [] 0.260g (2m
mol) was suspended in 2 ml of hexamethyldisilazane [] and refluxed in an oil bath (135°C) for 2 hours. After the reaction solution was left at room temperature overnight, excess hexamethyldisilazane was distilled off under reduced pressure. The operation of adding anhydrous benzene to the residue and distilling it off under reduced pressure was repeated three times, and then the residue was vacuum-dried to obtain the title compound [] ₁
was obtained as an oil and used directly in the next reaction. 2 Synthesis of compound [] ₁ Dissolve 1.02 g (2 mmol) of compound [] and 0.82 g (3 mmol) of compound [] ₁ in 20 ml of CH ₃ CN,
0.55 g (2 mmol) of Ag ₂ CO ₃ was added to this mixture and reacted at room temperature for 21 hours. After the reaction is complete,
Add 1 g of NaHCO ₃ and 2 ml of water and stir for 30 minutes.
The catalyst (Ag ₂ CO ₃ ) in the reaction solution was separated. The liquid was dried under reduced pressure, the residue was added to CHCl ₃ , and after separating the CHCl ₃ insoluble portion, CHCl ₃ was distilled off under reduced pressure, and the residue was vacuum-dried to obtain 0.98 g of pale yellow powder. Next, this product was separated and purified by silica gel chromatography using chloroform:methanol (10:1) fractionation to obtain _0.48 g (yield: 40%) of the target compound []1. mp: 105℃ (foaming) [α] ²⁵ _D : -33.4 (C = 1.0, in methanol) Mass spectrometry (EI): 603 (M ⁺ ) Elemental analysis: as C ₂₄ H ₃₀ O ₁₄ N ₃ F Theoretical value: C, 45.72; H, 4.79; N, 6.66 Actual value: C, 45.69; H, 4.78; N, 6.47 IRν ^KBr _nax (cm ^-1 ): 1730, 1660, 1535 ¹ H NMR (CDCl ₃ ), δ _H ( TMS), 90MHz 1.91 (3H, s, N Ac ), 1.95−2.18 (12H, O
Ac×4) 3.26 (1H, dd, J=4.5/13.5Hz, 3'-Heq) 3.76 (3H, s, COOC H ₃ ), 6.03 (1H, d, J=9.0Hz, AcN H ) 7.80 (1H , d, J=7.0Hz, 6-H), 9.86 (1H, broad s, NH ) Example 2 Methyl 5-(acetylamino)-2,3,5-
Trideoxy-4,7,8,9-tetra-O-
Acetyl-2-(2,4-dioxo-1,2,
Synthesis of 3,4-tetrahydro-pyrimidin-1-yl)-D-glycero-α-D-galacto-nonulosonate 1 2,4-bis-trimethylsilyloxy-pyrimidine [] ₂ 2.24 g (20 mmol) of uracil [] was suspended in 30 ml of hexamethyldisilazane [] and reacted on an oil bath at 90°C for 5 hours. After the reaction was completed, excess hexamethyldisilazane was distilled off under reduced pressure, and the residue was dried in vacuo to obtain the target compound [] ₂ as an oil, which was used as it was in the next reaction. 2 Synthesis of compound [] ₂ 1.02 g (2 mmol) of the compound [] ₂ and 1.03 g (4 mmol) of the above compound [] 2 were dissolved in 20 ml of CH ₃ CN, and 0.55 g (2 mmol) of Ag ₂ CO ₃ was added thereto and reacted at room temperature for 26 hours. After the reaction is complete, add
1 g of NaHCO ₃ and 2 ml of water were added and stirred for 30 minutes. After separating the precipitate, the liquid was dried under reduced pressure and the residue was dissolved in CHCl ₃ . After separating the CHCl ₃ insoluble components, the liquid was dried with MgSO ₄ and then CHCl ₃ was distilled off under reduced pressure. 0.93g of light brown powder was obtained. This product was purified by silica gel chromatography to obtain the title compound [] ₂ 0.46g (yield 39.6%)
I got it. mp: 108−112℃ [α] ²⁹ _D : −34.8 (C=0.50, in methanol) IRν ^KBr _nax (cm ^-1 ): 1735, 1650, 1540 ¹ H NMR (CDCl ₃ ) δ _H (TMS), 60MHz 1.90 (3H, s, N Ac ), 1.93−2.20 (12H, O Ac
×4) 3.34 (1H, dd, J = 6.0/14.2Hz, 3'-Heq) 3.76 (3H, s, COOC H ₃ ), 5.80 (1H, d, J = 8.4Hz, 5-H) 7.68 (1H , d, J=8.4Hz, 6-H) Example 3 Methyl 5-(acetylamino)-2,3,5-
Trideoxy-4,7,8,9-tetra-O-
Acetyl-2-[1,6-dihydro-6-oxo-9-(2,3,5-tri-O-acetyl-
β-D-ribofuranosyl)-purin-1-yl]
Synthesis of -D-glycero-α-D-galacto-nonulosonate 1 2',3',5'-tri-O-acetyl-inosine [] 10.73 g (40 mmol) of inosine [] and 40 ml of acetic anhydride were reacted in 60 ml of pyridine at room temperature for 8.5 hours. After the reaction was completed, methanol was added to decompose excess acetic anhydride, and the mixture was dried under reduced pressure.
The obtained white residue was recrystallized from methanol,
11.19 g (yield 70.9%) of colorless prism crystals were obtained. mp: 238-239℃ 2 Synthesis of compound [] ₃ Compound obtained in 1 above [] 1.18 g (3 mmol)
6 ml of hexamethyldisilazane was added to the mixture, and the mixture was refluxed on an oil bath for 4 hours. After drying the reaction solution under reduced pressure,
After vacuum drying, the desired product [] ₃ was obtained as an oily substance, which was used as it was in the next reaction. 3 Synthesis of compound [] ₃ Compound [] 1.02g (2mmol) and compound
1.40g (3mmol) was dissolved in 20ml of CH ₃ CN,
0.55 g (2 mmol) of Ag ₂ CO ₃ was added and reacted at room temperature for 47 hours. After the reaction is complete, add 1g of NaHCO ₃ to the reaction solution.
and 2 ml of water were added and stirred for 15 minutes. Next, the reaction solution was dried under reduced pressure, ethyl acetate was added to the residue, and after separating the ethyl acetate-insoluble matter, ethyl acetate was distilled off under reduced pressure to obtain 0.70 g of pale yellow powder. This product was scraped off and purified by TLC to obtain 0.085 g (yield: 4.9%) of the target product [] ₃ . mp: 143℃ (decomposition) [α] ²⁹ _D : -32.8 (C = 0.67, in methanol) Mass spectrometry (FD): 867 (M ⁺ ) IRν ^KBr _nax (cm ^-1 ): 1730, 1645, 1545 ¹ H NMR (CDCl ₃ ), δ _H (TMS), 90MHz 1.71−2.22 (24H, Ac ×8) 3.50 (1H, dd, J=4.5/13.5Hz, 3″−Heq) 3.75 (3H, s, COO CH ₃ ), 6.12 (1H, d, J = 4.8Hz, 1'-H) 7.94 (1H, s, 2-H), 8.52 (1H, s, 8-
H) Example 4 5-(acetylamino)-2,3,5-trideoxy-2-(5-fluoro-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidin-1-yl)- D-glycero-α-D-galacto-nonulosonic acid The above compound [] ₁ 0.100g (0.17mmol) in 2N
-Dissolved in 2 ml of NaOH and reacted at room temperature for 2 hours. After the reaction, add 8 ml of water to the reaction solution and add DowX-50.
(H ⁺ type) to remove the resin, and the liquid was treated with activated carbon and concentrated under reduced pressure on a water bath at 37°C. The residue was dried in vacuo to obtain 0.01 g of white powder. mp: 111℃ (foaming) [α] ²⁹ _D : −53.7 (C = 0.67, in methanol) Mass spectrometry (FD): 421 (M ⁺ ) ¹ H NMR (D ₂ O), δ _H (DSS), 60 MHz 2.00 (3H, s, N Ac ) 3.01 (1H, dd, J=4.0/14.0Hz, 3'-Heq)

Claims (1)

[Claims] 1. General formula: However, in the general formula [], R ¹ represents a hydrogen atom or an acetyl group, R ² represents a hydrogen atom or a methyl group, and Z represents [Formula] [Formula] or [Formula] -acetylneuraminic acid derivatives. 2 General formula: However, Ac represents an acetyl group, and a compound represented by the following formula [] ₁ , [] ₂ or [] ₃ : [Formula] [Formula] [Formula] is reacted, and if necessary, General formula, characterized by deacetylation: However, Z represents [Formula] [Formula] or [Formula], R ¹ represents a hydrogen atom or an acetyl group, and R ² represents a hydrogen atom or a methyl group. Manufacturing method. 3. The method according to claim 2, characterized in that the compound of formula [ ₁ ] is obtained by reaction of 5-fluorouracil and hexamethyldisilazane. 4. The method according to claim 2, characterized in that the compound of formula [ _2] is obtained by reaction of uracil and hexamethyldisilazane. 5. The method according to claim 2, wherein the compound [] ₃ is obtained by acetylating inosine and then reacting it with hexamethyldisilazane. 6 In the above reaction, Ag ₂ CO ₃ as a catalyst,
6. Process according to claim 2, characterized in that Hg(CN) ₂ , _HgBr2 and also acetonitrile, benzene, methylene chloride are used as reaction solvents.