JPH03842B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to aldose derivatives having the ability to inactivate plant viruses.

More specifically, the present invention relates to an aldose substituted with an O -arylcarbamoyl group or a derivative thereof having the ability to inactivate plant viruses.

In recent years, research into compounds that cleave nucleic acids as virus inactivators has become active, and simple compounds such as peroxides, quinones, and mercaptans have been proposed, but they are still far from being fully effective. Not yet.

The present inventors focused on the fact that the nucleic acids that form the main body of the virus are cleaved by oxygen radicals, and developed aldose, which itself does not have the ability to generate oxygen radicals, as a compound that has the ability to generate oxygen radicals. We conducted intensive research on its derivatives as compounds and discovered a new derivative that has the ability to cleave nucleic acids, that is, it has the ability to inactivate viruses.
This led to the present invention.

That is, the gist of the present invention is a virus inactivating agent for plants containing as an active ingredient O -arylcarbamoylaldose or O -arylcarbamoylaldouronic acid in which the aryl group is a phenyl group or a naphthyl group.

It has been revealed that this derivative has the ability to generate oxygen radicals and also exhibits a nucleic acid cleaving action in vitro.

Furthermore, the inactivating effect of this derivative on tobacco mosaic virus and bacteriophage φ×174 was tested in vitro, and it was found to be effective.

In the present invention, the number of carbon atoms in the main chain of the aldose moiety of O -arylcarbamoylaldose or the aldouronic acid moiety of O -carbamoylalduronic acid is preferably 5 or 6.

The formula of O -arylcarbamoylaldose or O -arylcarbamoylaldouronic acid in the present invention, for example, for pentose is as follows. However, this formula is not based on Fischer's formula.

Here R ¹ , R ² and R ³ are OH or -OCO-NH
-A, R ⁴ is -CH ₂ OH, -COOH or -
It represents CH ₂ --OCO-NH-A, R ¹ to R ⁴ as a whole contain at least one --OCO-NH-A, and A represents an aryl group. Examples of the aryl group A include a phenyl group and a naphthyl group (including groups in which H on the benzene nucleus thereof is substituted).

For example, the formula for hexose is as follows. However, this formula is not based on Fischer's formula.

Here R ⁵ , R ⁶ , R ⁷ and R ⁸ are OH or -OCO-
NH-A, R ⁹ represents -CH ₂ H, -COOH or -CH ₂ -OCO-NH-A, and R ⁵ to R ⁹ collectively represent at least one -OCO-NH-
Contains A, where A has the same meaning as above.

The O -arylcarbamoyl aldose of the present invention can be synthesized by reacting aldose or alduronic acid with allyl isocyanate in a suitable solvent.

When carbamoylating a specific site of an aldose, first determine the hydroxyl group at the site to be carbamoylated, protect other reactive hydroxyl groups,
Allyl isocyanate is reacted in a suitable solvent, the protected product is separated, the protecting group is removed, and the product is then separated and purified.

Etherification, acetalization, esterification, intramolecular lactonization, etc. can all be used to protect hydroxyl groups where carbamoylation is not desired, but isopropylidene acetalization (acetonidation) and intramolecular lactone formation are It has the advantage of being convenient for handling and being stable against subsequent silica gel chromatography treatment. However, intramolecular lactonization is possible only in the case of oxides of the primary alcohol moiety of aldoses, such as glucuronic acid.

As the aryl isocyanate, phenyl isocyanate, naphthalene isocyanate, and nuclear substituted derivatives thereof can be used. The nuclear substituents of the nuclear substituted derivatives include p-nitrophenyl group, m-nitrophenyl group, o-nitrophenyl group, p-, m- or o-nitronaphthyl group, p-nitrophenyl group, m-nitrophenyl group, o-nitrophenyl group,
-, m-, or o-halogenophenyl group, p-,
Mention may be made of the m- or o-halogenonaphthyl group.

As the solvent for carbamate formation with isocyanate, any solvent can be used as long as it has no effect on the carbamate reaction, including aliphatic hydrocarbons,
Basic aromatic compounds such as alicyclic hydrocarbons, aromatic hydrocarbons, and pyridine can be used. In particular, the above-mentioned basic aromatic compounds are advantageous because the reaction time can be shortened.

The concentration of aldose or alduronic acid (including protected ones) in the solvent is from 1 to
A range of 5g/100ml is preferred. The concentration of the arylisocyanate is preferably in the range of 1.5 to 2.0 molar equivalents relative to the aldose or alduronic acid.

The carbamate formation reaction is preferably carried out in a dry atmosphere to prevent the isocyanate from being destroyed. Although it may be carried out in an air atmosphere, it is preferably carried out in an inert gas such as N ₂ .

The carbamate reaction can be carried out at a temperature of about 10° C. to around the boiling point of the solvent.

The carbamate-forming reaction can be carried out at normal pressure, but it may also be carried out under reduced pressure or under increased pressure as long as the solvent does not evaporate.

The carbamate reaction time is 1 hour or more24
The reaction time can be about 1 hour, but from the viewpoint of yield and suppression of side reactions, about 5 to 15 hours is preferable.

Regarding the separation of the protected reaction product, it is preferable to separate it by chromatography because the reaction product generally has a high boiling point and is difficult to separate due to the risk of decomposition by distillation. The packing material for this chromatography is silica gel,
Although inorganic substances such as alumina or polysaccharides can be used, it is efficient to use silica gel. A mixed system of an aromatic hydrocarbon and an ester is suitable as the eluent. In particular, a benzene-ethyl acetate mixture is excellent.

Generally speaking, dioxane-
Although a hydrochloric acid mixture, 60% acetic acid, an ion exchange resin, etc. can be used, it is appropriate to treat with an acetone-water mixture in the presence of an ion exchange resin.

The product from which the protecting group has been removed is preferably separated and purified by silica gel chromatography as described above. As the eluent, a combination of a polar solvent and an alcohol is suitable, but a chloroform-methanol mixture can be used particularly efficiently.

Examples are shown below, but the present invention is not limited thereto.

Reference example 1 [Synthesis of 3- O -phenylcarbamoyl-D-glucose (1)] 1,2,5,6-di- O -isopropylidene-
After reacting α-D-glucofuranose (7.0 g), anhydrous pyridine (30 ml), and phenyl isocyanate (14 ml) at room temperature for 15 hours with stirring, the solvent and excess reagent were distilled off, and the product was recovered. Furthermore, it was subjected to silica gel column chromatography, and benzene:
Elution was performed with a mixture of ethyl acetate (20:1 V/V),
R _F 0.45 (benzene:ethyl acetate, 5:1V/
V) 1,2,5,6-di- O -isopropylidene-3- O -phenylcarbamoyl-α-D
−Glucofuranose (Silup; [α] _D +6l ^o ;
νmax 3300, 2980, 1730 cm ^-1 ) were obtained with a yield of 98%. Dissolve this compound (5 g) in a 10% acetone aqueous solution (100 ml) and use Amberlite (trademark).
IR120B (H ⁺ ) (strongly acidic ion exchange resin; H ⁺ type)
(hereinafter abbreviated as “IR120B(H ⁺ )”) (3g),
After stirring at 95°C for 5 hours, the resin was filtered, the filtrate was concentrated under reduced pressure, and the resulting syrup was subjected to silica gel column chromatography (chloroform:methanol, 20:1 V/V) and TLC (chloroform: methanol, 20:1 V/V).
Methanol, 6:1V/V) 0.22 fractions were collected,
Concentration under reduced pressure yielded the crystalline target product [1] in 79%.

mp: 110~120℃ [α] _D : +19.6°~+12.5° νmax [cm ^-1 ]: 3500, 2900, 1720 PMR (CDCl ₃ ) δ (ppm): 5.9 (H-l) 6.25 ( H-3) 7.5 (Aromatic H) UVλmax: 193nm (ε7450) 232nm (ε1320) Elemental analysis (C ₁₃ H ₁₇ O ₇ N): Calculated value (%) Actual value (%) N 4.51 4.68 Reference example 2 [3 -O- (α-naphthylcarbamoyl)-D-
Synthesis of glucose ()] 1,2,5,6-di- O -isopropylidene-
α-D-glucofuranose (6.5g), anhydrous pyridine (40ml), α-naphthyl isocyanate (8.2
ml) was reacted with stirring at room temperature for 5 hours in the absence of light, the solvent was distilled off under reduced pressure, and the resulting syrup was separated by silica gel column chromatography (benzene: ethyl acetate, 5:1 V/V). The fractions with R _F 0.55 were collected, concentrated under reduced pressure, and 1.
2,5,6-di- O -isopropiden-3- O-
(α-naphthylcarbamoyl)-α-D-glucofuranose (silap; [α] _D +72°; νmax3300,
2980, 1780cm ^-1 ; PMR (CDCl ₃ ) δ1.2, 1.3, 1.5,
1.6, 5.9, 7.5ppm) were obtained in 96% yield. This compound (4 g) was suspended in 100 ml of 10% acetone aqueous solution, IR120B (H ⁺ ) (3 g) was added, stirred at 97℃ for 10 hours, the resin was filtered, the liquid was concentrated under reduced pressure, and chromatographed on silica gel. Fractions showing R _F 0.21 (chloroform:methanol, 6:1 V/V) by graphie were collected, and the desired product was obtained in a yield of 82%.

mp: 196 to 198℃ [α _D ]: +13.5° to +11.2° νmax [cm ^-1 ]: 3500, 1720 PMR (DMSO) δ (ppm): 4, 68, 5.21,
7.5UVλmax: 222nm (ε45700), 287nm ((ε4850) Elemental analysis (C ₁₇ H ₁₉ O ₇ N): Calculated value (%) Actual value (%) N 3.95 3.99 Reference example 3 [3- O - (α-naphthyl carbamoyl)-D-
Synthesis of allose ()] 1,2,5,6-di- O -isopropylidene-
α-D-Allofuranose (6.5g), anhydrous pyridine (40ml), α-naphthylisocyanate (8.2ml)
After reacting with stirring at room temperature for 10 hours while shielding from light, the solvent was distilled off under reduced pressure, and the resulting syrup was fractionated and purified by silica gel chromatography (benzene: ethyl acetate, 5:1 V/V). Fractions with R _F 0.39 were collected, concentrated under reduced pressure, and 1,2,5,6-
Di- O -isopropylidene-3- O- (α-naphthylcarbamoyl)-α-D-allofuranose (silap; [α] _D +71.4°; νmax3300, 2980,
1780cm ^-1 ; PMR (CDCl ₃ ) δ1.2, 1.3, 1.5, 1.6,
5.9, 7.5ppm) with a yield of 97%. This compound (4 g) was suspended in 100 ml of 10% acetone aqueous solution,
Add IR120B (H ⁺ ) (3 g), stir at 85°C for 5 hours, filter the resin, concentrate the liquid under reduced pressure, and perform silica gel chromatography to R _F 0.27 (chloroform:methanol, 6:1V/V). ), and the desired product was obtained with a yield of 60%.

mp: 89 to 91℃ [α _D ]: +8.2° to 3.3° νmax [cm ^-1 ]: 3500, 1720 PMR (DMSO−d ₆ ) δ (ppm): 4.68, 5.22, 7.5 Elemental analysis (C ₁₇ H ₁₉ O ₇ N): Calculated value (%) Actual value (%) N 3.99 3.99 Reference example 4 [5- O -(α-naphthylcarbamoyl)-D-
Synthesis of glucuronic acid ()] 3,6-D-glucuronolactone (10g)
Dissolve IR120B in 400ml of anhydrous acetone by heating.
(H ⁺ ) (6 g) was added and heated under reflux for 15 hours. After the reaction, IR120B (H ⁺ ) was filtered off, concentrated, and extracted with chloroform. This was distilled off under reduced pressure. The syrup was dissolved in a small amount of ethyl acetate, and hexane was added to crystallize it . -isopropylidene-3,6
-D-glucuronolactone (mp120-121℃;
[α] _D +7.0°; νmax 3380, 1770 cm ^-1 ) was obtained with a yield of 50%. This compound (3.3g) and α-naphthylisocyanate (6.5ml) were mixed with anhydrous pyridine (40%
ml), reacted for 6 hours in the dark and distilled under reduced pressure to obtain a syrup, which was crystallized from methanol to give 1,2- O -isopropylidene-5- O- (α
-naphthylcarbamoyl)-3,6-D-glucuronolactone (mp174-176℃; νmax3420,
1800, 1720cm ^-1 ; PMR (CDCl ₃ ) δ7.8, 5, 2,
5.1, 4.9, 1.5, 1.3 ppm; [α] _D +75°) were obtained in 80% yield. This compound (3g) was suspended in a 10% acetone aqueous solution, and IR120B (H ⁺ ) (1g) was added to it for 80 minutes.
After hydrolysis at ℃ for 24 hours, the resin was filtered off, treated with decolorizing charcoal, concentrated under reduced pressure, and the product was crystallized from hot ethyl acetate to obtain 5- O- (α-naphthylcarbamoyl)-D-glucuronic acid. Obtained with a yield of 80%.

mp: 189 to 190℃ [α _D ]: −25° to −7.5° νmax (cm ^-1 ): 3480, 1730 PMR (DMSO−d ₆ ) δ (ppm): 8.1, 7.6, 5.6 Reference example 5 [5 - Synthesis of O -phenylcarbamoyl-D-ribose (v)] D-ribose (10g), 76ml of anhydrous acetone-anhydrous methanol mixture (1/1, V/V),
IR120B (H ⁺ ) (5 g) was reacted under reflux for 2 hours. The resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain syrup. Furthermore, the product was extracted and purified with chloroform to obtain methyl 2,3- O -isopropylidene-β-D-riboside in a yield of 60%. 1 g of this compound and 2.5 ml of phenyl isocyanate in 30 ml of pyridine,
The reaction was carried out with stirring at room temperature for 10 hours, and after distilling off the solvent, silica gel chromatography (benzene:
Fractions with R _F 0.65 were collected with ethyl acetate; 5:lV/V) to give methyl 2,6- O -isopropylidene-5- O -phenylcarbamoyl-β-D with a yield of 98%.
−ribofuranoside ([α] _D −33.6°; νmax3420,
1700cm ^-1 ; PMR (CDCl ₃ ) δ7.4, 4.9, 3.4, 1.5,
1.6ppm). 1 g of this compound was suspended in a 10% acetone aqueous solution, IR120B (H ⁺ ) (200 mg) was added, and after heating under reflux for 10 hours, the resin was filtered off, the filtrate was concentrated under reduced pressure, and crystallized from ethyl acetate. , the desired product was obtained in 80% yield.

mp: 106 to 107℃ [α _D ]: +20° to +26.3° νmax (cm ^-1 ): 3460, 3420, 3030, 1700, 1230 PMR (DMSO−d ₆ ) δ (ppm): 7.4, 5.02 Element Analysis (C ₁₂ H ₁₇ NO ₆ ): Calculated value (%) Actual value (%) N 5.20 5.30 Reference example 6 [5- O -(α-naphthylcarbamoyl)-D-
Synthesis of ribose ()] Methyl 2,3- O -isopropylidene-β-D-loribofuranoside (4 g), α-naphthyl isocyanate (5 ml), and pyridine (50 ml) used in the preparation of (v) were stirred at room temperature for 15 hours in the absence of light. After that, the solvent was distilled off and methyl 2,3- O -isopropylidene-5- O- (α-
naphthylcarbamoyl)-β-D-riboside (νmax3440, 1750, 1240cm ^-1 ; [α] _D -36.6°;
PMR (CDCl ₃ ) δ7, 6, 4.98, 3.28, 1.4,
1.2 ppm) was obtained in 100% yield. 5g of this compound
Soak in 10% acetone aqueous solution, IR120B (H ⁺ )
(2 g) was added and reacted by heating under reflux at 90° for 5 hours. After that, the resin was filtered off, the filtrate was concentrated, and R _{The F} 0.35 fraction was purified, concentrated, and crystallized from ethyl acetate to obtain the desired product in 85% yield.

mp: 113~114℃ [α] _D : +14.3°~+18.6° νmax (cm ^-1 ): 3460, 3400, 3030 1700 PMR (DMSO−d ₆ ) δ (ppm): 7.4, 5.02, Element Analysis (C ₁₃ H ₁₉ NO ₆ ): Calculated value (%) Actual value (%) N 4.39 4.34 Reference example 7 [Oxygen radical generation ability of compound ~] 5 × 10 ^-5 M cytochrome C solution (PH8.0,
Compound ~(10 ^-2 M) in 0.2M phosphate buffer)
When dissolved, the increase in absorbance at 550 nm was immediately examined at 25°C, and the result was: 6.95×10 ^-4 ;: 2.85×
The following results were obtained: 10 ^-4 ;:6.62×10 ^-4 ; :1.44×10 ^-2 ;v:5.67×10 ^-3 ; :3.09×10 ^-3 (ΔA550/min). Glucose (D
-glucose) and D-ribose are each 0.75×
10 ^-4 and 1.5×10 ^-4 (20% ethanol solution).

Reference Example 8 [In vitro nucleic acid cleavage activity of compound and] Compound and in vitro nucleic acid cleaving activity of compound and
7.2), double-stranded φ×174DNA200ng, sugar lnM,
Treated with Cu ²⁺ 10μM at 37℃ for 3 hours to cause single strand cleavage (Form) and linear DNA (Form)
When we investigated the generation of , each was 18.0%;
86.2, 8.4%; 84.3, 5.8%.

Example 1 [In vitro virus inactivation effect of compound ~] The in vitro virus inactivation effect of compound ~ was investigated using tobacco mosaic virus and bacteriophage φx174. The former has a sugar concentration of 10 ^-2 M in phosphate buffer (0.2 M, PH8.1),
The virus was reacted with Cu ²⁺ 10 ^-4 M at 37° C. for 3 hours, and the survival rate (%) of the virus was assayed by the half-leaf method using N. tapacum. The latter is φ × 174 at a sugar concentration of 10 ^-3 M and Cu ²⁺ 10 ^-6 M in phosphate buffer.
were treated at 37° C. for 3 hours, and the rate of plaque formation relative to 0 hours of treatment was defined as survival rate (%). Compound,
The inactivating ability of TMV was demonstrated by survival rates of 51, 14, and 60%, respectively.
On the other hand, compounds showed 33.8, 38.4, and 44.2% for φ×174, respectively.

The above three in vitro reactivity and biological activity results (Reference Examples 6, 7 and Example 1) indicate that these derivatives have a reactivity that is not observed in unsubstituted sugars (aldohexoses) or is greater than in unsubstituted sugars. This indicates that the substance has biological activity or biological activity.

Claims (1)

[Scope of Claims] 1 O-arylcarbamoyl albatose or O -arylcarbamoyl albatose in which the aryl group is a phenyl group or a naphthyl group
- A virus inactivator for plants containing arylcarbamoylaldouronic acid as an active ingredient. 2. The virus inactivating agent for plants according to item 1, wherein the main chain of the aldose moiety or the alduronic acid moiety has 5 or 6 carbon atoms.