JPS6251618A - Virus inactivator - Google Patents

Abstract

PURPOSE:A virus inactivator, containing a novel O-arylcarbamoylaldose as an active constituent, having oxgen radical forming ability and virus inactivating ability and capable of exhibiting nucleic acid cleaving action in vitro. CONSTITUTION:A virus inactivator obtained by using preferably an O- aralcarbamoylaldose having pentasaccharide and hexasaccharide in the aldose part as an active constituent. The O-arylcarbamoylaldose has nucleic acid cleaving action and is capable of inactivating viruses, e.g. tobacco mosaic virus or bacteriophage phiX174, in vitro. The O-arylcarbamoylaldose can be obtained by reacting aldose with an aryl isocyanate in a solvent. Specific examples thereof include compounds expressed by formulas I, II, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an aldose derivative (
= related.

Further (-specifically, it relates to an aldose substituted with a 9-arylcarbamoyl group having virus inactivation ability).

In recent years, compounds that cleave nucleic acids as virus inactivators (:
Research on this topic has become active, and simple compounds such as peroxides, quinones, and mercaptans have been proposed, but no one has yet achieved sufficient effects.

The present inventors discovered that the nucleic acids that form the main body of the virus are cleaved by oxygen radicals (secondary focus is on aldose, which itself does not have the ability to generate oxygen radicals, as a compound that has the ability to generate oxygen radicals). As a basic compound, we conducted extensive research on derivatives thereof, and found a new derivative that has a nucleic acid cleaving action, that is, an ability to inactivate viruses, and thus the present invention 1 was achieved.

That is, the gist of the present invention is a virus inactivating agent containing 9-arylcarbamoylaldose as an active ingredient.

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

Furthermore, the inactivating effect of these two derivatives on tobacco mosaic virus and bacterium ophage X1F4i was tested in vitro, and it was found to be effective.

The aldose moiety of the 9-arylcarbamoyl aldose in the present invention is preferably a pentose or a hexose.

Moreover, this aldose is a concept that includes an oxide of a primary alcohol moiety, such as glucuronic acid.

The formula of q-arylcarbamoylaldose in the present invention, for example, for pentose sugar is as follows. However, this formula is not based on Fisher's formula.

H-C=0 H-C-R'H-C-R" ■ H-C-R3 几4 Here ζ: R1, B2 and R3 are OH or -0CO-NH
-A is the front side, R')j -CH20H, -COOH
Also represents bar CH,0CO-NH-A, R1 to B4
The whole group includes at least one CO-NH-A and represents an aryl group. Examples of the aryl group A include a phenyl group, a naphthyl group, and a group in which the benzene nucleus b○H thereof is substituted.

For example, the formula for six carbon sugars is as follows. However, this formula is not based on Fisher's formula (=).

H-(,-0 H-C-1'L'H-C-R'H-C-R'H-C-R" where 1':, R', R', R' and R8 are OH or -0CO- represents one positive person, R9 is one pH, -COO
H or -OH, -000-N) I-A wo table L,,R
'Kala R' Mateha as a whole contains at least one 1000-NH-A and has the same meaning as above.

The present invention C: synthesizes the 9-arylcarbamoyl aldose (2 can be achieved by reacting the aldose (nyallylisonanate) in a suitable solvent (2).

When carbamoylating a specific site of aldose C:
First, determine the hydroxyl group at the site to be carbamoylated, and
Protect other reactive hydroxyl groups, ■ React with allylisonanate in a suitable solvent, ■ Separate the protected product, ■ Remove the protecting group, and then ■ Separate and purify the product. .

For the purpose of protecting hydroxyl groups for which carbamoylation is not desired (= can be used for any of etherification, acetalization, esterification, intramolecular lactonization, etc., but acetalization special C
: Improvylidene acetalization (acetonidation) and intramolecular lactonization are advantageous in terms of handling, and they also have the advantage of being more stable than the subsequent silica gel chromatography treatment (2). However, intramolecular lactonization This is possible only in the case of oxides of the primary alcohol moiety of aldoses.

As the arylisonanate, phenylisonanate naphthaleneisonanate and its nuclear substituted derivatives can be used. The nuclear substituents of the nuclear substituted derivatives include p-nitrophenyl group, m-nitrophenyl group,
o-=)lophenyl group may be mentioned.

Carbamate formation with carbon dioxide (carbamate reaction C: Any solvent can be used as long as the solvent is free of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, and bases such as pyridine. A basic aromatic compound can be used. In particular, the basic aromatic compound is advantageous because the reaction time can be shortened.

The concentration of the aldose (including protected ones) in the solvent is preferably in the range of 1 to 5 t/10O-. The concentration of the arylisonanate is 1.5 to 2.0 mol per 2 Equivalent ranges are preferred.

The carbamate formation reaction is preferably carried out in a dry atmosphere to prevent the inanate from being destroyed. Although it may be carried out in an air atmosphere, it is preferably carried out in an inert gas such as N2.

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 within a range in which the solvent does not evaporate.

The carbamate reaction time can vary from 1 hour to 24 hours, but is preferably about 5 to 15 hours from the viewpoint of yield and suppression of side reactions.

Regarding the separation of the protected reaction product, it is preferable to separate it by chromatography because the boiling point of the reaction product is generally high and there is a risk of decomposition by distillation, so it is preferable to separate it by chromatography. Although inorganic substances such as silica gel and 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 an eluent. In particular, a benzene-ethyl acetate mixture is excellent.

As a method for removing the protecting group, a general method such as a dioquinane-hydrochloric acid mixture, 60% acetic acid, an ion exchange resin, etc. can be used, but it is appropriate to treat with an acetone-water mixture in the presence of an ion exchange resin. It is.

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 to these.

Reference Example 1 (Synthesis of 3-0-phenylcarbamoyl-H-glucose (summer)) H-C=O H-C-OH H-C-OH ■ H20H 1,2,5,6-di-O-isopropylidene -α-P-
Glucofuranose (7,Of)・Anhydrous pyridine (30
-), phenylinane) (14-) was reacted at room temperature for 15 hours with stirring, and the solvent and excess reagent were distilled off. ) Benzene: Ethyl acetate (2o:lV
1,2,5, which shows R7o, +5 (benzene: ethyl acetate, 5: IV/V) by TLC.
6-di-9-isopropylidene-3-9-phenylcarbamoyl-α-glucofuranose (nrap; [α
]. +61°; νmax 3300% 2980% 173
0, -1) was obtained in 98% yield. This compound (5f)
Dissolve in a 10% acetone aqueous solution (Loom), add Amberlite (trademark) IR120B (H+) (strongly acidic ion exchange resin: H+ type) (hereinafter abbreviated as 1 liter 12QB(H+)J) (3f), After stirring at 95°C for 5 hours, the resin was filtered, and the residue was concentrated under reduced pressure. The resulting syrup was subjected to silica gel column chromatography (chloroform: )9. /-l, 20: lj'/V) Nikake, TLC (chloroform:methanol, 6: l
V/V) 0.22 were collected and concentrated under reduced pressure to obtain the crystalline target product [! ] was obtained in 79%.

m, p,: 110 to 120 °C [α) D: +1
9.6°~+12#5°y max (cm-1):
3500% 2900% 1720PMR (CDCI
! , ) δ (ppm): 5.9 (H-1) 6.25
(H-3) 7.5 (Aromatic H) UV, 2 max: l 93 nm (g 745
0) 232 nm (g 1320) Elemental analysis ("13H1707N): Calculated value (a) Actual value 驎N 4.51 4.68 Reference example 2 (3-!2-(α-naphthylcarbamoyl)-P-glucose ( Synthesis of 1)] H-C=0 H-C-OH H-C-OH OH20H 1,2,5,6-di-9-impropylidene-α-♀-
Glucofuranose (6,5f)・Anhydrous pyridine (40
After stirring and reacting α-naphthylisonanate (8,2d) at room temperature for 5 hours with continuous light, the solvent was distilled off under reduced pressure, and the resulting syrup was subjected to silica gel column chromatography (benzene column chromatography). :Ethyl acetate, 5:lV/V
), 0.55 fractions were collected and concentrated under reduced pressure to give 1,2,5.6-di-9-imbropian-3-0-(α-naphthylcarbamoyl)-α-D. −
Glucofuranose (nrap; [α]0+72, ν
max3300.2980.1780crn.

PM le (CDCl!,) δ1.2, 1,3.1.5.
1.6.5.9, 7.5 ppm) was obtained in 96% yield. This compound (4f) was dissolved in 10% acetone aqueous solution 1
00-(: Kendakushi, I 120B (H+) (3F
) was stirred at 97°C for 10 hours, the resin was filtered, the liquid was concentrated under reduced pressure, and silica gel chromatography was performed to obtain R,0,21 (chloroform; methanol, 6:
The fractions showing IV/V) were collected, and the desired product was obtained with a yield of 82%.

(It, Ri: 196~198℃ [α.): +13.5°~+11.2゜ymax(i'
]: 3500% 1720PMR (DMSO) δ(p
pm): 4.6B, 5.21.7.5UVλmax
: 222 nm (g 45700), 287 n
m (ε4850) Elemental analysis (C17H1,07N): Calculated value (a) Actual value (to) N 3.95 3.99 Reference example 3 (3-0-(α-naphthylcarbamoyl)-P-allose (1) Synthesis] H-C=0 H-C-OH H-C-OH OH20H 1,2,5,6-di-9-isopropylidene-α-♀-
Allofuranose (5,5f), anhydrous pyridine (40
After reacting 7ri・α-naphthalene) (8,2-) with stirring at room temperature for 10 hours under continuous light, the solvent was distilled off under reduced pressure, and the resulting wrap was subjected to phosphor gel chromatography. (benzene: ethyl acetate, '5: IV
/V) l"l-, both fractions of RFO and 39 were collected, and vacuum am was applied to obtain 1,2°5.6-di-9-isopropylidene-3-9-(α-naphthylcarbamoyl). )-α-D-7cr furanose (unlap; [α),
+71.40; νmax3300-2980-17
80 cm-”: PMR(CDCj3)δ1.
2.1.3.1.5.1.6.5.9.7.5 ppm
) was obtained in a yield of 977o. This compound (4f) is 1
Kendaku in 0% acetone aqueous solution 100-, L-120
B (H+) (3t) was added, stirred at 85°C for 5 hours, the resin was filtered, the liquid was concentrated under reduced pressure, and R was purified by silica gel chromatography.

0.27 (chloroform:methanol, 6:IV/V
) were collected, and the desired product was obtained in a yield of 60%.

m, p,: 89-91°C [α,): +8.2°-3.3°! / max (z-'): 3500.1720
PM (DMSO-d,) δ (ppm):
4.68.5.22.7.5 Elemental analysis (C17H19
0□N): Calculated value (Actual value (a) N 3.99 3.99 Reference example 4 (Synthesis of 5-0-(α-naphthylcarbamoyl)-P-glucuronic acid (ff)) H-C= 0 H-C-OH HCO-Co-NH-C1゜R7 0OH 3,6-D-glucuronolactone (10F) to 400-
of anhydrous acetone (=dissolved by heating, lR12゛○B(l(
'+) (6f) was added and heated under reflux for 15 hours. After the reaction, 1R120B (H+) was filtered off, concentrated, and extracted with chloroform, which was distilled off under reduced pressure, and the syrup was dissolved in Shaolong's acetic ether and added with hexane for crystallization.1.
2-0-inpropylidene-3,6-D-glucuronolactone (m, p.

120-121℃: [α)I) + 7.0'; v
max3380.1770 crn-') was obtained in a yield of 5o%. This compound (3,3F) and α-naphthylisonanate (6,5G) were mixed with anhydrous pyridine (40d).
1.2-0-
Isopropylidene-5-0-(α-naphthylcarbamoyl)-3,5-D-glucuronolactone (m, p, 1
74-176℃; νmax3420.1800-172
0 crpr , P M R (CDC4) δ7, day,
5.2.5.1, 4.9.1.5.1.3 ppm
: [α), +75°) was obtained in 80% yield. This compound (3f) was dissolved in a 10% acetone aqueous solution (10% acetone aqueous solution), and 1R12oB(H+) (1t) was added thereto at 80℃ for 2 hours.
After 4 hours of hydrolysis and reaction, the resin was filtered and treated with decolorizing charcoal.
Concentrate under reduced pressure and crystallize the product from hot ethyl acetate to give 5-
0-(α-naphthylcarbamoyl)-D-glucuronic acid was obtained with a yield of 80%.

mJl,: 189~190℃ [α,) Knee 25°~-7.5゜W maX (cfn-”): 3480.17
30PMR(I)MSO-d, )δ(9tm):
8.1% 7.6, 5.6 Reference Example 5 (5-0-phenylcarbamoyl-P~rehose (v)
Synthesis] H-C=0 summer-C-OH H-C-OH (v) -C-OH H-C-0-Co-zu-C6H5 p-ribose (10f)/anhydrous acetone-anhydrous) 9/
-/l/mixture (1/l, V/v) 75 mt.

El 120B (H+) (5r) was reacted under reflux for 2 hours. The resin was filtered off, and the filtrate was concentrated under reduced pressure to obtain a lump. Further, extract and purify with chloroform to obtain methyl 2.
3-0-isopropylidene-β 60%
Obtained from Yield 1. This compound 1f and 2.5 tnt of phenylisonanate were reacted in 30-mer pyridine with stirring at room temperature for 10 hours, and after distilling off the solvent, silica gel chromatography (benzene:acetic acid ether; 5:1V/
V) Both fractions of teRFO,65 were collected and methyl 2.3-0-isopropylidene-5-0-phenylcarbamoyl-β-p-ribofuranoside ([α)D-
33,6°; y max 3420%17006n
, PMR(CDCt3) δ 7.4.4.9.
3.4.1.5, 1.629 m) was obtained. This compound 1f was dissolved in a 10% acetone aqueous solution (Nikentakushi, 1R1
After adding 2OB(H+) (200++v) and heating under reflux for 10 hours, the resin was filtered off, the filtrate was concentrated under reduced pressure, and crystallized from ethyl acetate to obtain the desired product in a yield of 80%.

m, P,: 105 ~ 107 °C [α,): + 20' ~ + 215.3 °W
InaX (tyy(-1): 3460 m 34
20 s 303051700, 1230 PMR (DMSO-d, ) δ (ppm): 7.
4.5.02 Elemental analysis (01□H1, No, ): Calculated value Actual value ~ N 5.20 5.30 Reference example 6 (5-0-(α-naphthylcarbamoyl) once-liposu ] Methyl 2 used in the preparation of H-C=0 H-C-OH (V) 3-0-isopropylidene-β-■-ribofuranondo (4f), α-naphdylisonanate (5 mA), pyridine After stirring (50-) at room temperature for 15 hours in the absence of light, the solvent was distilled off and a syrup was prepared using methyl 2.3-0-isopropylidene-5.
-2-(α-Napht-rucarpamoyl)-β once restarted (v max 3440.1750,1240z-1
; (α]0-36.6°: PMJCDC/3) δ7
．． 6.4.98.3.28.1.4.1.2 ppm
) was obtained in 100% yield. This compound 5f was dissolved in a 10% acetone aqueous solution (Kentakushi, 1R120B(H”)).
2F) and heated under reflux at 900°C for 5 hours to react.
Thereafter, the resin was filtered off, the filtrate was concentrated, and silica gel column chromatography (chloroform:methanol;
1 O: IV/V) TeRFO, 35 fractions were purified, concentrated, and crystallized from ethyl acetate to obtain 85% of the target product.
yield was obtained.

m, p,: 113~114°C [α)D: +14.3°~+18.6°νmax
(Confucian-'): 3450.3400.30301PMR(
DM80-d,) a (pPm): 7.4.5.
02 elemental analysis (C1, H engineering, No.): Calculated value (to) Actual value (to) N 4,39 4.34 Reference example 7 [Oxygen radical generation ability of compound 1-11] 5 × 10 M
Butoclo-4C solution (pH 8, 0° 0.2M phosphate buffer) (1 mM (10-''M) of the two compounds was dissolved,
Immediately C:, when the increase in absorbance at 550 nm (7) at 25°C was examined, I: 6.95XIO-4; 1:2.
85X10, summer: 5,62Xl
○.

ff: 1.44X10, V: 5,67X10.

A result of w: 3,09XIO (Δλ550/m1n) was obtained. Grape dumplings (,H
-glucose) and P-ribose are each 0.75
I O-'# and 1.5 X I O-' (20
% ethanol solution).

Reference Example 8 [In vitro nucleic acid cleavage activity of compounds T, V and M] Compounds 1, V and K in phosphate buffer (pH 7, 2)
, double-stranded φx 174 DNA 200 nf,
When treated with 1 mM of sugar and 10 pM of Cu'' at 37°C for 3 hours, the production of wood-like single-site cleavage (Form I) and linear DNA (Forml) was examined, and the results were 18.0%; 86.2.8.4%; 84.
It was 3.5.8%.

Example 1 [In vitro virus inactivation effect of compounds 1 to 1 page] The in vitro virus inactivation effect of compounds i to M was demonstrated using tobacco mosaic virus and bacteriophage φ×1.
The study was conducted using 74. The former is a phosphate buffer (0.2M
, pH 8.1), sugar concentration 10"M, Cu"I
Q-' M was used to react with the virus at 37°C for 3 hours, and the survival rate (a) of the virus was assayed by the fresh leaf method using N and tabacum.

The latter was carried out at a sugar concentration of 10 −3 M in phosphate buffer, Cu10
φX174 was treated with M at 37℃ for 3 hours, and 0 hours of treatment (
The rate of plaque formation for the two was defined as the survival rate (a). The inactivation of Compounds 1, ■, and ■ against TMV was demonstrated with survival rates of 51.14% and 60%, respectively.

On the other hand, compounds ①, V, and W showed 33.8%, 38.4%, and 44.2% of φX174, respectively.

The above three in vitro reactivity and biological activity results←
Reference Example 6.7 and Example 1) show that these derivatives have greater reactivity or biological activity than the unsubstituted 451 (aldohexose) (2 is absent or unsubstituted). .

that's all

Claims (2)

[Claims] (1) A virus inactivator containing ¥O¥-arylcarbamoylaldose as an active ingredient. (2) The virus inactivating agent according to item 1, wherein the aldose moiety is a pentose or a hexose.