JP3286340B2 - Sialic acid derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel chemically and enzymatically stable sialic acid derivative which is useful as a drug such as a therapeutic agent for neurological diseases.

[0002]

2. Description of the Related Art Sialic acid contained in gangliosides and glycoproteins is present on the cell surface of cells or bacteria, and has recently become a medical substance as a substance involved in immunity, cancer, inflammation, viral infection, cell differentiation, hormone receptors and the like. In addition, pharmacologically, various derivatives containing sialic acid have been synthesized. It has been reported that these physiologically active sialic acid derivatives have pharmacological effects such as a therapeutic effect on a neuropathy disease, a cancer metastasis inhibitory effect, an anti-inflammatory effect, and a demyelinating disease therapeutic effect.

[0003]

An object of the present invention is to provide a novel chemically and enzymatically stable sialic acid derivative having a hydroxyl group at the 3-position.

[0004]

The compound of the present invention is a sialic acid derivative containing a group represented by the following general formula (I).

Embedded image In the above general formula, X is hydrogen, methyl, ethyl, n-
Carbon number 1 such as propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, etc.
4 to lower alkyl or benzyl, Y represents hydrogen or an acetyl group, preferably hydrogen, and Ac represents an acetyl group. The wavy line indicates that both the α-anomer form and the β-anomer form are included.

[0005] Many substances containing sialic acid as a constituent such as ganglioside, sialosyl cholesterol, sialosylglycerolipids, sialosylceramides, sialoglycoprotein, etc., are present in nature or chemically synthesized. Is normal sialic acid in which the 3-position is hydrogen. On the other hand, the sialic acid contained in the compound of the present invention has a hydroxyl group at the 3-position, and the sialic acid derivative composed of such 3-hydroxysialic acid is a novel compound.

The compound of the present invention can be specifically represented by the following general formula (II). Wherein, X is the same as the general formula (I), R is alkyl
Luglycerol, dialkylglycerol or diacyl
Represents glycerol .

The sialic acid derivative of the present invention includes a pharmaceutically acceptable salt of the compound represented by the general formula (I), for example, an alkali metal such as sodium and potassium;
Alkaline earth metals such as calcium, magnesium and barium, and other metal salts with aluminum or the like, or addition salts with acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, citric acid and lactic acid, or ammonia And salts with organic bases such as organic amines. These salts can be produced from the free sialic acid derivative of the present invention by known methods, or can be mutually converted. When an optical isomer is present in the compound of the present invention, the present invention includes any of the isomers.

Next, a method for producing the compound of the present invention will be described. For example, 5-acetamide-4,7,8,9-tetra-O-acetyl-2-halogeno-2,5-dideoxy-β-D-erythro-L-gluco-2-nonulopyranosonate alkyl or benzyl The compound of the present invention can be produced by a glycosidation reaction using an ester as a donor. Examples of the alkyl ester include methyl, ethyl, n-propyl, iso-propyl, n-butyl,
Examples thereof include esters of lower alkyl groups having 1 to 4 carbon atoms, such as iso-butyl, sec-butyl, and t-butyl.
The halogen at the 2-position is fluorine, chlorine, bromine, iodine or the like, with bromine being preferred. As an acceptor to be reacted with the glycosidation reaction donor, a compound having a hydroxyl group represented by the general formula R-OH (R is the same as in the above general formula (II))
Can be used.

The glycosidation reaction can be performed under the following reaction conditions. As the solvent, an appropriate solvent which does not hinder the reaction such as toluene, benzene or a mixed solvent of toluene and 1,2-dichloroethane can be used, the catalyst is silver trifluoromethanesulfonate, the neutralizing agent is disodium phosphate, phosphoric acid. Dialkali phosphate such as dipotassium or silver carbonate is preferred. The reaction temperature can be appropriately set depending on the target substance because the production ratio of the α-anomer and the β-anomer changes depending on the temperature. If the reaction time is long, decomposition of the product may occur due to side reactions, so that about 5 minutes to 1 hour is sufficient.

Also, 5-acetamide-4,7,8,9-tetra-O-acetyl-2,3-anhydro-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranoso. The glycosidation reaction using an alkyl or benzyl ester as a donor gives β
-Anomers can be selectively produced. Examples of the alkyl ester include the same lower alkyl esters as described above, and the reaction conditions such as a solvent and a catalyst in the glycosidation reaction can be appropriately set according to the above method. Methods for producing these glycosidation reaction donors and the like are described in the literature. [Okamoto et al., Bull. Chem. Soc. Jpn., 60, 631-
636 (1987))

Deacetylation after completion of the glycosidation reaction is carried out in a suitable solvent such as methanol or ethanol in the presence of a basic catalyst such as an alkyl metal alkoxide such as potassium t-butoxide or sodium methoxide. Can be used. It may be necessary to protect a group that does not participate in the reaction of the glycosidation reaction donor.For example, when glucose is used as a donor, it is preferable to protect a hydroxyl group other than a hydroxyl group that reacts with the acceptor with a benzyl group or the like. . In addition, the donor may be appropriately protected with an appropriate protecting group, and the protecting group may be removed according to a usual method used in the art. For example, debenzylation includes a method of catalytic reduction in a suitable solvent such as methanol in the presence of palladium-carbon.

Further, the sialic acid derivative of the present invention wherein X is lower alkyl or benzyl in the above general formula (I) can be easily converted to the compound of the present invention wherein X is hydrogen by a usual hydrolysis treatment. The compound produced in the present invention is purified by ordinary means such as distillation, chromatography, recrystallization and the like, and TLC, elemental analysis, melting point measurement, specific rotation (sodium D line), IR, NMR, UV, mass spectrum The identification was performed by the above method.

[0013]

【Example】

Embodiment 1 FIG. (1) 6.6 g of methyl 5-acetamide-4, 7, 8,
9-tetra-O-acetyl-2-bromo-2,5-dideoxy-β-D-erythro-L-gluco-2-nonulopyranosonate (raw material compound A), 5.0 g of 5-cholesten-3β -All, 6.6 g of anhydrous disodium hydrogen phosphate and 140 ml of dry benzene were mixed and stirred.
To this was added 3.1 g of silver trifluoromethanesulfonate dissolved in 90 ml of dry benzene at room temperature under an argon atmosphere. After stirring for 10 minutes, insolubles were removed by filtration through a filter aid and washed with ethyl acetate. The combined filtrate and washings were washed with 5% aqueous sodium thiosulfate, 5% aqueous sodium bicarbonate, and saturated saline, and then evaporated to dryness. (5-cholesten-3β-yl 5-acetamide-4,7,8,9-tetra-O-acetyl-5-deoxy-D-erythro-L-gluco-2-nonulopyranoside) onate is converted to an α-anomer and β -Anomer mixture (α: β = 6: 1). Yield: 61% Elemental analysis: as C ₄₇ H ₇₃ NO ₁₄

(2) 2.2 g of the above product and a catalytic amount of potassium t-butoxide were dissolved in 100 ml of anhydrous methanol and stirred at room temperature for 1 hour under an argon atmosphere. After neutralization by adding a cation exchange resin, the resin was filtered off and washed with methanol. The filtrate and the washing solution were combined and concentrated under reduced pressure.
Recrystallization from methanol-ether gave methyl (5-cholesten-3β-yl 5-acetamido-5-deoxy-).
An α-anomeric form of D-erythro-L-gluco-2-nonulopyranoside) onate was obtained as white crystals. After concentrating the recrystallized filtrate, the β-anomer and the remaining α-anomer were separated and purified using a silica gel column. (Α-anomer) Yield: 80% Melting point: 229-231 ° C [α] ²⁵ : -23.7 ° (C = 0.65, chloroform / methanol = 5
/ 1) IR (KBr): 3470, 3400, 2950, 1730, 1620, 1548, 14
35, 1373, 1315, 1070, 1033 cm ^-1 Mass (SIMS): m / z 708 (M + H), 322 (M-cholesterol + 1) Elemental analysis: As C ₃₉ H ₆₅ NO ₁₀ ¹ H-NMR (CDCl ₃ / CD ₃ OD = 1/1, δ): cholesterol moiety
0.69 (s, 3H), 0.87 and0.88 (2d, J = 6.6Hz, 2x3H), 0.93 (d,
J = 6.4Hz, 3H), 1.00 (s, 3H), 3.68-3.75 (m, 1H), 5.31-5.3
5 (m, 1H); Neu5Ac moiety 2.05 (s, 3H), 3.47 (dd, J = 1.2,
8.4Hz, 1H), 3.61 (d, J = 9.1Hz, 1H), 3.77 (dd, J = 8.2,9.1H
z, 1H), 3.81 (s, 3H), 3.93 (dd, J = 1.2,8.2Hz, 1H), 3.96 (d
d, J = 8.2,8.2Hz, 1H) (β-anomer) Yield: 13% Melting point: 272-275 ℃ [α] ²⁵ : -53.7 ° (C = 0.51, chloroform / methanol = 5
/ 1) IR (KBr): 3420, 2920, 1740, 1614, 1570, 1430, 13
80, 1315, 1095,1024 cm ^-1 Mass (SIMS): m / z 708 (M + H), 322 (M-cholesterol + 1) Elemental analysis: As C ₃₉ H ₆₅ NO ₁₀ ¹ H-NMR (CDCl ₃ / CD ₃ OD = 1/1, δ): cholesterol moiety
0.69 (s, 3H), 0.86 and0.87 (2d, J = 6.6Hz, 2x3H), 0.93 (d,
J = 6.5Hz, 3H), 1.01 (s, 3H), 3.65-3.74 (m, 1H), 5.25-5.2
9 (m, 1H); Neu5Ac moiety 2.04 (s, 3H), 3.44 (dd, J = 0.5,
8.8Hz, 1H), 3.55 (d, J = 9.0Hz, 1H), 3.83 (s, 3H), 3.84 (d
d, J = 9.0,9.1Hz, 1H), 3.92 (dd, J = 0.5,8.5Hz, 1H), 3.95
(dd, J = 8.5,9.1Hz, 1H)

(3) 1.2 g of the α-anomer compound, 1
After mixing 0 ml of 1N sodium hydroxide aqueous solution and 200 ml of methanol and stirring at room temperature for 2 hours, a cation exchange resin was added at 0 ° C. for neutralization. After the resin was filtered off and washed with methanol, the filtrate and the washing were combined and concentrated. The obtained solid crude product was washed with ether and recrystallized from methanol-chloroform-hexane to give 5-
Cholesten-3β-yl 5-acetamide-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosidonic acid (Compound 1α) was obtained as white crystals. Yield: 97% Melting point: 191-193 ° C [α] ²⁵ : -21.4 ° (C = 0.53, chloroform / methanol = 1
/ 1) IR (KBr): 3250, 2945, 1715, 1620, 1560, 1460, 13
68, 1103, 1066, 1021 cm ^-1 Mass (SIMS): m / z 694 (M + H), 308 (M-cholesterol + 1) Elemental analysis: As C ₃₈ H ₆₃ NO ₁₀ ¹ H-NMR (CD ₃ OD, δ): cholesterol moiety 0.71 (s, 3
H), 0.87 and 0.88 (2d, J = 6.5Hz, 2x3H), 0.94 (d, J = 6.4H
z, 3H), 1.02 (s, 3H), 3.67-3.76 (m, 1H), 5.33-5.37 (m, 1
H); Neu5Ac moiety 2.02 (s, 3H), 3.49 (dd, J = 1.5,8.7H
z, 1H), 3.56 (d, J = 9.5Hz, 1H), 3.64 (dd, J = 6.3, 12.1Hz, 1
H), 3.70 (dd, J = 9.5, 10.2Hz, 1H), 3.81 (dd, J = 12.1Hz, 1
H), 3.82 (dd, J = 1.5, 10.3Hz, 1H), 3.82 (ddd, J = 6.3,8.7
Hz, 1H), 3.93 (dd, J = 10.2, 10.3Hz, 1H)

Similarly, the β-anomeric 5-cholesten-3β-yl 5-acetamido-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranoside acid (compound 1β) was obtained. Yield: 97% Melting point: 193-195 ° C [α] ²⁵ : -56.9 ° (C = 0.54, chloroform / methanol = 1
/ 1) IR (KBr): 3400, 3260, 2920, 1757, 1618, 1578, 13
75, 1160, 1085, 1020 cm ^-1 Mass (SIMS): m / z 694 (M + H), 308 (M-cholesterol + 1) Elemental analysis: As C ₃₈ H ₆₃ NO ₁₀ ¹ H-NMR (CD ₃ OD, δ): cholesterol moiety 0.71 (s, 3
H), 0.87 and 0.88 (2d, J = 6.6Hz, 2x3H), 0.94 (d, J = 6.5H
z, 3H), 1.02 (s, 3H), 3.73-3.81 (m, 1H), 5.26-5.31 (m, 1
H); Neu5Ac moiety 2.00 (s, 3H), 3.43 (d, J = 9.1Hz, 1H),
3.48 (dd, J = 0.5, 9.0Hz, 1H), 3.64 (dd, J = 4.8, 11.0Hz, 1
H), 3.75 (ddd, J = 2.5, 4.8, 9.0Hz, 1H), 3.79 (dd, J = 2.5,
11.0Hz, 1H), 3.85 (dd, J = 9.1,9.2Hz, 1H), 3.96 (dd, J = 8.
5, 9.2Hz, 1H), 3.99 (dd, J = 0.5,8.5Hz, 1H)

Embodiment 2 FIG. Starting compound A and methyl 2,
3,4-tri-O-benzyl-α-D-glucoside is
Example 1 under silver trifluoromethanesulfonate catalyst
The same glycosidation reaction as in (1) was performed. Obtained α
-Anomer and β-anomer were prepared in Example 1 respectively.
After deacetylation in the same manner as in (2), debenzylation was performed by catalytic reduction according to a conventional method. Next, Example 1
After hydrolyzing the methyl ester in the same manner as (3),
The following compounds were obtained after neutralization with an acidic resin. Methyl 6-O- (5-acetamido-5-deoxy-α-
D-erythro-L-gluco-2-nonulopyranosylonate) -α-D-glucopyranoside (compound 2α) Melting point: 85-87 ° C [α] ²⁵ : + 26.8 ° (C = 1.1, H ₂ O ) IR (KBr): 3350, 1735, 1640, 1555, 1370, 1030 cm
^{-1 Mass (SIMS): m /} z 502 (M + H), 308 (M-Glc) Elementary analysis: as _{C 18 H 31 NO 15 · 3H} 2 O ¹ H-NMR (D ₂ O / TSP, δ): Glc moiety 3.41 (s, 3H), 3.47
(dd, J = 9.2, 9.3Hz, 1H), 3.56 (dd, J = 3.7, 9.8Hz, 1H), 3.
64 (dd, J = 9.2,9.8Hz, 1H), 3.76 (ddd, J = 9.3Hz, 1H), 3.98
(m, 2H), 4.78 (d, J = 3.7Hz, 1H); Neu5Ac moiety 2.03 (s, 3
H)

Methyl 6-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonate) -α-D-glucopyranoside (compound 2β) Melting point: 79 − 81 ° C [α] ²⁵ : + 17.7 ° (C = 1.1, H ₂ O) IR (KBr): 3400, 1730, 1635, 1550, 1368, 1070, 10
28 cm ^-1 Mass (SIMS): m / z 502 (M + H), 524 (M + Na), 308 (M-Gl
c) Elementary analysis: as _{C 18 H 31 NO 15 · 2H} 2 O ¹ H-NMR (D ₂ O / TSP, δ): Glc moiety 3.41 (s, 3H), 3.61
(dd, J = 3.8,8.9Hz, 1H), 3.77 (ddd, J = 1.3,3.5Hz, 1H),
3.85 (dd, J = 3.5,10.6Hz, 1H), 3.92 (dd, J = 1.3,10.6Hz, 1
H), 4.81 (d, J = 3.8Hz, 1H); Neu5Ac moiety 2.05 (s, 3H),
3.54 (d, J = 9.1Hz, 1H), 3.83 (m, 1H), 3.84 (d, J = 9.8Hz, 1
H), 3.90 (dd, J = 9.8, 10.3Hz, 1H), 3.98 (d, J = 10.6Hz, 1
H), 4.06 (dd, J = 10.3,10.6Hz, 1H)

Embodiment 3 FIG. Example 1 was carried out using starting compound A and 1,2 (or 2,3) -di-O-alkyl-sn-glycerol in the presence of a silver trifluoromethanesulfonate catalyst.
The same glycosidation reaction as in (1) was performed to obtain a mixture of an α-anomer and a β-anomer. Although this mixture could not be separated by silica gel chromatography, benzoyl chloride was reacted with pyridine in a pyridine solvent at room temperature by argon vaporization to benzoylate the hydroxyl group at the 3-position. Could be separated. Separated α
-Anomer and β-anomer are converted to Zem according to a conventional method.
After deacylation by the plen method, Example 1
The methyl ester was hydrolyzed in the same manner as in (3), and then neutralized with an acidic resin to obtain the following compound.

3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -1,2-di-O-octyl-sn-glycerol (compound 3α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-octyl-sn-glycerol (compound 3β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-decyl-sn-glycerol (compound 4α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-decyl-sn-glycerol (compound 4β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-dodecyl-sn-glycerol (compound 5α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-dodecyl-sn-glycerol (compound 5β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-tetradecyl-sn-glycerol (compound 6α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-tetradecyl-sn-glycerol (compound 6β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-hexadecyl-sn-glycerol (compound 7α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-hexadecyl-sn-glycerol (compound 7β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-octadecyl-sn-glycerol (compound 8α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-octadecyl-sn-glycerol (compound 8β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
2,3-di-O-tetradecyl-sn-glycerol (compound 9α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
2,3-di-O-tetradecyl-sn-glycerol (compound 9β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-tetradecyl-rac-glycerol (compound 10α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)-
1,2-di-O-tetradecyl-rac-glycerol (compound 10β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -1
-O-dodecyl-2-O-tetradecyl-sn-glycerol (compound 11α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)- 1
-O-dodecyl-2-O-tetradecyl-sn-glycerol (compound 11β) 3-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid)- 2
-O-dodecyl-1-O-tetradecyl-sn-glycerol (compound 12α) 3-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid)- 2
-O-dodecyl-1-O-tetradecyl-sn-glycerol (compound 12β)

The physical properties of the above compound obtained in Example 3 are shown in Tables 1 and 2.

[Table 1]

[Table 2]

Embodiment 4 FIG. The raw material compound A and 3-O-alkyl-sn-glycerol were subjected to a glycosidation reaction in the same manner as in Example 1 (1) under a silver trifluoromethanesulfonate catalyst to give a mixture of α-anomer and β-anomer. Obtained. This mixture could not be separated by silica gel chromatography, but was treated in acetonitrile with 4-
By treating with dimethylaminopyridine and lactonizing in the molecule, both anomers could be separated by silica gel chromatography. Separated α
-Anomer and β-anomer are converted to Zem according to a conventional method.
After deacylation by the plen method, Example 1
The methyl ester was hydrolyzed in the same manner as in (3), and then neutralized with an acidic resin to obtain the following compound. 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-dodecyl-sn-glycerol (compound 13α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-dodecyl-sn-glycerol (compound 13β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-tetradecyl-sn-glycerol (Compound 14
α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-tetradecyl-sn-glycerol (Compound 14
β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-hexadecyl-sn-glycerol (compound 15
α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-hexadecyl-sn-glycerol (compound 15
β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-octadecyl-sn-glycerol (compound 16
α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-octadecyl-sn-glycerol (compound 16
β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-Icosyl-sn-glycerol (compound 17α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-Icosyl-sn-glycerol (compound 17β) 1-O- (5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-docosyl-sn-glycerol (compound 18α) 1-O- (5-acetamido-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3
-O-docosyl-sn-glycerol (compound 18β)

The physical properties of the above compound obtained in Example 4 are shown in Table 3.

[Table 3]

Embodiment 5 FIG. The raw material compound A and the alkyl alcohol were subjected to glycosylation and deacetylation in the same manner as in Example 1, followed by hydrolysis of the methyl ester. In this reaction, the α-anomer was synthesized predominantly, and the following compound was obtained. Tetradecyl 5-acetamide-5-deoxy-α-D-
Erythro-L-gluco-2-nonulopyranosylonic acid (compound 19α) hexadecyl 5-acetamide-5-deoxy-α-D-
Erythro-L-gluco-2-nonulopyranosylonic acid (compound 20α)

Example 6. Uridine in which the 2 ', 3'-hydroxyl group was protected with a t-butyldimethylsilyl group and starting compound A
Was subjected to the same glycosidation reaction as in Example 1 (1).
A mixture of the obtained α-anomer and β-anomer is
After acetylation by the action of acetic anhydride in pyridine,
Both anomers were separated by silica gel chromatography. The separated α-anomer and β-anomer were deacylated by the Zemplen method according to a conventional method, and then desilylated by tetra-n-butylammonium fluoride. Next, the methyl ester was hydrolyzed in the same manner as in Example 1 (3) to obtain the following compound. 5'-O- (5-acetamido-5-deoxy-α-D-
Erythro-L-gluco-2-nonulopyranosylonic acid)-
Uridine (compound 21α) 5′-O- (5-acetamido-5-deoxy-β-D-
Erythro-L-gluco-2-nonulopyranosylonic acid)-
Uridine (compound 21β)

Embodiment 7 FIG. (1) 1.0 g of compound 1α was suspended in 100 ml of ethanol, and 121 mg of sodium hydrogen carbonate dissolved in 10 ml of water was added at room temperature. After stirring for 0.5 hour and the reaction solution became transparent, it was concentrated to obtain a syrupy crude product. This was dissolved in 100 ml of water, filtered through a microfilter, and the filtrate was concentrated to dryness to give sodium (5-cholesten-3β-yl 5-acetamide-5-yl).
Deoxy-α-D-erythro-L-gluco-2-nonulopyranoside) onate (compound 22α) was obtained as an amorphous powder. Yield: 95% Melting point: 201-205 ° C [α] ²⁵ : -14.9 ° (C = 0.2, CH ₃ OH) IR (KBr): 3380, 2930, 1615, 1555, 1460, 1430, 13
78, 1270, 1112, 1072, 1020 cm ^-1 Mass (SIMS): m / z 716 (M + Na) Elemental analysis: as C ₃₈ H ₆₂ NO ₁₀ Na ¹ H-NMR (CD ₃ OD, δ): cholesterol moiety 0.70 (s, 3
H), 0.87 and 0.88 (2d, J = 6.6Hz, 2x3H), 0.93 (d, J = 6.5H
z, 3H), 1.00 (s, 3H), 3.83-3.90 (m, 1H), 5.33-5.39 (m, 1
H); Neu5Ac moiety 2.04 (s, 3H), 3.37 (d, J = 9.6Hz, 1H),
3.52 (dd, J = 1.7,8.9Hz, 1H), 3.54 (dd, J = 9.6,9.6Hz, 1
H), 3.58 (dd, J = 1.7,10.5Hz, 1H), 3.66 (dd, J = 5.2,11.4
Hz, 1H), 3.81 (dd, J = 9.6,10.5Hz, 1H), 3.84 (dd, J = 2.5,1
1.4Hz, 1H), 3.88 (ddd, J = 2.5,5.2,8.9Hz, 1H)

(2) Compound 1β was used in place of compound 1α, and sodium (5-cholesten-3β-yl 5-acetamido-5-deoxy-β-D-erythro-
L-gluco-2-nonulopyranoside) onate (compound 22β) was obtained. Yield: 92% Melting point: 226-229 ° C [α] ²⁵ : -51.8 ° (C = 0.5, CH ₃ OH) IR (KBr): 3400, 2925, 1618, 1550, 1460, 1430, 13
75, 1160, 1063, 1030 cm ^-1 Mass (SIMS): m / z 716 (M + Na) Elemental analysis: As C ₃₈ H ₆₂ NO ₁₀ Na ¹ H-NMR (CD ₃ OD, δ): cholesterol moiety 0.71 (s, 3
H), 0.87 and 0.88 (2d, J = 6.5Hz, 2x3H), 0.93 (d, J = 6.4H
z, 3H), 1.00 (s, 3H), 3.65-3.72 (m, 1H), 5.27-5.32 (m, 1
H); Neu5Ac moiety 1.97 (s, 3H), 3.37 (d, J = 9.3Hz, 1H),
3.39 (dd, J = 0.5, 9.1Hz, 1H), 3.66 (dd, J = 4.7,11.4Hz, 1
H), 3.77 (ddd, J = 3.0, 4.7, 9.1Hz, 1H), 3.80 (dd, J = 3.0,
11.4Hz, 1H), 3.85 (dd, J = 9.3.9.5Hz, 1H), 3.97 (dd, J =
0.5, 9.8Hz, 1H), 4.03 (dd, J = 9.5,9.8Hz, 1H)

(3) The compounds (compounds 3α to 21β) obtained in Examples 3 to 6 were treated in the same manner as described above to obtain the following compounds. 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-octyl-sn-glycerol (compound 23α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-octyl-sn-glycerol (compound 23β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-decyl-sn-glycerol (compound 24α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-decyl-sn-glycerol (compound 24β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-dodecyl-sn-glycerol (compound 25α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-dodecyl-sn-glycerol (compound 25β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-tetradecyl-sn-glycerol (compound 26α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-tetradecyl-sn-glycerol (compound 26β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-hexadecyl-sn-glycerol (compound 27α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-hexadecyl-sn-glycerol (compound 27β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-octadecyl-sn-glycerol (compound 28α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-octadecyl-sn-glycerol (compound 28β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -2,3-di-O-tetradecyl-sn-glycerol (compound 29α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -2,3-di-O-tetradecyl-sn-glycerol (compound 29β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-tetradecyl-rac-glycerol (compound 30α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1,2-di-O-tetradecyl-rac-glycerol (compound 30β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -1-O-dodecyl-2-O-tetradecyl-
sn-glycerol (compound 31α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -1-O-dodecyl-2-O-tetradecyl-
sn-glycerol (compound 31β) 3-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -2-O-dodecyl-1-O-tetradecyl-
sn-glycerol (compound 32α) 3-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -2-O-dodecyl-1-O-tetradecyl-
sn-glycerol (compound 32β) 1-O- (sodium 5-acetamido-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-dodecyl-sn-glycerol (compound 33α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonic acid) -3-O-dodecyl-sn-glycerol (compound 33β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-tetradecyl-sn-glycerol (compound 34α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-tetradecyl-sn-glycerol (compound 34β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-hexadecyl-sn-glycerol (compound 35α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-hexadecyl-sn-glycerol (compound 35β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-octadecyl-sn-glycerol (compound 36α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-octadecyl-sn-glycerol (compound 36β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-icosyl-sn-glycerol (compound 37α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-icosyl-sn-glycerol (compound 37β) 1-O- (sodium 5-acetamide-5-deoxy-
α-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-docosyl-sn-glycerol (compound 38α) 1-O- (sodium 5-acetamide-5-deoxy-
β-D-erythro-L-gluco-2-nonulopyranosylonate) -3-O-docosyl-sn-glycerol (compound 38β) Sodium (tetradecyl 5-acetamide-5-deoxy-α-D-erythro-L -Gluco-2-nonulopyranoside) onate (compound 39α) sodium (hexadecyl 5-acetamido-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranoside) onate (compound 40α) 5′-O- [sodium ( 5-Acetamide-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosyl) onate] -uridine (compound 41α) 5′-O- [sodium (5-acetamide-5-deoxy-β-D- Erythro-L-gluco-2-nonulopyranosyl) onate] -uridine (compound 41β)

Embodiment 8 FIG. Starting compound A and 1,2-di-O
-Acyl-sn-glycerol was subjected to glycosidation in the same manner as in Example 1 (1). Next, the compound was acetylated by reacting with acetic anhydride in pyridine, and then separated into α-anomers and β-anomers by silica gel chromatography to obtain the following compounds. 3-O- [methyl 5-acetamide-3,4,7,8,9
-Penta-O-acetyl-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosyl) oate]
-1,2-di-O-dodecanoyl-sn-glycerol (compound 42α) 3-O- [methyl 5-acetamide-3,4,7,8,
9-penta-O-acetyl-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosyl) oate] -1,2-di-O-dodecanoyl-sn-glycerol (compound 42β) 3-O- [Methyl 5-acetamide-3,4,7,8,9
-Penta-O-acetyl-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosyl) oate]
-1,2-Di-O-tetradecanoyl-sn-glycerol (compound 43α) 3-O- [methyl 5-acetamide-3,4,7,8,9
-Penta-O-acetyl-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosyl) oate]
-1,2-Di-O-tetradecanoyl-sn-glycerol (compound 43β) 3-O- [methyl 5-acetamide-3,4,7,8,9
-Penta-O-acetyl-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosyl) oate]
-1,2-di-O-hexadecanoyl-sn-glycerol (compound 44α) 3-O- [methyl 5-acetamide-3,4,7,8,9
-Penta-O-acetyl-5-deoxy-β-D-erythro-L-gluco-2-nonulopyranosyl) oate]
-1,2-Di-O-hexadecanoyl-sn-glycerol (compound 44β)

The physical properties of the compounds obtained in Examples 5 to 8 are shown in Tables 4 to 7.

[Table 4]

[Table 5]

[Table 6]

[Table 7]

Example 9. 500 mg of methyl 5-acetamide-4,7,8,9-tetra-O-acetyl-2,3
-Anhydro-5-deoxy-β-D-erythro-L
-Gluco-2-nonulopyranosonate, 470 mg of 5
-Cholesten-3β-ol, 500 mg of disodium hydrogenphosphate anhydrous and 10 ml of dry 1,2-dichloroethane were mixed and stirred. To this was added 0.2 ml of trimethylsilyl trifluoromethanesulfonate at 5 ° C. under an argon atmosphere. After stirring for 12 minutes, the mixture was diluted with benzene and washed with water, saturated aqueous sodium hydrogen carbonate and saturated saline. After drying over sodium sulfate, the crude product obtained by evaporating to dryness under reduced pressure was purified by a silica gel column to give methyl (5-cholesten-3β-yl 5-acetamide-4,7,8,9- Tetra-O-acetyl-5-
Deoxy-β-D-erythro-L-gluco-2-nonulopyranoside) onate was obtained in a yield of 37%. Then
Deacetylation and hydrolysis of methyl ester were carried out in the same manner as in Examples 1 (2) and (3) to obtain compound 1β. Physical properties were consistent with those obtained in Example 1.

Example 10. 350 mg of 7-hydroxy-4-methylcoumarin sodium salt and 500 mg of the starting compound A were stirred in 5 ml of dry dimethylformamide for 1 hour at room temperature under an argon atmosphere. After evaporating the solvent under reduced pressure, water and chloroform were added to the residue and partitioned and extracted. The separated chloroform layer was washed with water, saturated saline, and dried sodium sulfate, and then concentrated. After purification on a silica gel column, hexane-ethyl acetate was added for crystallization, and methyl (4-methylcoumarin-7yl-5-acetamide-4,7,8,9-tetra-O-acetyl-5-deoxy-α -D-erythro-L-gluco-2-nonulopyranoside) onate was obtained as a white powder. This was deacetylated in the same manner as in Example 1 (2).
After hydrolyzing the methyl ester in the same manner as (3),
After neutralization with an acidic resin, 4-methylcoumarin-7yl 5-acetamide-5-deoxy-α-D-erythro-L-gluco-2-nonulopyranosidonic acid (4MU-3β-OH-
NeuAc) was obtained. Yield: 90% Melting point: 145-147 ° C [α] ²⁵ : + 43.2 ° (C = 1.1, H ₂ O) IR (KBr): 3400, 1720, 1700, 1603, 1552, 1380, 13
60, 1275, 1068, 1015 cm ^-1 Mass (SIMS): m / z 484 (M + H), 308 (M-4MU) Elemental analysis: As C ₂₁ H ₂₅ NO ₁₂

Embodiment 11 FIG. (1) Methyl 5-acetamide-4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-D-glycero-D-galact-2-nonulopyranosonate (25
Dimethyldioxirane (1.2 mol) dissolved in acetone was added to a 0 mg) dichloromethane solution (4 ml).
Added at ° C. After stirring the reaction mixture at 0 ° C. for 1 hour, it was further stirred at room temperature for 1 hour. Concentration under reduced pressure gave a solid which was recrystallized from hexane-ethyl acetate to give 25
2 mg of methyl 5-acetamide-4,7,8,9-tetra-O-acetyl-2,3-anhydro-5-deoxy-β-D-erythro-L-gluco-2-nonuropyranosonate Obtained as white crystals. According to the present method using a dialkyldioxirane such as dimethyldioxirane, the target substance can be obtained in high yield, and a purification step by chromatography or the like can be omitted.

(2) 1,2- of the above product (7.5 g)
To a dichloroethane solution (150 ml) was added a 30% hydrogen bromide acetic acid solution (3.8 ml) at room temperature. Reaction solution 1
After stirring for 0 minutes, the mixture was concentrated. The obtained residue was dissolved in ethyl acetate, and washed with sodium hydrogen carbonate and saturated saline. After drying over sodium sulfate, evaporate to dryness,
8.7 g of methyl 5-acetamide-4,7,8,9-tetra-O-acetyl-2-bromo-2,5-dideoxy-β-D-erythro-L-gluco-2-nonulopyranosonate I got Any of the compounds obtained in the above (1) and (2) can be used as a glycosidation reaction donor for producing the compound of the present invention.

[0035]

The chemical and enzymatic stability and physiological activity of the novel sialic acid derivative of the present invention are described below. (1) Chemical stability The sialic acid derivative of the present invention having a hydroxyl group at the 3-position (compound 2
α and 2β) and a known compound having no hydroxyl group were compared for chemical stability. As a target compound for comparison of the present compound 2α, methyl 6-O- (5-acetamide-
3,5-dideoxy-α-D-glycero-D-galacto-nonulopyranosylonate) -α-D-glucopyranoside, and methyl 6-O-
(5-acetamide-3,5-dideoxy-β-D-glycero-D-galacto-nonulopyranosylonate) -α-
D-glucopyranoside was used. Each test compound solution (water or 0.01 N sulfuric acid) of 250 μg / ml is put in a water bath at 80 ° C., and 5 μl is collected over time, separated by HPLC using a strongly acidic resin column, and detected by measuring UV 205 nm. The amount of sialic acid to be obtained was determined. Figure 1 shows an example of the results
And FIG.

FIG.

FIG. 2

(2) Enzymatic stability Sialidase is known as an enzyme that hydrolyzes sialic acid glycosidates such as gangliosides. So 3
The stability of a compound of the present invention containing a sialic acid having a hydroxyl group at a sialidase position was examined. Specifically, 4M
U-3β-OH-NeuAc and 4MU-NeuAc in which the 3-position is hydrogen are reacted with sialidase to produce 405n of 4-MU (4-methylumbelliferone).
The stability to sialidase was analyzed by measuring the fluorescence value at m. Since the substrate specificity of sialidase varies considerably depending on its origin, the test was performed using a total of four types of sialidases from bacteria and three sialidases from bovine brain, which are known to use most of gangliosides in human brain as substrates. went. Table 8 shows an example of the result.

[Table 8]

(3) Nerve growth promoting action Neuro2a cells (mouse neuroblastoma) purchased from ATCC were cultured in a medium obtained by adding 10% (V / V) of fetal bovine serum to Dulbecco's modified MEM. Neuro2a cells dispersed by trypsin treatment were diluted to 2 × 10 ³ cells / ml with the above medium, and this was diluted to 2 × 10 ³ cells / ml.
Each 1 ml was dispensed into a 4-well plate and cultured at 37 ° C in 5% carbon dioxide-95% air for 2 days. The culture medium of the cultured cells was removed by suction, 0.9 ml of a complete serum-free medium and 0.1 ml of an aqueous solution of a test compound were added, and the cells were cultured overnight under the same conditions. After the culture, five photographs were taken per well using a phase contrast microscope, and the ratio of cells having a projection of 50 μm or more was calculated. The nerve growth promoting effect of the test compound (mean ± standard error) was expressed as a ratio relative to the ratio of cells having processes in the control (control) as 1. In addition, the measurement results were determined by the T test, and a significant difference from the control was determined. (*: P <0.05, **: P <0.01, *
**: P <0.005)

Table 9 shows an example of the results.

[Table 9]

Further, by comparing the nerve growth promoting effect of compound 13α with compound 13α in which sialic acid at position 3 is hydrogen (subject compound), the difference due to the presence or absence of hydroxyl group at position 3 was examined. An example of the results is shown in Table 10.

[Table 10]

In general, glycosides of sialic acid are chemically unstable and difficult to handle, and natural α-glycosides are easily hydrolyzed both chemically and enzymatically (sialidase). On the other hand, the compound of the present invention containing a sialic acid in which the 3-position is a hydroxyl group is an extremely stable compound both chemically and enzymatically. As shown in FIGS. 1 and 2, α-glycosides and β-glycosides containing ordinary sialic acid are hydrolyzed with the passage of time. The compounds of the invention are chemically very stable and hardly decomposed even after 6 hours.

As shown in Table 8, 4MU-3β-OH-NeuAc containing 3-hydroxysialic acid was hardly hydrolyzed by sialidase such as sialidase from bacteria or sialidase from bovine brain, and 4MU of sialidase was used. -Did not inhibit the hydrolysis of NeuAc. As described above, it was revealed that the compound of the present invention containing sialic acid in which the 3-position is a hydroxyl group is very stable enzymatically.

Various known sialic acid derivatives have been known to have pharmacological effects such as a therapeutic effect on a neuropathy disease, an inhibitory effect on cancer metastasis, an anti-inflammatory effect, and a demyelinating disease treatment. Also, as shown in Table 10, it was revealed that the sialic acid derivative of the present invention in which the 3-position was substituted with a hydroxyl group also exhibited the same nerve growth effect as the target compound in which the 3-position was hydrogen. Moreover, 3-hydroxy sialic acid that is substantially the same conformation as the sialic acid was confirmed from the coupling constants in the ¹ H-NMR spectrum in the acetyl protecting body, which the present invention the sialic acid derivative 3-position is a hydroxyl group Positively suggests the above test results showing that the compound has the same physiological activity as a normal sialic acid-containing compound.

As described above, the sialic acid derivative of the present invention having a hydroxyl group at the 3-position has the same physiological activity as a conventional bioactive sialic acid derivative, and thus has dementia, Alzheimer's syndrome, facial paralysis, and sciatic paralysis. , Spinal muscular atrophy, muscular dystrophy, myasthenia gravis, multiple sclerosis,
Amyotrophic lateral sclerosis, acute disseminated encephalomyelitis, Guillain-Barre syndrome (polyneuropathy), post-vaccination encephalitis, sequelae of encephalitis, sequelae of head injury, cerebral ischemic disease, cerebral infarction, sequelae of cerebral hemorrhage, Compounds useful as pharmaceuticals such as therapeutic agents for peripheral or central nervous system diseases such as autonomic imbalance, diabetic neuropathy, smon or cancer metastasis inhibitors, anti-inflammatory agents, therapeutic agents for demyelinating diseases, anti-rheumatic agents, etc. It is. In addition, the compound of the present invention is chemically stable, so that it can be stored for a long period of time and is easy to handle, and does not undergo metabolism by sialidase, so that it has a long action time as compared with conventional bioactive sialic acid derivatives. Which is very useful.

[Brief description of the drawings]

FIG. 1 is a diagram showing test results comparing the chemical stability of a sialic acid derivative of the present invention having a hydroxyl group at the 3-position (α-anomeric compound) with a compound having no hydroxyl group.

FIG. 2 is a view showing the results of a test comparing the β-anomeric form of the sialic acid derivative of the present invention with the compound having no hydroxyl group at the 3-position, as in FIG.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-62-292790 (JP, A) JP-A-62-273992 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07H 15/04 A61K 31/7012 A61P 25/28 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A sialic acid derivative represented by the following general formula (II) and a pharmaceutically acceptable salt thereof. [In the formula, X is hydrogen, lower alkyl or benzyl, Y is hydrogen or acetyl group, Ac is acetyl group, R is alkyl group.
Lycerol, dialkyl glycerol or diacyl glycerol
Represents serol . ]