JPH0673084A - Glycoside and antihepatitic agent comprising the same - Google Patents

Abstract

PURPOSE:To obtain the subject new compound, composed of a glycoside having a specific molecular structure, capable of manifesting remarkable suppressing effects on hepatopathy, remarkably suppressing activities of aspartate transaminase (AST) and alanine transaminase (ALT) in a hepatopathic model induced by carbon tetrachloride with hardly any side effects and useful as an antihepatitic agent, etc. CONSTITUTION:The objective glycoside is expressed by formula III [R<1> is H, lower alkyl, (protected)monosaccharide residue or (protected)polysaccharide residue; R<2> is H, methyl or hydroxymethyl; R<3> and R<8> are OH, CH2OR<3>' (R<3>' is H or lower alkanoyl) or COOR<8>' (R<8>' is H or lower alkyl); either of R<4> and R<5>, either of R<6> and R<7> or either of R<9> and R<10> is H and the other is OH or lower alkanoyloxy; either of R<11> and R<12> or either of R<13> and R<14> is H and the other is OH, lower alkanoyloxy, etc.] is obtained by reacting a compound of formula I with a saccharide derivative such as formula II (Ac is acetyl) in methylene chloride in the presence of anhydrous calcium sulfate and a carbonate radical. This glycoside is capable of manifesting remarkable suppressing effects on hepatopathy and useful as an antihepatitic agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel glycoside and an anti-hepatitis agent containing the glycoside as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Naturally-occurring saponins are known to have various pharmacological or physiological activities, and expression of these activities requires only the structure of the saponin aglycone. Of course, it is also expected to depend on the type, number, conformation, binding mode, etc. of sugars in the molecule.

Of these, glycyrrhizin isolated from licorice, which is a crude drug, has an antiallergic action and an antiinflammatory action, and is currently used clinically as a drug for liver diseases. However, it has been reported that this glycyrrhizin causes edema and hypertension when administered to humans in large amounts (A. Kumagai, Y. Tamur.
a, C.I. Y. Ing, Contemporary Oriental Medicine, 12 , 38 (19
81)).

Therefore, there has been a demand for a drug for liver diseases in which such side effects are reduced.

[0005]

Under such circumstances, as a result of intensive studies conducted by the present inventors, the following general formula (A) was obtained.
The glycoside represented by has an excellent anti-hepatitis action,
Moreover, they have found that there are few side effects, and have completed the present invention.

That is, the present invention provides the following general formula (A):

[0007]

[Chemical 2]

[In the formula, R ¹ represents a monosaccharide residue, a disaccharide residue or a trisaccharide residue in which a hydrogen atom, a lower alkyl group or a hydroxy group may be protected by a lower alkanoyl group,
R ² represents a hydrogen atom, a methyl group or a hydroxymethyl group, R ³ and R ⁸ represent a hydroxy group, —CH ₂ OR ³ ′ or —COOR ⁸ ′ (wherein R ³ ′ represents a hydrogen atom or a lower alkanoyl group. , R ⁸ ′ represents a hydrogen atom or a lower alkyl group), and one of R ⁴ and R ⁵ , one of R ⁶ and R ⁷ , or one of R ⁹ and R ¹⁰ represents a hydrogen atom. , The other represents a hydroxy group or a lower alkanoyloxy group, and one of R ¹¹ and R ¹² or R
^{One of 13} and R ¹⁴ represents a hydrogen atom, and the other represents a hydroxy group, a lower alkanoyloxy group or a monosaccharide residue in which a hydroxy group may be protected by a lower alkanoyl group (provided that, One of which represents a hydroxy group or a lower alkanoyloxy group)] and an anti-hepatitis agent containing the glycoside as an active ingredient.

In the glycoside (A) of the present invention, examples of the lower alkyl group include linear or branched alkyl groups having 1 to 5 carbon atoms such as methyl group, ethyl group, propyl group and isopropyl group. The lower alkanoyl group is a linear or branched alkanoyl group having 2 to 5 carbon atoms,
Examples thereof include an acetyl group, a propionyl group, a butyryl group and the like. Further, as the lower alkanoyloxy group,
Examples thereof include linear or branched alkanoyloxy groups having 2 to 5 carbon atoms, such as acetoxy group, propionyloxy group and butyryloxy group. Examples of the monosaccharide residue include a glucopyranosyl group, a galactopyranosyl group, and a glucuronopyranosyl group. Examples of the disaccharide residue and the trisaccharide residue include groups in which two or three of these monosaccharides are bonded.

Of the glycosides (A) of the present invention, those in which R ¹ is a sugar (C) can be produced, for example, according to the following method.

[0011]

[Chemical 3]

(In the formula, R ¹ ′ represents a monosaccharide residue, a disaccharide residue or a trisaccharide residue in which a hydroxy group may be protected by a lower alkanoyl group, and R ^{2 to} R ¹⁴ are the same as above. Has meaning)

That is, the glycoside (C) of the present invention can be produced by subjecting the compound (B) to a glycosylation reaction using the sugar derivatives (1) to (3) by a conventional method. In addition, among glycosides (C),
R ¹ 'thing monosaccharides, R ^1' what is disaccharide, by hydrolysis with a β- glucosidase, it can also be produced. Examples of β-glucosidase used here include emulsin and the like.

Among the glycosides (A) of the present invention,
When R ¹ is a hydrogen atom or a lower alkyl group, it can be produced, for example, by the following method.

[0015]

[Chemical 4]

[0016]

[Chemical 5]

That is, methyl oleanolate (19)
And a sugar β-chloride (20) are reacted with each other to produce a compound (2
Compound (25) can be obtained by obtaining 1), detrichloroacetylating this to give compound (23), and then reacting this with sugar bromide (24). Further, the compound (26) can be obtained by further performing a deprotecting group reaction.

When the compound (A) of the present invention obtained as described above is used as an anti-hepatitis agent, its dose varies depending on the patient's weight, age, sex, administration method, physical condition, medical condition, etc. In the case of administration, it is 0 per day per kg of body weight.
4 mg to 200 mg, and in the case of parenteral administration, about 0.08 mg to 40 mg per kg body weight per day is appropriate.

The anti-hepatitis agent of the present invention is a tablet prepared by a conventional method.
It can be made into various dosage forms such as granules, powders, capsules, suspensions, injections and suppositories. To produce an oral solid preparation, the compound (A) is used as an excipient, and if necessary, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, a bulking agent, a coating agent, a sugar coating agent. After adding, etc., tablets,
Granules, powders, capsules, suppositories and the like are preferable. When preparing an injection, it is preferable to dissolve, disperse, or emulsify the compound (A) in an aqueous carrier such as distilled water for injection.

[0020]

The compound (A) of the present invention markedly inhibits AST and ALT activity in a model model of experimental liver injury induced by carbon tetrachloride, as is clear from the test examples shown below, and causes significant liver injury. Shows a suppressive effect. Therefore, the anti-hepatitis agent of the present invention containing the compound (A) of the present invention as an active ingredient has an excellent anti-hepatitis action and has few side effects.

[0021]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples. Example 1

[0022]

[Chemical 6]

(1) Ester glycosidation 1.0 g of the compound (4) was dissolved in 20 ml of dry methylene chloride, and anhydrous calcium sulfate (WA Hammond D) was added.
rierite Co. Made) 6.0 g and silver carbonate 850
After adding mg and stirring for 1 hour, compound (2) 4.0 g Silver trifluoromethanesulfonate (AgOTf) 384 mg
And 1,1,3,3-tetramethylurea (TMU) 21
5 μl was added and the mixture was stirred again for 36 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous solution of sodium bicarbonate and water in this order, dried over magnesium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (benzene-acetone, 7.5% gradient) to give an oil. 4.88 g of the residue of the product was obtained, and further high performance liquid chromatography [ODS 10 mmφ × 250 mm
(20% water-methanol)] to obtain 830 mg (yield 51.3%) of β-glycoside (5) and 73 mg (yield 4.5%) of α-glycoside (5 ′). Compound (5): FAB-MS m / z:. 1643 [M + Na] + 1 H-NMR (CDCl 3): Table _{^{1 13 C-NMR (C 5}} D 5 N): Table 2 Anal.Calcd for C ₇₉ H ₁₁₂ O ₃₅ : C, 58.51; H, 6.96 Found C, 58.51; H, 7.01 α-isomer (5 ′) of compound (5): FAB-MS m / z: 1643 [M + Na] ⁺ . ¹ H-NMR (CDCl ₃ ): Table 1 ¹³ C-NMR (C ₅ D ₅ N): Table 2 Anal.Calcd for C ₇₉ H ₁₁₂ O ₃₅ : C, 58.51; H, 6.96 Found C, 58.19; H, 7.11

(2) Deacetylation Compound (5) (450 mg) was dissolved in 5.0% potassium hydroxide methanol solution (2 ml), allowed to stand at room temperature for 1 hour, and then neutralized with acetic acid (pH 6 to 7). Then, it was applied to an ion exchange resin (Amberlite MB-3), pyridine was added to the eluate and the mixture was concentrated under reduced pressure, and the obtained residue was subjected to column chromatography (chloroform: methanol: water = 65: 35: 1).
0) and 240 mg of compound (6) (yield 80.4
%) Was obtained. FAB-MS m / z:. 1097 [M + Na] + 13 C-NMR (C 5 D 5 N): Tables 3 and 4 Reference Example

[0025]

[Chemical 7]

(1) Synthesis of Compound (8) 20 g of D-glucose was dissolved in 300 ml of dry pyridine, 34 g of triphenylmethyl chloride was added, and the mixture was stirred to dissolve and then left overnight. Acetic anhydride 30 in the reaction solution
0 ml was added and the mixture was again stirred overnight. The reaction solution was poured into ice water, extracted with methylene chloride, washed with a saturated aqueous solution of sodium hydrogencarbonate and water in this order, dried over magnesium sulfate, and concentrated under reduced pressure.
Dissolve the obtained residue in 200 ml of 70% acetic acid aqueous solution and
After refluxing for hours and filtration, the filtrate was extracted with methylene chloride, washed with a saturated aqueous solution of sodium bicarbonate and water in this order, dried over magnesium sulfate, and then concentrated under reduced pressure to obtain a residue. this,
32.07 g of compound (7) by recrystallization from diethyl ether / petroleum ether (mp. 40 ° C, yield 82.9%)
Got Compound (7) 5.4 g was dried with methylene chloride 20.
Dissolve it in ml, add 6.0 g of anhydrous calcium sulfate and 4.3 g of silver carbonate, and stir for 1 hour. Then, compound (2) 1
3.0 g was added and the mixture was stirred again overnight. The reaction solution is filtered,
The filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order, dried over magnesium sulfate, concentrated under reduced pressure, and subjected to column chromatography (CH ₂ Cl ₂ —MeOH, 1.0% gradient) to give compound (8) (7.3 g). (Yield 48.7%) was obtained. Compound (8): FAB-MS m / z: 989 [M + Na] ⁺ . (2) Synthesis of compound (3) Under ice cooling, 5.6 g of compound (8) was added to 150 ml of 30% hydrobromic acetic acid solution. After dissolution and stirring for 30 minutes, the reaction solution was poured into ice water, extracted with methylene chloride, and washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order. This was dried over magnesium sulfate and then concentrated under reduced pressure, and the obtained residue was subjected to column chromatography (benzene-acetone, 13% gradient) to give 3.4 g of compound (3) (yield 59.6%). Obtained. FAB-MS m / z: 1009 [M + Na] ⁺ .

Example 2

[0028]

[Chemical 8]

(1) Ester glycosidation: 1.0 g of the compound (4) was dissolved in 10 ml of dry methylene chloride, 7.0 g of anhydrous calcium sulfate and 1.37 g of silver carbonate.
Was added and the mixture was stirred for 1 hour, 800 mg of the compound (3) was added, and the mixture was stirred again for 28 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous solution of sodium hydrogencarbonate and water in this order, dried over magnesium sulfate, and then concentrated under reduced pressure. The obtained residue is subjected to silica gel column chromatography (benzene-acetone, 2
4.5% gradient), and 280 mg of compound (9) (yield 1
4.7%) was obtained. ¹ H-NMR (CDCl ₃ ): Table 1

(2) Deacetylation Compound (9) (280 mg) was dissolved in 5.0% potassium hydroxide methanol solution (2 ml), allowed to stand at room temperature for 1 hour, and then neutralized (pH 6 to 7) with acetic acid. It was attached to an ion exchange resin Amberlite MB-3, pyridine was added to the eluate and the mixture was concentrated under reduced pressure, and the obtained residue was subjected to column chromatography (chloroform: methanol: water = 65: 35: 1).
Compound (10) 75 mg (yield 41.3%)
Got FAB-MS m / z:. 1259 [M + Na] + 13 C-NMR (C 5 D 5 N): Tables 3 and 4

Example 3

[0032]

[Chemical 9]

Compound (11) [in the formula (D), R ¹⁵ = -β
-D-Glc (1 → 6) -β-D-Glc, R ¹⁶ = H,
R ¹⁷ = -CH ₃ ] 200 mg was added to a 0.1 M acetate buffer solution (pH
4.7) Dissolve in 4 ml of Triton X-100
0.5 ml and emursin (prepared from almonds) 50 mg
Was added and incubated at 37 ° C. for 14 hours. After adding 4 ml of ethanol to the reaction mixture and heating at 80 ° C for 5 minutes,
After filtration, the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (chloroform: methanol: water = 65: 3).
5: 20-10), the compound (12) [formula (D)
^{Among, R 15 = -β-D-} Glc, R 16 = H, R 17 = -CH
₃ ] 150 mg (yield 88.6%) was obtained. FAB-MS m / z:. 919 [M + Na] + 13 C-NMR (C 5 D 5 N): Table 3, Table 5

Example 4 Compound (13) [in the formula (D), R ¹⁵ = -β-D-Glc
(1 → 6) -β-D-Glc, R ¹⁶ = -β-D-Gl
c, R ¹⁷ = -CH _3] 200mg of 0.1M acetate buffer (p
H4.7) dissolved in 4 ml, Triton X-100
0.5 ml and emursin (prepared from almonds) 50 mg
Was added and incubated at 37 ° C. for 14 hours. After adding 4 ml of ethanol to the reaction mixture and heating at 80 ° C for 5 minutes,
After filtration, the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography (chloroform: methanol: water = 65: 3).
5: 20-10), the compound (14) [formula (D)
^{Among, R 15 = -β-D-} Glc, R 16 = -β-D-Gl
c, R ¹⁷ = -CH ₃ ] 130 mg (yield 75.0%) was obtained. FAB-MS m / z:. 1081 [M + Na] + 13 C-NMR (C 5 D 5 N): Table 3, Table 5

Example 5 (1) Glycosidation of compound (15) and compound (16) 1.0 g of methyl oleanolate (15) was dissolved in 5 ml of dry methylene chloride, and 2.0 g of anhydrous calcium sulfate and silver carbonate 1 were added. 0.5 g was added, and the surroundings of the container were shielded from light and stirred for 1 hour, and then 7.5 g of sugar β-chloride (16), 855 mg of silver trifluoromethanesulfonate (Ag-OTf) and 1,1,3,3- Tetramethylurea (TMU) 405
μl was added, and the mixture was further stirred at room temperature for 20 hours. The reaction solution was filtered, washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order, and dried over magnesium sulfate. After filtration, it was concentrated to obtain an oily residue. Column chromatography (benzene-ethyl acetate, 1.5% gradient) gave a crude product of compound (17) and a crude product of (18).

(2) Detrichloroacetylation of Crude of Compound (17) Crude of Compound (17) was saturated with ammonia ether solution 2
It was dissolved in 0 ml, stirred under ice cooling for 5 minutes, the solvent was distilled off under reduced pressure, and each residue was subjected to column chromatography (benzene-ethyl acetate, 6.0% gradient) to give compound (19).
Got Crystallized from methanol (19) 700 mg
[Yield 43.4% (yield from compound (15)]] was obtained. .. mp> 300 ℃ FAB- MS m / z: 781 [M + Na] + 1 H-NMR (CDCl 3): Table _{6 Anal.Calcd for C 43 H 66 O} 11: C, 68.05; H, 8.76 Found C, 67.84; H, 8.83

(3) Glycosidation of compound (19) and compound (20) 280 mg of compound (19) was dissolved in dry methylene chloride,
In the dark, 300 mg of anhydrous calcium sulfate, 314 mg of mercury (II) cyanide and 452 mg of mercury bromide were added, and the mixture was stirred for 1 hour. Then, 550 mg of sugar bromide (20) was added and the mixture was added again to 3
It was stirred for a day. The reaction solution was filtered, and the filtrate was washed with a saturated aqueous solution of sodium hydrogen carbonate and water in that order, dried over magnesium sulfate, and then subjected to column chromatography (benzene-ethyl acetate, 7.0% gradient) and high performance liquid chromatography [ODS 10 mmφ ×. 250 mm (20% water-acetone)], 210 mg of compound (21) (yield 52.9)
%) Was obtained. . FAB-MS m / z: 1097 [M + Na] + 1 H-NMR (CDCl 3): Table _{8 Anal.Calcd for C 56 H 82 O} 20: C, 62.55; H, 7.69 Found C, 62.35; H , 7.75

(4) Deprotection group reaction of compound (21) 980 mg of compound (21) at 170 ° C. in 2 ml of γ-collidine
And then cooled to 120 ° C. to obtain lithium iodide 50
A solution prepared by heating 0 mg of 2 mg of γ-collidine in advance was added, heated again to 170 ° C. under an argon stream and stirred for 2 hours, cooled, and then neutralized with acetic acid (pH 6 to 7).
Then, the residue obtained by concentration under reduced pressure was subjected to column chromatography (chloroform: methanol: water = 65: 3).
5: 20-15), and the glycoside (22) of the present invention.
340 mg (yield 46.9%) was obtained. ^{_{[α] D 20 -32.4 ° (}} c = 1.85, pyridine) FAB-MS m / z: . 817 [M + Na] + 13 C-NMR (C 5 D 5 N): Table 10

Example 6 (1) Detrichloroacetylation of crude compound (18)

[0040]

[Chemical 10]

The crude product of compound (18) was saturated with ammonia.
It was dissolved in 20 ml of ether solution and stirred for 5 minutes under ice cooling.
After that, the solvent was distilled off under reduced pressure. Column chromatograph each residue
Tography (benzene-ethyl acetate, 6.0% gradient)
And crystallized from methanol to give compound (23) [formula
(E) Medium, R¹⁸= OAc, R¹⁹= R^{twenty one}= H, R²⁰= CH
₂OAc] 630 mg [yield 39.1% (compound (15)
From the above)) was obtained. mp.> 300 ℃ FAB-MS m / z: 781 [M + Na]⁺.¹ H-NMR (CDCl₃): Table 6 Anal.Calcd for C₄₃H₆₆O₁₁: C, 68.05; H, 8.76 Found C, 67.69; H, 8.91

(2) Glycosidation of compound (23) and compound (20)

[0043]

[Chemical 11]

472 mg of the compound (23) was dissolved in dry methylene chloride, shielded from light and 300 mg of anhydrous calcium sulfate,
Mercury (II) cyanide 360 mg and mercury (II) bromide 490
After adding mg and stirring for 1 hour, sugar bromide (20) 76
0 mg was added and the mixture was stirred again for 1 day. After filtering the reaction solution, the filtrate was washed with a saturated aqueous solution of sodium bicarbonate and water in that order, dried over magnesium sulfate, and then subjected to column chromatography (benzene-ethyl acetate, 10.0% gradient) and high performance liquid chromatography [ODS 10 mmφ. × 250 mm
(20% water-acetone)], and the glycoside (24) of the present invention [in the formula (F), R ²² = Ac, R ²³ = OAc, R
^{24 = H, R 25 = CH} 2 OAc, R 26 = CH 3 ] 350mg
(Yield 52.3%) was obtained. . FAB-MS m / z: 1097 [M + Na] + 1 H-NMR (CDCl 3): Table _{8 Anal.Calcd for C 56 H 82 O} 20: C, 62.55; H, 7.69 Found C, 62.41; H , 7.87

(3) Deprotection group reaction of compound (24) 115 mg of compound (24) at 170 ° C. in 2 ml of γ-collidine
Lithium iodide 500
A solution prepared by previously heating and dissolving 2 mg of γ-collidine in 2 ml was added, the mixture was stirred again at 170 ° C. for 2 hours under an argon stream, cooled, and then neutralized with acetic acid (pH 6 to 7). Then, the residue obtained by concentration under reduced pressure was subjected to column chromatography (chloroform: methanol: water = 65: 35: 20-15) to give the glycoside (25) of the present invention [R ^{22 in the} formula (F)].
= R ²⁴ = R ²⁶ = H, R ²³ = OH, R ²⁵ = CH ₂ OH] 6
3 mg (yield 74.1%) was obtained. FAB-MS m / z:. 817 [M + Na] + 13 C-NMR (C 5 D 5 N): Table 10

Example 7 (1) Glycosidation of compound (15) and compound (26)

[0047]

[Chemical 12]

2.0 g of methyl oleanolate (15) was dissolved in 3 ml of dry methylene chloride, 1.5 g of anhydrous calcium sulfate and 1.1 g of silver carbonate were added, and the surroundings of the container were shielded from light and stirred for 1 hour. Sugar β-chloride (26) 1
8.0 g, silver trifluoromethanesulfonate (Ag-O
Tf) (6.6 g) and 1,1,3,3-tetramethylurea (TMU) (3.7 ml) were added, and the mixture was stirred at room temperature for 3 days. After filtering the reaction solution, it was washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order,
Dry over magnesium sulfate. After filtration, it was concentrated to obtain an oily residue. The residue was subjected to column chromatography (benzene-acetone, 0.6% gradient) to give a crude product of compound (27) [in the formula (G), R ²⁷ = OAc, R ²⁸ =
_{^{H, R 29 = COOCH 3,}} R 30 = COCCl 3 ] and (2
8) Crude product [in the formula (E), R ¹⁸ = OAc, R ¹⁹ = H, R
²⁰ = COOCH ₃ , R ²¹ = COCCl ₃ ].

(2) Detrichloroacetylation of crude compound (27) The compound (27) was dissolved in 10 ml of a saturated ammonia ether solution and stirred for 5 minutes under ice cooling, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography (benzene-ethyl acetate, 6.0% gradient) to give an oily compound (29) [in the formula (G), R ²⁷ = OAc, R ²⁸ = H,
R ²⁹ = COOCH ₃ , R ³⁰ = H] 1.42 g [yield 4
4.9% (yield from compound (15))] was obtained. . FAB-MS m / z: 767 [M + Na] + 1 H-NMR (CDCl 3): Table _{7 Anal.Calcd for C 42 H 64 O} 11: C, 67.72; H, 8.66 Found C, 67.41; H , 8.83

(3) Glycosidation of compound (29) and compound (20)

[0051]

[Chemical 13]

To a solution of 350 mg of the compound (29) in 2 ml of dry methylene chloride, 200 mg of anhydrous calcium sulfate, 238 mg of mercury (II) cyanide and 336 mg of mercury (II) bromide were added, and the mixture was stirred for 1 hour. ) 700 mg was added and stirred for 4 days. After filtering the reaction solution, the filtrate was washed with a saturated aqueous solution of sodium bicarbonate and water in this order, dried over magnesium sulfate, and then concentrated under reduced pressure to obtain a residue, which was subjected to column chromatography (benzene-ethyl acetate, 6.0% gradient). The glycoside (30) of the present invention [in the formula (H), R ³¹ =
Ac, R ³² = OAc, R ³³ = H, R ³⁴ = COOCH ₃ ,
R ³⁵ = CH ₃ ] 270 mg (yield 54.2%) was obtained. . FAB-MS m / z: 1083 [M + Na] + 1 H-NMR (CDCl 3): Table _{9 Anal.Calcd for C 55 H 80 O} 20: C, 62.25; H, 7.60 Found C, 62.13; H , 7.78

(4) Deprotection group reaction of compound (30) 150 mg of compound (30) was dissolved in 2 ml of 5.0% potassium hydroxide solution (ethanol: water = 1: 1) and refluxed for 5 hours. After the reaction, neutralize with acetic acid and concentrate under reduced pressure.
After dissolving in 2 ml of γ-collidine at 0 ° C and cooling to 120 ° C, a solution of 500 mg of lithium iodide preheated to 2 ml of γ-collidine was added, and the mixture was stirred again at 170 ° C for 2 hours under an argon stream. After cooling, neutralize with acetic acid (pH 6
~ 7) did. Then, the mixture was concentrated under reduced pressure, and the obtained residue was subjected to column chromatography (chloroform: methanol: water = 65: 35: 20-10) to give the glycoside (31) of the present invention [in the formula (H), R ³¹ = R ³³ = R ³⁵ = H, R ³²
= OH, R ³⁴ = COOH] 100 mg (yield 85.5%)
Got ^{_{[α] D 20 + 41.0 °}} (c = 1.34, pyridine) FAB-MS m / z: . 853 [M-1 + 2Na] + 13 C-NMR (C 5 D 5 N): Table 10

Example 8 (1) Detrichloroacetylation of Crude Compound (28) The crude compound (28) obtained in Example 7 (1) was dissolved in 10 ml of a saturated ammonia ether solution, and the mixture was cooled with ice. The residue obtained by stirring for 5 minutes and distilling off the solvent under reduced pressure was subjected to column chromatography (benzene-ethyl acetate, 6.0% gradient) to give an oily compound (32) [in formula (E) ,
R ¹⁸ = OAc, R ¹⁹ = R ²¹ = H, R ²⁰ = COOCH ₃ ]
730 mg [yield 23.3% (yield from compound (19)]] were obtained. . FAB-MS m / z: 767 [M + Na] + 1 H-NMR (CDCl 3): Table _{7 Anal.Calcd for C 42 H 64 O} 11: C, 67.72; H, 8.66 Found C, 67.29; H , 8.72

(2) Glycosidation of compound (32) and compound (20) 675 mg of compound (32) was dissolved in 18 ml of dry methylene chloride, and 600 mg of anhydrous calcium sulfate and mercury cyanide (I
457 mg of I) and 635 mg of mercury (II) bromide were added, and the mixture was stirred at room temperature for 1 hour, 1.4 g of compound (20) was added, and the mixture was further stirred for 5 days. After filtering the reaction solution, the filtrate was poured into ice water and extracted with methylene chloride. The extract was washed with a saturated aqueous solution of potassium hydrogen carbonate and water in this order, and then dried over magnesium sulfate. After filtration, the residue obtained by concentration is subjected to column chromatography (benzene-ethyl acetate, 12%
Gradient) followed by preparative high performance liquid chromatography (ODS-4251, methanol, 1 ml / min, 35
Glycoside (33) of the present invention [formula (F)
Medium, R ²² = Ac, R ²³ = OAc, R ²⁴ = H, R ²⁵ = CO
740 mg of OCH ₃ , R ²⁶ ═CH ₃ ]. (Yield 77.
0%). . FAB-MS m / z: 1083 [M + Na] + 1 H-NMR (CDCl 3): Table _{9 Anal.Calcd for C 55 H 80 O} 20 · 1 / 2H 2 O: C, 61.72; H, 7.63 .Found C, 61.73; H, 7.50

(3) Deprotection group reaction of compound (33) 670 mg of compound (33) was treated in the same manner as in Example 7 (4) to obtain 354 mg of deacetylated product. This product was dissolved in 7 ml of γ-collidine, and 500 mg of lithium iodide was previously added to γ.
-Adding the solution which was heated to 2 ml of cholinedi and dissolved, it was stirred at 160 ° C for 12.5 hours under an argon stream. The reaction solution was neutralized with acetic acid and then concentrated under reduced pressure to obtain a residue. The residue was subjected to column chromatography (chloroform: methanol: water = 63: 35: 20-10) to give the glycoside (34) of the present invention [in the formula (F), R ²² = R ²⁴ = R ²⁶ =
H, R ²³ = OH, R ²⁵ = COOH] 193.6 mg (yield 37.91%) was obtained. FAB-MS m / z:. 853 [M-1 + 2Na] + 13 C-NMR (C 5 D 5 N): Table 10

[0057]

[Chemical 14]

Example 9 (1) Glycosidation of compound (15) and compound (35) 5.0 g of methyl oleanolate (15) in 10 ml of dry methylene chloride was added to 5.0 g of anhydrous calcium sulfate and silver carbonate 2. After adding 8 g and shielding from light around the container and stirring for 1 hour, 27.0 g of sugar β-chloride (35) and silver trifluoromethanesulfonate (Ag-OTf) 13.9.
g and 1,1,3,3-tetramethylurea (TMU)
7.6 ml was added, and the mixture was stirred at room temperature for 4 days. The reaction solution was filtered, washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order, and dried over magnesium sulfate. After filtration, it was concentrated to obtain an oily residue. The residue was subjected to column chromatography (benzene-acetone, 4.0% gradient) to give a crude product of compound (36) [in the formula (G), R ²⁷ = H, R ²⁸ = OAc, R ²⁹ = CH _2].
OAc, R ³⁰ = COCCl ₃ ] and a crude compound (37) [in the formula (E), R ¹⁸ = H, R ¹⁹ = OAc, R ²⁰ = CH].
₂ OAc, R ²¹ = COCCl ₃ ].

(2) Detrichloroacetylation of Crude of Compound (36) Crude of Compound (36) was added to saturated ammonia ether solution 1
It was dissolved in 0 ml, stirred for 5 minutes under ice cooling, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (benzene-ethyl acetate, 6.0% gradient) to give an oily compound (38) [in the formula (G), R ²⁷ = H, R ²⁸ = OAc,
R ²⁹ = CH ₂ OAc, R ³⁰ = H] 3.86 g [yield 4
7.9% (yield from compound (15))] was obtained. . FAB-MS m / z: 781 [M + Na] + 1 H-NMR (CDCl 3): Table _{6 Anal.Calcd for C 43 H 66 O} 11: C, 68.05; H, 8.76 Found C, 67.92; H , 8.82

(3) Glycosidation of compound (38) and compound (20) 1.0 g of compound (38) was dissolved in dry methylene chloride,
Anhydrous calcium sulfate 1.0 g, mercury (II) cyanide 6.
After 3 g and 9.1 g of mercury (II) bromide were added and stirred for 1 hour, 11.6 g of sugar bromide (20) was added and stirred again for 3 days. After filtering the reaction solution, the filtrate was washed with a saturated aqueous solution of sodium bicarbonate and water in this order, dried over magnesium sulfate, and then the residue obtained by concentration under reduced pressure was subjected to column chromatography (benzene-ethyl acetate, 10.0% gradient) and Subjected to high performance liquid chromatography (ODS 10 mmφ × 250 mm (methanol)], the glycoside of the present invention (3
9) [in the formula (H), R ³¹ = Ac, R ³² = H, R ³³ = OA
c, to give _{^{R 34 = CH 2 OAc, R}} 35 = CH 3 ] 1.1g of (77.6% yield). . FAB-MS m / z: 1097 [M + Na] + 1 H-NMR (CDCl 3): Table _{8 Anal.Calcd for C 55 H 82 O} 20: C, 62.55; H, 7.69 Found C, 62.43; H , 7.79

(4) Deprotection group reaction of compound (39) 1.1 g of compound (39) was refluxed for 5 hours with 5 ml of 5.0% potassium hydroxide solution (ethanol: water = 1: 1), and after cooling, The residue obtained by neutralizing with acetic acid and concentrating under reduced pressure was dissolved in 10 ml of γ-collidine at 170 ° C and cooled to 120 ° C.
Lithium iodide (500 mg) was previously mixed with γ-collidine (2 ml).
Add the solution that was heated and melted, and again under argon flow,
Stir at 170 ° C for 2 hours, cool, and then neutralize with acetic acid (pH 6-
7) I did. Then, the residue obtained by concentration under reduced pressure was subjected to column chromatography (chloroform: methanol: water =
63: 35: 20-15), the glycoside (40) of the present invention [in the formula (H), R ³¹ = R ³² = R ³⁵ = H, R ³³ =
OH, R ³⁴ = CH ₂ OH] 490 mg (yield 60.3%)
Got ^{_{[α] D 20 + 7.3 °}} (c = 2.06, pyridine) FAB-MS m / z: . 817 [M + Na] + 13 C-NMR (C 5 D 5 N): Table 10

Example 10 (1) Synthesis of compound (41) The crude product of compound (37) obtained in Example 9 (1) was dissolved in 10 ml of saturated ammonia ether solution under ice cooling and stirred for 5 minutes. . The reaction solution was concentrated under reduced pressure and the residue was subjected to silica gel column chromatography (benzene-ethyl acetate, 6.0).
% Gradient) to give an oily compound (41) [formula (E)
Medium, R ¹⁸ = H, R ¹⁹ = OAc, R ²⁰ = CH ₂ OAc, R
²¹ = OH] 3.86 g [yield 47.9% (compound (1
The yield from 5))] was obtained. FAB-MS m / z: 781 [M + Na] ⁺ .Ana.Calcd for C ₄₃ H ₆₆ O ₁₁ : C, 68.05; H, 8.76 Found C, 67.92; H, 8.82

(2) Glycosidation of compound (41) and compound (20) 1.2 g of compound (41) was dissolved in dry methylene chloride,
Anhydrous calcium sulfate 1.0 g, mercury (II) cyanide 4.
After adding 9 g and 7.3 g of mercury (II) bromide and stirring for 1 hour, 8.8 g of sugar bromide (20) was added and the mixture was stirred again for 2 days. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution and water in this order and dried over magnesium sulfate,
The residue obtained by concentration under reduced pressure was subjected to column chromatography (benzene-ethyl acetate, 12.0% gradient) and high performance liquid chromatography HPLC [ODS 10 mmφ × 25.
0 mm (methanol)], and an oily glycoside (42) of the present invention [in the formula (F), R ²² = Ac, R ²³ = H, R
^{24 = OAc, R 25 = CH} 2 OAc, R 26 = CH 3 ] 99
0 mg (yield 58.2%) was obtained. . FAB-MS m / z: 1097 [M + Na] + 1 H-NMR (CDCl 3): Table _{9 Anal.Calcd for C 56 H 82 O} 20: C, 62.55; H, 7.69 Found C, 62.33; H , 7.88

(2) Deprotection group reaction of compound (42) 990 mg of compound (42) was refluxed with 5 ml of 5.0% potassium hydroxide solution (ethanol: water = 1: 1) for 5 hours,
It was neutralized with acetic acid and concentrated under reduced pressure. The residue was dissolved in 8 ml of γ-collidine at 170 ° C., cooled to 120 ° C., 2 ml of a solution prepared by previously heating 500 mg of lithium iodide to γ-collidine was added, and the mixture was again heated to 170 ° C. in an argon stream.
Heat to room temperature and stir for 2 hours. After cooling, neutralize with acetic acid (pH 6-
7) I did. Then, the residue obtained by concentration under reduced pressure was subjected to column chromatography (chloroform: methanol: water = 6).
5: 35: 20-15), the glycoside of the present invention (4
3) [In the formula (F), R ²² = R ²³ = R ²⁶ = H, R ²⁴ = O
H, R ²⁵ = CH ₂ OH] 420 mg (yield 57.4%) was obtained. ^{_{[α] D 20 + 75.0 °}} (c = 1.40, pyridine) FAB-MS m / z: . 817 [M + Na] + 1 H-NMR (C 5 D 5 N): Table 10

[0065]

[Table 1]

[0066]

[Table 2]

[0067]

[Table 3]

[0068]

[Table 4]

[0069]

[Table 5]

[0070]

[Table 6]

[0071]

[Table 7]

[0072]

[Table 8]

[0073]

[Table 9]

[0074]

[Table 10]

Test Example The glycosides of the present invention obtained in the examples were examined for pharmacological activity as follows. That is, the method of Segren et al. [Methods in Cell Bio
l. , 28, 432, (1970)], the hepatocytes isolated from rat liver were adjusted to 2 × 10 ⁶ cells / ml, and the hepatocytes were placed in a Kolben filled with 50% carbon tetrachloride volatilized and incubated. After inducing experimental hepatitis, only 0.1 ml of Hanks 'solution was added to a solution prepared by dissolving 1 mg of each saponin in 0.1 ml of Hanks' solution and control (C), and incubated at 37 ° C. for 1 hour. Then, the reaction suspension was subjected to centrifugation (10000 rpm × 19 minutes), and aspartate transferase (AS
T), alanine transferase (ALT) activity was measured. This experiment uses the fact that a large amount of enzymes such as AST and ALT are released from hepatocytes suffering from hepatitis, and those with small AST and ALT activities have a great effect on liver protection. . 10 compounds were used for each compound and control, and the data are shown as the average value. The results are shown in Fig. 1.

[Brief description of drawings]

FIG. 1 shows AST and AL of compounds of the present invention in test examples.
It is a figure which shows the result of having measured T activity.

Claims (2)

[Claims] 1. A compound represented by the general formula (A): [In the formula, R ¹ represents a hydrogen atom, a monosaccharide residue, a disaccharide residue or a trisaccharide residue in which a lower alkyl group or a hydroxy group may be protected by a lower alkanoyl group, and R ² represents a hydrogen atom, A methyl group or a hydroxymethyl group, R ³ and R ⁸ are a hydroxy group, —CH ₂ OR ³ ′ or —COO.
R ⁸ '(where R ^3' represents a hydrogen atom or a lower alkanoyl group, R ⁸ 'represents a hydrogen atom or a lower alkyl group)
And one of R ⁴ and R ⁵ , one of R ⁶ and R ⁷ , or one of R ⁹ and R ¹⁰ is a hydrogen atom, and the other is a hydroxy group or a lower alkanoyloxy group, ¹¹ or R ¹² or R ¹³ and R
^{One of 14} is a hydrogen atom, the other is a hydroxy group,
A glycoside represented by a lower alkanoyloxy group or a monosaccharide residue in which a hydroxy group may be protected by a lower alkanoyl group (wherein one of the other is a hydroxy group or a lower alkanoyloxy group)] . 2. An anti-hepatitis agent containing the glycoside according to claim 1 as an active ingredient.