JP2589431B2 - Sugar-modified polyglutamic acid derivative and production method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel sugar-modified polyglutamic acid derivative useful as an organ-directing drug delivery carrier, a synthetic intermediate thereof, and a method for producing the same.

[0002]

2. Description of the Related Art Attempts to use polyglutamic acid as a drug delivery carrier have been mainly studied as a delivery carrier for anticancer drugs, as shown in the following documents. 1) Goldberg, EP et al., Org. Coat Plast. Chem. 44 , 1
32-136 (1981) 2) Israel Patent No. 68449/3 3) Hurwitz, E. et al., J. Appl. Biochem. 2, 25-35 (198)
0) 4) Morimoto, Y. et al., J. Pharm. Dyn. 7, 688-698 (1984) 5) Kato, A. et al., Chem. Pharm. Bull. 30, 2951-2957 (1)
982) 6) Kato, Y. et al., Cancer Res. 44, 25-30 (1984) 7) Rowland, GF et al., Nature 255, 487-488 (1975) 8) Tsukada, Y. et al., J. Natl. Cancer Institute 73,
721-729 (1984)

The above-mentioned references 1) to 3) show examples in which doxorubicin or daunorubicin is bound to polyglutamic acid, 4) shows an example in which melphalan is bound, and 5) binds to mitomycin C. 6) shows an example in which arabinofuranosyl cytosine is bound, 7) shows an example in which phenylenediamine mustard and anti-lymphoma immunoglobulin are bound, and 8) shows Shows an example in which daunorubicin is bound to an anti-α-fetoprotein antibody.

[0004] As in these examples, an anticancer agent or an antitumor agent is bound to polyglutamic acid directly or to polyglutamic acid modified for drug binding, thereby enabling selective transport to cancer tissue or tumor cells. In order to
It has been proposed to further bind antibodies recognizing these tissues and cells. Therefore, it was predicted that polyglutamic acid could be a drug delivery carrier provided with organ tropism. However, there is no concrete attempt to realize this prediction, and it remains as a problem to be solved in drug delivery technology.

[0005]

In view of the above situation in the prior art, the present inventors have conducted various studies and found that modification of polyglutamic acid with a sugar imparts organ directivity. They have found and completed the present invention. That is, an object of the present invention is to provide a technique for imparting organ tropism to polyglutamic acid.

[0006]

(Material) The final target substance in the present invention is a sugar-modified polyglutamic acid derivative represented by the following formula (I).

[0007]

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(In the formula, R ₁ represents OR ′ or NHR ″, R ′ represents a hydrogen atom, an alkali metal atom or a physiologically active substance, and R ″ represents a hydrogen atom or a physiologically active substance.
R ₂ is a glycosyl group, and the hydroxyl group or the amino group at the 2-position may be substituted with an acetyl group. n is 7 ~
Represents an integer of 10. The molar ratio of m to k is from 1 to 100
Is)

Formula (I) shows that the sugar-modified polyglutamic acid derivative of the present invention is composed of two different glutamic acid units. The first unit (A) is an unmodified glutamic acid unit, and the second unit (B) is a glutamic acid unit modified by a sugar via an aminoalkyl chain as a spacer. Further, the formula (I) means that these two types of units (A) and (B) are contained in each molecule in k and m units, respectively. However, the description of formula (I) means that each unit (A) and (B) is randomly copolymerized.

Examples of the glycosyl group for R ₂ include mannopyranosyl, galactopyranosyl, xylopyranosyl, fucopyranosyl, 2-amino-2-deoxy-mannopyranosyl, 2-amino-2-deoxy-fucopyranosyl and the like. Examples of the hydroxyl group or amino group substituted with an acetyl group include 2,3,4,6-tetra-0-acetyl-mannopyranosyl and 2-acetamido-2-deoxy-fucopyranosyl.

The physiologically active substance is a drug that is delivered to a target organ and exhibits a pharmacological effect, and examples thereof include daunorubicin, triprolidine, doxorubicin, and methotrexate via a spacer having an amino group at the terminal.

The sugar-modified polyglutamic acid derivative (I) of the present invention comprises the unit (A) and the unit (B) as essential constitutional units. The third unit, for example, the sugar-modified polyglutamic acid derivative (I) ) Is required for the benefit of iodine labeling for animal testing (C)

[0013]

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May be included.

When the substance of formula (I) is used as a drug delivery carrier, the drug is bound to the first unit (A), ie some of the unmodified glutamic acid units. The bond may be made directly via the amino group of the drug or indirectly via the amino group of the spacer having an amino group, and may be carried out according to a usual method for forming an acid amide bond. In addition, a drug can be bound via an ester bond or an acid anhydride bond.

The substance of the formula (II) is useful when the substance of the formula (I) is synthesized by a copolymerization method and is a synthetic intermediate for this purpose.

[0017]

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(Wherein R ₂ and n are the same as described above).
The compounds of the formula I) are useful starting materials for the synthesis of the compounds of the formula (II) and are useful for protecting the amino group of glutamic acid by 9-.
The fluorenylmethoxycarbonyl group is particularly chosen. This selection is characterized in that a series of reaction steps leading to the substance of the formula (II) can be realized.

[0019]

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Wherein R ₃ represents a 9-fluorenylmethoxycarbonyl group and R ₄ represents a carboxyl-protecting group. Examples of the protecting group for R ₄ include t-butyl and the like.

The substance of the formula (IV) is useful for synthesizing the substance of the formula (II) or for synthesizing the substance of the formula (I) using polyglutamic acid as a raw material. It is prepared in advance as a sugar with a spacer having an amino group to achieve a certain sugar modification purpose.

[0022]

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(Production Method) In order to produce the substance of the formula (IV), a glycosyl halide in which the hydroxyl group of the glycoside is protected or the 2-position hydroxyl group is further changed to an azide group is added to the N- ( (Hydroxyalkyl) phthalimide, and further deprotected as necessary, or further reduce the azide group to an amino group, further acetylate the amino group, and then treat this with hydrazine to remove the phthalimide. It can be obtained by: The reaction is performed at a low temperature of −10 ° C. or lower in the presence of silver silicate or the like.

[0024]

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(Where n is the same as above).
Can be obtained, for example, by reacting an alkanediol having one terminal OH group protected with a 2,3-dihydropyranyl group with phthalimide, and then deprotecting to release the OH group.

To prepare the substance of formula (III), a reactive derivative of 9-fluorenylmethoxycarboxylic acid, such as succinic acid, is added to the α-amino group of glutamic acid having a protected γ-carboxyl group. Reaction with imide, and α-
It is obtained by protecting the carboxyl group and then removing the protecting group for the γ-carboxyl group.

To prepare the substance of the formula (II), the compound of the formula (III)
Is reacted with a glycosyloxyalkylamine represented by the formula (IV) or a salt thereof, an α-carboxyl protecting group and an α-amino group protecting group are eliminated, and then bis (trichloroform) Reaction of a carbonate such as methyl) carbonate gives the N-carboxyglutamic anhydride derivative.

There are two methods for producing the sugar-modified polyglutamic acid derivative of the formula (I). The first method is a method of copolymerizing an N-carboxyglutamic anhydride derivative represented by the formula (II) and an N-carboxyglutamic anhydride represented by the following formula (VI). , A method of copolymerizing N-carboxyglutamic anhydride of the formula (VI) and reacting a glycosyloxyalkylamine represented by the formula (IV) or a salt thereof with polyglutamic acid obtained by removing a protecting group for a carboxyl group. It is.

[0029]

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(Wherein R ₅ represents a protecting group for a carboxyl group such as a benzyl group) The first copolymerization method may be carried out in accordance with a conventional method of copolymerization, and different from the above formulas (II) and (VI) )
Are reacted in dimethylformamide in the presence of a condensing agent such as hexylamine, and then the carboxyl-protecting group is eliminated. By coupling the active substance, a substance of formula (I) is obtained.

In the second method, polyglutamic acid is added to glycosyloxyalkylamine or a salt thereof (IV) as a condensing agent, for example, 1,3-dicyclohexylcarbodiimide, 1-ethoxycarbonyl-2-ethoxy-1,2.
Reacting in dimethylformamide in the presence of dihydroquinoline and, if necessary, linking a bioactive substance to the γ-carboxyl group, gives the substance of formula (I).

If necessary, 2- (4-hydroxyphenyl) ethylamine is added together with glycosyloxyalkylamine or a salt thereof (IV), and the reaction is carried out in the same manner. A substance of formula (I) can be obtained.

In producing polyglutamic acid, N-carboxy-L-glutamic anhydride and N-carboxy-L-glutamic acid anhydride are used.
By copolymerizing -carboxy-D-glutamic anhydride, polyglutamic acid in which L-glutamic acid and D-glutamic acid are randomly arranged can be obtained.

(Characteristics of Substance) The molecular weight of the substance of the formula (I) can be measured by various methods. For example, a gel permeation chromatography method using polyethylene glycol as a standard substance (column: TSK-GEL) G4000PWXL + G3000PWXL, eluent:
The weight average molecular weight (Mw) can be determined from 0.2 M saline solution and elution rate: 0.5 ml / min). Formula (I)
Although the molecular weight of the substance is not particularly limited, it is usually 5,000.
It has a molecular weight of 0-150,000.

The sugar content of the substance of the formula (I) can be determined by colorimetry by the phenol-sulfuric acid method. Further, the hydroxyphenylethylamino group content of the unit (C) in the substance of the formula (I) can be determined by the absorbance at 275 nm.

The molar ratio of the unit (B) to the unit (A) is from 1 to 100, and when iodine labeling is necessary for animal experiments, the molar ratio of the unit (C) to the unit (A) is 1 to 15.

[0037]

The efficacy of the substance of the formula (I) as a drug carrier is, as shown in the experimental examples described later, in the case of the substance of the formula (I) modified with galactose as compared with the unmodified polyglutamic acid. It was found that the accumulation in the kidney was small and the accumulation in the liver was large. Since the existence of a lectin that recognizes specific galactose is known in the liver, it is presumed that the substance of the formula (I) modified with galactose was recognized by the lectin. Therefore, when an organ, a cancer cell, or the like has a lectin that recognizes a specific sugar, the substance of formula (I) can be an organ-directed drug delivery carrier.

[0038]

The present invention will be described more specifically with reference to examples. The reaction steps corresponding to the examples are shown in Reaction Formulas 1 to 17. The compound numbers in the reaction formulas correspond to the compound numbers described in Reference Examples and Examples.

[0039]

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[0040]

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[0041]

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[0042]

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[0043]

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[0044]

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[0045]

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[0046]

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[0047]

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[0048]

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[0049]

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[0050]

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[0051]

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[0052]

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[0053]

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[0054]

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[0055]

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[0056]

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Reference Example 1 1,8-Octanediol monotetrahydropyranyl ether (2) 36 ml (392 mmol) of 2,3-dihydropyran was
A solution of 50 g (342 mmol) of 1,8-octanediol (1) and 0.5 ml (6 mmol) of concentrated hydrochloric acid in 400 ml of ethylene glycol dimethyl ether was added with stirring. After further stirring at room temperature for 2 hours, the reaction solution was neutralized by adding 1 ml of triethylamine. After the precipitated triethylamine hydrochloride was removed by filtration, the filtrate was concentrated to dryness under reduced pressure. The residue obtained is purified by distillation under high vacuum,
32.1 g (41%) of the title compound (2) were obtained.

2: Bp _1.5 mmHg 140-150 ° C. IR (CHCl ₃ ) cm ^-1 : 3400 (OH), 1460, 1355, 1015 ¹ H-NMR (CDCl ₃ ): δ 4.55 (1H, m, -OCHO- )

Reference Example 2 8-phthalimidooctanol tetrahydropyranyl ether (3) 14.9 g (64.7 mmol) of compound (2), 18.67 g (71.2 mmol) of triphenylphosphine and 10.47 g (71. 2 mmol) in 300 ml of anhydrous tetrahydrofuran in a stream of nitrogen.
While stirring, 11.2 ml (71.2 mmol) of diethyl azodicarboxylate was slowly added dropwise at room temperature. After further stirring at room temperature for 4 hours, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate = 3: 1) to obtain 23 g (quantitative) of the title compound (3).

3: ¹ H-NMR (CDCl ₃ ): δ 7.9-7.7 (4H, m, aromatic H) 4.58 (1H, m, CH)

Reference Example 3 8-phthalimidooctanol (4) 47 g (137 mmol) of compound (3) were added to 80% acetic acid 5
After dissolving in 00 ml, the mixture was heated and stirred at 80 ° C. for 3 hours. After the reaction mixture was concentrated to dryness under reduced pressure, the obtained residue was partitioned between an aqueous layer and an ethyl acetate layer. The organic layer was separated and washed sequentially with water, aqueous sodium hydrogen carbonate solution and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate = 2: 1) to obtain 24.3 g (65%) of the title compound (4).

4: mp 64.5-65 ° C. IR (CHCl ₃ ) cm ⁻¹ : 3630 (OH), 1773, 1712, 1390 ¹ H-NMR (CDCl ₃ ): δ 7.9-7.65 (4H, m, aromatic H ) 3.8-3.6 (4H, m, -CH ₂ N, -CH ₂ O) 1.8-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 4 8-phthalimidooctyl 2
—O-acetyl-3,4,6-tri-O-benzyl-α
-D-mannopyranoside (6) The method described in the literature (T. OGAWA et al., Tetrahedron, 37, 2779).
(1981)), 1.6 g (31.58 mmol) of 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl chloride (5), 8- 8.7 g (31.58 mmol) of phthalimidooctanol (4) and 17 ml of 1,1,3,3-tetramethylurea (1
26.3 mmol) was dissolved in 200 ml of dichloroethane and cooled to -40 ° C under a stream of nitrogen. 8.93 g (34.73 mmol) of silver triflate was added to the mixture, and the mixture was stirred at the same temperature for 30 minutes. After removing the cooling bath, the mixture was further stirred at room temperature for 16 hours. After the reaction mixture was filtered using celite, the filtrate was washed successively with an aqueous sodium hydrogen carbonate solution and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate = 3: 1) to obtain 12.3 g (52%) of the title compound (6).

[0064] 6: syrup [α] ^{_{D 24 + 17.7 ° (c 0.61}} , CHCl 3) IR (CHCl 3) cm -1: 1710, 1522, 1477, 1423 1 H-NMR (CDCl 3): δ 7.81 ( 2H, d, J = 10Hz, aromatic H) 7.68 (2H, d, J = 10Hz, aromatic H) 7.4-7.2 (15H, m, aromatic H) 5.33 (1H, dd, J ₁ , ₂ = 1.5Hz, J _{2, 3 = 3.5Hz, H-} 2) 4.80 (1H, d, J 1, 2 = 1.5Hz, H-1) 3.96 (1H, dd, J 2, 3 = 3.5Hz, H-3) 3.86 (1H , t, J ₃ , ₄ = 10Hz, H-4) 2.13 (3H, s, OAc) 1.75-1.20 (12H, m,-(CH ₂ ) _6- ) 1.45 (9H, s, C (CH ₃ ) ₃ )

Reference Example 5 8-phthalimidooctyl
3,4,6-tri-O-benzyl-α-D-mannopyranoside (7) A mixture of 12.3 g (16.4 mmol) of compound (6) in 200 ml of methanol and 50 ml of tetrahydrofuran was added with
4.1 ml of M sodium methoxide-methanol solution was added, and the mixture was left at room temperature for 4 hours. After neutralizing the reaction solution by adding ion-exchange resin Amberlite IR120B (H ⁺ type), the resin was filtered off and washed with methanol. The filtrate and washing solution were combined, and the solvent was distilled off under reduced pressure.
1.4 g were obtained. The residue was purified by silica gel column chromatography (hexane-ethyl acetate = 2: 1),
8.5 g (73.3%) of the title compound (7) were obtained.

7: Syrup-like [α] _D ^26.5 + 31.4 ° (c 1.12, CHCl ₃ ) IR (film) cm ^-1 : 3467, 1772, 1712, 1395, 1365 ¹ H-NMR (CDCl ₃ ): δ 7.83 (2H, d, J = 10Hz, aromatic H) 7.70 (2H, d, J = 10Hz, aromatic H) 7.4-7.2 (15H, m, aromatic H) 5.33 (1H, dd, J ₁ , ₂ = 1.5Hz, J ₂ , ₃ = 3.5Hz, H-2) 4.87 (1H, d, J ₁ , ₂ = 1.5Hz, H-1)

Reference Example 6 8-aminooctyl 3,4
6-tri-O-benzyl-α-D-mannopyranoside (8) 8.5 g (12 mmol) of compound (7) in ethanol 6
Hydrazine hydrate 3 ml (60 mmol) in 0 ml solution
Was added and the mixture was heated under reflux on an oil bath for 3 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. Toluene was added to the residue, and the mixture was concentrated to dryness under reduced pressure to remove the remaining hydrazine, thereby obtaining 7.1 g (quantitative) of the title compound (8).

8: Amorphous powder [α] _D ²⁷ + 37.1 ° (c 0.21, MeOH) IR (film) cm ⁻¹ : 3300, 1496, 1454, 1363, 1103, 1058 ¹ H-NMR (CDCl ₃ ): δ 7.4-7.1 (15H, m, aromatic H) 4.75 (1H, d, J ₁ , ₂ = 1.5Hz, H-1) 4.00 (1H, dd J ₂ , ₃ = 3.5Hz, H-2) 2.62 (2H , t, J = 8Hz, -CH ₂ N) 1.6-1.25 (12H, m,-(CH ₂ ) _6- )

Reference Example 7 8-aminooctyl α-D-
Mannopyranoside (9) Compound (8) 6.9 g (12 mmol) of methanol 2
12 ml of 1 M hydrochloric acid and 10% palladium-
1 g of carbon was added and hydrogenated at room temperature for 16 hours. After removing the catalyst by filtration, the filtrate was concentrated to dryness under reduced pressure to give 3.99 g (quantitative) of the hydrochloride of the title compound (9).

9 (hydrochloride): amorphous powder [α] _D ²⁶ + 42.9 ° (c 1.13, MeOH) IR (Nujol) cm ^-1 : 3346, 1620, 1506, 1460, 1377 ¹ H-NMR (CD ₃ OD): δ 4.72 (1H, d, J 1, 2 = 2Hz, H-1) 3.81 (1H, dd J 5, 6a = 2.5Hz, J 6a, 6b = 11.5Hz, H-6a) 3.76 (1H, dd, J ₂ , ₃ = 3.5Hz, H-2) 3.59 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 3.51 (1H, m, H-5) 2.90 (2H, t, J = 8Hz, -CH ₂ N) 1.7-1.35 (12H, m,-(CH ₂ ) _6- )

The hydrochloride of the compound (9) obtained above 2.64
g was dissolved in aqueous methanol and passed through a column of 50 ml of ion exchange resin Amberlite IRA-400 (OH type). The column was washed with 500 ml of 50% aqueous methanol. The combined eluate and washings were concentrated to dryness under reduced pressure to give 2.31 g of the free base of the title compound (9).

9 (free base): amorphous powder [α] _D ²² + 51.5 ° (c 1.02, MeOH) ¹ H-NMR (CD ₃ OD): δ 4.69 (1H, d, J ₁ , ₂ = 1.5 Hz) , H-1) 3.78 (1H , dd J 5, 6a = 2.5Hz, J 6a, 6b = 12Hz, H-6a) 3.74 (1H, dd, J 2, 3 = 3Hz, H-2) 3.57 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 3.49 (1H, ddd, H-5) 2.59 (2H, t, J = 7.5Hz, -CH ₂ N) 1.6-1.25 (12H, m,-( CH ₂ ) _6- )

Reference Example 8 8-phthalimidooctyl
2,3,4,6-tetra-O-benzyl-β-D-mannopyranoside (11) and 8-phthalimidooctyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (12) ( i) The method described in the literature (Bull. Soc. Chem. Jpn, 49, 26)
39 (1976)), 2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl chloride (10)
A solution of 1.7 g (2.89 mmol) in 10 ml of toluene was stirred at −18 ° C. under a nitrogen stream at −18 ° C. at 795 mg (2.89 mmol) of 8-phthalimidooctanol (4), 2 g of powdered molecular sieve 4A and 2 g of silver silicate 2
g of toluene suspension. The cooling bath was removed and the mixture was further stirred at room temperature for 18 hours. After the reaction mixture was filtered using celite, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was subjected to a row bar column C (hexane-ethyl acetate = 4:
Purification in 1) gave 1.51 g of the title compound (11) (6
5.4%) and 180 mg (7.8) of the title compound (12).
%).

[0074] 11: syrup [α] ^{_{D 26 - 39.2 ° (c 0.13}} , CHCl 3) IR (film) cm -1: 1770, 1712, 1496, 1467, 1396, 1365, 1105, 1068 1 H-NMR ( CDCl ₃ ): δ 7.82 (2H, dd, aromatic H) 7.68 (2H, dd, aromatic H) 7.5-7.2 (20H, m, aromatic H) 3.96 (1H, s, H-1) 1.7-1.2 (12H, m,-(CH ₂ ) _6- )

12: Syrup-like [α] _D ²⁶ + 26.2 ° C. (c 0.13, CHCl ₃ ) IR (film) cm ⁻¹ : 1770, 1712, 1496, 1467, 1346, 1365, 1105, 1068 ¹ H-NMR ( CDCl ₃ ): δ 7.82 (2H, dd, aromatic H) 7.68 (2H, dd, aromatic H) 7.4-7.2 (20H, m, aromatic H) 4.62 (1H, s, H-1) 3.97 (1H, t, _{J 3, 4 = 9.5Hz, H} -4) 3.80 (1H, dd, J 2, 3 = 3Hz, J 3, 4 = 9.5Hz, H-3) 3.31 (1H, m, -CH 2 N) 1.7- 1.2 (12H, m,-(CH ₂ ) _6- )

(Ii) 2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl chloride (10)
A solution of 9 g (14.5 mmol) in 20 ml of methylene chloride was added at room temperature under a stream of argon at 10.0 g (36.25 mmol) of 8-phthalimidooctanol (4), 7.8 g (26.1 mmol) of silver carbonate and The solution was added dropwise to a suspension of 7.8 g of calcium sulfate in 50 ml of methylene chloride. The reaction mixture was stirred at room temperature for 19 hours, protected from light with aluminum foil. After filtering the reaction mixture using Celite,
The filtrate was concentrated to dryness under reduced pressure. The obtained residue is subjected to silica gel column chromatography (hexane-ethyl acetate =
4: 1) to give the same title compound (11) as above
7.49 g (65.7%) were obtained.

Reference Example 9 8-aminooctyl 2,3
4,6-Tetra-O-benzyl-β-D-mannopyranoside (13) 1.39 g (1.74 mmol) of compound (11) and 8.0 g (87 mmol) of hydrazine acetate were dissolved in 90 ml of methanol. The mixture was heated and refluxed on an oil bath for 4 hours. After cooling to room temperature, the reaction mixture was poured into a large amount of water, and the precipitated product was extracted with ethyl acetate. The organic layer was separated, washed with water, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. Ether was added to the obtained crystalline residue and the mixture was collected by filtration to obtain 1.1 g (95%) of compound (13).

[0078] 13: mp 89-90 ° C. [α] ^{_{D 23 - 42.2 ° (c 0.49}} , MeOH) IR (film) cm -1: 1661, 1603, 1497, 1454, 1302, 1113, 1059 1 H-NMR ( _{CDCl 3): δ 7.5-7.1 (20H} , m, aromatic H) 4.37 (1H, s, H-1) 3.90 (1H, dd, J 2, 3 = 2.5Hz, H-2) 3.85 (1H, t, _{J 3, 4 = 9.5Hz, H} -4) 3.50 (1H, dd, J 2, 3 = 2.5Hz, J 3, 4 = 9.5Hz, H-3) 2.66 (2H, t, -CH 2 N) 1.7 -1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 10 8-benzyloxycarbonylaminooctyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (14) 0.29 g of compound (13) and 0.03 g of triethylamine
0.044 g of benzyloxycarbonyl chloride was added to a solution of 7 g of chloroform in 20 ml while stirring at room temperature. After stirring at room temperature for 3 hours, the reaction mixture was washed with water. The organic layer was separated, dried over sodium sulfate, and concentrated to dryness under reduced pressure. The obtained residue is subjected to silica gel column chromatography (hexane-ethyl acetate = 4: 1).
Then, 0.29 g (83%) of the title compound (14) was obtained.

[0080] 14: amorphous powder [α] ^{_{D 22 - 38.7 ° (c 1.27}} , CHCl 3) 1 H-NMR (CDCl 3): δ 7.16-7.48 (25H, m, aromatic H) 4.38 (1H, s, H-1) 3.17 (2H, m) 1.67-1.25 (12H, m,-(CH ₂ ) _6- )

Reference Example 11 8-aminooctyl β-D
-Mannopyranoside (15) hydrochloride To a solution of 200 mg (0.299 mmol) of compound (13) in 15 ml of methanol were added 0.3 ml of 1 M hydrochloric acid and 50 mg of 10% palladium-carbon, and hydrogenated at room temperature for 8 hours. After removing the catalyst by filtration, the filtrate was concentrated to dryness under reduced pressure to obtain 153 mg of the hydrochloride of the title compound (15).

15 (hydrochloride): amorphous powder [α] _D ²⁴ -10.3 ° (c 0.62, MeOH) IR (KBr) cm ^-1 : 3358, 1614, 1074 ¹ H-NMR (CD ₃ OD): δ 4.49 (1H, s, H- 1) 3.44 (1H, dd, J 5, 6b = 3.5Hz, H-6b) 3.19 (1H, ddd, H-5) 2.90 (2H, t, J = 7.5Hz, - (CH ₂ N) 1.7-1.3 (12H, m,-(CH ₂ ) _6- )

Reference Example 12 8-aminooctyl β-D
-P-Toluenesulfonic acid salt of mannopyranoside (15) 2.64 g (3.3 mmol) of compound (14) and p-
After dissolving 0.63 g (3.3 mmol) of toluenesulfonic acid in 20 ml of methanol, 50 mg of 10% palladium-carbon was added and hydrogenated at room temperature for 3 hours. After filtration of the catalyst, the filtrate was concentrated to dryness under reduced pressure to give the title compound (1
1.57 g of p-toluenesulfonic acid salt of 5) (quantitative)
I got

[0084] 15 (p-toluenesulfonate): amorphous powder [α] ^{_{D 24 - 10.3 ° (c 0.62}} , MeOH) IR (KBr) cm -1: 3358, 1614, 1074 1 H-NMR (CD 3 OD): δ 7.71 (2H, d, J = 7.8Hz, aromatic H) 7.23 (2H, d, J = 7.8Hz, aromatic H) 4.49 (1H, d, J ₁ , ₂ = 0.7Hz, H-1) 3.24 (2H, m) 2.90 (2H, t, J = 7.5Hz, -CH ₂ N) 1.65-1.4 (12H, m,-(CH ₂ ) _6- )

Reference Example 13 8-aminooctyl-β-D
-Mannopyranoside (15) 8-aminooctyl β- synthesized by the method of Reference Example 11
After dissolving the hydrochloride salt of D-mannopyranoside (15) in aqueous methanol, the ion exchange resin IRC-50 (NH ₄
Mold) (4.5 cm × 40 cm). 800 water
After elution with 1 ml, the column was eluted with 1,700 ml of 6% aqueous ammonia. The fractions after 1,000 ml of the eluate were collected and concentrated to dryness under reduced pressure to obtain 1.305 g (58%) of the free base of the title compound (15).

[0086] 15 (free base): amorphous powder [α] ^{_{D 21 - 38.9 ° (c 1.01}} , MeOH) 1 H-NMR (D 2 O): δ 4.66 (1H, d, J 1, 2 = 1.0Hz , H-1) 3.97 (1H , d, J 2, 3 = 3.0Hz, H-2) 3.92 (1H, dd, J 5, 6a = 2.2Hz, J 6a, 6b = 12Hz, H-6a) 3.87 ( 1H, dd, J = 3.2Hz, 10Hz, -CH 2 O) 3.72 (1H, dd, J 5, 6b = 6.6Hz, H-6b) 3.65 (1H, m, -CH 2 O) 3.63 (1H, dd , J ₂ , ₃ = 3.2, J ₃ , ₄ = 9.8Hz, H-3) 3.56 (1H, t, J ₃ , ₄ = 9.8Hz, H-4) 3.35 (1H, ddd, H-5) 2.68 ( 2H, t, J = 7.0Hz, -CH ₂ N) 1.60-1.30 (12H, m,-(CH ₂ ) _6- )

Reference Example 14 8-phthalimidooctyl
3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside (17) and 8-phthalimidooctyl 3,4,6-tri-O-acetyl-2-azido-2 -Deoxy-α-D-mannopyranoside (18)

The method described in the literature (Carbohydr. Res., 136,
153 (1985)), 9.5 g (24.1 mmol) of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranosyl bromide (16) Is stirred at -15 ° C under a nitrogen stream.
6.63 g of 8-phthalimidooctanol (4)
(24.09 mmol), powdered molecular sieve 4
A was added dropwise to a suspension of 10 g of silver silicate and 10 g of silver silicate in 80 ml of toluene. The reaction mixture was stirred at the same temperature for 3 hours.
After removing the cooling bath, the mixture was further stirred at room temperature for 15 hours. After the reaction mixture was filtered using celite, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was subjected to row bar column C (hexane-
Purify with ethyl acetate = 2: 1) to give the title compound (17)
5.01 g (35.6%) and the title compound (18)
84 g (12.4%) were obtained.

[0089] 17: syrup [α] ^{_{D 23 - 49 ° (c 0.5}} , CHCl 3) IR (CHCl 3) cm -1: 2250, 1750, 1715, 1398, 1370 1 H-NMR (CDCl 3): δ 7.82 (2H, dd, aromatic H ) 7.69 (2H, dd, aromatic H) 5.23 (1H, t, J 3, 4 = 9.5Hz, H-4) 4.96 (1H, dd, J 2, 3 = 3.5Hz, H-3) 4.64 (1H, d, J 1, 2 = 1.5Hz, H-1) 4.25 (1H, dd, J 5, 6a = 5Hz, J 6a, 6b = 12.5Hz, H-6a) 4.11 (1H , dd, J ₅ , _6b = 2.5Hz, H-6b) 4.07 (1H, dd, J ₂ , ₃ = 3.5Hz, H-2) 3.89 (1H, m, CH ₂ N) 3.65 (1H, t, J = 7.5Hz, -CH ₂ O) 3.58 (1H, ddd, H-5) 3.48 (1H, m, -CH ₂ N) 2.09 (3H, s, OAc) 2.07 (3H, s, OAc) 2.02 (3H, s, OAc) 1.7-1.2 (12H, m,-(CH ₂ ) _6- )

18: Syrup-like [α] _D ²³ + 47.7 ° (c 0.56, CHCl ₃ ) IR (CHCl ₃ ) cm ^-1 : 2250, 1750, 1715, 1398, 1370 ¹ H-NMR (CDCl ₃ ): δ 7.83 (2H, dd, aromatic H) 7.70 (2H, dd, aromatic H) 5.38 (1H, dd, J ₂ , ₃ = 4Hz, J ₃ , ₄ = 9.5Hz, H-3) 5.30 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 4.81 (1H, d, J ₁ , ₂ = 1.5Hz, H-1) 4.24 (1H, dd, J ₅ , _6a = 5Hz, J _6a , _6b = 12.5Hz, H-6a) 4.07 (1H, dd, J 5, 6b = 2.5Hz, H-6b) 4.00 (1H, dd, J 2, 3 = 3.5Hz, H-2) 3.72-3.62 (3H, m, -CH ₂ O, -CH ₂ N) 3.41 (1H, m, -CH ₂ N) 3.58 (1H, ddd, H-5) 2.09 (3H, s, OAc) 2.08 (3H, s, OAc) 2.04 (3H, s , OAc) 1.7-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 15 8-phthalimidooctyl
2-Azido-2-deoxy-β-D-mannopyranoside (19) To a solution of 4.0 g (6.795 mmol) of compound (17) in 55 ml of methanol was added 1.7 ml of a 1M sodium methoxide-methanol solution, and the mixture was allowed to stand at room temperature for 16 hours. I left it. Add the ion exchange resin Amberlite IR-120B (H
After neutralizing by adding ( ⁺ type), the resin was filtered off and washed with methanol. The filtrate and washings were combined and concentrated to dryness under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform-methanol = 10: 1) to obtain 1.82 g (58%) of the title compound (19).

[0092] 19: syrup [α] ^{_{D 28 - 72.5 ° (c 1.03}} , CHCl 3) IR (CHCl 3) cm -1: 3500, 2114, 1772, 1711, 1398, 1072 1 H-NMR (CD 3 OD ): δ 7.77 (2H, dd, aromatic H) 7.83 (2H, dd, aromatic H) 4.64 (1H, d, J ₁ , ₂ = 1.5Hz, H-1) 3.89 (1H, m, -CH ₂ N) 3.56 (1H, dd, J ₂ , ₃ = 3.5Hz, J ₃ , ₄ = 9.5Hz, H-3) 3.50 (1H, m, -CH ₂ N) 3.39 (1H, t, J = 9.5Hz, H- 4) 3.16 (1H, ddd, J ₅ , _6a = 2.5Hz, J ₅ , _6b = 6.3Hz, H-5) 1.7-1.25 (12H, m,-(CH ₂ ) _6- )

Reference Example 16 8-phthalimidooctyl
2-acetamido-2-deoxy-β-D-mannopyranoside (20) 380 mg of nickel chloride hexahydrate was added to a solution of 1.68 g (3.63 mmol) of compound (19) in 43 ml of ethanol.
After adding 0.68 ml of a solution of (1.6 mmol) in 10 ml of ethanol, sodium borohydride 412 was added.
A solution of mg (10.89 mmol) dissolved in 40 ml of ethanol was slowly added dropwise with stirring. After stirring at room temperature for 30 minutes, the reaction solution was neutralized by adding acetic acid. 2 ml of acetic anhydride was added to the reaction mixture, and the mixture was left at room temperature for 18 hours. After the reaction mixture was concentrated to dryness under reduced pressure, the obtained residue was purified by silica gel column chromatography (chloroform-methanol = 7: 1) to give the title compound (20) 5.
87 mg (34%) were obtained.

[0094] 20: amorphous powder [α] ^{_{D 26.5 - 28.2 ° (c 1.42}} , MeOH) IR (KBr) cm -1: 3425, 1672, 1060 1 H-NMR (CD 3 OD): δ 7.7-7.5 ( 4H, m, aromatic H) 4.58 (1H, d, J 1, 2 = 1.5Hz, H-1) 4.36 (1H, dd, J 2, 3 = 3.5Hz, H-2) 3.48 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 2.00 (3H, s, NAc) 1.8-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 17 8-aminooctyl 2-acetamido-2-deoxy-β-D-mannopyranoside (21) To a solution of 580 g (1.212 mmol) of compound (20) in 10 ml of ethanol was added 2 ml of hydrazine hydrate. In addition, the mixture was heated and refluxed at 70 ° C. for 5 hours. After cooling to room temperature, the reaction solution was diluted by adding toluene, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform-methanol = 7: 1),
140 mg (34%) of the title compound (21) were obtained. At the same time, 121 mg of compound (20) was recovered.

[0096] 21: amorphous powder [α] ^{_{D 21 - 35.8 ° (c 0.45}} , MeOH) IR (KBr) cm -1: 3350, 1715, 1660, 1545, 1370, 1070 1 H-NMR (CD 3 OD) : δ 4.47 (1H, s, H-1) 4.56 (1H, dd, J 1, 2 = 1.5Hz, J 2, 3 = 3.5Hz, H-2) 1.98 (3H, s, NAc) 1.6-1.2 ( 12H, m,-(CH ₂ ) _6- )

Reference Example 18 8-phthalimidooctyl
2.75 g (10) of 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (23) 8-phthalimidooctanol (4)
Mmol), 2.53 g (10 mmol) of second silver cyanide and 5 g of anhydrous calcium sulfate in 50 ml of dry benzene.
A suspension of 50 ml of dry nitromethane was stirred at room temperature for 30 minutes under a stream of argon. 2,3,4,6
A solution of 6 g (14.6 mmol) of tetra-O-acetyl-α-D-galactopyranosyl bromide (22) in 20 ml of dry benzene was added dropwise at room temperature with stirring. The reaction mixture was stirred at room temperature for 12 hours. After the reaction mixture was filtered using celite, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was purified by flash column chromatography (ethyl acetate-hexane = 1: 1),
5.48 g (90.5%) of the title compound (23) were obtained as a colorless oil.

[0098] 23: syrup [α] _{^{D 27 - 1.02 ° (c 2.25}} , CHCl 3) IR (CHCl 3) cm -1: 1750, 1710 1 H-NMR (CDCl 3): δ 7.80-7.85 (2H, m, aromatic H) 7.65-7.70 (2H, m, aromatic H) 5.36 (1H, d, J ₃ , ₄ = 3.4Hz, H-4) 5.18 (1H, dd, J ₂ , ₃ = 10.5Hz, J _{1 , 2 = 7.8Hz, H-2} ) 4.99 (1H, dd, J 2, 3 = 10.5Hz, J 3, 4 = 3.4Hz, H-3) 4.43 (1H, d, J 1, 2 = 7.8Hz, H-1) 4.05-4.20 (2H, m, -CH 2 O) 3.82-3.90 (2H, m, -CH 2 O) 3.65 (2H, t, J = 7.6Hz, -CH 2 N) 3.40-3.50 ( 1H, m, H-5) 2.12 (3H, s, OAc) 2.03 (6H, s, OAc × 2) 1.96 (3H, s, OAc) 1.50-1.70 (4H, m, CH ₂ × 2) 1.20-1.40 (8H, m, CH ₂ × 4)

Reference Example 19 8-phthalimidooctyl
β-D-galactopyranoside (24) To a solution of 5.4 g (8.9 mmol) of compound (23) in 100 ml of methanol was added 0.5 ml of a 28% sodium methoxide-methanol solution, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure. The residue obtained is washed with water 5
After dissolving in 0 ml, ion-exchange resin Amberlite IR
-120B (H ⁺ type) was added for neutralization. After filtering off the resin, the filtrate was concentrated to dryness under reduced pressure to give the title compound (2
4) 1.61 g (41.3%) was obtained.

[0100] 24: mp 123-125 ° C. [α] ^{_{D 27 - 11.4 ° (c 1.09}} , CHCl 3) 1 H-NMR (CD 3 OD): δ 7.80-7.90 (2H, m, aromatic H) 7.75-7.80 (2H, m, aromatic H) 4.16 (1H, d, J ₁ , ₂ = 7.6Hz, H-1) 3.82-3.88 (1H, m) 3.79 (1H, d, J = 2.7Hz) 3.65-3.75 (2H , m) 3.62 (2H, t, J = 7.3Hz, -CH ₂ O) 3.40-3.52 (4H, m) 1.50-1.70 (4H, m, CH ₂ × 2) FAB-Mass: m / z 460 (M + Na) ⁺ , 438 (M + H) ⁺ , 276, 258, 16
0

Reference Example 20 8-aminooctyl β-D
-Galactopyranoside (25) To a solution of 970 mg (2.22 mmol) of compound (24) in 30 ml of ethanol was added 500 mg of hydrazine hydrate, and the mixture was heated and refluxed on an oil bath for 66 hours. After cooling to room temperature,
The resulting insolubles were removed by filtration. The filtrate was concentrated to dryness under reduced pressure. The obtained crude product was dissolved in 30 ml of purified water, and neutralized by adding concentrated hydrochloric acid. The reaction mixture was treated with ion exchange resin I.
The solution was passed through a 300 ml column of RC-50 (NH ₄ ⁺ type). The desired product was eluted with 800 ml of 2M aqueous ammonia. The obtained eluate was concentrated to dryness under reduced pressure to obtain 440 mg (64.6%) of the title compound (25).

25: mp 135-142 ° C. (EtOH-isopropyl ether) [α] _D ²² -13.5 ° (c 0.55, MeOH) ¹ H-NMR (CD ₃ OD): δ 4.19 (1H, d, J ₁ , ₂ = 7.6Hz, H-1) 3.85-3.92 (1H, m) 3.81 (2H, d, J = 3.2Hz) 3.42-3.55 (4H, m) 3.70-3.76 (2H, q) 2.63 (2H, t, J = 7.1Hz, -CH ₂ N) 1.58-1.65 (2H, m, -CH ₂ ) 1.42-1.50 (2H, m, -CH ₂ ) 1.35-1.42 (2H, m, -CH ₂ ) 1.33 (6H, m, -CH ₂ × 3) FAB-Mass: m / z 308 (M + H) ⁺ , 146

Reference Example 21 8-phthalimidooctyl
2,3,4-tri-O-acetyl-α-D-xylopyranoside (27), 8-phthalimidooctyl 2,3
4-tri-O-acetyl-β-D-xylopyranoside (28) and 8-phthalimidooctyl 3,4-di-O-acetyl-α-D-xylopyranoside (29) 2,3,4-tri-O- To a solution of 1.85 g (5.45 mmol) of acetyl-α-D-xylopyranosyl bromide (26) in 30 ml of benzene, 1.3 mercuric cyanide was added.
8 g (5.45 mmol), powdered molecular sieve 4A (3.5 g) and 1.50 g (5.45 mmol) of 8-phthalimidooctanol (4) were added, and the mixture was stirred at room temperature for 18 hours in a stream of argon. After the reaction mixture was filtered to remove insolubles, 40 ml of ethyl acetate was added to the filtrate. The reaction solution was washed with 40 ml of water, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The resulting syrupy residue was purified by silica gel column chromatography (toluene-ethyl acetate = 10: 1) to give 694 mg (23.9%) of the title compound (27) and compound (28) 31.
7 mg (10.9%) and 313 mg (1
1.7%).

27: [α] _D + 81.5 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2940, 1760, 1710, 1230, 1050 ¹ H-NMR (CDCl ₃ ): δ 7.70-7.72 (2H, m , aromatic H) 7.83-7.85 (2H, m, aromatic H) 5.47 (1H, t, J 2, 3 = 10.2Hz, H-3) 4.97 (1H, d, J 1, 2 = 3.7Hz, H-1 ) 4.9-5.0 (1H, m, H -4) 4.78 (1H, dd, J 2, 3 = 10.2Hz, J 1, 2 = 3.7Hz, H-2) 3.76 (1H, dd, J = 11.0Hz, J = 6.1Hz, H-5b) 3.6-3.8 (4H, m, H-5a, -CH ₂ O and -CH ₂ N) 3.5-3.6 (1H, m, -CH ₂ O) 2.02 (3H, s, OAc) 2.03 (3H, s, OAc) 2.05 (3H, s, OAc) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.3-1.4 (8H, m, -CH ₂ × 4)

28: [α] _D -22.4 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2940, 1760, 1710, 1230, 1050 ¹ H-NMR (CDCl ₃ ): δ 7.83-7.85 (2H, m , aromatic H) 7.70-7.72 (2H, m, aromatic H) 5.12 (1H, t, J = 8.6Hz, H-3) 4.9-5.0 (1H, m, H-4) 4.90 (1H, dd, J ₂ , ₃ = 8.6Hz, J ₁ , ₂ = 6.8Hz, H-2) 4.45 (1H, d, J = 6.8Hz, H-1) 4.11 (1H, dd, J = 11.8Hz, J = 5.1Hz, H -5b) 3.7-3.8 (1H, m, -CH 2 O) 3.67 (2H, t, J = 7.1Hz, -CH 2 N) 3.4-3.5 (1H, m, -CH 2 O) 3.35 (1H, dd , J = 11.8Hz, J = 8.8Hz, H-5a) 2.03 (3H, s, OAc) 2.04 (3H, s, OAc) 2.05 (3H, s, OAc) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.3-1.4 (8H, m, -CH ₂ × 4)

29: [α] _D + 79.8 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2940, 1760, 1710, 1230, 1050 ¹ H-NMR (CDCl ₃ ): δ 7.83-7.85 (2H, m , aromatic H) 7.70-7.72 (2H, m, aromatic H) 5.22 (1H, t, J = 9.5Hz, H-3) 4.7-4.8 (1H, m, H-4) 3.6-3.8 (4H, m, _{H-5b, -CH 2 O,} -CH 2 N) 3.5-3.6 (2H, m, H-2, H-5a) 4.82 (1H, d, J = 3.7Hz, H-1) 3.4-3.5 (1H , m, -CH ₂ O) 2.03 (3H, s, OAc) 2.09 (3H, s, OAc) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.3-1.4 (8H, m, -CH ₂ × Four)

Reference Example 22 8-aminooctyl β-D
-Xylopyranoside (30) To a solution of 680 mg (1.27 mmol) of compound (27) in 10 ml of methanol was added 0.16 ml of a 28% sodium methoxide-methanol solution, and the mixture was stirred at room temperature for 2 hours. The reaction solution was neutralized with Amberlite IR-120B (H ⁺ ). After filtering off the resin, the filtrate was concentrated to dryness under reduced pressure.
After dissolving the obtained syrup-like residue in 1 ml of ethanol, 318 mg (6.35 mmol) of hydrazine hydrate was obtained.
Was added and the mixture was refluxed at 100 ° C. for 2 hours. Further, 318 mg (6.35 mmol) of hydrazine hydrate was added, and the mixture was heated under reflux for 2 hours. After filtering off the precipitated insoluble matter, the filtrate was concentrated to dryness under reduced pressure. The obtained syrup-like residue was purified by silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 10: 10: 1). Fractions positive for the ninhydrin reagent were collected and the solvent was distilled off under reduced pressure. Dissolve the obtained powdery substance in 30 ml of water,
The mixture was neutralized by adding 3M hydrochloric acid. The solution was passed through a 100 ml column of Amberlite IRC-50 (NH ₄ ⁺ type) and washed with 200 ml of water. Then eluted with 2M aqueous ammonia. The eluate was collected and the solvent was distilled off under reduced pressure to obtain 274 mg (77.8%) of the title compound (30).

30: [α] _D + 72.5 ° (H ₂ O) IR (KBr) cm ^-1 : 3300, 2940, 1580, 1030 ¹ H-NMR (D ₂ O): δ 4.88 (1H, d, J ₁ , ₂ = 3.4Hz, H-1) 3.5-3.7 (7H, m, H-2, H-3, H-4, H-5, -CH ₂ O) 2.98 (2H, t, J = 7.8Hz , -CH ₂ N) 1.6-1.7 (4H, m, -CH ₂ × 2) 1.3-1.5 (8H, m, -CH ₂ × 4)

Reference Example 23 8-phthalimidooctyl
2,3,4-tri-O-benzyl-β-L-fucopyranoside (32) and 8-phthalimidooctyl 2,3,4
-Tri-O-benzyl-α-L-fucopyranoside (3
3) 4,26 g (8.57 mmol) of 2,3,4-tri-O-benzyl-α-L-fucopyranosyl bromide (31)
2.17 g of mercuric cyanide in a 40 ml solution of benzene
(8.57 mmol), powdered molecular sieve 4A
4.0 g and 8-phthalimidooctanol (4)
2.36 g (8.57 mmol) was added, and the mixture was stirred at room temperature in an argon stream for 22 hours. After the reaction mixture was filtered to remove insolubles, 30 ml of ethyl acetate was added to the filtrate. The reaction solution was washed with 80 ml of water, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained syrupy residue was purified by silica gel column chromatography (hexane-ethyl acetate = 6: 1) to give the title compound (32)
1.83 g (30.9%) and the title compound (33) 84
2 mg (14.2%) were obtained.

32: [α] _D + 4.0 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2980, 1770, 1710, 1400, 1069 ¹ H-NMR (CDCl ₃ ): δ 7.6-7.7 (2H, m , aromatic H) 7.8-7.9 (2H, m, aromatic H) 7.2-7.4 (15H, m, aromatic H) 4.9-5.0 (2H, m, -CH ₂ ph) 4.6-4.8 (4H, m, -CH ₂ ph) 4.29 (1H, d, J ₁ , ₂ = 7.6Hz, H-1) 3.78 (1H, dd, J ₂ , ₃ = 9.8Hz, J ₁ , ₂ = 7.6Hz, H-2) 3.65 (1H, t, J = 7.3Hz, -CH ₂ N) 3.54 (1H, d, J ₃ , ₄ = 2.9Hz, H-4) 3.50 (1H, dd, J ₂ , ₃ = 9.8Hz, J ₃ , ₄ = 2.9 Hz, H-3) 3.4-3.5 (2H, m, H-5 and -CH ₂ O) 3.9-4.0 (1H, m, -CH ₂ O) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.2-1.4 (8H, m, -CH ₂ × 4) 1.16 (3H, d, J = 6.4Hz, H-6)

33: [α] _D -35.2 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2970, 1770, 1400, 1100 ¹ H-NMR (CDCl ₃ ): δ 7.8-7.9 (2H, m, aromatic H) 7.6-7.7 (2H, m, aromatic H) 7.2-7.4 (15H, m, aromatic H) 4.77 (1H, d, J ₁ , ₂ = 3.4Hz, H-1) 4.6-5.0 (6H, m, -CH ₂ ph × 3) 4.01 (1H, dd, J ₂ , ₃ = 10.3Hz, J ₁ , ₂ = 3.4Hz, H-2) 3.93 (1H, dd, J ₂ , ₃ = 10.3Hz, J ₃ , ₄ = 2.7Hz, H-3) 3.86 (1H, q, J = 6.4Hz, H-5) 3.6-3.7 (3H, m, H-4 and -CH ₂ N) 3.5-3.6 (1H, m,- (CH ₂ O) 3.4-3.5 (1H, m, -CH ₂ O) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.3-1.4 (8H, m, -CH ₂ × 4) 1.09 (3H, d , J ₅ , ₆ = 6.4Hz, H-6)

Reference Example 24 8-phthalimidooctyl
β-L-fucopyranoside (34) To a solution of 1.70 g (2.46 mmol) of compound (32) in 10 ml of ethanol, 1 ml of acetic acid and 10% palladium-
1 g of carbon was added, and catalytic reduction was carried out using a Parr apparatus (50 psi). After filtering off the catalyst, the filtrate was concentrated to dryness under reduced pressure.
The obtained syrupy residue was purified by silica gel column chromatography (chloroform-methanol = 40: 1) to obtain 894 mg (86.2%) of the title compound (34).

34: [α] _D + 12.5 ° (MeOH) IR (KBr) cm ⁻¹ : 3510, 2940, 1690, 1400, 1080 ¹ H-NMR (CD ₃ OD): δ 7.8-7.9 (4H, m , aromatic H) 4.16 (1H, d, J ₁ , ₂ = 7.6Hz, H-1) 3.8-3.9 (1H, m, -CH ₂ O) 3.66 (2H, t, J = 7.1Hz, -CH ₂ N ) 3.5-3.6 (2H, m, H-4 and H-5) 3.4-3.5 (3H, m, H-2 H-3 and -CH ₂ O) 1.6-1.7 (4H, m, -CH ₂ × 2 ) 1.3-1.4 (8H, m, -CH ₂ × 4) 1.25 (3H, d, J ₅ , ₆ = 6.4Hz, H-6)

Reference Example 25 8-aminooctyl β-L
-Fucopyranoside (35) To a solution of 800 mg (1.90 mmol) of compound (34) in 10 ml of ethanol, 476 mg of hydrazine hydrate (9.
(50 mmol), and the mixture was refluxed at 100 ° C. for 2 hours. Further, 476 mg (9.50 mmol) of hydrazine hydrate was added, and the mixture was heated under reflux at the same temperature for 22 hours. After filtering off the precipitated insoluble matter, the filtrate was concentrated to dryness under reduced pressure. The obtained syrupy residue is subjected to silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 2).
0: 10: 1). The ninhydrin reagent positive fractions were collected and the solvent was distilled off under reduced pressure. The residue obtained is washed with water 1
After dissolving in 0 ml, the mixture was neutralized by adding 3M hydrochloric acid. This solution was passed through a 50 ml column of Amberlite IRC-50 (NH ₄ ⁺ type). After the column was washed with 100 ml of water, the desired product was eluted with 2M aqueous ammonia. The eluate was concentrated to dryness under reduced pressure to give 505 mg of the title compound (35) (9
(1.3%).

35: [α] _D + 10.6 ° (H ₂ O) IR (KBr) cm ⁻¹ : 3480, 2980, 1610, 1380, 1080 ¹ H-NMR (D ₂ O): δ 4.36 (1H, d , J ₁ , ₂ = 8.1Hz, H-1) 3.88 (1H, dt, J = 10.0Hz, J = 6.8Hz, -CH ₂ O) 3.78 (1H, q, J = 6.8Hz, H-5) 3.74 (1H, d, J ₃ , ₄ = 3.2Hz, H-4) 3.65 (1H, dt, J = 10.0Hz, J = 6.8Hz, -CH ₂ O) 3.63 (1H, dd, J ₂ , ₃ = 10.0 _{Hz, J 3, 4 = 3.2Hz} , H-3) 3.45 (1H, dd, J 1, 2 = 8.1Hz, J 2, 3 = 3.2Hz, H-2) 2.98 (2H, t, J = 7.8Hz , -CH ₂ N) 1.6-1.7 (4H, m, -CH ₂ × 2) 1.3-1.4 (8H, m, -CH ₂ × 4) 1.25 (3H, d, J ₅ , ₆ = 6.4Hz, H- 6)

Reference Example 26 8-phthalimidooctyl
3,4-di-O-acetyl-2-azido-2-deoxy-α-L-fucopyranoside (37) and 8-phthalimidooctyl 3,4-di-O-acetyl-2-azido-
2-Deoxy-β-L-fucopyranoside (38) The method described in the literature (RU Lemieux et al., Canadian. J. Che.
m., 57, 1244 (1979)), 911 mg (2.71 mmol) of 3,4-di-O-acetyl-2-azido-2-deoxy-β-L-fucopyranosyl bromide (36). Of mercuric cyanide in 10 ml of benzene
(2.71 mmol), powdered molecular sieve 4A
1 g and 8-phthalimidooctanol (4) 746
mg (2.71 mmol) was added, and the mixture was stirred at room temperature in an argon stream for 15 hours. After the reaction mixture was filtered to remove insolubles, 20 ml of ethyl acetate was added to the filtrate. Water 3
After washing with 0 ml, it was dried over magnesium sulfate and concentrated to dryness under reduced pressure. The resulting syrupy residue is subjected to silica gel column chromatography (hexane-ethyl acetate =
4: 1) to give 308 mg (2) of the title compound (37).
1.4%) and 610 mg (42.4) of the title compound (38).
%).

37: [α] _D -110.4 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 2980, 2110, 1750, 1234 ¹ H-NMR (CDCl ₃ ): δ 7.70 (2H, dd, J = 5.4) Hz, J = 2.9Hz, aromatic H ) 7.84 (2H, dd, J = 5.4Hz, J = 2.9Hz, aromatic H) 5.36 (1H, dd, J 2, 3 = 11.2Hz, J 3, 4 = 3.2Hz , H-3) 5.30 (1H, d, J ₃ , ₄ = 3.2Hz, H-4) 4.93 (1H, d, J ₁ , ₂ = 3.4Hz, H-1) 4.14 (1H, q, J = 6.6 Hz, H-5) 3.6-3.7 (3H, m, -CH ₂ N and -CH ₂ O) 3.57 (1H, dd, J ₂ , ₃ = 11.2Hz, J ₁ , ₂ = 3.4Hz, H-2) 3.4-3.5 (1H, m, -CH ₂ O) 2.05 (3H, s, OAc) 2.16 (3H, s, OAc) 1.6-1.7 (4H, m, -CH ₂ × 2) 1.13 (3H, d, J ₅ , ₆ = 6.5Hz, H-6) 1.2-1.4 (8H, m, -CH ₂ × 4)

38: [α] _D + 9.1 ° (CHCl ₃ ) IR (KBr) cm ^-1 : 3020, 2110, 1750, 1242 ¹ H-NMR (CDCl ₃ ): δ 7.70 (2H, dd, J = 5.4) Hz, J = 2.9Hz, aromatic H ) 7.84 (2H, dd, J = 5.4Hz, J = 2.9Hz, aromatic H) 5.17 (1H, d, J 3, 4 = 3.2Hz, H-4) 4.75 (1H , dd, J ₂ , ₃ = 10.7Hz, J ₃ , ₄ = 3.2Hz, H-3) 4.31 (1H, d, J ₁ , ₂ = 7.8Hz, H-1) 3.9-4.0 (1H, m,- CH ₂ O) 3.73 (1H, q, J ₅ , ₆ = 6.6Hz, = 6.6, H-5) 3.6-3.7 (3H, m, H-2 and -CH ₂ N) 3.5-3.6 (1H, m, -CH ₂ O) 2.05 (3H, s, OAc) 2.16 (3H, s, OAc) 1.6-1.7 (4H, m, -CH ₂ × 2) 1.20 (3H, d, J = 6.4Hz, H-6) 1.2-1.4 (8H, m, -CH ₂ × 4)

Reference Example 27 8-aminooctyl 2-acetamido-2-deoxy-α-L-fucopyranoside (40) 28% sodium methoxide-methanol was added to a solution of 300 mg (0.565 mmol) of compound (37) in 5 ml of methanol. 0.08 ml of the solution was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was subjected to Amberlite IR-120B (H ⁺
(Type). After filtering off the resin, the filtrate was concentrated to dryness under reduced pressure. The obtained syrupy residue is diluted with ethanol 15
After dissolving in water, 64.1 mg of sodium borohydride
(1.70 mmol) and nickel chloride hexahydrate 40
0.5 ml of an ethanol solution of mg was added, and the mixture was stirred at room temperature for 2 hours. After the reaction solution was neutralized with 6M hydrochloric acid, the precipitate was removed by filtration. The filtrate was concentrated to dryness under reduced pressure, and the resulting syrupy residue was dissolved in 5 ml of methanol. 1 ml of acetic anhydride was added to this solution and stirred at room temperature for 3 hours. The reaction solution was concentrated to dryness under reduced pressure, and the syrupy residue obtained was subjected to silica gel column chromatography (chloroform-methanol = 3.
Purification by 0: 1) gave 257 mg of 8-phthalimidooctyl 2-acetamido-2-deoxy-α-L-fucopyranoside (39). Compound (39) (257 mg) was dissolved in ethanol (5 ml), and hydrazine hydrate (141 mg) was dissolved.
(2.83 mmol) was added and the mixture was heated under reflux at 100 ° C. for 2 hours. Further, 141 mg (2.83 mmol) of hydrazine hydrate was added, and the mixture was heated under reflux at the same temperature for 2 hours. After the reaction mixture was filtered to remove insolubles, the filtrate was concentrated to dryness under reduced pressure. The resulting syrupy residue was purified by silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 30: 10: 1). Ninhydrin-positive fractions were collected and concentrated to dryness under reduced pressure. The residue obtained was dissolved in 10 ml of water and neutralized with 3M hydrochloric acid. This solution was passed through a 50 ml column of Amberlite IRC-50 (NH ₄ ⁺ type). After washing with 100 ml of water, the desired product was eluted with 2M aqueous ammonia. The eluate is concentrated to dryness under reduced pressure,
41.1 mg (23%) of the title compound (40) were obtained.

40: [α] _D -74.8 ° (H ₂ O) IR (KBr) cm ^-1 : 3400, 1640, 1030 ¹ H-NMR (D ₂ O): δ 4.1-4.2 (2H, m, H -2 and H-5) 3.92 ( 1H, dd, J 2, 3 = 11.2Hz, J 3, 4 = 3.2Hz, H-3) 3.82 (1H, d, J 3, 4 = 3.2Hz, H-4 ) 3.6-3.7 (1H, m, -CH ₂ O) 3.4-3.5 (1H, m, -CH ₂ O) 3.00 (2H, t, J = 7.6Hz, -CH ₂ N) 2.05 (3H, s, NAc ) 1.5-1.7 (4H, m, -CH ₂ × 2) 1.24 (3H, d, J ₅ , ₆ = 6.6Hz, H-6) 1.2-1.4 (8H, m, -CH ₂ × 4)

Reference Example 28 Poly-L-glutamic acid (4
5) Commercially available poly-L-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 13,300) (41) 10 g
(0.751 mmol) in 200 ml of an aqueous solution under ice-cooling,
While stirring, 51 ml of 1M hydrochloric acid was added dropwise to adjust the pH of the solution to about 2. The deposited precipitate was collected by centrifugation. The precipitate is washed with a sufficient amount of cold water, suspended in purified water and freeze-dried to obtain poly-L-glutamic acid (45).
6.5 g were obtained. Table 1 shows the physical constants of the compound (45).

[0122]

[Table 1]

Reference Example 29 Poly-L-glutamic acid (4
6), (47) and (48) In the same manner as in Reference Example 28, commercially available sodium salt of poly-L-glutamic acid (average molecular weight distribution (viscosity method) = 3)
6,240) (42), poly-L-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 61,000)
(43), Poly-L-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 77,800) (44) is treated with 1 M hydrochloric acid in an aqueous solution to give a free acid to give the title compound (46), (47) ) And (48) were obtained. The physical constants of the obtained compound are shown in Table 1.

Reference Example 30 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (49) 100 mg of poly-L-glutamic acid (45) (0.007)
51 mmol), 10 mg (0.072 mmol) of 2- (4-hydroxyphenyl) ethylamine and 19 mg (0.0768 mmol) of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline in N, N-dimethyl It was dissolved in 3.5 ml of formamide and stirred at room temperature for 18 hours. Reaction solution 0.01M isotonic phosphate buffer (pH 7.4) 1
After dilution with 0 ml, the mixture was subjected to gel filtration column chromatography (Sephadex G-100, 50 cm × 2.5 cm) to elute 5 ml of each fraction. Fraction Nos. 15 to 27 were collected and collected as Spectra / Pore 3 (molecular weight cut-off = about 3,5
00) Dialysis was performed at 4 ° C. for 16 hours against purified water using a dialysis membrane. The dialysate was removed and lyophilized to give 75 mg of the title compound (49). When the amount of the introduced 2- (4-hydroxyphenyl) ethylamino residue was quantified using an absorbance of 275 nm, 5 mol of 2- (4-hydroxyphenyl) ethylamino group was introduced per 1 mol of poly-L-glutamic acid. Turned out to be. Table 2 shows the physical constants of compound (49).

[0125]

[Table 2]

Reference Example 31 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (50) Poly-L-glutamic acid (46) 100 mg (0.007)
51 mmol), 145 mg of O-sulfobenzimide
(0.791 mmol) and 141 mg (0.869 mmol) of N, N'-carbonyldiimidazole were added to N, N
-Dissolved in 3 ml of dimethylformamide and left at room temperature for 3 hours
Stirred for 0 minutes. To this solution was added 200 mg (1.458 mmol) of 2- (4-hydroxyphenyl) ethylamine and 0.19 ml of 1,1,3,3-tetramethylguanidine.
(1.514 mmol) in 4 ml of N, N-dimethylformamide (0.07 ml, 0.025 mmol) was added, and the mixture was stirred at room temperature for 2 days. The reaction mixture was added to 0.
After dilution with 01M isotonic phosphate buffer (pH 7.4), Spectra / Pore 1 (molecular weight cut off = 6,000-8,
000) Using a dialysis membrane, dialyzed against purified water at 4 ° C. for 48 hours. The dialysate was removed and lyophilized to give 78 mg of the title compound (50). The amount of the introduced 2- (4-hydroxyphenyl) ethylamino residue was measured at an absorbance of 275 nm.
Quantitative analysis revealed that 5 moles of 2- (4-hydroxyphenyl) ethylamino groups were introduced per mole of poly-L-glutamic acid. Table 2 shows the physical constants of compound (50).

Reference Example 32 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (51) and (52) Poly-L-glutamic acid was prepared in the same manner as in Reference Example 31. (47) and (48) show 10 molar equivalents of 2
2 using-(4-hydroxyphenyl) ethylamine
The introduction of-(4-hydroxyphenyl) ethylamino group gave the title compounds (51) and (52). Table 2 shows the amount (molar ratio) of the introduced 2- (4-hydroxyphenyl) ethylamino group and the physical constants of the obtained compound.

Reference Example 33 Poly-D-glutamic acid (5
6), (57) and (58) In the same manner as in Reference Example 28, commercially available sodium salt of poly-D-glutamic acid (average molecular weight distribution (viscosity method) = 1)
0,600) (53), poly-D-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 41,000)
(54), sodium poly-L-glutamate (average molecular weight distribution (viscosity method) = 95,100) (55) was treated with 1 M hydrochloric acid in an aqueous solution to obtain a free acid, and the title compound (56), (57) ) And (58) were obtained. Table 3 shows the physical constants of the obtained compound.

[0129]

[Table 3]

Reference Example 34 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D-glutamic acid sodium salt (59), (60) and (61) (56), (5
A 2- (4-hydroxyphenyl) ethylamino group was introduced into each of 7) and (58) to give the title compound (5
9), (60) and (61) were obtained. Introduced 2-
Table 4 shows the amount (molar ratio) of (4-hydroxyphenyl) ethylamino group and the physical constants of the obtained compound.

[0131]

[Table 4]

Example 1 Poly-γ- [8- (β-D-galactopyranosyloxy) octylamino] -γ- [2
-(4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (62) 75 mg (0.0066) of poly-L-glutamic acid (45)
Mmol) of dry N, N-dimethylformamide 1.5
180 mg (0.586 mmol) of 8-aminooctyl β-D-galactopyranoside (25), 2-
7.2 mg of (4-hydroxyphenyl) ethylamine
(0.0547 mmol) and 80.5 mg (0.7 mmol) of N-hydroxysuccinimide in 1.5 ml of dry N, N-dimethylformamide were added, and the mixture was cooled with ice. This reaction mixture was stirred with 1-ethyl-3-
A solution of 135 mg (0.7 mmol) of (3-dimethylaminopropyl) -carbodiimide hydrochloride and 10 ml of dry N, N-dimethylformamide was added. After removing the cooling bath,
Stirred overnight at room temperature. 30 ml of purified water was added to the reaction mixture, and the precipitated insolubles were removed by centrifugation. The supernatant was used as Spectra / Pore 3 (molecular weight cut off = about 3,50).
0) Dialysis was performed at 4 ° C. for 24 hours against purified water using a dialysis membrane. The dialysate was taken out and freeze-dried. The obtained residue was dissolved in a 0.01 M isotonic phosphate buffer (pH 7.4) and purified by gel filtration column chromatography (Sephadex G-100, 5 cm × 30 cm). Each eluted fraction is subjected to high performance liquid chromatography (column: TSK-
GEL G3000PWXL, eluent: 0.2 M saline, elution rate: 0.5 ml / min, detection: ultraviolet absorption (28
0 nm)). The fractions containing the poly-L-glutamic acid derivative were collected, and again dialyzed against purified water for 48 hours at 4 ° C. using a Spectra / Pore 3 (molecular weight cut off = about 3,500) dialysis membrane. The dialysate was removed and lyophilized to give 89.2 mg of the title compound (62). When the content of the introduced 8- (β-D-galactopyranosyloxy) octylamino residue was colorimetrically determined by the phenol-sulfuric acid method, the amount introduced was 36 mol per mol of polyglutamic acid. Further, when the amount of 2- (4-hydroxyphenyl) ethylamino group was quantified using the absorbance of 275 nm, the introduced amount (molar ratio) was 3. The sugar content of the compound and 2-
Table 4 shows the content of (4-hydroxyphenyl) ethylamino group.
It was shown to. Table 6 shows physical constants of the compounds.

[0133]

[Table 5]

[0134]

[Table 6]

Example 2 Poly-γ- [8- (β-D-mannopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (63) 50 mg (0.0037) of poly-L-glutamic acid (45)
Mmol) in 2 ml of dry N, N-dimethylformamide, 2-ethoxy-1-ethoxycarbonyl-1,2.
-Dihydroquinoline (152 mg, 0.615 mmol) was added, and the mixture was stirred at room temperature for 1 hour. To this solution was added hydrochloride of 8-aminooctyl β-D-mannopyranoside (15).
90 mg (0.56 mmol) and 0.1% triethylamine.
48 ml (0.56 mmol) of a 2 ml solution of N, N-dimethylformamide were added and stirred at room temperature for 1.5 hours. Then, 2- (4-hydroxyphenyl) ethylamine 5
mg was added and the mixture was further stirred for 15.5 hours. N, N
-After dilution by adding 0.5 ml of dimethylformamide,
Stirring was continued for another 25 hours. To the reaction mixture was added 0.5 ml of a 0.1 M aqueous sodium hydroxide solution and 15 ml of a 0.01 M isotonic phosphate buffer. After removing the precipitated insoluble matter by centrifugation, the supernatant was filtered using Millipore filter paper. The filtrate was purified by gel filtration column chromatography (Sephadex G-100, 2.5 cm × 40 cm). Poly-L
-Glutamic acid derivative-containing fractions were collected and dialyzed against purified water for 48 hours using a Spectra / Pore 3 (molecular weight cut-off = about 3,500) dialysis membrane. The dialysis solution is taken out and lyophilized to give a colorless amorphous compound (6
3) 35 mg was obtained. Colorimetric determination of the sugar content by the phenol-sulfuric acid method revealed that 7 moles of 8- (β-D-mannopyranosyloxy) octylamino group was introduced per mole of poly-L-glutamic acid. Also, 2- (4-
The amount of (hydroxyphenyl) ethylamino group is measured at an absorbance of 275.
When quantified using nm, it was found that 3 mol of 2- (4-hydroxyphenyl) ethylamino groups had been introduced. The physical constants of the obtained compound are shown in Table 6.

Example 3 Poly-γ- [8- (β-D-mannopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (64) Poly-L-glutamic acid (45) 50 mg (0.0037
Mmol) of N, N-dimethylformamide in 100 ml of 1-ethoxycarbonyl-2-ethoxy-1,2.
-Dihydroquinoline (152 mg, 0.615 mmol) was added, and the mixture was stirred for 1 hour and 40 minutes. Then, hydrochloride of 8-aminooctyl β-D-mannopyranoside (15) 190
mg (0.56 mmol) and 0.78 triethylamine
ml (0.56 mmol) of 50 ml of N, N-dimethylformamide and 2- (4-hydroxyphenyl)
Ethylamine 5 mg N, N-dimethylformamide 10
ml solution was added and stirred at room temperature for 45 hours. The reaction mixture was purified by a method similar to that described in Example 2 to obtain the title compound (64). Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. Table 6 shows physical constants of the compounds.

Example 4 Poly-γ- [8- (β-D-mannopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (65) 100 mg (0.075) of poly-L-glutamic acid (45)
1. mmol) anhydrous N, N-dimethylformamide
96 mg of O-sulfobenzimide (0.52
4 mmol), N, N'-carbonyldiimidazole 9
3 mg (0.573 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. 20 mg (0.146 mmol) of 2- (4-hydroxyphenyl) ethylamine and 0.19 ml of 1,1,3,3-tetramethylguanidine are added to this mixture.
(1.514 mmol) in 4 ml of N, N-dimethylformamide (2.0 ml) and 8-aminooctyl
153 mg of hydrochloride salt of β-D-mannopyranoside (15)
(0.461 mmol) in 1 ml of N, N-dimethylformamide was added and stirred at room temperature for 24 hours. The reaction mixture was purified in the same manner as described in Example 2,
The title compound (65) was obtained. Sugar content of compound and 2
Table 5 shows the content of-(4-hydroxyphenyl) ethylamino group. Table 6 shows physical constants of the compounds.

Example 5 Poly-γ- [8- (β-D-mannopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (66) In the same manner as described in Example 1, poly-L-glutamic acid (45) was added to 8-aminooctyl α-D-mannopyranoside ( Using 9) and 2- (4-hydroxyphenyl) ethylamine, 8- (β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced, The title compound (66) was obtained. The sugar content of the obtained compound and 2-
Table 4 shows the content of (4-hydroxyphenyl) ethylamino group.
It was shown to. Table 6 shows physical constants of the compounds.

Example 6 Poly-γ- [8- (α-D-mannopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (67) Poly-L-glutamic acid (45) 75 mg (0.0056
10 mmol of dry N, N-dimethylformamide
180 mg (0.611 mmol) of 8-aminooctyl α-D-mannopyranoside (9), 7.5 mg (0.00546 mmol) of 2- (4-hydroxyphenyl) ethylamine and 1-ethoxycarbonyl- A solution of 98 mg (0.0396 mmol) of 2-ethoxy-1,2-dihydroquinoline in 3 ml of dry N, N-dimethylformamide was added, and the mixture was stirred at room temperature for 16 hours. After diluting the reaction mixture with 30 ml of 0.01 M isotonic phosphate buffer, the solution was purified by using a Spectra / Pore 3 (molecular weight cut-off = about 3,500) dialysis membrane at 4 ° C. against purified water.
Dialysis was performed for 4 hours. The dialysate was taken out and freeze-dried.
The obtained residue is subjected to a 0.01 M isotonic phosphate buffer (pH 7.4).
And purified by gel filtration column chromatography (Sephadex G100, 5 cm × 30 cm). Each fraction eluted is subjected to high performance liquid chromatography (column: T
SK-GEL G3000PWXL, eluate: 0.2M
Saline, dissolution rate: 0.5 ml / min. , Detection: monitored by ultraviolet absorption (280 nm). The fractions containing the poly-L-glutamic acid derivative were collected and re-specified to Spectra / Pore 3
(Molecular weight cut-off = about 3,500)
Dialysis was performed at 4 ° C. for 48 hours against purified water. The dialysate was removed and lyophilized to give 80 mg of the title compound (67). Table 5 shows the sugar content and 2- (4-hydroxyphenyl) ethylamino group content of the obtained compound. Also,
The physical constants of the compounds are shown in Table 6.

Example 7 Poly-γ- [8- (α-D-xylopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (68) In the same manner as described in Example 3, poly-L-glutamic acid (45) was added to 8-aminooctyl α-D-xylopyranoside ( 30) and 2- (4-hydroxyphenyl) ethylamine to introduce an 8- (α-D-xylopyranosyloxy) octylamino group and a 2- (4-hydroxyphenyl) ethylamino group. Compound (68) was obtained. Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. Table 6 shows physical constants of the compounds.

Example 8 Poly-γ- [8- (β-L-fucopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (69) In the same manner as described in Example 3, poly-L-glutamic acid (45) was added to compound (34) and 2- (4- Using (hydroxyphenyl) ethylamine, 8- (β-
L-fucopyranosyloxy) octylamino group and 2-
Introduction of a (4-hydroxyphenyl) ethylamino group gave the title compound (69). Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. Table 6 shows physical constants of the compounds.

Example 9 Poly-γ- [8- (2-acetamido-2-deoxy-α-L-fucopyranosyloxy)
Octylamino] -γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (70) In the same manner as described in Example 3, poly-L-glutamic acid (45) was added to the compound Using (40) and 2- (4-hydroxyphenyl) ethylamine, 8- (2-
Acetamide-2-deoxy-α-L-fucopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced to give the title compound (7
0) was obtained. In order to determine the sugar content of compound (70),
The mixture was hydrolyzed by heating and stirring with 4M hydrochloric acid at 100 ° C. for 4 hours. When the obtained hydrolysis product was colorimetrically determined by the Elson-Morgan method, 8- (2-acetamido-2-deoxy-α-) was obtained.
L-fucopyranosyloxy) octylamino group content is
It was 3 moles per mole of poly-L-glutamic acid. Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. Table 6 shows physical constants of the compounds.

Example 10 Poly-γ- [8- (2-acetamido-2-deoxy-β-D-mannopyranosyloxy) octylamino] -γ- [2- (4-hydroxyphenyl) ethylamino ] -L-glutamic acid sodium salt (71) In the same manner as described in Example 9, compound (21) and 2- (4-hydroxyphenyl) ethylamine were used for poly-L-glutamic acid (45), 8- (2-
Acetamide-2-deoxy-β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced to give the title compound (7
1) was obtained. The sugar content of compound (71) was hydrolyzed with 4M hydrochloric acid in the same manner as described in Reference Example 27, and then colorimetrically determined by the Elson-Morgan method. Table 5 shows the sugar content and 2- (4-hydroxyphenyl) ethylamino group content of the compound. Table 6 shows physical constants of the compounds.

Reference Example 35 Poly-L-glutamic acid sodium salt (73) A method described in the literature (J. Am. Chem. Soc., 78, 941 (1956))
2.63 g (10.0 mmol) of γ-benzyl-N, O-carbonyl-L-glutamate (72) synthesized in the above was dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a nitrogen stream. To this solution, 6.6 μl of hexylamine was added to dry N, N-dimethylformamide 100
A solution dissolved in μl was added, and the mixture was stirred at room temperature under a nitrogen stream for 24 hours. 500 ml of isopropyl ether was added to the reaction mixture.
While stirring, and the deposited precipitate was collected by filtration. After the precipitate was thoroughly washed with ethanol, it was dried under reduced pressure.
The obtained precipitate was dissolved in a mixture of 17 ml of trifluoroacetic acid and 3 ml of anisole, and 17 ml of methanesulfonic acid was added under a nitrogen stream. The reaction mixture was stirred under ice cooling for 20 minutes. The cooling bath was removed, and the mixture was further stirred at room temperature for 30 minutes. The reaction solution was poured into 300 ml of isopropyl ether while stirring, and the deposited precipitate was collected by filtration. The precipitate was washed with isopropyl ether, added to 20 ml of a 5% aqueous sodium hydrogen carbonate solution, and stirred at room temperature for 30 minutes. The obtained solution was dialyzed against purified water at 4 ° C. for 2 days using a Spectra / pore dialysis membrane. The dialysate was taken out and freeze-dried to obtain 1.1 g of the title compound (73). Gel permeation chromatography using polyethylene glycol as a standard (column: TSK-GEL
G4000PWXL + G3000PWXL, eluent:
The weight average molecular weight (Mw) determined by 0.2M saline solution and elution rate: 0.5 ml / min) was 22,000. Table 7 shows the physical constants of the obtained compound.

[0145]

[Table 7]

Reference Example 36 Poly-L-glutamic acid (7
4) In the same manner as in Reference Example 35, γ-benzyl-N, O
N, using -carbonyl-L-glutamate (72)
Polymerization was carried out in N-dimethylformamide using triethylamine as a polymerization initiator. The product was debenzylated with trifluoroacetic acid, anisole and methanesulfonic acid in the same manner as in Reference Example 35, and then purified to obtain the title compound (74). The weight average molecular weight (Mw) of the compound (74) was determined by gel permeation chromatography using polyethylene glycol as a standard (column: TSK-GEL G4000PWXL + G3
000PWXL, eluate: 0.2M saline, elution rate:
(0.5 ml / min) was 57,200. Table 7 shows the physical constants of the obtained compound.

Example 11 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (75) Obtained by treating the compound (73) synthesized in Reference Example 35 with 1M hydrochloric acid in the same manner as in Reference Example 28 To a solution of 129.0 mg of the free acid of compound (73) in 1.0 ml of N, N-dimethylformamide was added 51.6 mg (0.25 mmol) of N, N'-dicyclohexylcarbodiimide and N-N-dicyclohexylcarbodiimide.
A solution prepared by dissolving 35.5 mg (0.31 mmol) of hydroxysuccinimide in 0.5 ml of N, N-dimethylformamide was added, and the mixture was stirred at 4 ° C. for 2 hours. 2-
6.8 mg of (4-hydroxyphenyl) ethylamine
(0.05 mmol), 8-aminooctyl β-D-
Mannopyranoside (15) p-toluenesulfonate 9
6.0 mg (0.20 mmol) and triethylamine 2
A solution prepared by dissolving 8 µl of the mixture in 0.5 ml of N, N-dimethylformamide was added thereto, followed by stirring at 4 ° C for 2 hours. Except for the cooling bath,
The mixture was further stirred at room temperature for 2 hours. 5 ml of a 5% aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and precipitated insolubles were removed by centrifugation. The obtained supernatant was filtered using Millipore filter paper. The filtrate was purified by gel filtration column chromatography (Sephadex G-100, 2.5 cm × 20 cm). Fractions containing the poly-L-glutamic acid derivative were collected and purified using purified water using a Spectra / pore dialysis membrane.
It was dialyzed for 2 days at ° C. The dialysate was taken out and freeze-dried to obtain 127.5 mg of the title compound (75). The sugar content of compound (75) was determined by colorimetry using the phenol-sulfuric acid method. The 2- (4-hydroxyphenyl) ethylamino group content was quantified using an absorbance of 275 nm. Table 8 shows the obtained results. Table 9 shows the physical constants of the compounds.

[0148]

[Table 8]

[0149]

[Table 9]

Example 12 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (76) In the same manner as in Example 11, the free acid of compound (74) was added to the p-toluenesulfonic acid salt of compound (15) and 2
8 using-(4-hydroxyphenyl) ethylamine
Introduction of-(β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group gave the title compound (76). Compound (7
Table 8 shows the sugar content and 2- (4-hydroxyphenyl) ethylamino group content of 6). Table 9 shows the physical constants of the compounds.

Reference Example 37 γ-benzyl-D-glutamate (78) 75.0 g (694 mmol) of benzyl alcohol was added to 75 ml of a 60% sulfuric acid solution of 100 g (680 mmol) of commercially available D-glutamic acid (77) under ice-cooling. added. After stirring the reaction mixture at 70 ° C. for 1 hour, the generated water was distilled off under reduced pressure. 120 g of sodium hydrogen carbonate was added to the reaction mixture.
Was poured into a solution dissolved in 400 ml of cold water while stirring. Further, 200 ml of cold water was added and left for a while. The precipitated crystals were collected by filtration and washed well with cold water. The product is 2
The crystals were recrystallized twice and the crystals were collected by filtration. Crystals are ethanol 100
After washing with 100 ml of ether and 100 ml of ether, the residue was dried under reduced pressure to obtain 52.1 g (32%) of the title compound (78).

[0152] 78: mp 164-164.5 ℃ [α] ^{_{D 25 - 19.4 ° (c 1.29}} , AcOH) IR (KBr) cm -1: 3000, 1724, 1582, 1517, 1173

REFERENCE EXAMPLE 38 γ-benzyl-N, O-carbonyl-D-glutamate (79) A suspension of 24 g (101 mmol) of compound (78) in 150 ml of anhydrous tetrahydrofuran was stirred under a nitrogen stream with stirring under a nitrogen stream. Methyl carbonate) 11 g (3
(7.1 mmol) in 40 ml of anhydrous tetrahydrofuran was added dropwise over 5 minutes. After stirring the reaction mixture at room temperature for 15 minutes, heating was started and the mixture was stirred at an internal temperature of 40-50 ° C for 4 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. 150 ml of hexane was added to the obtained crystalline residue, and the crystals were collected by filtration. Recrystallization from a hexane-ethyl acetate mixture gave 17.7 g (66%) of the title compound (79).

79: mp 84-85 ° C. [α] _D ²⁵ + 16.7 ° (c 1.05, AcOEt) IR (KBr) cm ^-1 : 1867, 1716, 1704, 1258, 928 ¹ H-NMR (CDCl ₃ ): δ 7.4 (5H, m, aromatic H) 6.18 (1H, s, NH) 5.14 (2H, s, CH ₂ ph) 4.36 (1H, t, J = 5.5Hz, α-CH) 2.60 (2H, m,- CH ₂ CO) 2.28 (1H, m, Glu-CH ₂ ) 2.10 (1H, m, Glu-CH ₂ )

Reference Example 39 Irregular copolymer of L-glutamic acid and D-glutamic acid (80) 2.37 g (90 mmol) of γ-benzyl-N, O-carbonyl-L-glutamate (72) and γ-benzyl Benzyl-N, O-carbonyl-D-glutamate (79) 26
0 mg (10 mmol) was dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a stream of nitrogen. To this solution, 13.2 μl of hexylamine was added to dry N, N-
A solution dissolved in 100 μl of dimethylformamide was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was poured into 500 ml of isopropyl ether with stirring. The precipitated precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained precipitate was dissolved in a mixture of 17 ml of trifluoroacetic acid and 3 ml of anisole while cooling with ice. Then, 17 ml of methanesulfonic acid was added and the mixture was stirred at the same temperature for 20 minutes. The cooling bath was removed, and the mixture was further stirred at room temperature for 30 minutes. 300 ml of isopropyl ether while stirring the reaction mixture
And the resulting precipitate was collected by filtration. The obtained precipitate is added to 20 ml of a 5% aqueous sodium hydrogen carbonate solution, and the mixture is added at room temperature for 3 hours.
Stirred for 0 minutes. The obtained solution was dialyzed against purified water at 4 ° C. for 2 days using a Spectra / pore dialysis membrane. Take out the dialysis solution, freeze-dry and obtain the title compound (80)
1.10 g were obtained. When the average molecular weight of the compound (80) was determined by gel permeation chromatography using polyethylene glycol as a standard substance, the weight average molecular weight (Mw) was 13,000. Table 10 shows the physical constants of the obtained compound.

[0156]

[Table 10]

REFERENCE EXAMPLE 40 Irregular copolymers of L-glutamic acid and D-glutamic acid (81) and (82) In the same manner as in Reference Example 39, γ-benzyl-N, N-dimethylformamide in N, N-dimethylformamide was used. O-carbonyl-L-
Glutamate (72) and γ-benzyl-N, O-carbonyl-D-glutamate (79) were subjected to a polymerization reaction at a molar ratio of 1: 1 and 1: 9, followed by depolymerization. After benzylation, the title compounds (81) and (8
2) was obtained. When the weight-average molecular weight of the obtained compounds was determined in the same manner as in Reference Example 39, all the compounds were 13,000. Table 10 shows the physical constants of the obtained compound.

Reference Example 41 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D, L-glutamic acid sodium salt (83) 38.7 mg (0.003 mmol) of compound (80) in N, N -Dimethylformamide 0.5 ml solution, N, N '
1.85 mg of dicyclohexylcarbodiimide (0.0
0009 mmol), 1.73 mg (0.00015 mmol) of N-hydroxysuccinimide and 2- (4-
2.06 mg of hydroxyphenyl) ethylamine (0.0
0015 mmol) in 0.5 ml of N, N-dimethylformamide and stirred at room temperature for 20 hours. 5 ml of a 5% aqueous sodium hydrogen carbonate solution was added to the reaction mixture. After removing the precipitated insoluble matter by centrifugation, the supernatant was filtered using Millipore filter paper. The filtrate was subjected to gel filtration column chromatography (Sephadex G-100,
(2.5 cm x 20 cm). Fractions containing the polyglutamic acid derivative were collected and dialyzed against purified water at 4 ° C. for 2 days using a Spectra / pore dialysis membrane. The dialysate was removed and lyophilized to give the title compound (83). The 2- (4-hydroxyphenyl) ethylamino group content of the compound (83) was quantified using an absorbance of 275 nm. Table 11 shows the obtained 2- (4-hydroxyphenyl) ethylamino group content and physical constants.

[0159]

[Table 11]

Reference Example 42 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D, L-glutamic acid sodium salt (84) and (85) The compound was prepared in the same manner as described in Reference Example 41. (8
In 1) and (82), 2- (4-hydroxyphenyl)
An ethylamino group was introduced, and the title compound (8
4) and (85) were obtained. 2- (4-
Table 11 shows the content of (hydroxyphenyl) ethylamino group and physical constants.

Reference Example 43 γ-benzyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (87) The method described in the literature (ER BLOUT AND RH KARTSON, J. A.
m. Chem. Soc., 78, 941 (1956)), 6.0 g (25.28 mmol) of γ-benzyl-L-glutamate (86) and 2.68 g (25.28 mmol) of sodium carbonate. Was dissolved in a mixture of 120 ml of water and 60 ml of acetonitrile, followed by cooling with ice. Then N- (9-fluorenylmethoxycarbonyloxy) succinimide 8.5
A solution of 3 g (25.28 mmol) in 36 ml of acetonitrile was added dropwise with stirring. After removing the cooling bath, the mixture was further stirred at room temperature for 2 hours. After the reaction solution was concentrated to a small amount, a large amount of water was added, and the precipitated insoluble material was removed by filtration. After the filtrate was acidified with diluted hydrochloric acid, the precipitated product was extracted with ethyl acetate. The extract was washed with water, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 12.3 g of a residue. The residue was washed with a mixture of ethyl acetate-hexane,
The product was collected and 9.4 g (81%) of the title compound (87).
I got

87: Amorphous powder [α] _D ²⁴ + 9.3 ° (c 0.92, CHCl ₃ ) IR (KBr) cm ^-1 : 1728, 1688, 1512 ¹ H-NMR (CDCl ₃ -CD ₃ OD): δ 7.8-7.2 (13H, m, aromatic H) 5.06 (2H, s, CH ₂ ph) 4.37 (1H, dd, J = 7.5Hz, Fmoc-CH ₂ ) 4.34 (1H, dd, J = 7.5Hz, 10Hz, Fmoc-CH ₂ ) 4.26 (1H, t, J = 10.5Hz, Fmoc-CH ₂ ) 4.01 (1H, m, Glu-CH) 2.44 (2H, m, -CH ₂ COO-) 2.19 (1H, m, Glu -CH ₂ ) 1.95 (1H, m, Glu-CH ₂ )

Reference Example 44 γ-benzyl-α-tert-butyl-N- (9-fluorenylmethoxycarbonyl)-
L-glutamate (88) After dissolving 18 g (39.17 mmol) of compound (87) in 375 ml of methylene chloride, the mixture was cooled to -18 ° C under a nitrogen stream. After adding 1.95 ml (36.58 mmol) of concentrated sulfuric acid to this solution, about 6% of isobutene gas was added with stirring.
0 ml was blown. After sealing the reaction vessel, allow
Left for hours. 5.1 ml of triethylamine was added to the reaction mixture.
Then, the mixture was neutralized, and the solvent was distilled off under reduced pressure. The obtained residue was partitioned between an aqueous layer and an ethyl acetate layer. The organic layer was separated and washed sequentially with water, aqueous sodium hydrogen carbonate solution and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The resulting syrupy residue is subjected to silica gel column chromatography (hexane-ethyl acetate = 4: 1).
And purified by 18.1 g (89%) of the title compound (88).
I got

88: Syrup-like [α] _D ²⁴ + 50.7 ° (c 1.34, CHCl ₃ ) IR (film) cm ^-1 : 1732, 1516, 1450 ¹ H-NMR (CDCl ₃ ): δ 7.8-7.2 (13H , m, aromatic H) 5.11 (2H, s, CH ₂ ph) 4.37 (2H, dd, J = 7Hz, Fmoc-CH ₂ ) 4.32 (1H, m, Glu-CH ₂ ) 4.20 (1H, t, J = 7Hz, Fmoc-CH) 2.42 ( 2H, m, -CH 2 COO-) 2.22 (1H, m, Glu-CH 2) 1.97 (1H, m, Glu-CH 2) 1.45 (9H, s, C (CH 3 ) ₃ )

Reference Example 45 α-tert-butyl-N- (9
-Fluorenylmethoxycarbonyl) -L-glutamate (89) A mixture of 15 g (29.09 mmol) of compound (88) in 156 ml of methanol and 156 ml of tetrahydrofuran was added to:
5.63 g of 10% palladium-carbon was added, and the mixture was cooled to 10 ° C under a nitrogen stream. Then ammonium formate 5.6
3 g (89.28 mmol) was added, and the mixture was vigorously stirred at the same temperature for 2 hours. After filtering off the catalyst, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform-methanol = 10: 1) to obtain 8.9 g (75%) of the title compound (89).

89: mp 94.5-95.5 ° C. [α] _D ²⁴ + 0.4 ° (c 1.34, CHCl ₃ ) IR (film) cm ⁻¹ : 1728, 1688, 1512 ¹ H-NMR (CDCl ₃ ): δ 7.8- 7.2 (13H, m, aromatic H ) 4.38 (2H, dd, J = 6Hz, Fmoc-CH 2) 4.35 (2H, dd, J = 6Hz, Fmoc-CH 2) 4.28 (1H, m, Glu-CH) 4.18 (1H, t, J = 7Hz, Fmoc-CH) 2.45 (1H, br.signal, OH) 2.40 (2H, m, -CH ₂ COO-) 2.17 (1H, m, Glu-CH ₂ ) 1.91 (1H, m, Glu-CH ₂ ) 1.45 (9H, s, C (CH ₃ ) ₃ )

Reference Example 46 8- [α-tert-butyl-N
-(9-Fluorenylmethoxycarbonyl) -L-glutamide] octyl α-D-mannopyranoside (90) α-tert-butyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (89) 42g
To a solution of (3444 mmol) and 385 mg (3.344 mmol) of N-hydroxysuccinimide in 8 ml of N, N-dimethylformamide, 690 mg (3.344 mmol) of N, N'-dicyclolohexylcarbodiimide was added. Stirred at room temperature for 2 hours. After the precipitated insoluble matter was removed by filtration, the filtrate was treated with 895 mg (3.04 mmol) of 8-aminooctyl α-D-mannopyranoside (9) in N, N-
The solution was added to a 5 ml solution of dimethylformamide and stirred at room temperature for 16 hours. After filtering off the precipitated insoluble matter, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was subjected to silica gel column chromatography (chloroform-methanol = 10:
Purification in 1), 1.56 g (73) of the title compound (90)
%).

90: amorphous powder [α] _D ²⁸ + 21.0 ° (c 0.79, CHCl ₃ ) IR (CHCl ₃ ) cm ^-1 : 1716, 1660 ¹ H-NMR (CD ₃ OD): δ 7.9-7.3 ( 8H, m, aromatic H) 4.69 (1H, d, J 1, 2 = 1.5Hz, H-1) 4.39 (1H, dd, J = 7Hz, Fmoc-CH 2) 4.29 (1H, dd, J = 7Hz, _{Fmoc-CH 2) 4.21 (1H} , t, J = 7Hz, Fmoc-CH) 4.03 (1H, dd, J = 4.5Hz, Glu-CH) 3.80 (1H, dd, J 5, 6a = 2.5Hz, J 6a _{, 6b = 12.5Hz, H-6a} ) 3.76 (1H, dd, J 1, 2 = 1.5Hz, J 2, 3 = 3.5Hz, H-2) 3.59 (1H, t, J 3, 4 = 9.5Hz, H-4) 3.50 (1H, ddd, H-5) 2.30 (2H, m, -CH 2 COO-) 2.10 (1H, m, Glu-CH 2) 1.90 (1H, m, Glu-CH 2) 1.45 ( 9H, s, C (CH ₃ ) ₃ )

Reference Example 47 8- [α-tert-butyl-N
-(9-Fluorenylmethoxycarbonyl) -L-glutamide] octyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (91) 1.77 g of compound (90) (2.52 mmol) 5) Acetic anhydride (5 ml) was added to a pyridine (15 ml) solution, and the mixture was allowed to stand at room temperature for 16 hours. After pouring the reaction mixture into a large amount of water, the precipitated product was extracted with ethyl acetate. The organic layer was washed sequentially with diluted hydrochloric acid, a sodium hydrogen carbonate solution and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 2.06 g (94%) of the title compound (91).

91: Amorphous powder [α] _D ^22.5 + 19.2 ° (c 0.5, CHCl ₃ ) IR (CHCl ₃ ) cm ^-1 : 1744, 1664, 1514, 1450, 1371 ¹ H-NMR (CDCl ₃ ): δ 7.8-7.2 (8H, m, aromatic H) 5.98 (1H, s, NH) 5.62 (1H, d, J = 7.5Hz, NH) 5.34 (1H, dd, J 2, 3 = 3.5Hz, J 3, _{4 = 10Hz, H-3)} 5.27 (1H, t, J 3, 4 = 10Hz, H-4) 5.22 (1H, dd, J 1, 2 = 1.5Hz, J 2, 3 = 3.5Hz, H-2 ) 4.78 (1H, d, J ₁ , ₂ = 1.5 Hz, H-1) 4.39 (2H, m, Fmoc-CH ₂ ) 4.26 (1H, dd, J ₅ , _6a = 5.5 Hz, J _6a , _6b = 12.5 Hz, H-6a) 4.21 ( 2H, t, J = 7Hz, Fmoc-CH Glu-CH) 4.10 (1H, dd, J 5, 6b = 2.5Hz, H-6b) 3.97 (1H, ddd, H-5 ) 3.65 (1H, m, -CH ₂ O) 3.42 (1H, m, -CH ₂ O) 3.23 (2H, m, -CH ₂ N) 2.20 (2H, m, -CH ₂ COO-) 2.15 (3H, s, OAc) 2.09 (3H, s, OAc) 2.04 (3H, s, OAc) 1.98 (3H, s, OAc) 1.47 (9H, s, C (CH ₃ ) ₃ ) 1.7-1.2 (12H, m,- (CH ₂ ) _6- )

Reference Example 48 8- [N- (9-fluorenylmethoxycarbonyl) -L-glutamide] octyl
2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (92) 85 g of 2.0 g (2.29 mmol) of compound (91)
It was dissolved in 57 ml of formic acid-methanol solution and left at room temperature for 16 hours. After diluting the reaction mixture with toluene, it was concentrated to dryness under reduced pressure. The obtained residue is subjected to silica gel column chromatography (chloroform-methanol = 7: 1).
To give 1.90 g (quantitative) of the title compound (92).
I got

92: Amorphous powder [α] _D ^26.5 + 26.2 ° (c 0.5, CHCl ₃ ) IR (CHCl ₃ ) cm ^-1 : 3400, 1745, 1371, 1084, 1051 ¹ H-NMR (CDCl ₃ ): δ 7.8-7.2 (8H, m, aromatic H) 6.18 (1H, s, NH) 6.03 (1H, d, J = 7.5Hz, NH) 5.32 (1H, dd, J 2, 3 = 3.5Hz, J 3, _{4 = 10Hz, H-3)} 5.26 (1H, t, J 3, 4 = 10Hz, H-4) 5.21 (1H, dd, J 1, 2 = 1.5Hz, J 2, 3 = 3.5Hz, H-2 ) 4.33 (3H, m, Fmoc-CH ₂ , Glu-CH) 4.25 (1H, dd, J ₅ , _6a = 5.5Hz, J _6a , _6b = 12.5Hz, H-6a) 4.20 (1H, t, J = 7Hz, Fmoc-CH) 4.10 ( 1H, dd, J 5, 6b = 2.5Hz, H-6b) 3.88 (1H, ddd, H-5) 3.66 (1H, m, -CH 2 O) 3.42 (1H, m _{, -CH 2 O) 3.27 (2H} , m, -CH 2 N) 2.56 (1H, m, Glu-CH 2) 2.45 (1H, m, Glu-CH 2) 2.14 (3H, s, OAc) 2.09 (3H , s, OAc) 2.03 (3H, s, OAc) 1.98 (3H, s, OAc) 1.85 (1H, br.s, OH) 1.6-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 49 8- [γ-L-glutamide]
Octyl 2,3,4,6-tetra-O-acetyl-α
-D-Mannopyranoside (93) 1.9 g (2.341 mmol) of compound (92) was dissolved in 20 ml of morpholine, and the mixture was allowed to stand at room temperature for 40 minutes.
After diluting the reaction mixture with toluene, it was concentrated to dryness under reduced pressure. The remaining morpholine was completely removed by repeating the addition of toluene to the residue and concentration to dryness. The obtained residue was purified by silica gel column chromatography (chloroform-methanol = 5: 1) to obtain 590 mg (43%) of the title compound (93).

93: Amorphous powder IR (KBr) cm ^-1 : 3000, 1750, 1640, 1595, 1530, 1323, 1226, 1136, 1089, 1047

Example 13 8- [γ- (N, O-dicarbonyl) -L-glutamide] octyl 2,3,4,6-
Tetra-O-acetyl-α-D-mannopyranoside (9
4) To a solution of 490 mg (0.828 mmol) of compound (93) in 20 ml of anhydrous tetrahydrofuran was added 86 mg (0.29 mmol) of bis (trichloromethyl carbonate), and the mixture was heated and stirred at 60 ° C. for 3 hours under a nitrogen stream. After cooling to room temperature, the solvent was distilled off under reduced pressure. Toluene was added to the residue, and the mixture was concentrated to dryness. The generated hydrogen chloride was completely removed, and 500 mg of the title compound (94) (9
8%).

94: Amorphous powder IR (KBr) cm ^-1 : 1857, 1787, 1747, 1659, 1369, 1088 ¹ H-NMR (CDCl ₃ ): δ 4.76 (1H, s, H-1) 2.12 (3H , s, OAc) 2.06 (3H, s, OAc) 2.01 (3H, s, OAc) 0.96 (3H, s, OAc)

Reference Example 50 Poly-γ- [8- (2,3,
4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy) octylamino] -γ-benzyl-L-glutamate (95) Compound (94) 500 mg (0.828 mmol) and described in the literature Γ-benzyl- (N, O-carbonyl) -L- synthesized by the method (J. Am. Chem. Soc., 78, 941 (1956)).
Glutamate (72) 220 mg (0.828 mmol)
296 mg (2.92 mmol) of n-hexylamine was added to 10 ml of anhydrous N, N-dimethylformamide.
0.02 solution dissolved in 10 ml of N-dimethylformamide
8 ml (0.0082 mmol) was added and the mixture was stirred at room temperature for 24 hours. Pour the reaction mixture into 150 ml of ethanol,
The deposited precipitate was collected by centrifugation. The precipitate was thoroughly washed with ethanol, and dried by heating at 50 ° C. under reduced pressure for 4 hours to obtain 140 mg of the title compound (95).

Example 14 Poly-γ- [8- (α-D-
Mannopyranosyloxy) octylamino] -L-glutamic acid sodium salt (96) 140 mg of the compound (95) obtained in Reference Example 50 was dissolved in 30 ml of anhydrous tetrahydrofuran, and 1 ml of a 1M sodium methoxide-methanol solution was added. Stir at room temperature for 3 hours. After the reaction mixture was concentrated to dryness under reduced pressure, the obtained residue was dissolved in 4 ml of trifluoroacetic acid containing 0.6 ml of anisole under ice cooling. To this solution was added 4 ml of methanesulfonic acid, and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was poured into 200 ml of isopropyl ether with stirring, and the deposited precipitate was collected by centrifugation. The precipitate was dissolved in 10 ml of a saturated aqueous solution of sodium hydrogen carbonate, and dialyzed against purified water for 48 hours using a Spectra / Pore 3 (molecular weight cut-off = about 3,500) dialysis membrane. The dialysis solution was taken out, freeze-dried, and the title compound (96) 6
0 mg was obtained. The average molecular weight distribution of the compound (96) was determined by gel permeation chromatography (column: TSK-
GEL G4000PWXL + G3000PWXL, eluent: 0.2 M saline, elution rate: 0.5 ml / min), using polyethylene glycol as a standard substance, number average molecular weight (Mn) = 10,368, weight average molecular weight (Mw) = 11,320, molecular weight distribution (Mw /
Mn) = 1.09. Further, the mannose content of the compound (96) was determined by colorimetry by the phenol-sulfuric acid method, and it was found that the content was 1% by weight.

[0179] 96: amorphous powder [α] ^{_{D 24 - 61.1 ° (c 0.35}} , 0.01MPBS) IR (KBr) cm -1: 3300, 1720, 1655, 1549, 1410, 1258

Example 15 Poly-γ- [8- (α-D-
Xylopyranosyloxy) octylamino] -γ- [2
-(4-hydroxyphenyl) ethylamino] -doxorubicin-L-glutamic acid sodium salt (97) 13.0 mg of the compound (68) obtained in Example 7 was dissolved in 5 ml of water, and 3 ml of an aqueous solution of 5.0 mg of doxorubicin hydrochloride was dissolved. Was added to adjust the pH to 7.0 with 1N hydrochloric acid. 10 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added to the obtained solution, and the mixture was stirred at room temperature for 15 hours. The reaction mixture was purified using a SpectraPore / Pore 3 (molecular weight cut-off = about 3,500) dialysis membrane to give 0.01
M 4 each for phosphate buffer (pH 7.0) and water
Dialysis at 24 ° C. for 24 hours. The reaction solution was taken out and freeze-dried. When the amount of doxorubicin obtained was quantified using an absorbance of 490 nm, it was found that 1 mol of doxorubicin had been introduced.

Experimental Example 1. Sample The polyglutamic acid obtained in Example 1 (molecular weight: 13,30
The sample obtained by modifying 36 galactoses in 0) was used as a sample. Unmodified polyglutamic acid (molecular weight 13,300) was used as a control sample.

2. Methods Samples were labeled with ¹²⁵ I prior to animal experiments. ¹²⁵ I
Labeling was carried out by the chloramine T method as usual. The reaction solution was subjected to gel filtration through a Sephadex G-25 column to obtain a polymer fraction. To this fraction was added 1% of bovine serum albumin and an unlabeled compound to prepare a 1 mg / ml administration solution, which was used in the following animal experiments. Rats (SD male, 7 weeks old) were given 1 mg / kg of ¹²⁵ I-labeled sample or control sample in femoral vein, and blood was collected from the carotid artery 1, 2, and 3 minutes after administration, and 5 minutes after administration. All blood was collected from the abdominal aorta and sacrificed, and eleven major organs were removed. Blood is the plasma after centrifugation, and the excised organ is weighed as it is,
Radioactivity was measured using a gamma counter, and the plasma concentration and the concentration in each organ were calculated. Similarly, the concentrations in the main organs 1 hour and 4 hours after the administration of the sample or control sample were measured in the same manner as described above. Each animal experiment was performed with three animals per group.

3. Results and Discussion The results of the blood concentration up to 5 minutes after administration are shown in FIG. The sample was found to disappear from the blood more quickly than the control. Five minutes after the administration, in the organ distribution (FIG. 2), as compared with the control, the accumulation of the kidney was reduced in the specimen, and the accumulation in the liver was confirmed. Furthermore, the concentration in other organs was lower in the sample than in the control, indicating that the galactose modification increased the directivity of polyglutamic acid to the liver.

FIGS. 3 and 4 show the time course of the concentrations of the specimen and the control in the liver and kidney 5 minutes, 1 hour and 4 hours after administration. In the kidney, the AUC (area under the curve) of the sample was smaller than that of the control, but in the liver, the AUC of the sample was extremely large, and the sample tended to accumulate in the liver.

Conventionally, the presence of a lectin that recognizes specific galactose is known in the liver, and it was thought that galactose-modified polyglutamic acid was recognized by this lectin and accumulated in the liver. Therefore, it is suggested that the substance of the present invention is very useful as a carrier for a drug delivery system (DDS) to the liver. In addition, the other sugar-modified polyglutamic acid in the present invention is useful as an organ-directing DDS carrier if a lectin that recognizes a sugar specific to a certain organ or cancer cell is present.

[Brief description of the drawings]

FIG. 1 is a graph showing changes in blood after intravenous administration.

FIG. 2 is a graph showing the concentration in each organ 5 minutes after administration.

FIG. 3 is a graph showing changes in the concentration in the kidney.

FIG. 4 is a graph showing changes in concentration in the liver.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroshi Suzaki 3-1-16-1 Someji, Chofu-shi, Tokyo E-106 Tamagawa Residence E-106 (72) Inventor Akinori Gonmasa 3-chome, Chiyoda, Kashiwa-shi, Chiba 7 Vinus Kashiwa 203

Claims (5)

(57) [Claims] 1. A structural unit (A) represented by the following formula (I)
And a sugar-modified polyglutamic acid derivative comprising (B). Embedded image (Wherein R ₁ represents OR ′ or NHR ″, R ′ represents a hydrogen atom, an alkali metal atom or a physiologically active substance,
R ″ represents a hydrogen atom or a physiologically active substance. R ₂ is a glycosyl group, whose hydroxyl group or 2-position amino group may be substituted with an acetyl group. N represents an integer of 7 to 10. The molar ratio of m to k is from 1 to 100) 2. An N-carboxyglutamic anhydride derivative represented by the following formula (II): Embedded image (Wherein, R ₂ and n are the same as described above) 3. A compound of the formula (III) (Wherein R ₃ represents a 9-fluorenylmethoxycarbonyl group and R ₄ represents a carboxyl-protecting group), to a glutamic acid derivative represented by the formula (IV): (Wherein R ₂ and n are the same as defined above), and then reacting with a glycosyloxyalkylamine or a salt thereof.
A formula (II) characterized in that the protecting group for the carboxyl group and the protecting group for the α-amino group are eliminated, followed by reaction with a carbonate ester. (Wherein, R ₂ and n are the same as described above). 4. A compound of the formula (II) Wherein R ₂ and n are the same as defined above, and a derivative of the formula (VI): (Wherein, R ₅ represents a carboxyl-protecting group) with N-carboxyglutamic anhydride,
Next, the protecting group of the carboxyl group is eliminated, and if necessary, a physiologically active substance is bonded to the carboxyl group. (Wherein, R ₁ , R ₂ n, k and m are the same as those described above). 5. The polyglutamic acid has the formula (IV): Wherein R ₂ and n are the same as defined above, and reacting a glycosyloxyalkylamine or a salt thereof, and if necessary, binding a bioactive substance to a carboxyl group. Formula 10 (Wherein, R ₁ , R ₂ n, k and m are the same as those described above).