JPH05178986A - Sugar-modified polyglutamic acid derivative - Google Patents

Abstract

PURPOSE:To provide the title deriv. having an organ specificity and useful as a pharmacological transport carrier by selecting a specific sugar-modified polyglutamic acid deriv. CONSTITUTION:A sugar-modified polyglutamic acid deriv. comprising structural units A and B of formula I [wherein R1 is OR' or NHR'' (wherein R'' is H or a physiologically active substance; and R' is R'' or an alkali metal); R2 is glucosyl provided that its amino group at the 2-position or OH group may be substd. by an acetyl group; n is 7-10; and the molar ratio of k to m is 1-100] is selected. The deriv. is prepd. by reacting an N-carboxyglutamic anhydride deriv. of formula II with an N-caroxyglutamic anhydride of formula N (wherein R5 is a protective group of carboxyl group), separating the protective group from the carboxyl group, and, if necessary, bonding a physiologically active substance to the carboxyl group.

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 them.

[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 agents, as shown in the following documents. 1) Goldberg, EP et al., Org. Coat Plast.chem. 44 , 132-
136 (1981) 2) Israel Patent No. 68449/3 3) Hurwitz, E. et al., J. Appl. Biochem. 2, 25-35 (1980) 4) Morimoto, Y. et al., J. Pharm. Dyn. 7 , 688-698 (1984) 5) Kato, A. et al., Chem. Pharm. Bull. 30, 2951-2957 (198
2) 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, 721-7
29 (1984)

[0003] Documents 1) to 3) above show examples in which doxorubicin or daunorubicin is bound to polyglutamic acid, 4) examples in which melphalan is bound, and 5) bind mitomycin C. 6) shows an example in which arabinofuranosyl cytosine is bound, 7) shows an example in which phenylenediamine mustard and an anti-lymphoma immunoglobulin are bound, and 8) Shows an example in which daunorubicin is bound to an anti-α-fetoprotein antibody.

As in these examples, an anticancer agent or an antitumor agent can be bound to polyglutamic acid directly or to polyglutamic acid obtained by modifying the binding for drug binding to enable selective delivery to cancer tissues or tumor cells. It has been proposed to further bind an antibody that recognizes these tissues and cells for the purpose of Therefore, it was predicted that polyglutamic acid could serve as a drug delivery carrier with organ-directivity. 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 light of the above situation in the prior art, the present inventors have made various investigations and found that modification of polyglutamic acid with sugar imparts organ-directivity. They have found the present invention and completed the present invention. That is, an object of the present invention is to provide a technique for imparting organ-directivity to polyglutamic acid.

[0006]

[Means for Solving the Problems]

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

[0007]

[Chemical 16]

(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 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 with a sugar via an aminoalkyl chain as a spacer. Further, the formula (I) means that these two kinds of units (A) and (B) are contained in one molecule in k and m units, respectively. But,
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 which 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 has the unit (A) and the unit (B) as essential constituent units, and the third unit, for example, the sugar-modified polyglutamic acid derivative (I). ) Is a constituent unit (C) necessary for the benefit of iodine labeling for animal experiments.

[0013]

[Chemical 17]

May be included.

When the substance of formula (I) is used as a drug delivery carrier, the drug is bound to some of the first units (A), ie the unmodified glutamic acid units. The coupling may be carried out 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 the usual method for forming an acid amide bond. It is also possible to bond a drug through an ester bond or an acid anhydride bond.

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

[0017]

[Chemical 18]

(Wherein R ₂ and n are the same as described above) Formula (II
The substance of I) is a useful starting material for synthesizing the substance of formula (II), and is used for the protection of the amino group of glutamic acid.
The fluorenylmethoxycarbonyl group is particularly selected. This selection is characterized in that a series of reaction steps leading to the substance of formula (II) can be realized.

[0019]

[Chemical 19]

(In the formula, R ₃ represents a 9-fluorenylmethoxycarbonyl group, and R ₄ represents a protecting group for a carboxyl group). Examples of the protecting group for R ₄ include t-butyl and the like.

The substance of formula (IV) is useful for synthesizing the substance of formula (II) or for synthesizing the substance of formula (I) from polyglutamic acid as a raw material, and is a feature of the present invention. It is prepared in advance as a sugar with a spacer having an amino group for the purpose of achieving a certain sugar modification.

[0022]

[Chemical 20]

(Production Method) In order to produce the substance of the formula (IV), a glycosyl halide in which the hydroxyl group of glycoside is protected or the 2-position hydroxyl group is further replaced with an azide group is added to N- () of the following formula (V). (Hydroxyalkyl) phthalimide is reacted and further deprotected if necessary, or further azido group is further reduced to an amino group, and the amino group is further acetylated, and then this is treated with hydrazine to eliminate phthalimide. It is obtained by The reaction is carried out at a low temperature of −10 ° C. or lower in the presence of silver silicate or the like.

[0024]

[Chemical 21]

(Wherein n is the same as above)
This substance can be obtained, for example, by reacting an alkanediol in which one terminal OH group is protected with a 2,3-dihydropyranyl group with phthalimide, and then deprotecting to release the OH group.

To prepare the substance of the formula (III), a reactive derivative of 9-fluorenylmethoxycarboxylic acid, such as succinic acid, can be added to the α-amino group of glutamic acid having a protected γ-carboxyl group. The imide is reacted, 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 formula (II), the formula (III)
The glutamic acid derivative represented by the formula (IV) is reacted with the glycosyloxyalkylamine represented by the formula (IV) or a salt thereof to further eliminate the protecting group for the α-carboxyl group and the protecting group for the α-amino group. The N-carboxyglutamic anhydride derivative is obtained by reacting a carbonic acid ester such as methyl) carbonate.

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

[0029]

[Chemical formula 22]

The first copolymerization method (in the formula, R ₅ represents a protective group for a carboxyl group such as a benzyl group) may be carried out according to a conventional method of copolymerization, and different formulas (II) and (VI )
Of the two N-carboxyglutamic anhydrides are reacted in dimethylformamide in the presence of a condensing agent such as hexylamine, then the protecting group of the carboxyl group is eliminated, and if necessary, the carboxyl group is converted into a physiological group. By coupling the active substance, a substance of formula (I) is obtained.

The second method is to use polyglutamic acid with glycosyloxyalkylamine or its salt (IV) as a condensing agent such as 1,3-dicyclohexylcarbodiimide, 1-ethoxycarbonyl-2-ethoxy-1,2.
By reacting in dimethylformamide in the presence of dihydroquinoline and optionally attaching a physiologically active substance to the γ-carboxyl group, a substance of formula (I) is obtained.

If necessary, 2- (4-hydroxyphenyl) ethylamine may be added together with glycosyloxyalkylamine or its salt (IV) and reacted in the same manner to contain the third unit (C). A substance of formula (I) can be obtained.

In producing polyglutamic acid, if N-carboxy-L-glutamic acid anhydride and N-carboxy-D-glutamic acid anhydride are copolymerized, L-glutamic acid and D-glutamic acid are randomly arranged. Polyglutamic acid is obtained.

(Characteristics of substances) The molecular weight of the substance of the formula (I) can be measured by various methods. For example, gel permeation chromatography (column: TSK-GEL) using polyethylene glycol as a standard substance. G4000P
WXL + G3000PWXL, eluent: 0.2M saline, elution rate:
The weight average molecular weight (Mw) can be determined by 0.5 ml / min. The molecular weight of the substance of formula (I) is not particularly limited, but is usually 5,000 to 150,000.

The sugar content in the substance of formula (I) can be determined by colorimetric determination by the phenol-sulfuric acid method. Further, the content of the hydroxyphenylethylamino group 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 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 potency of the substance of formula (I) as a drug carrier is shown in the following experimental examples in the case of the substance of formula (I) modified with galactose as compared with unmodified polyglutamic acid. , It was found that the accumulation in the kidney was small and conversely the accumulation in the liver was large. Since the existence of a lectin that specifically recognizes galactose is known in the liver, it is presumed that the substance of formula (I) modified with galactose is recognized by the lectin. Therefore,
When the organ or cancer cell or the like has a lectin that recognizes a specific sugar, the substance of formula (I) can serve as an organ-directed drug delivery carrier.

[0038]

EXAMPLES The present invention will be described in more detail by way of 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]

[Chemical formula 23]

[0040]

[Chemical formula 24]

[0041]

[Chemical 25]

[0042]

[Chemical formula 26]

[0043]

[Chemical 27]

[0044]

[Chemical 28]

[0045]

[Chemical 29]

[0046]

[Chemical 30]

[0047]

[Chemical 31]

[0048]

[Chemical 32]

[0049]

[Chemical 33]

[0050]

[Chemical 34]

[0051]

[Chemical 35]

[0052]

[Chemical 36]

[0053]

[Chemical 37]

[0054]

[Chemical 38]

[0055]

[Chemical Formula 39]

[0056]

[Chemical 40]

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

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) Compound (2) 14.9 g (64.7 mmol), triphenylphosphine 18.67 g (71.2 mmol) and phthalimide 10.47 g (71. To a solution of 2 mmol) in 300 ml of anhydrous tetrahydrofuran, 11.2 ml (71.2 mmol) of diethylazodicarboxylic acid ester was slowly added dropwise at room temperature while stirring in a nitrogen stream. 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 the compound (3) was added to 80% acetic acid 5%.
After dissolving in 00 ml, the mixture was heated with stirring at 80 ° C. for 3 hours. The reaction mixture was concentrated to dryness under reduced pressure, and the obtained residue was partitioned between an aqueous layer and an ethyl acetate layer. The organic layer was separated and washed successively 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) Method described in the literature (T.OGAWA et al., Tetrahedron, 37, 2779 (19
81)) synthesized 2-O-acetyl-3,4,6-tri-
1.6 g (31.58 mmol) of O-benzyl-α-D-mannopyranosyl chloride (5), 8.7 g (31.58 mmol) of 8-phthalimidooctanol (4) and 1,1,3,3. 3-tetramethylurea 17 ml
(126.3 mmol) was dissolved in 200 ml of dichloroethane and then cooled to -40 ° C under a nitrogen stream. To this mixture was added 8.93 g (34.73 mmol) of silver triflate 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. The reaction mixture was filtered through Celite, and the filtrate was washed successively with aqueous sodium hydrogen carbonate solution and water. After drying with magnesium sulfate,
The solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (hexane-ethyl acetate = 3:
Purified in 1), 12.3 g (52%) of the title compound (6).
%) Was obtained.

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) 12.3 g (16.4 mmol) of compound (6) in 200 ml of methanol and 50 ml of tetrahydrofuran,
4.1 ml of 1M sodium methoxide-methanol solution was added, and the mixture was left at room temperature for 4 hours. Ion exchange resin Amberlite IR120B (H ⁺ type) was added to the reaction solution for neutralization, then the resin was filtered off and washed with methanol. The filtrate and the washing solution were combined and the solvent was distilled off under reduced pressure to obtain 11.4 g of a syrupy residue. The residue was purified by silica gel column chromatography (hexane-ethyl acetate = 2: 1) to obtain 8.5 g (73.3%) of the title compound (7).

7: Syrup [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) Compound (7) 8.5 g (12 mmol) of ethanol 6
3 ml of hydrazine hydrate (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 residue was concentrated to dryness under reduced pressure to remove the remaining hydrazine to obtain 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- )

Example 1 8-Aminooctyl α-D-
Mannopyranoside (9) Compound (8) 6.9 g (12 mmol) of methanol 2
To a 00 ml solution, 12 ml of 1M hydrochloric acid and 1 g of 10% palladium-carbon were 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 obtain 3.99 g (quantitative) of hydrochloride of the title compound (9).

9 (hydrochloric acid salt): 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- )

Hydrochloride of compound (9) obtained above 2.64
g was dissolved in water-containing 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% hydrous methanol. The eluate and the washing solution were combined and concentrated under reduced pressure to dryness to obtain 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 = 1.5Hz , 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 7 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) Method described in 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 to prepare 795 mg (2.89 mmol) of 8-phthalimidooctanol (4) and 2 g of powdered molecular sieve 4A. And 2 g of silver silicate were added dropwise to a suspension of toluene. The cooling bath was removed, and the mixture was further stirred at room temperature for 18 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to dryness. The obtained residue was rovers column C (hexane-ethyl acetate = 4:
Purified in 1), 1.51 g (6) of the title compound (11).
5.4%) and 180 mg (7.8) of the title compound (12).
%) Was obtained.

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 [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) 8.
A solution of 9 g (14.5 mmol) of methylene chloride in 20 ml of a solution of 10.0 g (36.25 mmol) of 8-phthalimidooctanol (4), 7.8 g (26.1 mmol) of silver carbonate at room temperature under a stream of argon. It was added dropwise to a suspension of 7.8 g of calcium sulfate in 50 ml of methylene chloride. The reaction mixture was protected from light with an aluminum foil and further stirred at room temperature for 19 hours. After filtering the reaction mixture through 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) and the same title compound (11) as above
7.49 g (65.7%) were obtained.

Reference Example 8 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, Heated to reflux 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 evaporated under reduced pressure. Ether was added to the obtained crystalline residue and filtered to obtain 1.1 g (95%) of compound (13).

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 9 8-Benzyloxycarbonylaminooctyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (14) 0.29 g of compound (13) and triethylamine.
To a solution of 07 g of chloroform in 20 ml, 0.044 g of benzyloxycarbonyl chloride was added with stirring at room temperature. After stirring at room temperature for 3 hours, the reaction mixture was washed with water. After separating the organic layer and drying with sodium sulfate,
It was concentrated to dryness under reduced pressure. The obtained residue was subjected to silica gel column chromatography (hexane-ethyl acetate = 4:
Purified in 1), 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- )

Example 2 8-Aminooctyl β-D-
Mannopyranoside (15) Hydrochloride compound (13) 20
To a solution of 0 mg (0.299 mmol) in 15 ml of methanol, 0.3 ml of 1M hydrochloric acid and 10% palladium on carbon 5
0 mg was added and hydrogenated at room temperature for 8 hours. After removing the catalyst by filtration, the filtrate was concentrated to dryness under reduced pressure to give the title compound (15).
153 mg of the hydrochloride of was obtained.

[0082] 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- )

Example 3 8-aminooctyl β-D-
P-Toluenesulfonic acid salt of mannopyranoside (15) 2.64 g (3.3 mmol) of compound (14) and p
-Toluenesulfonic acid (0.63 g, 3.3 mmol) was dissolved in methanol (20 ml), 10% palladium-carbon (50 mg) was added, and the mixture was hydrogenated at room temperature for 3 hours. After filtering the catalyst, the filtrate was concentrated to dryness under reduced pressure to obtain 1.57 g (quantitative) of p-toluenesulfonate of the title compound (15).

[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- )

Example 4 8-Aminooctyl-β-D-
Mannopyranoside (15) 8-aminooctyl β-D synthesized by the method of Example 2
After dissolving the hydrochloride of mannopyranoside (15) in hydrous methanol, the ion exchange resin IRC-50 (NH ₄
Type) column (4.5 cm × 40 cm). 800 water
After eluting with 1 ml of 6% aqueous ammonia, the mixture was eluted with 1700 ml of 6% aqueous ammonia. Fractions after flowing 1000 ml of eluate were collected and concentrated to dryness under reduced pressure to give 1.305 g (58%) of the free base of the title compound (15).

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 10 8-phthalimidooctyl
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside (17) and 8-
Phthalimide octyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranoside (18)

Methods described in literature (Carbohydr. Res., 136,
153 (1985)) 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranosyl bromide (16) 9.5 g (24.1 mmol) Toluene 80ml solution of -15 ℃ under nitrogen stream with stirring
Then, 6.63 g of 8-phthalimidooctanol (4)
(24.09 mmol), powdered molecular sieve 4
10 g of A and 10 g of silver silicate were added dropwise to a suspension of 80 ml of toluene. The reaction mixture was stirred at the same temperature for 3 hours.
The cooling bath was removed, and the mixture was further stirred at room temperature for 15 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to dryness. The obtained residue is applied to a 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)
1.84 g (12.4%) was obtained.

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 [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 11 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. Ion exchange resin Amberlite IR-120B (H
⁽⁺ Type) was added for neutralization, and then 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).

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 12 8-phthalimidooctyl
2-Acetamido-2-deoxy-β-D-mannopyranoside (20) Compound (19) 1.68 g (3.63 mmol) in ethanol 43 ml solution, nickel chloride hexahydrate 380 mg
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, acetic acid was added to the reaction solution to neutralize it. 2 ml of acetic anhydride was added to the reaction mixture and the mixture was allowed to stand at room temperature for 18 hours. The reaction mixture was concentrated to dryness under reduced pressure, and 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- )

Example 5 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 under reflux at 70 ° C. for 5 hours. After cooling to room temperature, toluene was added to the reaction solution to dilute it, 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) was obtained. At the same time, 121 mg of compound (20) was recovered.

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 13 8-phthalimidooctyl
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside (23) 8-phthalimidooctanol (4) 2.75 g (10)
50 mg of dry benzene, 2.53 g (10 mmol) of silver cyanocyanide and 5 g of anhydrous calcium sulfate.
A suspension of 50 ml of dry nitromethane was stirred for 30 minutes at room temperature under a stream of argon. 2, 3, 4, to this suspension
A solution of 6 g (14.6 mmol) of 6-tetra-O-acetyl-α-D-galactopyranosyl bromide (22) in 20 ml of dry benzene was added dropwise with stirring at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to dryness. The obtained residue was purified by flash column chromatography (ethyl acetate-hexane = 1: 1),
5.48 g (90.5%) of the title compound (23) was 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 x 2) 1.96 (3H, s, OAc) 1.50-1.70 (4H, m, CH ₂ x 2) 1.20-1.40 (8H, m, CH ₂ × 4)

Reference Example 14 8-phthalimidooctyl
β-D-galactopyranoside (24) 0.5 ml of 28% sodium methoxide-methanol solution was added to 100 ml of methanol containing 5.4 g (8.9 mmol) of compound (23), 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 treated with water 5
After dissolving in 0 ml, ion exchange resin Amberlite IR
Neutralization was carried out by adding -120B (H ⁺ type). After the resin was filtered off, 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 ℃ [ 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

Example 6 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 under reflux on an oil bath for 66 hours. After cooling to room temperature,
The resulting insoluble material was filtered off. The filtrate was concentrated to dryness under reduced pressure. The obtained crude product was dissolved in 30 ml of purified water, and concentrated hydrochloric acid was added to neutralize it. Ion exchange resin I
RC-50 (NH ₄ ⁺ type) was passed through a 300 ml column. The target 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).

[0102] 25: mp 135 - 142 ℃ ( 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 15 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-acetyl-α-D-xylopyranosyl bromide (26) 1.85 g (5.45 mmol) of benzene 30 ml In solution, mercuric cyanide 1.3
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 an argon stream. After the reaction mixture was filtered to remove insoluble matter, 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 obtained 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 the compound (28) 31.
7 mg (10.9%) and compound (29) 313 mg (1
1.7%) was obtained.

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)

Example 7 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 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 removing the resin by filtration, the filtrate was concentrated to dryness under reduced pressure. The obtained syrupy residue was dissolved in 1 ml of ethanol, 318 mg (6.35 mmol) of hydrazine hydrate was added, and the mixture was heated under reflux 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. The precipitated insoluble material was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The resulting syrupy residue was subjected to silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 10: 10: 1).
Purified in. Fractions positive to the ninhydrin reagent were collected and the solvent was distilled off under reduced pressure. The obtained powdery substance was dissolved in 30 ml of water and neutralized by adding 3M hydrochloric acid. This solution was passed through a column of 100 ml of Amberlite IRC-50 (NH ₄ ⁺ type) and washed with 200 ml of water. It was then eluted with 2M aqueous ammonia. The eluates were 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 16 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 40 ml of benzene solution
(8.57 mmol), powdered molecular sieve 4A
And 4.0 g of 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 insoluble matter, 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 title compound (33) 8
42 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 17 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 1 g of 10% palladium-carbon were added, and catalytic reduction was carried out by a Parr apparatus (50 psi). After the catalyst was filtered off, the filtrate was concentrated to dryness under reduced pressure. The obtained syrupy residue was subjected to silica gel column chromatography (chloroform-methanol = 40: 1).
After purification with 894 mg of the title compound (34) (86.2).
%) Was obtained.

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)

Example 8 8-Aminooctyl β-L-
Fucopyranoside (35) Compound (34) 800 mg (1.90 mmol) in ethanol 10 ml solution, hydrazine hydrate 476 mg (9.
(50 mmol) was added and the mixture was heated under reflux 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. The precipitated insoluble material was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The obtained syrupy residue was subjected to silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 2.
It was purified at 0: 10: 1). The ninhydrin reagent positive fractions were collected and the solvent was distilled off under reduced pressure. The residue obtained is treated with water 1
After dissolving in 0 ml, 3M hydrochloric acid was added to neutralize. The solution was passed through a column of 50 ml Amberlite IRC-50 (NH ₄ ⁺ type). After washing the column 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 (9) of the title compound (35).
1.3%) was obtained.

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 18 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) Method described in literature (RULemieux et al., Canadian. J. Che.
m., 57, 1244 (1979)) 3,4-di-O-acetyl-2-azido-2-deoxy-β-L-fucopyranosyl bromide (36) 911 mg (2.71 mmol) 265 mg of mercuric cyanide (2.
71 mmol), 1 g of powdered molecular sieve 4A
And 8-phthalimidooctanol (4) 746 mg
(2.71 mmol) was added, and the mixture was stirred at room temperature in an argon stream for 1 hour.
Stir for 5 hours. After the reaction mixture was filtered to remove insoluble matter, 20 ml of ethyl acetate was added to the filtrate. The reaction solution is water 30
After washing with ml, it was dried over magnesium sulfate and concentrated to dryness under reduced pressure. The obtained syrupy residue was subjected to silica gel column chromatography (hexane-ethyl acetate = 4:
Purified in 1), 308 mg (21.
4%) and 610 mg (42.4) of the title compound (38).
%) Was obtained.

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)

Example 9 8-Aminooctyl 2-acetamido-2-deoxy-α-L-fucopyranoside (4
0) To a solution of 300 mg (0.565 mmol) of the compound (37) in 5 ml of methanol was added 0.08 ml of 28% sodium methoxide-methanol solution, and the mixture was stirred at room temperature for 24 hours. Amberlite IR-120B (H ⁺
Type). After removing the resin by filtration, the filtrate was concentrated to dryness under reduced pressure. The syrup-like residue obtained was mixed with ethanol 15
After dissolving in ml, sodium borohydride 64.1mg
(1.70 mmol) and nickel chloride hexahydrate 4
0.5 ml of 0 mg ethanol solution 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 filtered off. 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 =
30: 1) to give 8-phthalimidooctyl 2-
Acetamide-2-deoxy-α-L-fucopyranoside (39) (257 mg) was obtained. 257 mg of compound (39) was dissolved in 5 ml of ethanol, and hydrazine hydrate 141 mg
(2.83 mmol) was added, and the mixture was heated under reflux at 100 ° C. for 2 hours. Furthermore, 141 mg of hydrazine hydrate (2.83
Was added and the mixture was heated under reflux at the same temperature for 2 hours. After the reaction mixture was filtered to remove insoluble matter, 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), and ninhydrin-positive fractions were collected and concentrated to dryness under reduced pressure. The obtained residue was dissolved in 10 ml of water and neutralized with 3M hydrochloric acid. This solution was passed through a column of 50 ml Amberlite IRC-50 (NH ₄ ⁺ type). After washing with 100 ml of water, the desired product was eluted with 2M aqueous ammonia. The eluate was concentrated to dryness under reduced pressure to obtain 41.1 mg (23%) of the title compound (40).

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

[0122]

[Table 1]

Reference Example 20 Poly-L-glutamic acid (4
6), (47) and (48) In the same manner as in Reference Example 19, 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) was treated with 1M hydrochloric acid in an aqueous solution to give the free acid to give the title compounds (46) and (47). ) And (48) were obtained. The physical constants of the obtained compound are shown in Table 1.

Reference Example 21 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (49) Poly-L-glutamic acid (45) 100 mg (0.007)
51 mmol), 2- (4-hydroxyphenyl) ethylamine 10 mg (0.072 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline 19 mg (0.0768 mmol), 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) After diluting with 10 ml, gel filtration column chromatography (Sephadex G-100, 50 cm × 2.5 c
m) and eluted with 5 ml of each fraction. Fraction No. 15 ~
Collect 27 and Spectra / Pore 3 (Molecular weight cutoff =
Using a dialysis membrane (about 3,500), it was dialyzed against purified water at 4 ° C. for 16 hours. The dialyzed solution was taken out and freeze-dried to obtain 75 mg of the title compound (49). Introduced 2- (4
-Hydroxyphenyl) ethylamino residue amount of absorbance 2
Quantification using 75 nm revealed that 5 mol of 2- (4-hydroxyphenyl) ethylamino group was introduced per mol of poly-L-glutamic acid. The physical constants of compound (49) are shown in Table 2.

[0125]

[Table 2]

Reference Example 22 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) N, N'-carbonyldiimidazole were added to N,
It was dissolved in 3 ml of N-dimethylformamide and stirred at room temperature for 1 hour and 30 minutes. To this solution was added 200 mg (1.458 mmol) of 2- (4-hydroxyphenyl) ethylamine and 0.19 of 1,1,3,3-tetramethylguanidine.
0.07 ml (0.025 mmol) of a solution prepared by dissolving 4 ml of N, N-dimethylformamide in ml (1.514 mmol) was added, and the mixture was stirred at room temperature for 2 days. The reaction mixture was charged to 0.
After dilution with 01M isotonic phosphate buffer (pH 7.4), Spectra / Pore 1 (molecular weight cutoff = 6,000-
8,000) dialysis membrane at 4 ℃ against purified water 4
It was dialyzed for 8 hours. The dialyzed solution was taken out and freeze-dried to obtain 78 mg of the title compound (50). Introduced 2- (4-
Hydroxyphenyl) ethylamino residue amount of absorbance 27
When quantified using 5 nm, poly-L-glutamic acid 1
It was found that 5 mol of 2- (4-hydroxyphenyl) ethylamino groups were introduced per mol. Compound (5
The physical constants of 0) are shown in Table 2.

Reference Example 23 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (51) and (52) In the same manner as in Reference Example 22, poly-L-glutamic acid. A 2- (4-hydroxyphenyl) ethylamino group was introduced into (47) and (48) by using 10 molar equivalents of 2- (4-hydroxyphenyl) ethylamine, and the title compounds (51) and (52) were introduced. ) Got. Table 2 shows the amount of 2- (4-hydroxyphenyl) ethylamino group introduced (molar ratio) and the physical constant of the obtained compound.
It was shown to.

Reference Example 24 Poly-D-glutamic acid (5
6), (57) and (58) In the same manner as in Reference Example 19, 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), poly-L-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 95,100) (55) was treated with 1M hydrochloric acid in an aqueous solution to give a free acid, and the title compounds (56) and (57) were obtained. ) And (58) were obtained. The physical constants of the obtained compound are shown in Table 3.

[0129]

[Table 3]

Reference Example 25 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D-glutamic acid sodium salt (59), (60) and (61) A compound was prepared in the same manner as in Reference Example 21. (56), (5
A 2- (4-hydroxyphenyl) ethylamino group was introduced into each of 7) and (58) to obtain the title compounds (59), (60) and (61). Table 4 shows the amount of introduced 2- (4-hydroxyphenyl) ethylamino groups (molar ratio) and the physical constants of the obtained compound.

[0131]

[Table 4]

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

[0133]

[Table 5]

[0134]

[Table 6]

Example 11 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (63) Poly-L-glutamic acid (45) 50 mg (0.0037
2-ethoxy-1-ethoxycarbonyl-1,2 in 2 ml of dry N, N-dimethylformamide.
-152 mg (0.615 mmol) of dihydroquinoline was added, and the mixture was stirred at room temperature for 1 hour. To this solution was added 8-aminooctyl β-D-mannopyranoside (15) hydrochloride 1
A solution of 90 mg (0.56 mmol) and 0.48 ml (0.56 mmol) of triethylamine in 2 ml of N, N-dimethylformamide was added, and the mixture was stirred at room temperature for 1.5 hours. Then, 5 mg of 2- (4-hydroxyphenyl) ethylamine was added, and the mixture was further stirred for 15.5 hours. After adding 0.5 ml of N, N-dimethylformamide to the reaction solution to dilute it, stirring was continued for another 25 hours. 0.1 to the reaction mixture
0.5 ml of an aqueous solution of sodium hydroxide M and 15 ml of 0.01 M isotonic phosphate buffer were added. The precipitated insoluble matter was removed by centrifugation, and 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).
Fractions containing the poly-L-glutamic acid derivative were collected and dialyzed against purified water for 48 hours using a Spectra / Pore 3 (molecular weight cutoff = about 3,500) dialysis membrane. The dialyzed solution was taken out and freeze-dried to obtain 35 mg of a colorless amorphous compound (63). Colorimetric determination of the sugar content by the phenol-sulfuric acid method revealed that 7 moles of 8- (β-D-mannopyranosyloxy) octylamino group were introduced per mole of poly-L-glutamic acid. Also, 2
When the amount of-(4-hydroxyphenyl) ethylamino group was quantified using an absorbance of 275 nm, 3 mol of 2- (4-
It was found that a hydroxyphenyl) ethylamino group was introduced. The physical constants of the obtained compound are shown in Table 6.

Example 12 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
1-ethoxycarbonyl-2-ethoxy-1,2 in 100 ml of N, N-dimethylformamide.
-152 mg (0.615 mmol) of dihydroquinoline was added and stirred for 1 hour and 40 minutes. Then 8-aminooctyl β-D-mannopyranoside (15) hydrochloride 190
mg (0.56 mmol) and triethylamine 0.7
A solution of 8 ml (0.56 mmol) of N, N-dimethylformamide in 50 ml and a solution of 5-mg of 2- (4-hydroxyphenyl) ethylamine in 10 ml of N, N-dimethylformamide were added, and the mixture was stirred at room temperature for 45 hours. The reaction mixture was purified by a method similar to the method described in Example 11 to obtain the title compound (64). Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. The physical constants of the compounds are shown in Table 6.

Example 13 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (65) Poly-L-glutamic acid (45) 100 mg (0.075
1 mmol) anhydrous N, N-dimethylformamide 3.
96 mg (0.52) of O-sulfobenzimide was added to a 5 ml solution.
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 were added to this mixture.
2.0 ml of a solution of (1.514 mmol) in 4 ml of N, N-dimethylformamide and 153 mg of 8-aminooctyl β-D-mannopyranoside (15) hydrochloride.
A solution of (0.461 mmol) in N, N-dimethylformamide (1 ml) was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was purified by the same method as described in Example 11 to obtain the title compound (65). Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the compound. The physical constants of the compounds are shown in Table 6.

Example 14 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (66) In the same manner as in the method described in Example 10, poly-L-glutamic acid (45) was added to 8-aminooctyl α-D-.
Using mannopyranoside (9) and 2- (4-hydroxyphenyl) ethylamine, 8- (β-D-mannopyranosyloxy) octylamino group and 2- (4-
A hydroxyphenyl) ethylamino group was introduced to obtain the title compound (66). Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the obtained compound. The physical constants of the compounds are shown in Table 6.

Example 15 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 ml of dry N, N-dimethylformamide
To the solution, with stirring, 180 mg (0.611 mmol) 8-aminooctyl α-D-mannopyranoside (9), 7.5 mg (0.00546 mmol) 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 0.01M isotonic phosphate buffer, Spectra / Pore 3 (molecular weight cutoff =
Using a dialysis membrane (about 3,500), it was dialyzed against purified water at 4 ° C. for 24 hours. The dialyzed solution was taken out and freeze-dried. The obtained residue is mixed with 0.01 M isotonic phosphate buffer (pH
It was dissolved in 7.4) and purified by gel filtration column chromatography (Sephadex G100, 5 cm × 30 cm). Each eluted fraction was monitored by high performance liquid chromatography (column: TSK-GELG3000PWXL, eluent: 0.2M saline, elution rate: 0.5 ml / min., Detection: ultraviolet absorption (280 nm)). Fractions containing the poly-L-glutamic acid derivative were collected and again dialyzed against purified water at 4 ° C. for 48 hours using a Spectra / Pore 3 (molecular weight cutoff = about 3,500) dialysis membrane. The dialyzed solution was taken out and freeze-dried to give the title compound (6
7) 80 mg was obtained. The sugar content of the obtained compound and 2-
The content of (4-hydroxyphenyl) ethylamino group is shown in Table 5.
It was shown to. The physical constants of the compounds are shown in Table 6.

Example 16 Poly-γ- [8- (α-D-
Xylopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (68) In the same manner as in the method described in Example 12, poly-L-glutamic acid (45) was added with 8-aminooctyl α-D-.
Using xylopyranoside (30) and 2- (4-hydroxyphenyl) ethylamine, 8- (α-D-xylopyranosyloxy) octylamino group and 2- (4
-Hydroxyphenyl) ethylamino group is introduced,
The title compound (68) was obtained. Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. The physical constants of the compounds are shown in Table 6.

Example 17 Poly-γ- [8- (β-L-
Fucopyranosyloxy) octylamino] -γ- [2-
(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (69) In the same manner as in the method described in Example 12, poly-L-glutamic acid (45) was added to compounds (34) and 2- (4).
8-hydroxyphenyl) ethylamine
A (β-L-fucopyranosyloxy) octylamino group and a 2- (4-hydroxyphenyl) ethylamino group were introduced to obtain the title compound (69). 2-of the compound
Table 5 shows the (4-hydroxyphenyl) ethylamino group content and the sugar content. The physical constants of the compounds are shown in Table 6.

Example 18 Poly-γ- [8- (2-acetamido-2-deoxy-α-L-fucopyranosyloxy) octylamino] -γ- [2- (4-hydroxyphenyl) ethylamino] -L-Glutamic acid sodium salt (69) In the same manner as in the method described in Example 12, poly-L-glutamic acid (45) was added to compounds (40) and 2- (4).
8-hydroxyphenyl) ethylamine
A (2-acetamido-2-deoxy-α-L-fucopyranosyloxy) octylamino group and a 2- (4-hydroxyphenyl) ethylamino group were introduced to obtain the title compound (70). In order to determine the sugar content of the compound (70), it was hydrolyzed with 4 M hydrochloric acid by heating with stirring at 100 ° C. for 4 hours. Colorimetric determination of the obtained hydrolysis product by Elson-Morgan method gave 8- (2-acetamido-2-deoxy-
The content of α-L-fucopyranosyloxy) octylamino group was 3 mol per mol of poly-L-glutamic acid. Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. The physical constants of the compounds are shown in Table 6.

Example 19 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 in the method described in Example 10, poly-L-glutamic acid (45) was added to compounds (21) and 2- (4).
8-hydroxyphenyl) ethylamine
The (2-acetamido-2-deoxy-β-D-mannopyranosyloxy) octylamino group and the 2- (4-hydroxyphenyl) ethylamino group were introduced to obtain the title compound (71). The sugar content of the compound (71) was hydrolyzed with 4M hydrochloric acid by the same method as described in Example 18, and then colorimetrically determined by the Elson-Morgan method. Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the compound. The physical constants of the compounds are shown in Table 6.

Reference Example 26 Poly-L-glutamic acid sodium salt (73) γ-benzyl-N, O-carbonyl-synthesized by the method described in the literature (J. Am. Chem. Soc., 78, 941 (1956)). 2.63 g (10.0 mmol) of L-glutamate (72) was dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a nitrogen stream. Hexylamine 6.
6 μl of dried N, N-dimethylformamide 100 μl
The solution dissolved in was added, and the mixture was stirred under a nitrogen stream at room temperature for 24 hours. The reaction mixture was poured into 500 ml of isopropyl ether with stirring, and the deposited precipitate was collected by filtration. The precipitate was washed well with ethanol and then dried under reduced pressure. The obtained precipitate was mixed with 17 ml of trifluoroacetic acid and anisole 3
After dissolving in a mixed solution of ml, 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 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 dialyzed solution 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 substance (column: TSK-GEL
G4000PWXL + G3000PWXL, eluent:
The weight average molecular weight (Mw) determined with 0.2 M saline solution, elution rate: 0.5 ml / min) was 22,000. The physical constants of the obtained compound are shown in Table 7.

[0145]

[Table 7]

Reference Example 27 Poly-L-glutamic acid (74) In the same manner as in Reference Example 26, γ-benzyl-N, O.
N-, using carbonyl-L-glutamate (72)
Polymerization was carried out using triethylamine as a polymerization initiator in N-dimethylformamide. The product was debenzylated with trifluoroacetic acid, anisole and methanesulfonic acid in the same manner as in Reference Example 26, and then purified to obtain the title compound (74). The weight average molecular weight (Mw) of the compound (74) was measured by gel permeation chromatography using polyethylene glycol as a standard substance (column: TSK-GEL G4000PWXL + G3).
000PWXL, eluent: 0.2M saline, elution rate:
0.5 ml / min), it was 57,200. The physical constants of the obtained compound are shown in Table 7.

Example 20 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (75) The compound (73) synthesized in Reference Example 26 was used and obtained by treating with 1M hydrochloric acid in the same manner as in Reference Example 19. To a solution of 129.0 mg of the free acid of compound (73) in 1.0 ml of N, N-dimethylformamide, 51.6 mg (0.25 mmol) of N, N'-dicyclohexylcarbodiimide and N were added.
A solution of 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 in this solution
-(4-hydroxyphenyl) ethylamine 6.8mg
(0.05 mmol), 8-aminooctyl β-D-
Mannopyranoside (15) p-toluenesulfonate 9
A solution prepared by dissolving 6.0 mg (0.20 mmol) and 28 μl of triethylamine in 0.5 ml of N, N-dimethylformamide was added, and the mixture was stirred at 4 ° C. for 2 hours. The cooling bath was removed, and the mixture was further stirred at room temperature for 2 hours. 5 ml of 5% aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the precipitated insoluble matter was removed by centrifugation. The resulting 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).
Purified in. Fractions containing the poly-L-glutamic acid derivative were collected and dialyzed against purified water at 4 ° C. for 2 days using a Spectra / Pore dialysis membrane. The dialyzed solution was taken out and freeze-dried to obtain 127.5 mg of the title compound (75). The sugar content of the compound (75) was colorimetrically determined by the phenol-sulfuric acid method. The content of 2- (4-hydroxyphenyl) ethylamino group was quantified by using an absorbance of 275 nm. The obtained results are shown in Table 8. The physical constants of the compounds are shown in Table 9.

[0148]

[Table 8]

[0149]

[Table 9]

Example 21 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 20, the free acid of compound (74) was mixed with p-toluenesulfonate of compound (15) and 2 The 8- (β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced using-(4-hydroxyphenyl) ethylamine to give the title compound (76). Got Table 8 shows the sugar content and 2- (4-hydroxyphenyl) ethylamino group content of the compound (76). The physical constants of the compounds are shown in Table 9.

Reference Example 28 γ-Benzyl-D-glutamate (78) 75.0 g (694 mmol) of benzyl alcohol was added to 75 ml of a 60% sulfuric acid solution containing 100 g (680 mmol) of commercially available D-glutamic acid (77) under ice cooling. added. The reaction mixture was stirred at 70 ° C. for 1 hour, and then the produced water was distilled off under reduced pressure. 120 g of sodium hydrogen carbonate as the reaction mixture
Was poured into a liquid 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. 2 products from hot water
The crystals were recrystallized twice, and the crystals were collected by filtration. Crystallized with ethanol 100
After washing with 100 ml of ether and 100 ml of ether, it was dried under reduced pressure to obtain 52.1 g (32%) of the title compound (78).

78: mp 164-14.5 ° C. [α] _D ²⁵ -19.4 ° (c 1.29, AcOH) IR (KBr) cm ^-1 : 3000, 1724, 1582, 1517, 1173

Reference Example 29 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 while stirring for bis (trichloro). Methyl carbonate) 11 g (3
40 ml of anhydrous tetrahydrofuran (7.1 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 15 minutes, then heated and stirred at an internal temperature of 40 to 50 ° C for 4 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. To the obtained crystalline residue, 150 ml of hexane was added and the crystals were collected by filtration. Recrystallization from a hexane-ethyl acetate mixed solution 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 30 Disordered 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-N, O-carbonyl-D-glutamate (79) 2
60 mg (10 mmol) were dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a stream of nitrogen.
To this solution was added 13.2 μl of hexylamine 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 deposited precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure. The obtained precipitate was dissolved in a mixed solution of 17 ml of trifluoroacetic acid and 3 ml of anisole while cooling with ice. Next, 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.
Isopropyl ether 300 while stirring the reaction mixture
The resulting precipitate was collected by filtration. The obtained precipitate was added to 20 ml of a 5% aqueous sodium hydrogen carbonate solution, and the mixture was 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 dialyzed solution was taken out and freeze-dried to give the title compound (8
0) 1.10 g was 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. The physical constants of the obtained compound are shown in Table 10.

[0156]

[Table 10]

Reference Example 31 Disordered Copolymers of L-Glutamic Acid and D-Glutamic Acid (81) and (82) In the same manner as in Reference Example 30, γ-benzyl-N, in N, N-dimethylformamide was used. O-carbonyl-L-
After the polymerization reaction of glutamate (72) and γ-benzyl-N, O-carbonyl-D-glutamate (79) was carried out at a molar ratio of 1: 1 and 1: 9,
Debenzylation gave the title compounds (81) and (82), respectively. When the weight average molecular weight of the obtained compound was determined in the same manner as in Reference Example 30, all the compounds were 13,000. The physical constants of the obtained compound are shown in Table 10.

Reference Example 32 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D, L-glutamic acid sodium salt (83) Compound (80) 38.7 mg (0.003 mmol) of N, N -Dimethylformamide 0.5 ml solution, N, N '
-Dicyclohexylcarbodiimide 1.85 mg (0.0
0009 mmol), 1.73 mg (0.00015 mmol) N-hydroxysuccinimide and 2- (4
-Hydroxyphenyl) ethylamine 2.06 mg (0.
(00015 mmol) was added to 0.5 ml of N, N-dimethylformamide, and the mixture was stirred at room temperature for 20 hours. To the reaction mixture was added 5 ml of 5% aqueous sodium hydrogen carbonate solution. The precipitated insoluble matter was removed by centrifugation, and the supernatant was filtered using Millipore filter paper. The filtrate was subjected to gel filtration column chromatography (Sephadex G-100,
2.5 cm × 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 dialyzed solution was taken out and freeze-dried to obtain the title compound (83). The 2- (4-hydroxyphenyl) ethylamino group content of the compound (83) was quantified by using the absorbance at 275 nm. Table 11 shows the obtained 2- (4-hydroxyphenyl) ethylamino group content and physical constants.

[0159]

[Table 11]

Reference Example 33 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D, L-glutamic acid sodium salt (84) and (85) Compounds were prepared in the same manner as in Reference Example 32. (8
A 2- (4-hydroxyphenyl) ethylamino group was introduced into 1) and (82) to obtain the title compounds (84) and (85), respectively. 2-of the obtained compound
Table 11 shows (4-hydroxyphenyl) ethylamino group content and physical constants.

Reference Example 34 γ-benzyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (87) The method described in the literature (ERBLOUT AND RHKARTSON, J. Am.C.
Chem. Soc., 78, 941 (1956)).
6.0 g (25.28 mmol) of L-glutamate (86) and 2.68 g (25.28 mmol) of sodium carbonate were dissolved in a mixture of 120 ml of water and 60 ml of acetonitrile and then cooled with ice. Then N- (9-fluorenylmethoxycarbonyloxy) succinimide 8.5
A solution of 3 g (25.28 mmol) dissolved 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. The reaction solution was concentrated to a small amount, a large amount of water was added, and the precipitated insoluble material was filtered off. The filtrate was acidified with dilute hydrochloric acid, and 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. Ethyl acetate-hexane mixture was added to the residue for washing,
The product was collected and 9.4 g (81%) of the title compound (87) was obtained.
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 35 γ-benzyl-α-tert-butyl-N- (9-fluorenylmethoxycarbonyl)-
L-Glutamate (88) 18 g (39.17 mmol) of the compound (87) was dissolved in 375 ml of methylene chloride and then 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 parts of isobutene gas was added with stirring.
Blow 0 ml. After sealing the reaction vessel tightly, bring it to room temperature 16
Left for hours. 5.1 ml of triethylamine in the reaction mixture
After neutralizing by adding, the solvent was distilled off under reduced pressure. The obtained residue was partitioned into an aqueous layer and an ethyl acetate layer. The organic layer was separated and washed successively with water, aqueous sodium hydrogen carbonate solution and water. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained syrupy residue was subjected to silica gel column chromatography (hexane-ethyl acetate = 4:
Purified in 1), 18.1 g (89) of the title compound (88).
%) Was obtained.

88: Syrup [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 )

Example 22 α-tert-butyl-N- (9
-Fluorenylmethoxycarbonyl) -L-glutamate (89) To a mixed solution of 15 g (29.09 mmol) of compound (88) in 156 ml of methanol and 156 ml of tetrahydrofuran was added 10% palladium-carbon (5.63 g), and a nitrogen stream was added. Cooled to 10 ° C. Next, 5.63 g (89.28 mmol) of ammonium formate was added, and the mixture was stirred at the same temperature for 2 minutes.
Stir vigorously for hours. After the catalyst was filtered off, the filtrate was concentrated to dryness under reduced pressure. The obtained residue was subjected to silica gel column chromatography (chloroform-methanol = 10:
Purified in 1), 8.9 g (75) of the title compound (89)
%) Was obtained.

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 36 8- [α-tert-butyl-N
-(9-Fluorenylmethoxycarbonyl) -L-glutamide] octyl α-D-mannopyranoside (90) α-tert-butyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (89) 1. 42 g
(3.344 mmol) and N-hydroxysuccinimide 385 mg (3.344 mmol) in N, N-dimethylformamide 8 ml solution, N, N'-dicyclolohexylcarbodiimide 690 mg (3.344 mmol)
Was added and the mixture was stirred at room temperature for 2 hours. The precipitated insoluble material was filtered off, and the filtrate was treated with 8-aminooctyl α-D-mannopyranoside (9) (895 mg, 3.04 mmol) of N, N.
-Adding to a 5 ml solution of dimethylformamide, stirring was continued for 16 hours at room temperature. The precipitated insoluble material was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The obtained residue was subjected to silica gel column chromatography (chloroform-methanol = 10:
Purified in 1), 1.56 g (73) of the title compound (90).
%) Was obtained.

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-6
a) 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 37 8- [α-tert-butyl-N
-(9-Fluorenylmethoxycarbonyl) -L-glutamide] octyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (91) Compound (90) 1.77 g (2.52 mmol) 5 ml of acetic anhydride was added to a 15 ml solution of pyridine) and left 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 successively with dilute hydrochloric acid, 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 = 1.5Hz, H-1) 4.39 (2H, m, Fmoc-CH ₂ ) 4.26 (1H, dd, J ₅ , _6a = 5.5Hz, J _6a , _6b = 12.5 Hz, H-6
a) 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 38 8- [N- (9-fluorenylmethoxycarbonyl) -L-glutamide] octyl
85% of 2.0 g (2.29 mmol) of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (92) compound (91)
It was dissolved in 57 ml of formic acid-methanol solution and left at room temperature for 16 hours. The reaction mixture was diluted with toluene and then concentrated to dryness under reduced pressure. The obtained residue is subjected to silica gel column chromatography (chloroform-methanol = 7: 1).
After purification with 1.90 g of the title compound (92) (quantitative)
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-6
a) 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 39 8- [γ-L-glutamide]
Octyl 2,3,4,6-tetra-O-acetyl-α
-D-Mannopyranoside (93) 1.9 g (2.341 mmol) of the compound (92) was dissolved in 20 ml of morpholine and then left at room temperature for 40 minutes.
The reaction mixture was diluted with toluene and then concentrated to dryness under reduced pressure. Toluene was added to the residue and concentrated to dryness was repeated to completely remove the residual morpholine. 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, 1323,1
226, 1136, 1089, 1047

Example 23 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 the compound (93) in 20 ml of anhydrous tetrahydrofuran was added 86 mg (0.29 mmol) of bis (trichloromethyl carbonate), and the mixture was heated with stirring 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 concentrated to dryness was repeated to completely remove the produced hydrogen chloride, and the title compound (94) 500 mg (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 40 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)).
220 mg (0.828 mmol) of glutamate (72)
N-hexylamine (296 mg, 2.92 mmol) was added to anhydrous N, N-dimethylformamide (10 ml).
Liquid 0.02 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 then dried by heating at 50 ° C. for 4 hours under reduced pressure to obtain 140 mg of the title compound (95).

Example 24 Poly-γ- [8- (α-D-
Mannopyranosyloxy) octylamino] -L-glutamic acid sodium salt (96) 140 mg of the compound (95) obtained in Reference Example 40 was dissolved in 30 ml of anhydrous tetrahydrofuran, and 1 ml of 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, 4 ml of trifluoroacetic acid containing 0.6 ml of anisole was added to and dissolved in the obtained residue under ice cooling. 4 ml of methanesulfonic acid was added to this solution, and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was poured into 200 ml of isopropyl ether while stirring, and the deposited precipitate was collected by centrifugation. The precipitate was dissolved in 10 ml of a saturated aqueous sodium hydrogen carbonate solution, and then dialyzed against purified water for 48 hours using a Spectra / Pore 3 (molecular weight cutoff = about 3,500) dialysis membrane. The dialyzed solution was taken out and freeze-dried to give the title compound (96) 6
0 mg was obtained. The average molecular weight distribution of the compound (96) was measured 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 /
It was found that Mn) = 1.09. Further, the mannose content of the compound (96) was colorimetrically determined 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 25 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 16 was dissolved in 5 ml of water, and 3 ml of an aqueous solution of 5.0 mg of doxorubicin hydrochloride. 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 resulting solution, and the mixture was stirred at room temperature for 15 hours. The reaction mixture was treated with Spectrapore / Pore 3 (molecular weight cutoff = about 3,500) dialysis membrane to obtain 0.01
4 each for M phosphate buffer (pH 7.0) and water
It dialyzed at 24 degreeC for 24 hours. The liquid in the reaction 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 was introduced.

Experimental Example 1. Sample Polyglutamic acid obtained in Example 10 (molecular weight 13,30
The sample sample was prepared by modifying 36) of galactose in 0). As a control sample, unmodified polyglutamic acid (molecular weight 13,300) was used.

2. Method ¹²⁵ I labeling of samples was performed prior to animal experiments. ¹²⁵ I
The labeling was carried out by the chloramine T method as usual. The reaction solution was subjected to gel filtration with a Sephadex G-25 column to obtain a polymer fraction. To this fraction, 1% bovine serum albumin and an unlabeled product were added to prepare a 1 mg / ml administration solution, which was used in the following animal experiments. A ¹²⁵ I-labeled sample or control sample was administered at 1 mg / kg into the femoral vein of a rat (SD male, 7 weeks old), and blood was collected from the carotid artery 1, 2 and 3 minutes after the administration, and 5 minutes later. Whole blood was collected from the abdominal aorta and killed, and 11 major organs were excised.
Blood was centrifuged, plasma was weighed, the excised organ was weighed as it was, and radioactivity was measured by a gamma counter to calculate plasma concentration and each organ concentration. Similarly, the concentrations in the main organs 1 hour and 4 hours after the administration of the sample sample or the control sample were also measured in the same manner as above. Each animal experiment was performed with 3 animals per group.

3. Results and Discussion Results of blood concentration up to 5 minutes after administration are shown in FIG. It was found that the specimen disappeared from the blood more quickly than the control. Further, 5 minutes after the administration, in the organ distribution (Fig. 2), the accumulation of the kidney was decreased and the accumulation in the liver was confirmed in the sample as compared with the accumulation of the control in the kidney. Furthermore, the concentrations in other organs were also lower in the sample than in the control, which revealed that the galactose modification increased the tropism of polyglutamic acid to the liver.

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

The existence of a lectin that specifically recognizes galactose has been known in the liver, and it was considered 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. The other sugar-modified polyglutamic acid in the present invention is also useful as an organ-oriented DDS carrier if a lectin that recognizes a sugar specific to an organ or cancer cell is present.

[Brief description of 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 concentration in the kidney.

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

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hideo Nogusa 2-9-10 Akihara, Kashiwa-shi, Chiba Pilot House Kashiwa 103 (72) Inventor Hiroshi Susaki 3-1-16-1 Senji, Chofu-shi, Tokyo Tamagawa Housing Ho-8 −106 (72) Inventor Akinori Gonsho 3-8-7 Chiyoda, Kashiwa City, Chiba Vinus Kashiwa 203

Claims (9)

[Claims] 1. A structural unit (A) represented by the following formula (I):
And a sugar-modified polyglutamic acid derivative consisting of (B). [Chemical 1] (In the formula, 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 represents a glycosyl group which may have a hydroxyl group or an amino group at the 2-position substituted with an acetyl group. N represents an integer of 7 to 10. The molar ratio of m to k is 1 to 100). 2. An N-carboxyglutamic acid anhydride derivative represented by the following formula (II). [Chemical 2] (In the formula, R ₂ and n are the same as above) 3. A glutamic acid derivative represented by the following formula (III): [Chemical 3] (In the formula, R ₃ represents a 9-fluorenylmethoxycarbonyl group, and R ₄ represents a protecting group for a carboxyl group.) 4. A glycosyloxyalkylamine represented by the following formula (IV) or a salt thereof. [Chemical 4] (In the formula, R ₂ and n are the same as above) 5. A glycosyl halide in which a hydroxyl group of a glycoside is protected or a hydroxyl group at the 2-position is replaced with an azido group is represented by the formula (V): (In the formula, n is the same as above), N- (hydroxyalkyl) phthalimide is reacted, and if necessary, deprotection is further performed, or the azido group is further reduced to an amino group, and the amino group is further converted into an acetyl group. And then treating it with hydrazine to eliminate the phthalimide. (Wherein R ₂ and n are the same as above), and a method for producing a glycosyloxyalkylamine or a salt thereof. 6. A glutamic acid having a protected γ-carboxyl group is reacted with a reactive derivative of 9-fluorenylmethoxycarboxylic acid to further protect the α-carboxyl group, and then a protective group for the γ-carboxyl group. Formula (III) characterized by desorbing (In the formula, R ₃ is the same as above, and R ₄ represents a protecting group for a carboxyl group). 7. Formula (III): (In the formula, R ₃ and R ₄ are the same as the above), the glutamic acid derivative represented by the formula (IV): (In the formula, R ₂ and n are the same as above), a glycosyloxyalkylamine or a salt thereof is reacted, and α
A compound of formula (II) embedded image characterized in that the protecting group for the carboxyl group and the protecting group for the α-amino group are eliminated and then a carbonate ester is reacted. (In formula, R < ₂ > and n are the same as the above) The manufacturing method of the N- carboxy glutamic acid anhydride derivative shown by. 8. Formula (II): (Wherein R ₂ and n are the same as above), and an N-carboxyglutamic acid anhydride derivative represented by the formula (VI): (Wherein R ₅ represents a protecting group for a carboxyl group) is reacted with N-carboxyglutamic anhydride,
Next, the protecting group of the carboxyl group is eliminated, and if necessary, a physiologically active substance is bound to the carboxyl group, which is represented by the formula (I): (In the formula, R ₁ , R ₂ n, k, and m are the same as the above), and a method for producing the sugar-modified polyglutamic acid derivative. 9. Polyglutamic acid has the formula (IV): (Wherein R ₂ and n are the same as above), a glycosyloxyalkylamine or a salt thereof is reacted, and if necessary, a physiologically active substance is bound to a carboxyl group, wherein Chemical 15] (In the formula, R ₁ , R ₂ n, k, and m are the same as the above), and a method for producing the sugar-modified polyglutamic acid derivative.