JPH08337566A - Glutamic acid derivative and its production - Google Patents

Abstract

PURPOSE: To obtain a glutamic acid derivative usable as a synthetic intermediate for a sugar-modified polyglutamic acid derivative useful as a drug delivery carrier having organotropic property. CONSTITUTION: This glutamic acid derivative is expressed by formula I (R3 is 9-fluorenylmethoxycarbonyl; R4 is a carboxyl-protecting group). The compound is produced by reacting the α-amino group of glutamic acid having protected γ-carboxyl group with a reactive derivative of 9-fluorenylmethoxycarboxylic acid, e.g. by reacting the acid with succinimide, protecting the α-carboxyl group and eliminating the protective group of the γ-carboxyl group. The compound of formula I can be converted to a compound of formula II (R2 is glucosyl; (n) is 7-10) by selecting the 9-fluorenylmethoxycarbonyl for the protection of the amino group of glutamic acid. Further, the compound of formula II is converted to a sugar-modified polyglutamic acid derivative of formula III (R1 is OR' or NHR"; R' is H, an alkali metal, etc.; R" is H or a physiologically active substance; the molar ratio of (m) to (k) is 1-100).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a glutamic acid derivative which is a synthetic intermediate of a sugar-modified polyglutamic acid derivative useful as an organ-directing drug delivery carrier.

[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 , 1
32-136 (1981) 2) Israel Patent No. 68449/3 3) Hurwitz, E. et al., J. Appl. Biochem. 2, 25-35 (198
0) 4) Morimoto, Y. et al., J. Pharm. Dyn. 7, 688-698 (1984) 5) Kato, A. et al., Chem. Pharm. Bull. 30, 2951-2957 (1
982) 6) Kato, Y. et al., Cancer Res. 44, 25-30 (1984) 7) Rowland, GF et al., Nature 255, 487-488 (1975) 8) Tsukada, Y. et al., J. Natl. Cancer Institute 73,
721-729 (1984)

References 1) to 3) above show examples of doxorubicin or daunorubicin bound to polyglutamic acid, 4) examples of melphalan binding, and 5) mitomycin C binding. 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 is bound to polyglutamic acid directly or to polyglutamic acid modified for drug binding to enable selective delivery to cancer tissues or tumor cells. For the purpose of
It has been proposed to further bind an antibody that recognizes these tissues and cells. Therefore, it was predicted that polyglutamic acid could serve as a drug delivery carrier with organ-directivity. However, there is no specific 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 studies and found that modification of polyglutamic acid with sugar imparts organ-directivity. They have found the present invention and completed the present invention.

[0006]

[Means for Solving the Problems]

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

[0007]

Embedded image

(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 its hydroxyl group or 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. However, the description of formula (I) means that each unit (A) and (B) is randomly copolymerized.

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

The physiologically active substance is a drug 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 a unit (A) and a unit (B) as essential constitutional units, and a 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 4]

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 a 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]

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(Wherein R ₂ and n are the same as above) The substance of the formula (III) is a useful starting material for synthesizing the substance of the formula (II), and is used for protecting 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 6]

(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.

[0022]

[Chemical 7]

(Production Method) 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 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]

Embedded image

(In the formula, n is the same as the above) In the substance of the formula (V), for example, an alkane diol in which one terminal OH group is protected with a 2,3-dihydropyranyl group is reacted with phthalimide, It can be obtained by deprotection to liberate the OH group.

To prepare the substance of 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 (4) is reacted with the glycosyloxyalkylamine represented by the formula (IV) or a salt thereof to remove 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 protecting group of a carboxyl group with a glycosyloxyalkylamine represented by the formula (IV) or a salt thereof. Is.

[0029]

[Chemical 9]

(In the formula, R ₅ represents a protecting group for a carboxyl group such as benzyl group) The first copolymerization method may be carried out according to a conventional method of copolymerization,
Reacting two different N-carboxyglutamic anhydrides of the above formulas (II) and (VI) in dimethylformamide in the presence of a condensing agent such as hexylamine,
Next, the protective group of the carboxyl group is eliminated, and if necessary, a physiologically active substance is bound to the carboxyl group to obtain the substance of formula (I).

The second method is to use polyglutamic acid with glycosyloxyalkylamine or a salt thereof (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, N-carboxy-L-glutamic acid anhydride and N
By copolymerizing -carboxy-D-glutamic acid anhydride, polyglutamic acid in which L-glutamic acid and D-glutamic acid are irregularly arranged can be obtained.

[0034]

The efficacy of the substance of the formula (I) as a drug carrier is shown in the following experimental examples in the substance of the formula (I) modified with galactose as compared with unmodified polyglutamic acid. It was found that the accumulation in the kidney was small and the accumulation in the liver was large. Since the existence of a lectin that specifically recognizes galactose has been 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, cancer cell, or the like has a lectin that recognizes a specific sugar, the substance of formula (I) can serve as a drug delivery carrier having organ-directivity.

[0035]

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

[0036]

[Chemical 10]

[0037]

[Chemical 11]

[0038]

[Chemical 12]

[0039]

[Chemical 13]

[0040]

Embedded image

[0041]

[Chemical 15]

[0042]

Embedded image

[0043]

[Chemical 17]

[0044]

Embedded image

[0045]

[Chemical 19]

[0046]

Embedded image

[0047]

[Chemical 21]

[0048]

[Chemical formula 22]

[0049]

[Chemical formula 23]

[0050]

[Chemical formula 24]

[0051]

[Chemical 25]

[0052]

[Chemical formula 26]

[0053]

[Chemical 27]

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 residue obtained is purified by distillation under high vacuum,
32.1 g (41%) of the title compound (2) was obtained.

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

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

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

Reference Example 3 8-phthalimidooctanol (4) 47 g (137 mmol) of compound (3) 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 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 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 literature (T. OGAWA et al., Tetrahedron, 37, 2779).
(1981)) 2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl chloride (5) 1.6 g (31.58 mmol), 8- 8.7 g (31.58 mmol) of phthalimidooctanol (4) and 17 ml of 1,1,3,3-tetramethylurea (1
(26.3 mmol) was dissolved in 200 ml of dichloroethane and 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 over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane-ethyl acetate = 3: 1) to obtain 12.3 g (52%) of the title compound (6).

6: Syrup-like [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 were mixed with 1
M sodium methoxide-methanol solution (4.1 ml) was added and the mixture was allowed to stand 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 washings are combined and the solvent is distilled off under reduced pressure to give a syrup-like residue 1
1.4 g were obtained. The residue was purified by silica gel column chromatography (hexane-ethyl acetate = 2: 1),
8.5 g (73.3%) of the title compound (7) was obtained.

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 (60 mmol) of hydrazine hydrate 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- )

Reference Example 7 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 10% palladium-
1 g of carbon was added and hydrogenated at room temperature for 16 hours. After removing the catalyst by filtration, the filtrate was concentrated to dryness under reduced pressure to give 3.99 g (quantitative) of the hydrochloride of the title compound (9).

9 (hydrochloride): amorphous powder [α] _D ²⁶ + 42.9 ° (c 1.13, MeOH) IR (Nujol) cm ⁻¹ : 3346, 1620, 1506, 1460, 1377 ¹ H-NMR (CD ₃ OD): δ 4.72 (1H, d, J ₁ , ₂ = 2Hz, H-1) 3.81 (1H, dd J ₅ , _6a = 2.5Hz, J _6a , _6b = 11.5Hz, H-6a) 3.76 (1H, dd, J ₂ , ₃ = 3.5Hz, H-2) 3.59 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 3.51 (1H, m, H-5) 2.90 (2H, t, J = 8Hz, -CH ₂ N) 1.7-1.35 (12H, m,-(CH ₂ ) _6- )

Hydrochloride of compound (9) obtained above 2.64
g was dissolved in aqueous methanol and passed through a column of 50 ml of ion exchange resin Amberlite IRA-400 (OH type). The column was washed with 500 ml of 50% 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 8 8-phthalimidooctyl
2,3,4,6-Tetra-O-benzyl-β-D-mannopyranoside (11) and 8-phthalimidooctyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (12) ( i) 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) of toluene in 10 ml of nitrogen was stirred at -18 ° C at -18 ° C to prepare 795 mg (2.89 mmol) of 8-phthalimidooctanol (4), 2 g of powdery molecular sieve 4A and silver silicate. Two
g to a toluene suspension. 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 to dryness under reduced pressure. 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-like [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) in 20 ml of methylene chloride was added to 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 9 8-aminooctyl 2,3
4,6-Tetra-O-benzyl-β-D-mannopyranoside (13) 1.39 g (1.74 mmol) of compound (11) and 8.0 g (87 mmol) of hydrazine acetate were dissolved in 90 ml of methanol. The mixture was heated under 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 10 8-Benzyloxycarbonylaminooctyl 2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (14) 0.29 g of the compound (13) and 0.03 of triethylamine.
To a solution of 7 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. The organic layer was separated, dried over sodium sulfate, and then concentrated to dryness under reduced pressure. The obtained residue is subjected to silica gel column chromatography (hexane-ethyl acetate = 4: 1).
The title compound (14) (0.29 g, 83%) was obtained after purification.

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

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

[0079] 15 (hydrochloride): amorphous powder [α] ^{_{D 24 - 10.3 ° (c 0.62}} , MeOH) IR (KBr) cm -1: 3358, 1614, 1074 1 H-NMR (CD 3 OD): δ 4.49 (1H, s, H-1) 3.44 (1H, dd, J ₅ , _6b = 3.5Hz, H-6b) 3.19 (1H, ddd, H-5) 2.90 (2H, t, J = 7.5Hz,- CH ₂ N) 1.7-1.3 (12H, m,-(CH ₂ ) _6- )

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

[0081] 15 (p-toluenesulfonate): amorphous powder [α] ^{_{D 24 - 10.3 ° (c 0.62}} , MeOH) IR (KBr) cm -1: 3358, 1614, 1074 1 H-NMR (CD 3 OD): δ 7.71 (2H, d, J = 7.8Hz, aromatic H) 7.23 (2H, d, J = 7.8Hz, aromatic H) 4.49 (1H, d, J ₁ , ₂ = 0.7Hz, H-1) 3.24 (2H, m) 2.90 (2H, t, J = 7.5Hz, -CH ₂ N) 1.65-1.4 (12H, m,-(CH ₂ ) _6- )

Reference Example 13 8-Aminooctyl-β-D
-Mannopyranoside (15) 8-aminooctyl β- synthesized by the method of Reference Example 11
After dissolving the hydrochloride of D-mannopyranoside (15) in water-containing methanol, the ion-exchange resin IRC-50 (NH ₄
Type) column (4.5 cm × 40 cm). Water 800
After elution with 1 ml of 6% aqueous ammonia, the elution was performed with 1700 ml of 6% aqueous ammonia. Fractions were collected after flowing 1,000 ml of eluate 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 ( ₁ H, d, J ₁ , ₂ = 1.0 Hz) , H-1) 3.97 (1H, d, J ₂ , ₃ = 3.0Hz, H-2) 3.92 (1H, dd, J ₅ , _6a = 2.2Hz, J _6a , _6b = 12Hz, H-6a) 3.87 ( 1H, dd, J = 3.2Hz, 10Hz, -CH ₂ O) 3.72 (1H, dd, J ₅ , _6b = 6.6Hz, H-6b) 3.65 (1H, m, -CH ₂ O) 3.63 (1H, dd , J ₂ , ₃ = 3.2, J ₃ , ₄ = 9.8Hz, H-3) 3.56 (1H, t, J ₃ , ₄ = 9.8Hz, H-4) 3.35 (1H, ddd, H-5) 2.68 ( 2H, t, J = 7.0Hz, -CH ₂ N) 1.60-1.30 (12H, m,-(CH ₂ ) _6- )

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

Methods described in literature (Carbohydr. Res., 136,
153 (1985)), 9.5 g (24.1 mmol) of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranosyl bromide (16) Is stirred at -15 ° C under a nitrogen stream.
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 to dryness under reduced pressure. 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-like [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: syrupy [α] _D ²³ + 47.7 ° (c 0.56, CHCl ₃ ) IR (CHCl ₃ ) cm ^-1 : 2250, 1750, 1715, 1398, 1370 ¹ H-NMR (CDCl ₃ ): δ 7.83 (2H, dd, aromatic H) 7.70 (2H, dd, aromatic H) 5.38 (1H, dd, J ₂ , ₃ = 4Hz, J ₃ , ₄ = 9.5Hz, H-3) 5.30 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 4.81 (1H, d, J ₁ , ₂ = 1.5Hz, H-1) 4.24 (1H, dd, J ₅ , _6a = 5Hz, J _6a , _6b = 12.5Hz, H-6a) 4.07 (1H, dd, J ₅ , _6b = 2.5Hz, H-6b) 4.00 (1H, dd, J ₂ , ₃ = 3.5Hz, H-2) 3.72-3.62 (3H, m, -CH ₂ O, -CH ₂ N) 3.41 (1H, m, -CH ₂ N) 3.58 (1H, ddd, H-5) 2.09 (3H, s, OAc) 2.08 (3H, s, OAc) 2.04 (3H, s , OAc) 1.7-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 15 8-phthalimidooctyl
2-Azido-2-deoxy-β-D-mannopyranoside (19) To a solution of 4.0 g (6.795 mmol) of compound (17) in 55 ml of methanol was added 1.7 ml of a 1M sodium methoxide-methanol solution and the mixture was allowed to stand at room temperature for 16 hours. I left it. Ion exchange resin Amberlite IR-120B (H
⁽⁺ Type) was added to neutralize, 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 16 8-phthalimidooctyl
2-Acetamido-2-deoxy-β-D-mannopyranoside (20) To a solution of 1.68 g (3.63 mmol) of compound (19) in 43 ml of ethanol, 380 mg of nickel chloride hexahydrate was added.
After adding 0.68 ml of a solution prepared by dissolving (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 while 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. After the reaction mixture was concentrated to dryness under reduced pressure, the obtained residue was purified by silica gel column chromatography (chloroform-methanol = 7: 1) to give the title compound (20) 5.
87 mg (34%) were obtained.

[0091] 20: amorphous powder [α] ^{_{D 26.5 - 28.2 ° (c 1.42}} , MeOH) IR (KBr) cm -1: 3425, 1672, 1060 1 H-NMR (CD 3 OD): δ 7.7-7.5 ( 4H, m, aromatic H) 4.58 (1H, d, J ₁ , ₂ = 1.5Hz, H-1) 4.36 (1H, dd, J ₂ , ₃ = 3.5Hz, H-2) 3.48 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 2.00 (3H, s, NAc) 1.8-1.2 (12H, m,-(CH ₂ ) _6- )

Reference Example 17 8-aminooctyl 2-acetamido-2-deoxy-β-D-mannopyranoside (21) A solution of 580 g (1.212 mmol) of compound (20) in 10 ml of ethanol was mixed with 2 ml of hydrazine hydrate. In addition, the mixture was heated under reflux at 70 ° C for 5 hours. After cooling to room temperature, the reaction solution was diluted by adding toluene, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform-methanol = 7: 1),
140 mg (34%) of the title compound (21) 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 18 8-phthalimidooctyl
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside (23) 8-phthalimidooctanol (4) 2.75 g (10)
50 ml of dry benzene, 2.53 g (10 mmol) of silver cyano cyanide 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, 6 in this suspension
A solution of 6 g (14.6 mmol) of tetra-O-acetyl-α-D-galactopyranosyl bromide (22) in 20 ml of dry benzene was added dropwise at room temperature with stirring. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated to dryness under reduced pressure. The obtained residue was purified by flash column chromatography (ethyl acetate-hexane = 1: 1),
5.48 g (90.5%) of the title compound (23) was obtained as a colorless oil.

[0095] 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 19 8-phthalimidooctyl
β-D-galactopyranoside (24) 0.5 ml of 28% sodium methoxide-methanol solution was added to a solution of 5.4 g (8.9 mmol) of compound (23) in 100 ml of methanol, 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.

[0097] 24: mp 123-125 ° C. [α] ^{_{D 27 - 11.4 ° (c 1.09}} , CHCl 3) 1 H-NMR (CD 3 OD): δ 7.80-7.90 (2H, m, aromatic H) 7.75-7.80 (2H, m, aromatic H) 4.16 (1H, d, J ₁ , ₂ = 7.6Hz, H-1) 3.82-3.88 (1H, m) 3.79 (1H, d, J = 2.7Hz) 3.65-3.75 (2H , m) 3.62 (2H, t, J = 7.3Hz, -CH ₂ O) 3.40-3.52 (4H, m) 1.50-1.70 (4H, m, CH ₂ × 2) FAB-Mass: m / z 460 (M + N)
a) ⁺ , 438 (M + H) ⁺ , 276, 258,
160

Reference Example 20 8-aminooctyl β-D
-Galactopyranoside (25) To a solution of 970 mg (2.22 mmol) of compound (24) in 30 ml of ethanol was added 500 mg of hydrazine hydrate, and the mixture was heated 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. After dissolving the obtained crude product in 30 ml of purified water,
It was neutralized by adding concentrated hydrochloric acid. The reaction mixture was passed through a column of 300 ml of ion exchange resin IRC-50 (NH ₄ ⁺ type).
The target substance was eluted with 800 ml of 2M aqueous ammonia. The obtained eluate was concentrated to dryness under reduced pressure to give the title compound (25)
440 mg (64.6%) of

[0099] 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 21 8-phthalimidooctyl
2,3,4-tri-O-acetyl-α-D-xylopyranoside (27), 8-phthalimidooctyl 2,3
4-Tri-O-acetyl-β-D-xylopyranoside (28) and 8-phthalimidooctyl 3,4-di-O-acetyl-α-D-xylopyranoside (29) 2,3,4-tri-O- Acetyl-α-D-xylopyranosyl bromide (26) (1.85 g, 5.45 mmol) in benzene (30 ml) was added with 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 in an argon stream for 18 hours. 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)

Reference Example 22 8-Aminooctyl β-D
-Xylopyranoside (30) 0.16 ml of 28% sodium methoxide-methanol solution was added to a solution of 680 mg (1.27 mmol) of compound (27) in 10 ml of methanol, 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 syrup-like residue obtained was dissolved in 1 ml of ethanol and then 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 obtained syrupy residue was purified by silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 10: 10: 1). Fractions positive to the ninhydrin reagent were collected and the solvent was distilled off under reduced pressure. Dissolve the obtained powdery substance in 30 ml of water,
The mixture was neutralized by adding 3M hydrochloric acid. 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 evaporated under reduced pressure to obtain 274 mg (77.8%) of the title compound (30).

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

Reference Example 23 8-phthalimidooctyl
2,3,4-Tri-O-benzyl-β-L-fucopyranoside (32) and 8-phthalimidooctyl 2,3,4
-Tri-O-benzyl-α-L-fucopyranoside (3
3) 4.26 g (8.57 mmol) of 2,3,4-tri-O-benzyl-α-L-fucopyranosyl bromide (31)
2.17 g of mercuric cyanide in 40 ml of benzene solution
(8.57 mmol), powdery 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 resulting 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) 84
2 mg (14.2%) were obtained.

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

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

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

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

Reference Example 25 8-Aminooctyl β-L
Fucopyranoside (35) To a solution of 800 mg (1.90 mmol) of compound (34) in 10 ml of ethanol, 476 mg of hydrazine hydrate (9.
(50 mmol) 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 syrupy residue thus obtained was subjected to silica gel column chromatography (chloroform-methanol-concentrated aqueous ammonia = 2.
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. This 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%).

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

Reference Example 26 8-phthalimidooctyl
3,4-di-O-acetyl-2-azido-2-deoxy-α-L-fucopyranoside (37) and 8-phthalimidooctyl 3,4-di-O-acetyl-2-azido-
2-Deoxy-β-L-fucopyranoside (38) Reference method (RU Lemieux 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 in 10 ml of benzene solution
(2.71 mmol), powdery molecular sieve 4A
1 g and 8-phthalimidooctanol (4) 746
mg (2.71 mmol) was added, and the mixture was stirred at room temperature in an argon stream for 15 hours. After the reaction mixture was filtered to remove insoluble matter, 20 ml of ethyl acetate was added to the filtrate. The reaction solution is water 3
After washing with 0 ml, it was dried over magnesium sulfate and concentrated to dryness under reduced pressure. The resulting syrupy residue was subjected to silica gel column chromatography (hexane-ethyl acetate =
4: 1) and 308 mg (2) of the title compound (37).
1.4%) 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 x 4)

Reference Example 27 8-aminooctyl 2-acetamido-2-deoxy-α-L-fucopyranoside (40) Compound (37) 300 mg (0.565 mmol) in 5 ml of methanol was added with 28% sodium methoxide-methanol. A solution (0.08 ml) was added, 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 obtained syrupy residue is diluted with ethanol 15
After dissolving in ml, sodium borohydride 64.1mg
(1.70 mmol) and nickel chloride hexahydrate 40
0.5 mg of ethanol solution was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was neutralized with 6M hydrochloric acid, and 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 syrupy residue obtained by concentrating the reaction solution to dryness under reduced pressure was subjected to silica gel column chromatography (chloroform-methanol = 3).
Purification by 0: 1) gave 257 mg of 8-phthalimidooctyl 2-acetamido-2-deoxy-α-L-fucopyranoside (39). 257 mg of compound (39) was dissolved in 5 ml of ethanol, and 141 mg of hydrazine hydrate
(2.83 mmol) was added and the mixture was heated under reflux at 100 ° C. for 2 hours. Further, 141 mg (2.83 mmol) of hydrazine hydrate was added, and the mixture was heated under reflux at the same temperature for 2 hours. After the reaction mixture was filtered to remove 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,
41.1 mg (23%) of the title compound (40) was obtained.

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

Reference Example 28 Poly-L-glutamic acid (4
5) Commercially available poly-L-glutamic acid sodium salt (average molecular weight distribution (viscosity method) = 13,300) (41) 10 g
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 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.

[0119]

[Table 1]

Reference Example 29 Poly-L-glutamic acid (4
6), (47) and (48) In the same manner as in Reference Example 28, commercially available poly-L-glutamic acid sodium salt (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 1 M hydrochloric acid in an aqueous solution to give the free acid to give the title compounds (46), (47). ) And (48) were obtained. The physical constants of the obtained compound are shown in Table 1.

Reference Example 30 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (49) 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) 1
After diluting with 0 ml, it was subjected to gel filtration column chromatography (Sephadex G-100, 50 cm × 2.5 cm), and 5 ml of each fraction was eluted. Fraction Nos. 15 to 27 are collected to collect Spectra / Pore 3 (molecular weight cutoff = about 3.5
(00) Using a dialysis membrane, it was dialyzed against purified water at 4 ° C. for 16 hours. The dialysate was removed and lyophilized to give 75 mg of the title compound (49). When the amount of the introduced 2- (4-hydroxyphenyl) ethylamino residue was quantified using the absorbance of 275 nm, 5 mol of 2- (4-hydroxyphenyl) ethylamino group was introduced per mol of poly-L-glutamic acid. It turned out. The physical constants of compound (49) are shown in Table 2.

[0122]

[Table 2]

Reference Example 31 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (50) Poly-L-glutamic acid (46) 100 mg (0.007)
51 mmol), 145 mg of O-sulfobenzimide
(0.791 mmol) and 141 mg (0.869 mmol) of N, N'-carbonyldiimidazole were added to N, N
-Dissolve in 3 ml of dimethylformamide and stir for 3 hours at room temperature.
Stir for 0 minutes. 200 mg (1.458 mmol) of 2- (4-hydroxyphenyl) ethylamine and 0.19 ml of 1,1,3,3-tetramethylguanidine were added to this solution.
(1.514 mmol) was added to 0.07 ml (0.025 mmol) of a solution of 4 ml of N, N-dimethylformamide, 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) It was dialyzed against purified water at 4 ° C. for 48 hours using a dialysis membrane. The dialyzed solution was taken out and freeze-dried to obtain 78 mg of the title compound (50). The amount of introduced 2- (4-hydroxyphenyl) ethylamino residue is measured by absorbance at 275 nm.
It was found that 5 mol of 2- (4-hydroxyphenyl) ethylamino group was introduced per mol of poly-L-glutamic acid. The physical constants of compound (50) are shown in Table 2.

Reference Example 32 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (51) and (52) In the same manner as in Reference Example 31, poly-L-glutamic acid. In (47) and (48), 10 molar equivalents of 2 respectively
2 with-(4-hydroxyphenyl) ethylamine
A-(4-hydroxyphenyl) ethylamino group was introduced to obtain the title compounds (51) and (52). The amount of introduced 2- (4-hydroxyphenyl) ethylamino group (molar ratio) and the physical constants of the obtained compound are shown in Table 2.

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

[0126]

[Table 3]

Reference Example 34 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D-glutamic acid sodium salt (59), (60) and (61) In the same manner as in Reference Example 30, the compound was obtained. (56), (5
A 2- (4-hydroxyphenyl) ethylamino group was introduced into each of 7) and (58) to give the title compound (5
9), (60) and (61) were obtained. Introduced 2-
Table 4 shows the amount (molar ratio) of (4-hydroxyphenyl) ethylamino group and the physical constants of the obtained compound.

[0128]

[Table 4]

Reference Example 35 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-
7.2 mg of (4-hydroxyphenyl) ethylamine
(0.0547 mmol) and 80.5 mg (0.7 mmol) of N-hydroxysuccinimide were dissolved in 1.5 ml of dry N, N-dimethylformamide, and the mixture was ice-cooled. The reaction mixture was stirred with 1-ethyl-3-
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,
Stir 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) Dialysis was performed at 4 ° C. for 24 hours against purified water 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). Each eluted fraction was subjected to high performance liquid chromatography (column: TSK-
GEL G3000PWXL, eluent: 0.2M saline, elution rate: 0.5 ml / min, detection: ultraviolet absorption (28
0 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 obtain 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. Moreover, when the amount of 2- (4-hydroxyphenyl) ethylamino groups was quantified using the absorbance of 275 nm, the amount introduced (molar ratio) was 3. The sugar content of the compound and 2-
The content of (4-hydroxyphenyl) ethylamino group is shown in Table 5.
It was shown to. In addition, Table 6 shows the physical constants of the compounds.

[0130]

[Table 5]

[0131]

[Table 6]

Reference Example 36 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
90 mg (0.56 mmol) and triethylamine 0.
48 ml (0.56 mmol) of a 2 ml solution of N, N-dimethylformamide were added and stirred at room temperature for 1.5 hours. Then 2- (4-hydroxyphenyl) ethylamine 5
mg was added and the mixture was further stirred for 15.5 hours. N, N in the reaction solution
-After adding 0.5 ml of dimethylformamide for dilution,
Stirring was continued for another 25 hours. To the reaction mixture was added 0.5 ml of 0.1 M aqueous sodium hydroxide solution and 15 ml of 0.01 M isotonic phosphate buffer. 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). Poly-L
-Glutamic acid derivative-containing fractions were collected and dialyzed against purified water for 48 hours using a Spectra / Pore 3 (molecular weight cutoff = about 3,500) dialysis membrane. When the dialyzed solution is taken out and freeze-dried, a colorless amorphous compound (6
3) 35 mg was obtained. Colorimetric determination of the sugar content by the phenol-sulfuric acid method revealed that 7 moles of 8- (β-D-mannopyranosyloxy) octylamino group were introduced per mole of poly-L-glutamic acid. Also, 2- (4-
Hydroxyphenyl) ethylamino group amount of absorbance 275
When quantified using nm, it was found that 3 mol of 2- (4-hydroxyphenyl) ethylamino groups had been introduced. The physical constants of the obtained compound are shown in Table 6.

Reference Example 37 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (64) Poly-L-glutamic acid (45) 50 mg (0.0037
Mmol) of N, N-dimethylformamide in 100 ml of 1-ethoxycarbonyl-2-ethoxy-1,2.
-152 mg (0.615 mmol) of dihydroquinoline was added and stirred for 1 hour and 40 minutes. Then, the hydrochloride salt of 8-aminooctyl β-D-mannopyranoside (15) 190
mg (0.56 mmol) and triethylamine 0.78
ml (0.56 mmol) N, N-dimethylformamide 50 ml solution and 2- (4-hydroxyphenyl)
Ethylamine 5 mg N, N-dimethylformamide 10
ml solution was 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 Reference Example 36 to obtain the title compound (64). Table 2 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound.
It was shown to. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 38 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 ml (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. To this mixture was added 20 mg (0.146 mmol) of 2- (4-hydroxyphenyl) ethylamine and 0.19 ml of 1,1,3,3-tetramethylguanidine.
2.0 ml of a solution of (1.514 mmol) in 4 ml of N, N-dimethylformamide and 8-aminooctyl
β-D-mannopyranoside (15) hydrochloride 153 mg
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 in Reference Example 36 to obtain the title compound (65). Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the compound. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 39 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 Reference Example 35, poly-L-glutamic acid (45) was added to 8-aminooctyl α-D-.
Using mannopyranoside (9) and 2- (4-hydroxyphenyl) ethylamine, introduction of 8- (β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group was performed. The title compound (66) 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. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 40 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) of 8-aminooctyl α-D-mannopyranoside (9), 7.5 mg (0.00546 mmol) of 2- (4-hydroxyphenyl) ethylamine and 1-ethoxycarbonyl- A solution of 98 mg (0.0396 mmol) of 2-ethoxy-1,2-dihydroquinoline in 3 ml of dry N, N-dimethylformamide was added, and the mixture was stirred at room temperature for 16 hours. After diluting the reaction mixture with 30 ml of 0.01 M isotonic phosphate buffer, it was diluted with purified water using Spectra / Pore 3 (molecular weight cut-off = about 3,500) dialysis membrane at 4 ° C. at 2 ° C.
It was dialyzed for 4 hours. The dialyzed solution was taken out and freeze-dried.
The obtained residue is used as a 0.01 M isotonic phosphate buffer solution (pH 7.4).
It was dissolved in and purified by gel filtration column chromatography (Sephadex G100, 5 cm × 30 cm). Each eluted fraction was subjected to high performance liquid chromatography (column: T
SK-GEL G3000PWXL, eluent: 0.2M
Saline solution, dissolution rate: 0.5 ml / min. , Detection: ultraviolet absorption (280 nm)). Fractions containing the poly-L-glutamic acid derivative were collected and recollected in Spectra / Pore 3
(Molecular weight cutoff = about 3,500) Using a dialysis membrane,
Dialysis was performed at 4 ° C. for 48 hours against purified water. The dialyzed solution was taken out and freeze-dried to obtain 80 mg of the title compound (67). Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the obtained compound. Also,
The physical constants of the compound are shown in Table 6.

Reference Example 41 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 Reference Example 37, poly-L-glutamic acid (45) was added to 8-aminooctyl α-D-.
Using xylopyranoside (30) and 2- (4-hydroxyphenyl) ethylamine, 8- (α-D-xylopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced. , The title compound (68) was obtained. Table 5 shows the 2- (4-hydroxyphenyl) ethylamino group content and the sugar content of the compound. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 42 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 Reference Example 37, poly-L-glutamic acid (45) was added to compound (34) and 2- (4-).
Using hydroxyphenyl) ethylamine, 8- (β
-L-fucopyranosyloxy) octylamino group and 2
A-(4-hydroxyphenyl) ethylamino group was introduced to obtain the title compound (69). Compound 2- (4-
The hydroxyphenyl) ethylamino group content and sugar content are shown in Table 5. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 43 Poly-γ- [8- (2-acetamido-2-deoxy-α-L-fucopyranosyloxy) octylamino] -γ- [2- (4-hydroxyphenyl) ethylamino] -L-Glutamic acid sodium salt (70) In the same manner as in the method described in Reference Example 37, poly-L-glutamic acid (45) was added to compound (40) and 2- (4-).
Using hydroxyphenyl) ethylamine, 8- (2
-Acetamide-2-deoxy-α-L-fucopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced to give the title compound (7
0) was obtained. To determine the sugar content of compound (70),
It was hydrolyzed by heating and stirring with 4M hydrochloric acid at 100 ° C. for 4 hours. Colorimetric determination of the obtained hydrolysis product by the Elson-Morgan method gave 8- (2-acetamido-2-deoxy-α-
L-fucopyranosyloxy) octylamino group content is
It 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. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 44 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 Reference Example 35, poly-L-glutamic acid (45) was added to compound (21) and 2- (4-).
Using hydroxyphenyl) ethylamine, 8- (2
-Acetamide-2-deoxy-β-D-mannopyranosyloxy) octylamino group and 2- (4-hydroxyphenyl) ethylamino group were introduced to obtain the title compound (71). The sugar content of the compound (71) was used as in Reference Example 43.
After hydrolysis with 4M hydrochloric acid by the same method as described in 1., colorimetric determination was carried out by Elson-Morgan method. Table 5 shows the sugar content and the 2- (4-hydroxyphenyl) ethylamino group content of the compound. In addition, Table 6 shows the physical constants of the compounds.

Reference Example 45 Poly-L-glutamic acid sodium salt (73) The method described in the literature (J. Am. Chem. Soc., 78, 941 (1956)).
2.63 g (10.0 mmol) of .gamma.-benzyl-N, O-carbonyl-L-glutamate (72) synthesized in 1. was dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a nitrogen stream. To this solution was added 6.6 μl of hexylamine to dry N, N-dimethylformamide 100.
A solution dissolved in μl was added, and the mixture was stirred at room temperature under a nitrogen stream for 24 hours. 500 ml of isopropyl ether to the reaction mixture
While 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 dissolved in a mixed solution of 17 ml of trifluoroacetic acid and 3 ml of anisole, and then 17 ml of methanesulfonic acid was added under a nitrogen stream. The reaction mixture was stirred under ice cooling for 20 minutes. The cooling bath was removed, and the mixture was further stirred at room temperature for 30 minutes. The reaction solution was poured into 300 ml of isopropyl ether while stirring, and the deposited precipitate was collected by filtration. The precipitate was washed with isopropyl ether, added to 20 ml of a 5% aqueous sodium hydrogen carbonate solution, and stirred at room temperature for 30 minutes. The obtained solution was dialyzed against purified water at 4 ° C. for 2 days using a Spectra / Pore dialysis membrane. The 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, elution rate: 0.5 ml / min) was 22,000. The physical constants of the obtained compound are shown in Table 7.

[0142]

[Table 7]

Reference Example 46 Poly-L-glutamic acid (7
4) In the same manner as in Reference Example 45, γ-benzyl-N, O
N-, using carbonyl-L-glutamate (72)
Polymerization was performed 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 45, and then purified to obtain the title compound (74). The weight average molecular weight (Mw) of the compound (74) was determined by gel permeation chromatography using polyethylene glycol as a standard 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.

Reference Example 47 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 45 was used, and treated with 1M hydrochloric acid in the same manner as in Reference Example 28. To a solution of 129.0 mg of the free acid of compound (73) in 1.0 ml of N, N-dimethylformamide, 51.6 mg (0.25 mmol) of N, N'-dicyclohexylcarbodiimide and N-
A solution prepared by dissolving 35.5 mg (0.31 mmol) of hydroxysuccinimide in 0.5 ml of N, N-dimethylformamide was added, and the mixture was stirred at 4 ° C for 2 hours. 2-in this solution
(4-hydroxyphenyl) ethylamine 6.8 mg
(0.05 mmol), 8-aminooctyl β-D-
Mannopyranoside (15) p-toluenesulfonate 9
6.0 mg (0.20 mmol) and triethylamine 2
A solution prepared by dissolving 8 μl in 0.5 ml of N, N-dimethylformamide was added, and the mixture was stirred at 4 ° C. for 2 hours. Except the cooling bath,
The mixture was further stirred at room temperature for 2 hours. To the reaction mixture, 5 ml of 5% aqueous sodium hydrogen carbonate solution was added, and the precipitated insoluble material was removed by centrifugation. The resulting supernatant was filtered using Millipore filter paper. The filtrate was purified by gel filtration column chromatography (Sephadex G-100, 2.5 cm × 20 cm). Fractions containing the poly-L-glutamic acid derivative were collected and purified using purified water using a Spectra / pore dialysis membrane.
It was dialyzed for 2 days at ° C. The dialyzed solution was taken out and freeze-dried to obtain 127.5 mg of the title compound (75). The sugar content of compound (75) was determined by colorimetry using the phenol-sulfuric acid method. The content of 2- (4-hydroxyphenyl) ethylamino group was quantified 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.

[0145]

[Table 8]

[0146]

[Table 9]

Reference Example 48 Poly-γ- [8- (β-D-
Mannopyranosyloxy) octylamino] -γ- [2
-(4-Hydroxyphenyl) ethylamino] -L-glutamic acid sodium salt (76) In the same manner as in Reference Example 47, the free acid of compound (74) was added to p-toluenesulfonic acid salt of compound (15) and 2
8 with-(4-hydroxyphenyl) ethylamine
A-(β-D-mannopyranosyloxy) octylamino group and a 2- (4-hydroxyphenyl) ethylamino group were introduced to obtain the title compound (76). Compound (7
Table 8 shows the sugar content and 2- (4-hydroxyphenyl) ethylamino group content of 6). The physical constants of the compounds are shown in Table 9.

Reference Example 49 γ-Benzyl-D-glutamate (78) To 75 ml of a 60% sulfuric acid solution of 100 g (680 mmol) of commercially available D-glutamic acid (77) was added 75.0 g (694 mmol) of benzyl alcohol 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 the mixture was 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. Crystallize 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).

[0149] 78: mp 164-164.5 ℃ [α] ^{_{D 25 - 19.4 ° (c 1.29}} , AcOH) IR (KBr) cm -1: 3000, 1724, 1582, 1517, 1173

Reference Example 50 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. After stirring the reaction mixture at room temperature for 15 minutes, heating was started and the internal temperature was stirred at 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 51 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) 26
0 mg (10 mmol) was dissolved in 10 ml of dry N, N-dimethylformamide with stirring under a stream of nitrogen. Hexylamine (13.2 μl) was added to this solution with 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. 300 ml of isopropyl ether while stirring the reaction mixture
And the deposited precipitate was collected by filtration. The precipitate obtained was added to 20 ml of a 5% aqueous sodium hydrogen carbonate solution and the mixture was stirred at room temperature for 3
Stir for 0 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 (80).
1.10 g were obtained. When the average molecular weight of the compound (80) was determined by gel permeation chromatography using polyethylene glycol as a standard substance, the weight average molecular weight (Mw) was 13,000. The physical constants of the obtained compound are shown in Table 10.

[0153]

[Table 10]

Reference Example 52 L-Glutamic acid and D-glutamic acid irregular copolymers (81) and (82) In the same manner as in Reference Example 51, γ-benzyl-N, in N, N-dimethylformamide was used. O-carbonyl-L-
Glutamate (72) and γ-benzyl-N, O-carbonyl-D-glutamate (79) were subjected to a polymerization reaction at a molar ratio of 1: 1 and 1: 9, followed by depolymerization. After benzylation, the title compounds (81) and (8
2) was obtained. When the weight average molecular weight of the obtained compounds was determined in the same manner as in Reference Example 51, all the compounds were 13,000. The physical constants of the obtained compound are shown in Table 10.

Reference Example 53 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 (0.0
(0015 mmol) was dissolved in 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 x 20 cm). Fractions containing the polyglutamic acid derivative were collected and dialyzed against purified water at 4 ° C. for 2 days using a Spectra / Pore dialysis membrane. The 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.

[0156]

[Table 11]

Reference Example 54 Poly-γ- [2- (4-hydroxyphenyl) ethylamino] -D, L-glutamic acid sodium salt (84) and (85) A compound was prepared in the same manner as in Reference Example 53. (8
2- (4-hydroxyphenyl) in 1) and (82)
An ethylamino group was introduced, and the title compound (8
4) and (85) were obtained. 2- (4- of the obtained compound
Table 11 shows the content of hydroxyphenyl) ethylamino group and the physical constants.

Reference Example 55 γ-benzyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (87) The method described in the literature (ER BLOUT AND RH KARTSON, J. A.
m. Chem. Soc., 78, 941 (1956)), 6.0 g (25.28 mmol) of γ-benzyl-L-glutamate (86) and 2.68 g (25.28 mmol) of sodium carbonate. Was dissolved in a mixed solution of 120 ml of water and 60 ml of acetonitrile and then ice-cooled. 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 the title compound (87) 9.4 g (81%)
I got

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

Reference Example 56 γ-benzyl-α-tert
-Butyl-N- (9-fluorenylmethoxycarbonyl) -L-glutamate (88) 18 g (39.17 mmol) of compound (87) was dissolved in 375 ml of methylene chloride and then cooled to -18 ° C under a nitrogen stream. did. 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.
0 ml was blown. After sealing the reaction vessel tightly, bring it to room temperature 16
Left for hours. 5.1 ml of triethylamine was added to the reaction mixture.
Was added to neutralize, and 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: 1).
After purification with 18.1 g (89%) of the title compound (88).
I got

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

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

Reference Example 57 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
To a solution of 385 mg (3.344 mmol) of N-hydroxysuccinimide (3.344 mmol) and 8 ml of N, N-dimethylformamide was added 690 mg (3.344 mmol) of N, N'-dicyclolohexylcarbodiimide, Stir at room temperature for 2 hours. The precipitated insoluble material was removed by filtration, and the filtrate was treated with 8-aminooctyl α-D-mannopyranoside (9) (895 mg, 3.04 mmol) of N, N-.
It was added to a 5 ml solution of dimethylformamide and stirred at room temperature for 16 hours. 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-6a) 3.76 (1H, dd, J ₁ , ₂ = 1.5Hz, J ₂ , ₃ = 3.5Hz, H-2) 3.59 (1H, t, J ₃ , ₄ = 9.5Hz, H-4) 3.50 (1H, ddd, H-5) 2.30 (2H, m, -CH ₂ COO-) 2.10 (1H, m, Glu-CH ₂ ) 1.90 (1H, m, Glu-CH ₂ ) 1.45 ( 9H, s, C (CH ₃ ) ₃ )

Reference Example 58 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 solution of 15) in pyridine and the mixture was allowed to stand at room temperature for 16 hours. After pouring the reaction mixture into a large amount of water, the precipitated product was extracted with ethyl acetate. The organic layer was washed 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-6a) 4.21 (2H, t, J = 7Hz, Fmoc-CH Glu-CH) 4.10 (1H, dd, J ₅ , _6b = 2.5Hz, H-6b) 3.97 (1H, ddd, H-5 ) 3.65 (1H, m, -CH ₂ O) 3.42 (1H, m, -CH ₂ O) 3.23 (2H, m, -CH ₂ N) 2.20 (2H, m, -CH ₂ COO-) 2.15 (3H, s, OAc) 2.09 (3H, s, OAc) 2.04 (3H, s, OAc) 1.98 (3H, s, OAc) 1.47 (9H, s, C (CH ₃ ) ₃ ) 1.7-1.2 (12H, m,- (CH ₂ ) _6- )

Reference Example 59 8- [N- (9-fluorenylmethoxycarbonyl) -L-glutamide] octyl
85% of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (92) Compound (91) 2.0 g (2.29 mmol)
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)
I got

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

Reference Example 60 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 the mixture was allowed to stand 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 remaining 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

Reference Example 61 8- [γ- (N, O-dicarbonyl) -L-glutamide] octyl 2,3,4,6-
Tetra-O-acetyl-α-D-mannopyranoside (9
4) To a solution of 490 mg (0.828 mmol) of compound (93) in 20 ml of anhydrous tetrahydrofuran was added 86 mg (0.29 mmol) of bis (trichloromethyl carbonate), and the mixture was heated and stirred at 60 ° C. for 3 hours under a nitrogen stream. After cooling to room temperature, the solvent was distilled off under reduced pressure. Toluene was added to the residue, and the mixture was concentrated to dryness. The generated hydrogen chloride was completely removed, and 500 mg of the title compound (94) (9
8%).

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

Reference Example 62 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 under reduced pressure at 50 ° C. for 4 hours to obtain 140 mg of the title compound (95).

Reference Example 63 Poly-γ- [8- (α-D-
Mannopyranosyloxy) octylamino] -L-glutamic acid sodium salt (96) 140 mg of the compound (95) obtained in Reference Example 62 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. The reaction mixture was concentrated to dryness under reduced pressure, and 4 ml of trifluoroacetic acid containing 0.6 ml of anisole was added to and dissolved in the resulting 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 compound (96) was determined by gel permeation chromatography (column: TSK-
GEL G4000PWXL + G3000PWXL, eluent: 0.2M 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 found to be 1% by weight.

[0176] 96: amorphous powder [α] ^{_{D 24 - 61.1 ° (c 0.35}} , 0.01MPBS) IR (KBr) cm -1: 3300, 1720, 1655, 1549, 1410, 1258

Reference Example 64 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 Reference Example 41 was dissolved in 5 ml of water, and a solution of 5.0 mg of doxorubicin hydrochloride in 3 ml of water. 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 reaction solution was taken out and freeze-dried. When the amount of doxorubicin obtained was quantified using an absorbance of 490 nm, it was found that 1 mol of doxorubicin had been introduced.

Experimental Example 1. Sample Polyglutamic acid obtained in Reference Example 35 (molecular weight 13, 30
The sample sample was prepared by modifying 0) with 36 galactoses. Unmodified polyglutamic acid (molecular weight 13,300) was used as a control sample.

2. Methods Samples were labeled with ¹²⁵ I prior to animal experiments. ¹²⁵ I
The labeling was performed by the chloramine T method in the usual way. 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. The whole blood was collected from the abdominal aorta and killed, and 11 major organs were extracted. Blood is plasma after centrifugation, and the extracted organs are weighed as they are,
Radioactivity was measured with a gamma counter to calculate plasma concentration and concentration in each organ. Similarly, the concentrations in the main organs 1 hour and 4 hours after the administration of the specimen 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, compared with the accumulation of the control in the kidney. Furthermore, even in the concentrations in other organs, the amount of the sample was lower than that of 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 of the concentrations of the specimen and the control in the liver and kidney 5 minutes, 1 hour and 4 hours after administration. In the kidney, the AUC (area under the curve) of the sample was smaller than that of the control, whereas in the liver, the AUC of the sample was conversely very large, and the sample tended to accumulate in the liver.

The presence 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-directed DDS carrier if a lectin that recognizes a sugar specific to a certain 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 front page (72) Hideo Nogusa 2-9-10 Akihara, Kashiwa-shi, Chiba Pilot House Kashiwa 103 (72) Inventor Hiroshi Susaki 3-1-166 Senji, Chofu-shi, Tokyo Tamagawa Housing Ho-8 −106 (72) Inventor Akinori Gonsho 3-8-7 Chiyoda, Kashiwa City, Chiba Vinus Kashiwa 203

Claims (2)

[Claims] 1. A glutamic acid derivative represented by the following formula (III): Embedded image (In the formula, R ₃ represents a 9-fluorenylmethoxycarbonyl group, and R ₄ represents a protecting group for a carboxyl group.) 2. 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 and R ₄ are the same as the above), and a method for producing a glutamic acid derivative.