JPH0885703A - Polysaccharide derivative having organotropy and drug carrier - Google Patents

Abstract

PURPOSE: To obtain a specific compound useful as a drug carrier which transfers a drug such as an antitumor or antiinflammatory drug to a specific organ. CONSTITUTION: This compound is represented by the formula E-T<1> -T<2> -F [wherein E is a monosaccharide selected from among glucose, fructose, mannose, fucose, neuraminic acid, uronic acid, galactosamine, glucosamine, ribose, deoxyribose, arabinose, and xylose, a monosaccharide derivative selected from among N- acylation, O-acylation, O-alkylation, and esterification products of these monosaccharides, or an oligosaccharide consisting of two to six molecules of any of these monosaccharides (and derivatives); T<1> is CH2 CH2 (OCH2 CH2 )m (where (m) is 1 to 10), (CH2 )n (where (n) is 2 to 16), or a group represented by the formula (where X is NHCOCH3 , OH, or NH2 ); T<2> is NH, NHCO, CONH, or NHCONH; and F is a polysaccharide selected from among chitosan, pullulan, dextran, mannoglucan, heparin, hyaluronic acid, and chondroitin sulfate or a derivative of any of these polysaccharides].

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel compound having an organ-localizing property and a drug carrier.

[0002] In recent years, polysaccharide type water-soluble polymer was used as a carrier, an attempt to perform the drug delivery has been made to specific organs which drug is chemically bonded to. Examples of such drug carriers include:
Japanese Patent Application No. 4-234846, PCT / JP94 / 003
No. 22, WO92 / 14759, which can efficiently deliver to the tumor a drug having side effects in other organs or a drug with limited expression of drug efficacy in the tumor. However, these leave room for further improvement regarding site-specific transferability, particularly organ transferability. On the other hand, in connection with organ translocation, sialyl Lewis X binds to ELAM-1 (Phillips, ML, et al. (1990) Science 250,
1130-1132), sialyl Lewis X is present in neutrophils, and E
Reacting with LAM-1 (Bevilacqua, MP, et al. (1
989) Science 243,1160-1165) and sialyl Lewis X
Suppresses neutrophil infiltration (Mulligan, MS, et al. (1
993) J. Exp. Med. 178, 623-631) is known.

[0003]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a drug carrier exhibiting organ-directivity.

The compound and drug carrier according to the present invention are represented by the following general formula (I). ^{E-T 1 -T 2 -F (} I) ( In the formula, E is chosen glucose, fructose, mannose, fucose, neuraminic acid, uronic acid, galactosamine, glucosamine, ribose, deoxyribose, arabinose and xylose Monosaccharides, or N- or O-acyl derivatives of these monosaccharides, O-
Represents an alkyl derivative or an ester derivative, or represents an oligosaccharide consisting of 2 to 6 of these monosaccharides or derivatives of monosaccharides, and T ¹ represents —CH ₂ CH ₂ (OCH ₂ C
H ₂ ) m- (m represents an integer of 1 to 10),-(CH
₂ ) n- (n represents an integer of 2 to 16) or the following group (II),

[0005]

Embedded image (In the above group, X is, -NHCOCH _3, -OH or -
It represents an NH ₂₎ ^{T 2} is, -NH -, - NHCO -, - C
Represents ONH- or -NHCONH-, and F represents a polysaccharide selected from chitosan, pullulan, dextran, mannoglucan, heparin, hyaluronic acid, and chondroitin sulfate, or a derivative of these polysaccharides)

[0006]

DETAILED DESCRIPTION OF THE INVENTION Compound of General Formula (I) In the general formula (I), E is hexose such as glucose, fructose, mannose, fucose, neuraminic acid and uronic acid, galactosamine, hexosamine such as glucosamine, It represents a monosaccharide selected from pentoses such as ribose, deoxyribose, arabinose and xylose, or represents an oligosaccharide composed of 2 to 6 of these monosaccharides. Here, the monosaccharide may be a derivative thereof, and examples thereof include N- or O-acyl derivatives, O-alkyl derivatives, ester derivatives with sulfuric acid, phosphoric acid and the like of the above monosaccharides.

Specific examples of these derivatives include sialic acid (eg, N-acetylneuraminic acid) which is an N- and / or O-acyl derivative of neuraminic acid, and N-acetylgalactosamine which is an N-acyl derivative of hexosamine. , N-acetylglucosamine, mannose-6-phosphate in which the 6-position hydroxyl group of mannose is phosphorylated, galactose-3-sulfate in which the 3-position of galactose is sulfated,
6-O-benzoyl-glucose in which the 6-position of glucose is benzoylated, 6-O-carboxymethyl-N-acetylglucosamine in which the 6-position hydroxyl group of N-acetylglucosamine is carboxymethylated, and 2 of glucosamine
2-N-benzyl-glucosamine and the like in which the amino group at the position is monobenzylated.

The oligosaccharides are 2 to 6 (preferably 2 to 6)
4) A linear or branched hetero-oligomer or homo-oligomer composed of the above monosaccharides or monosaccharide derivatives. Examples of such oligosaccharides include sucrose, sialyl Lewis A, sialyl Lewis X, lactose, maltose, Lewis X, and sulfated Lewis X. Here, "monosaccharide" and "oligosaccharide" mean
It means one in which one hydrogen atom of the hydroxyl group (preferably hydroxyl group at the anomer position) of the sugar molecule is removed.

In the general formula (I), E and T ¹ are O
Although they are linked by an -α-glycoside bond or an O-β-glycoside bond, the bond may be any of these.

In T ¹ , m represents an integer of 1 to 10,
It is preferably 2-5. Further, n represents an integer of 2 to 16, preferably 2 to 8. When T ¹ represents the group (II), preferable examples of the group (II) include those in which X is a hydroxyl group or an acetamide group.

In the general formula (I), F represents a polysaccharide such as chitosan, pullulan, dextran, mannoglucan, heparin, hyaluronic acid and chondroitin sulfate, and these polysaccharides may be derivatives thereof. For example, these carboxymethyl derivatives (carboxymethyl chitosan, carboxymethyl pullulan, carboxymethyl dextran, carboxymethyl annoglucan, etc.), desulfated derivatives (de-N-sulfated heparin, etc.), polyalcoholated derivatives (polyalcoholized mannoglucan, etc.) ) Is mentioned. In the case of a carboxymethyl derivative, the degree of carboxymethylation is preferably 0.5 to 0.7.

[0012] In addition to these, the polysaccharide derivative is further converted to have a carboxyl group or an amino group, and those carrying a pharmaceutical compound described below are also included in the polysaccharide derivative.

The molecular weight of the polysaccharide in which the pharmaceutical compound is not introduced is preferably 10 to 1,000 kD, more preferably 40 to 150 kD.
kD is particularly preferred.

The term "polysaccharides and derivatives thereof" as used herein means those from which functional groups used in the T ² form (eg, amino group, carboxyl group, hydroxyl group, etc.) have been removed.

As a preferred group of compounds of the general formula (I) according to the present invention, E is glucose, mannose, galactose, fucose, neuraminic acid, galactosamine, glucosamine, sialic acid, N-acetylgalactosamine, N-acetylglucosamine. 2 to 4 of the monosaccharides or derivatives of these monosaccharides, or the above monosaccharides or monosaccharide derivatives selected from sucrose, sialyl Lewis A, sialyl Lewis X, lactose, maltose, Lewis X and sulfated Lewis X. Represents a saccharide oligosaccharide consisting of 1 or more, F represents a saccharide derivative selected from carboxymethyl chitosan or carboxymethyl pullulan, or a derivative thereof in which a pharmaceutical compound is supported, and T ¹ is —CH ₂ CH ₂ (OCH ₂ CH ₂ ) m-
And T ² represents —NH— or —NHCO—.

As another preferred compound group, E and F have the same meanings as described above, and T ¹ is-(CH ₂ ).
and those in which T ² represents —CONH—.

In another preferred compound group, E and F have the same meanings as described above, and T ¹ is the above-mentioned group (II).
In which T ² represents —NH—.

The compound according to the present invention has an organ-localizing property of migrating to a specific organ. For example, E is galactose,
Alternatively, when the oligosaccharide has galactose at the non-reducing end (for example, lactose), the compound according to the present invention is transferred to the liver. Although not being bound by the following theory, it is considered that galactose exhibits organ translocation by being taken up by the galactose / N-acetylgalactose recognition lectin in the liver. In addition, in the present specification, “translocation to an organ” is meant to include the property to migrate to an inflammatory site and the property of avoiding reticuloendothelial tissue described later.

Further, the compound according to the present invention has inflammatory site-localizing property. For example, E is the cell adhesion molecule ELAM-1
When it is a sugar that binds to, the compound according to the present invention is translocated to the site of inflammation. In particular, the compound according to the present invention was found to have a high inflammatory site translocation property as compared with the case of simply administering sugar.

Furthermore, the compounds according to the invention have the property of avoiding reticuloendothelial tissues. For example, when E is N-acetylneuraminic acid, the compound according to the present invention avoids reticuloendothelial tissues such as liver and spleen.

Therefore, the compound according to the present invention can be used as a drug carrier for delivering a drug such as an antitumor agent or an antiinflammatory agent to a specific organ.

Here, examples of the compound that binds to the ELAM-1 include compounds represented by the following formula (III) or (IV).

[0023]

[Chemical 3]

[0024]

[Chemical 4] (In the formula, X is the same as defined above, Y represents a sialic acid or _-SO 2 OH)

In the above formula (III), X is --NHCO.
The compound that represents CH ₃ and Y represents sialic acid is sialyl Lewis X, X represents —NHCOCH ₃ , and Y represents —S.
The compound representing O ₂ OH is sulfated Lewis X. Also,
In the above formula (IV), X represents —NHCOCH ₃ , and Y
Sialyl Lewis A is a compound in which is sialic acid.

The structure of the compound according to the present invention can be further modified in consideration of the above-mentioned uses,
These modified compounds, such as polysaccharides carrying a pharmaceutical compound, are also included in the compound according to the present invention.

The pharmaceutical compound may be directly loaded or may be loaded via a suitable spacer. Here, this "spacer" can play the role of releasing the pharmaceutical compound in the living body at a constant and desired rate. A peptide is mentioned as a specific example of such a spacer. The peptide preferably has 2 to 10 amino acids, more preferably 2 to 4 amino acids. The amino acids constituting the peptide may be neutral amino acids, basic amino acids or acidic amino acids, and may be aliphatic amino acids or aromatic amino acids. It is also preferred that the peptide contains at least one heterologous amino acid.

The sequence of the peptide is not particularly limited in the present invention, and examples thereof include the following sequences: -Gly-Gly-Gly-DA.Tyr--Gly-Phe-Gly-Gly-DA. Tyr- -Phe-Phe-Gly-DA * Tyr- (DA * Tyr represents deamino tyrosine).

The pharmaceutical compound which can be carried by the compound according to the present invention includes methotrexate (MT
X), doxorubicin (DXR), mitomycin C
Examples thereof include, but are not limited to, antitumor agents such as (MMC) and anti-inflammatory agents such as dexamethasone and indomethacin.

Production of Compounds < Compounds of General Formula (I)> The compounds of the general formula (I) according to the present invention can be prepared, for example, by the formula (V): E ^* -R ¹ (V) (wherein E ^* is the above-mentioned The functional group of E is protected by a corresponding protecting group, and R ¹ is a bromine atom, a chlorine atom, an alkylthio group, an acyloxy group, —O—C (═NH) —CCl.
₃ ) and a compound of formula (VI): HO-T ¹ -R ² (VI) (wherein T ¹ has the same meaning as described above, R ² is a bromine atom,
A chlorine atom, a toluenesulfonyloxy group, or a methanesulfonyloxy group) is reacted with a compound under a reaction condition to form a glycosidic bond, the resulting compound is deprotected, and then a solvent not involved in the reaction (for example, hydrogen carbonate). Aqueous solution of sodium and polysaccharide with amino group at room temperature to 7
It can be obtained by reacting at a temperature of 0 ° C. for 12 to 180 hours.

Further, the compound of the general formula (I) according to the present invention includes a compound of the above formula (V) and a compound of the formula (VII): HO-T ¹ -R ³ (VII) (wherein T ¹ is as described above). Is synonymous, R ³ is -N ₃ ,-
NHW ¹ (W ¹ represents an amino-protecting group) is reacted with a compound under a reaction condition to form a glycosidic bond, the resulting compound is deprotected, and then in a solvent that does not participate in the reaction, It can be obtained by reacting a polysaccharide having a carboxyl group in the presence of a condensing agent at a temperature of 0 to 60 ° C. for 2 to 180 hours.

Further, the compound of the general formula (I) according to the present invention includes a compound of the above formula (V) and a compound of the formula (VIII): HO-T ¹ -R ⁴ (VIII) (wherein T ¹ is as described above). Are synonymous, and R ⁴ is -COOW
² (wherein W ² represents a protecting group for a carboxyl group) is reacted under reaction conditions to form a glycosidic bond, the resulting compound is deprotected, and then the carboxyl group is converted to an active ester. It can be obtained by reacting a polysaccharide having an amino group with a polysaccharide having an amino group at a temperature of 0 to 40 ° C. for 2 to 64 hours in a solvent that does not participate in the reaction (for example, an aqueous sodium hydrogen carbonate solution).

Further, the compound of the general formula (I) according to the present invention, in which T ¹ represents the group (II), is represented by the following formula (I
X) compounds:

[Chemical 5] (Wherein E and X have the same meanings as described above) and a polysaccharide having an amino group in a solvent that does not participate in the reaction (for example, an aqueous solution of acetic acid), sodium cyanoborohydride (NaBH ₃ C
It can be obtained by reacting in the presence of N) at a temperature of room temperature to 80 ° C.

The "reaction for forming a glycosidic bond" is
For example, the following (a) to (d) can be carried out: (a) a halogenated sugar in which the anomeric hydroxyl group of the sugar is substituted with a halogen and an aliphatic alcohol are mixed with a solvent ( For example, activator (silver silicate, silver carbonate, silver perchlorate, silver salt such as silver trifluoromethanesulfonate, mercury salt such as mercury oxide, tin salt) in dichloroethane, methylene chloride, benzene, toluene) In the presence of (a brominated sugar is obtained by treating a sugar with an acetylated hydroxyl group with hydrogen bromide / acetic acid, and a fluorinated sugar is a sugar with an unprotected hydroxyl group at the anomeric position as diethylaminosulfur trifluoro). (B) It is possible to obtain a sugar having a hydroxyl group acylated and a fatty acid alcohol in a solvent that does not participate in the reaction (for example, methylene chloride or dichloromethane). Among ethane), an acid catalyst (e.g., boron trifluoride-diethyl ether complex (BF ₃
Et ₂ O), trimethylsilyl trifluoromethane sulfonate (TMSOTf), pyridinium paratoluene sulfonic acid (PPTS), etc.), and reacting (c) a sugar in which the hydroxyl group at the anomeric position of the sugar is unprotected, 1,
After treatment with 8-diazabicyclo (5,4,0) -7-undecene (DBU), a base such as potassium carbonate, and trichloroacetonitrile to give an imidate, an acid catalyst (for example, BF ₃ .Et ₂ O, TMSOTf, PPT
S) and the like, and reacting with an aliphatic alcohol under the same conditions as in the above (b), (d) a sugar having a hydroxyl group converted to an alkylthio group and an aliphatic alcohol, an activator (for example, N -Reacting in the presence of iodosuccinimide (NIS) / trifluoromethanesulfonic acid (TfOH) etc.).

Carboxymethyl chitosan, which is a polysaccharide having a carboxyl group, can be obtained by reducing the molecular weight of chitosan with lysozyme, reducing it with sodium borohydride, and performing a partial deacetylation reaction.

Carboxymethyl pullulan is prepared by adding pullulan and chloroacetic acid in an aqueous sodium hydroxide solution at 0 ° C.
Can be obtained by reacting at room temperature for 1 to 24 hours. Carboxymethyl dextran and carboxymethyl mannoglucan can be prepared in the same manner.

The amino group-containing polysaccharide may be obtained by converting a carboxyl group-containing polysaccharide. For example, a polysaccharide having a carboxyl group and H ₂ N-Z-NHW
¹ (W ¹ has the same meaning as described above, and Z is-(CH ₂ ) p-
(P = 1 to 8) or _{-CH 2 - (OCH 2 CH 2} ) q
A polysaccharide having an amino group can be obtained by reacting a compound represented by — (q = 1 to 6)) in the presence of a condensing agent at a temperature of 0 to 60 ° C. for 2 to 180 hours.

The polyalcoholated derivative obtained by polyalcoholizing the polysaccharide can be obtained by oxidizing the polysaccharide with periodic acid to cleave the diol and then treating with a reducing agent such as sodium borohydride.

<Introduction of Pharmaceutical Compound> The polysaccharide derivative according to the present invention also includes those carrying a pharmaceutical compound.
The polysaccharide having an amino group into which the drug is introduced is (1) converted into an active ester from the carboxyl group of the drug having a carboxyl group, and then in a solvent that does not participate in the reaction with the polysaccharide having an amino group (for example, an aqueous solution of sodium hydrogen carbonate). (2) reacting a drug having a leaving group or a drug converted to have a leaving group with a polysaccharide having an amino group in a solvent that does not participate in the reaction (for example, an aqueous solution of sodium hydrogencarbonate). Or (3) the presence of sodium cyanoborohydride in a solvent that does not participate in the reaction between the drug having a formyl group or the drug converted to have a formyl group and the polysaccharide having an amino group (eg, acetic acid aqueous solution). It can be obtained by reacting under room temperature to 80 ° C.

The polysaccharide having a carboxyl group into which a drug is introduced can be obtained by reacting a polysaccharide having a carboxyl group with a drug having an amino group in the presence of a condensing agent.

The introduction of the pharmaceutical compound can be carried out not only by introducing it into the polysaccharide as described above prior to the synthesis of the general formula (I) but also by introducing it directly into the compound of the above formula (I).

The pharmaceutical compound can be introduced via a spacer. The polysaccharide derivative carrying a pharmaceutical compound via a spacer may be synthesized as described above using either a polysaccharide to which a spacer is bound or a pharmaceutical compound to which a spacer is bound.

[0043]

The present invention will be explained in more detail by the following examples, but the present invention is not limited to these examples. The compound numbers in the examples are the numbers shown in the schemes showing the synthetic processes described below.

Example 1 (1) Synthesis of Compound 1-1 It was synthesized according to the method described in Carbohydrate Research, 212 , 277-281 (1991).

(2) Synthesis of Compound 1-2 To a solution of hexaethylene glycol (23.8 g) in methylene chloride (200 ml) was added triethylamine (14.1 ml) and methanesulfonyl chloride (6.52 ml) at 0 ° C. In addition, the mixture was stirred at the same temperature for 1 hour. The reaction solution was diluted with methylene chloride, washed with 2% citric acid and saturated aqueous sodium hydrogen carbonate solution, and then dried, and the solvent was evaporated under reduced pressure (23.7 g). Subsequently, the residue was dissolved in methyl ethyl ketone (300 ml), lithium bromide (36.6 g) was added, and the mixture was stirred with heating under reflux for 1 hr. The reaction solution was cooled to room temperature, the precipitate was filtered off, the filtrate was added to saturated brine, and the mixture was extracted with ethyl acetate and ethylene chloride. After drying the extract, the solvent was distilled off, and the residue was then subjected to column chromatography using silica gel (500 g) (ethylene chloride-methanol).
The compound 1-2 (1
1.8 g) was obtained as a colorless oil.

¹ H-NMR (CDCl ₃ ) δ: 3.82
(2H, t, J = 6.3Hz), 3.75-3.70.
(2H, m), 3.70-3.64 (8H, m), 3.
63-3.60 (2H, m), 3.48 (2H, t, J
= 6.3 Hz). IR (CHCl ₃ ): 3500c
m ^-1 .

(3) Synthesis of Compound 1-3 and Compound 1-4 Compound 1-1 (761 m) was added to a solution of molecular sieves 3A (1.0 g) in acetonitrile (12 ml).
g) and compound 1-2 (345 mg) were added, and 2 was added at room temperature.
After stirring for an hour, N-iodosuccinimide (509 mg) and trifluoromethanesulfonic acid (21 μl) were added at −40 ° C., and the mixture was stirred at the same temperature for 2 hours. After filtering the reaction solution, the filtrate was washed with an aqueous solution of sodium thiosulfate and an aqueous solution of saturated sodium hydrogen carbonate, and then dried to remove the solvent. By purifying the obtained residue by silica gel column chromatography (150 g, toluene: acetone: methanol 500: 200: 7),
Compound 1-3 (368 mg) and compound 1-4 (357 m
g) was obtained.

Compound 1-3; colorless resin [α] _D ²⁵ -11.7 ° (c1.15, CHCl ₃ ) IR (CHCl ₃ ): 1745, 1690 cm ^{−1 1} H-NMR (CDCl ₃ ) δ : 5.38 (1H, dd
d, J = 8.5, 5.4, 2, 7 Hz), 5.32 (1
H, dd, J = 8.5, 2.0 Hz), 5.10 (1
H, d, J = 8.5 Hz), 4.66 (1H, ddd,
J = 12.7, 9.7, 4.6 Hz), 4.30 (1
H, dd, J = 12.5, 2.7 Hz), 4.09 (1
H, dd, J = 12.5, 5.6 Hz), 4.08-
4.02 (2H, m), 3.90 (1H, ddd, J =
11.0, 5.4, 3.7 Hz, 3.81 (2H,
t, J = 6.3 Hz), 3.80 (3H, s), 3.7
0-3.59 (18H, m), 3.48 (2H, t, J
= 6.3 Hz), 3.46 (1 H, m), 2.61 (1
H, dd, J = 12.7, 4.6 Hz), 2.14
2.14, 2.04, 2.03, 1.88 (each
3H, s), 1.98 (1H, dd, J = 12.9, 1
2.4 Hz).

Compound 1-4; colorless oil [α] _D ^20.5 + 3.0 ° (c1.02, CHCl ₃ ) IR (CHCl ₃ ): 1745, 1680 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 6 .30 (1H, d,
J = 10.3 Hz), 5.39 (1H, dd, J = 3.
2,2.4 Hz), 5.31 (1H, ddd, J = 8.
3, 3.2, 2.4 Hz), 5.23 (1H, m),
4.89 (1H, dd, J = 12.2, 2.4Hz),
4.61 (1H, dd, J = 10.7, 2.4Hz),
4.13 (1H, qlike), 4.12 (1H, d
d, J = 12.2, 8.3 Hz), 3.89 (1H, d
dd, J = 10.3, 5.6, 4.4 Hz), 3.85
-3.62 (18H, m), 3.81 (2H, t, J =
6.3 Hz), 3.80 (3H, s), 3.52 (1
H, m), 3.48 (2H, t, J = 6.3 Hz),
2.44 (1H, dd, J = 12.7, 4.4Hz),
2.15, 2.05, 2.03, 2.00, 1.88
(Each 3H, s), 1.90-1.84 (1H,
m).

(4) Synthesis of Compound 1-5 A solution of Compound 1-3 (195 mg) in methanol (2.0 ml) was dissolved in 28% sodium methoxide-
A methanol solution (200 μl) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was added to a cation exchange resin (Dowex 5
After being neutralized with ⁰ wH ⁺ , the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. Next, 1,4-dioxane (2.5 ml) and 0.1N sodium hydroxide aqueous solution (2.5 ml) were added to the obtained residue, and the mixture was stirred at room temperature for 10 minutes. Then, the compound was neutralized with a cation exchange resin (Dowex 50wH ⁺ ), the insoluble matter was filtered off, the filtrate was concentrated under reduced pressure, and purified by column chromatography (methanol) using a polymer gel (90 cc). 1-5
(139 mg) was obtained as a colorless powder.

[Α] _D ²⁷ -3.3 ° (c1.11, Me
OH) IR (KBr): 3375, 1616 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 3.92 (1H,
m), 3.86-3.81 (2H, m), 3.80 (2
H, t, J = 6.3 Hz), 3.78-3.55 (24
H, m), 3.51 (2H, t, J = 6.1 Hz),
2.73 (1H, dd, J = 12.4, 4.4Hz),
2.00 (3H, s), 1.74 (1H, dd, J = 1)
2.4, 11.7 Hz).

(5) Preparation of carboxymethyl chitosan As described in Japanese Patent Application No. 2-215803, commercially available carboxymethyl-chitin is partially hydrolyzed with lysozyme, and the non-reducing end is reduced with NaBH ₄ .
Carboxymethyl chitosan was obtained by partial deacetylation with aqueous sodium hydroxide.

(6) Synthesis of sialic acid-modified carboxymethyl chitosan (compound 1) Carboxymethyl chitosan (100 mg) and compound 1-
5 (382 mg) was dissolved in a 0.5% aqueous sodium hydrogen carbonate solution (6 ml), and then sodium hydrogen carbonate (51
mg) was added and the mixture was stirred at 60 ° C. for 160 hours. The reaction solution was added to 99.5% ethanol (35 ml) to precipitate a crude product, and the precipitate was mixed with 95% ethanol (40 m).
It was washed with 1 × 3 times), acetone (40 ml) and diethyl ether (40 ml) in this order, and then dried under reduced pressure (95 mg). Then, the crude product was dissolved in 10 ml of water, and then purified water (100 ml)
(00 ml) was used as an external solution and dialyzed for 12 hours at room temperature. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate a target substance, and the precipitate was washed with 95% ethanol (40 ml), acetone (40 ml) and diethyl ether (40 ml) in this order. Then, it is dried under reduced pressure to modify sialic acid-modified carboxymethyl chitosan (Compound 1, 47 mg, ds: 0.14, Neu content:
14%) was obtained (ds was calculated by the resorcinol hydrochloric acid method).

Example 2 (1) Synthesis of Compound 2-1 Compound 1-2 (1.38 g) and β-D-glucose pentaacetate (2.34 g) were dissolved in a methylene chloride solution (40 ml) at 0 ° C. Then, a boron trifluoride diethyl ether complex (1.48 ml) was added, and the mixture was stirred at the same temperature for 12 hours. The reaction mixture was diluted with methylene chloride, washed with water and saturated aqueous sodium hydrogen carbonate solution, and then dried, and the solvent was evaporated under reduced pressure. The resulting residue is treated with silica gel (1
Compound 2-1 (2.30 g) was obtained as a colorless oil by purification by column chromatography (ethylene chloride-methanol 50: 1) using 50 g).

[Α] _D ²⁵ -12.5 ° (c1.21, C
HCl ₃ ) IR (CHCl ₃ ): 1755 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 5.20 (1 H, d
d, J = 9.8, 9.5 Hz), 5.08 (1H, d
d, J = 9.8, 9.8 Hz), 4.92 (1H, d
d, J = 9.5, 7.8 Hz), 4.61 (1H, d,
J = 7.8 Hz), 4.26 (1H, dd, J = 12.
2, 4.6 Hz), 4.14 (1H, dd, J = 12.
2,2.4 Hz), 3.94 (1H, dt, J = 11.
5,4.4 Hz), 3.81 (2H, t, J = 6.3H)
z), 3.77-3.60 (21H, m), 3.48.
(2H, t, J = 6.3Hz), 2.09, 2.05
2.02, 2.01 (each 3H, s).

(2) Synthesis of Compound 2-2 Compound 2-1 (1.08 g) dissolved in methanol (1
5%) solution, 28% sodium methoxide-methanol solution (200 μl) was added, and the mixture was stirred at room temperature for 30 minutes. Cation exchange resin (Dowex 50wH)
⁺ ), The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was then washed with silica gel (150
The compound 2-2 (755 mg) was obtained as a colorless oil by purifying by column chromatography using (g) (ethylene chloride-methanol 6: 1).

[Α] _D ²⁶ -11.6 ° (c1.26, M
OH) IR (CHCl ₃ ): 3450 cm ^{−1 1} H-NMR (C ₅ D ₅ N + D ₂ O) δ: 4.85 (1
H, d, J = 7.5 Hz, 4.34 (1H, dd, J
= 12.0, 2.5 Hz), 4.25 (1H, dd, J
= 12.0, 5.5 Hz), 4.25 (1H, dt, J
= 11.0, 5.0 Hz), 4.22 (1H, dd, J
= 9.0, 9.0 Hz), 4.20 (1H, dd, J =
9.0, 9.0 Hz), 4.01 (1H, dd, J =
9.0, 7.5 Hz), 3.92 (1H, ddd, J =
9.0, 5.5, 2.5 Hz), 3.90 (1H, d
t, J = 11.0, 5.0 Hz), 3.77 (2H,
t, J = 6.0 Hz), 3.72 (2H, dd, J =
5.0, 5.0 Hz), 3.67-3.59 (16H,
m), 3.55 (2H, t, J = 6.0Hz).

(3) Synthesis of glucose-modified carboxymethyl chitosan (compound 2) Carboxymethyl chitosan (100 mg) and compound 2
(304 mg) was dissolved in a 0.5% aqueous sodium hydrogen carbonate solution (6 ml), and the mixture was stirred at 60 ° C. for 64 hours. The reaction solution was added to 99.5% ethanol (35 ml) to precipitate a crude product, and the precipitate was mixed with 95% ethanol (4 ml).
It was washed with 0 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml) in this order, and then dried under reduced pressure (112 mg).

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000) was used using a dialysis membrane (Spectra / Pore Co .: molecular weight exclusion limit 12,000 to 14000).
0 ml) was used as an external solution and dialyzed at room temperature for 12 hours. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate a target substance, and the precipitate was washed with 95% ethanol (40 ml), acetone (40 ml) and diethyl ether (40 ml) in this order. Then, it is dried under reduced pressure to modify glucose-modified carboxymethyl chitosan (compound 2, 90 mg, ds: 0.20, Glc content:
11%) was obtained (ds was calculated by the phenol-sulfuric acid method).

Example 3 (1) Synthesis of Compound 3-1 Compound 1-2 (2.07 g) and α-D-mannose pentaacetate (3.51 g) were dissolved in a methylene chloride solution (60 ml) at 0 ° C. Then, a boron trifluoride diethyl ether complex (2.94 ml) was added, and the mixture was stirred at room temperature for 48 hours. The reaction solution was diluted with methylene chloride, washed with water, dried and the solvent was distilled off under reduced pressure. Then, the obtained residue was purified by column chromatography using silica gel (330 g) (ethylene chloride-methanol 100: 1) to obtain Compound 3-1 (2.54 g) as a colorless oil.

[Α] _D ²⁷ + 26.9 ° (c1.09, C
HCl ₃ ) IR (CHCl ₃ ): 1747 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 5.36 (1H, d
d, J = 10.0, 3.4 Hz), 5.29 (1H, d
d, J = 10.0, 10.0 Hz), 5.27 (1H,
dd, J = 3.4, 1.7 Hz), 4.61 (1H,
d, J = 1.7 Hz), 4.30 (1H, dd, J = 1)
2.0, 4.6 Hz), 4.14 (1H, dd, J = 1)
2.0, 2.2 Hz), 3.81 (2H, t, J = 6.
3 Hz), 3.71-3.64 (20 H, m), 3.4
8 (2H, t, J = 6.3 Hz), 2.16, 2.1
1, 2.04, 1.99 (each 3H, s).

(2) Synthesis of Compound 3-2 Compound 3-1 (2.07 g) dissolved in methanol (2
0%) solution, 28% sodium methoxide-methanol solution (150 μl) was added, and the mixture was stirred at room temperature for 20 minutes. Cation exchange resin (Dowex 50wH)
⁺ ), The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. Subsequently, the resulting residue was treated with silica gel (150
The compound 3-2 (755 mg) was obtained as a colorless oil by purifying by column chromatography (ethylene chloride-methanol 6: 1) using g).

[Α] _D ²⁶ + 30.8 ° (c1.00, M
OH) IR (CHCl ₃ ): 3450 cm ^{−1 1} H-NMR (C ₅ D ₅ N + D ₂ O) δ: 5.38 (1
H, br. s), 4.63 (1H, dd, J = 9.3,
9.3 Hz), 4.56-4.52 (2H, m), 4.
52 (1H, dd, J = 11.5, 2.2Hz), 4.
37 (1H, dd, J = 11.5, 5.9Hz), 4.
32 (1H, ddd, J = 9.3, 5.9, 2.2H
z), 4.07 (1H, ddd, J = 10.7, 5.
1, 3.7 Hz), 3.80 (2H, t, J = 6.1H
z), 3.76 (1H, ddd, J = 10.7, 5.
9, 4.2 Hz), 3.72-3.60 (18H,
m), 3.58 (2H, t, J = 6.1Hz).

(3) Synthesis of Mannose-Modified Carboxymethyl Chitosan (Compound 3) Mannose-modified carboxymethyl chitosan (Compound 3, compound 3) was prepared by the same method as described in Example 2 (3).
(ds: 0.20, Man content: 11%) was obtained (ds was calculated by the phenol-sulfuric acid method).

Example 4 (1) Synthesis of Compound 4-1 Compound 1-2 (345 mg) and α-D-galactose pentaacetate (586 mg) were dissolved in a methylene chloride solution (10 ml) and trifluorinated at 0 ° C. Boron diethyl ether complex (369 μl) was added, and the mixture was stirred at room temperature for 14 hours. The reaction solution was diluted with methylene chloride, washed with water, dried and the solvent was distilled off under reduced pressure. Then, the obtained residue was purified by column chromatography using silica gel (200 g) (methylene chloride-methanol 20: 1) to obtain Compound 4-1 (473 mg) as a colorless oil.

[Α] _D ²¹ -4.8 ° (c1.03, CH
Cl ₃ ) IR (CHCl ₃ ): 1749, 1712 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 5.39 (1 H, d,
J = 3.4 Hz), 5.21 (1H, dd, J = 10.
5,8.1 Hz), 5.02 (1H, dd, J = 10.
5,3.4 Hz), 4.57 (1H, d, J = 8.1H
z), 4.17 (1H, dd, J = 11.2, 6.6H)
z), 4.13 (1H, dd, J = 12.2, 6.8H)
z), 3.96 (1H, ddd, J = 11.0, 9.
8, 4.2 Hz), 3.91 (1H, dd, J = 6.
8, 6.6 Hz), 3.81 (2H, t, J = 6.3H)
z), 3.75 (1H, ddd, J = 11.0, 7.
1,4.2 Hz), 3.70-3.62 (18H,
m), 3.48 (2H, t, J = 6.3Hz), 2.1
5, 2.06, 2.05, 1.99 (each 3H,
s).

(2) Synthesis of Compound 4-2 Compound 4-1 (1.73 g) dissolved in methanol (2
0%) solution, 28% sodium methoxide-methanol solution (150 μl) was added, and the mixture was stirred at room temperature for 20 minutes. Cation exchange resin (Dowex 50wH)
⁺ ), The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The residue obtained subsequently is silica gel (70 g).
Compound 4-2 was purified by column chromatography (ethylene chloride-methanol 6: 1) using
(1.16 g) was obtained as a colorless oil.

[0068] ^{_{[α] D 23.5 -3.8 ° (}} c1.01, M
OH) IR (CHCl ₃ ): 3450 cm ^{−1 1} H-NMR (C ₅ D ₅ N + D ₂ O) δ: 4.76 (1
H, d, J = 7.8 Hz), 4.49 (1H, d, J =
3.2 Hz), 4.39 (1H, dd, J = 9.5,
7.8 Hz), 4.38 (1H, dd, J = 11.2,
6.6 Hz), 4.35 (1H, dd, J = 11.5,
6.1 Hz), 4.25 (1 H, m), 4.13 (1
H, dd, J = 9.5, 3.2 Hz, 4.02 (1
H, dd, J = 6.6, 6.1 Hz), 3.93 (1
H, ddd, J = 10.7, 6.1, 4.4 Hz),
3.82 (2H, t, J = 5.9Hz), 3.74 (1
H, m), 3.71-3.62 (17H, m), 3.5
9 (2H, t, J = 5.9Hz).

(3) Synthesis of galactose-modified carboxymethyl chitosan (compound 4) Galactose-modified carboxymethyl chitosan (compound 4, ds: 0.23, Gal content) was prepared by the same method as described in Example 2 (3). : 13%) was obtained (d
s was calculated by the phenol-sulfuric acid method).

Example 5 (1) Synthesis of compound 5-2 2,3,4-tri-O-benzyl-1-O-paranitrobenzyl-fucopyranose (compound 5-1, α: β = 3)
6:64, 875 mg), zinc trifluoromethanesulfonate (545 mg) and compound 1-2 (345 m)
g) was dissolved in acetonitrile (20 ml) and chlorotrimethylsilane (190 μl) was added at 0 ° C. After stirring at the same temperature for 2 hours, the reaction solution was diluted with methylene chloride, washed with water and a saturated sodium hydrogen carbonate aqueous solution, and then dried, and the solvent was distilled off under reduced pressure. The residue was treated with silica gel (7
Column chromatography (toluene-
Compound 5 was obtained by purification with acetone 7: 1).
-2 (444 mg) as α-glycoside: β-glycoside = 7
Obtained as a 1:29 mixture (from ¹ H NMR integration ratio).

¹ H-NMR (CDCl ₃ ) δ: α-glycoside-derived −7.41−7.26 (15H, m), 4.9
8, 4.74 (each 1H, d, J = 11.5H
z), 4.87, 4.69 (each 1H, d, J =
12.2 Hz), 4.79, 4.65 (each 1
H, d, J = 12.0Hz, 4.86 (1H, d, J
= 3.7 Hz), 4.03 (1H, dd, J = 10.
3, 3.7 Hz), 3.94 (1H, dd, J = 10.
3,2.9 Hz), 3.93 (1H, q, J = 6.6H
z), 3.80 (2H, t, J = 6.3Hz), 3.7.
5-3.57 (21H, m), 3.46 (2H, t, J
= 6.3 Hz), 1.10 (3H, d, J = 6.6H)
z). Derived from β-glycoside-4.75 (1H, d, J =
7.5 Hz), 1.17 (3H, d, J = 6.3H)
z).

(2) Synthesis of compound 5 A solution of compound 5-2 (3.00 g) in tetrahydrofuran (120 ml) was added with palladium-carbon (10
%, 1.50 g), and under a medium pressure hydrogen stream (50 ps)
i), and stirred at room temperature for 12 hours. After the catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was treated with silica gel (600
The compound 5 (1.10 g) was obtained as a colorless oily substance by purification by column chromatography (ethylene chloride-methanol 15: 1) using g).

[Α] _D ²⁵ -65.6 ° (c1.35, M
OH) IR (CHCl ₃ ): 3570,3500 cm ^{−1 1} H-NMR (CD ₃ OD) δ: 4.78 (1 H, d,
J = 3.7 Hz), 4.01 (1H, q, J = 6.6H
z), 3.80 (2H, t, J = 6.1Hz), 3.8.
0 (1H, m), 3.74 (1H, dd, J = 10.
0, 3.2 Hz), 3.70 (1H, dd, J = 10.
0, 3.7 Hz), 3.70-3.59 (20H,
m), 3.51 (2H, t, J = 6.1Hz), 3.2
6 (1H, m), 1.20 (3H, d, J = 6.6H
z).

(3) Synthesis of fucose-modified carboxymethyl chitosan (Compound 5) By the same method as described in Example 2 (3), fucose-modified carboxymethyl chitosan (Compound 5, d).
(s: 0.20, Fuc content: 10.5%) was obtained (ds
Was calculated by the phenol-sulfuric acid method).

Example 6 (1) Synthesis of compound 6-1 β-D-galactosamine pentaacetate (2.20)
g) was dissolved in 1,2-ethylene chloride (30 ml), trimethylsilyltrifluoromethanesulfonate (1.19 ml) was added, and the mixture was stirred at 50 ° C for 1 hr.
Triethylamine (1.70 ml) was added at room temperature, the solvent was evaporated, and the residue was purified by column chromatography using silica gel (45 g) (ethylene chloride-methanol-triethylamine 40: 1: 4). Thus, compound 6-1 (1.90 g) was obtained as a crude product.

(2) Synthesis of Compound 6-2 Compound 1-2 (1.62 g) and Compound 6-1 (1.
90 g) was dissolved in a 1,2-ethylene chloride solution (12 ml) containing Molecular Sieves 4A (1.2 g), trimethylsilyltrifluoromethanesulfonate (870 μl) was added, and the mixture was stirred at 50 ° C. for 1 hr. Triethylamine (1.40 ml) was added at room temperature, the reaction solution was filtered, the filtrate was diluted with methylene chloride, washed with water, dried and the solvent was distilled off. Subsequently, the residue was subjected to column chromatography using silica gel (150 g) (toluene-acetone-methanol 500: 300:
Compound 6-2 (2.95 by purifying in 8).
g) was obtained as a colorless oil.

[Α] _D ²⁸ -19.9 ° (c0.98, C
HCl ₃ ) IR (CHCl ₃ ): 1745, 1678 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 6.53 (1 H, d,
J = 9.0 Hz), 5.32 (1H, br.d), 4.
99 (1H, dd, J = 11.2, 3.4Hz), 4.
79 (1H, d, J = 8.5 Hz), 4.25 (1H,
ddd, J = 11.2, 9.0, 8.5 Hz), 4.1
8 (1H, dd, J = 11.2, 6.6Hz), 4.1
8 (1H, dd, J = 11.2, 6.8Hz), 3.9
0 (1H, dd, J = 6.8, 6.6Hz), 3.87
(1H, m) 3.81 (2H, t, J = 6.3H
z), 3.75-3.59 (21H, m), 3.47.
(2H, t, J = 6.3Hz), 2.16, 2.05
1.99, 1.98 (each3H, s).

(3) Synthesis of compound 6 Compound 6-2 (2.20 g) dissolved in methanol (2
8%) solution, 28% sodium methoxide-methanol solution (200 μl) was added, and the mixture was stirred at room temperature for 20 minutes. Cation exchange resin (Dowex 50wH)
⁺ ), The insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The residue obtained subsequently is silica gel (70 g).
Was purified by column chromatography (methylene chloride-methanol 6: 1) using
(1.58 g) was obtained as a colorless oil.

[Α] _D ²⁶ -3.7 ° (c1.04, Me
OH) IR (KBr): 3370,1661 cm ^{-1 1} H-NMR (C ₅ D ₅ N + D ₂ O) δ: 5.04 (1
H, d, J = 8.3 Hz, 4.83 (1H, dd, J
= 10.5, 8.3 Hz), 4.46 (1H, br.
d), 4.38 (1H, dd, J = 11.2, 6.6H)
z), 4.34 (1H, dd, J = 11.2, 5.8H
z), 4.30 (1H, dd, J = 10.5, 3.2H)
z), 4.16 (1H, dt, J = 11.2, 4.4H
z), 3.90 (1H, dd, J = 6.6, 5.8H
z), 3.93 (1H, dt, J = 11.2, 5.3H
z), 3.81 (2H, t, J = 6.1Hz), 3.7.
2-3.62 (18H, m), 3.59 (2H, t, J
= 6.1 Hz), 2.16 (3H, s).

(4) Synthesis of N-acetylgalactosamine-modified carboxymethylchitosan (compound 6) N-acetylgalactosamine-modified carboxymethylchitosan (compound 6) was obtained by the same method as described in Example 2 (3).

Example 7 (1) Synthesis of compound 7-1 β-D-glucosamine pentaacetate (14.0 g)
Was dissolved in 1,2-ethylene chloride (180 ml), trimethylsilyltrifluoromethanesulfonate (6.96 ml) was added, and the mixture was stirred at 55 ° C for 4 hr.
Triethylamine (10.1 ml) was added at room temperature, the solvent was evaporated, and the residue was silica gel (200 g).
Compound 7-1 was purified by column chromatography (ethylene chloride-methanol-triethylamine 200: 1: 1) using a crude product (11.8).
Obtained as g).

(2) Synthesis of compound 7-2 Compound 7-1 (1.85 g) and compound 1-2 (1.1.
56 g) was dissolved in a 1,2-ethylene chloride solution (12 ml) containing Molecular Sieves 4A (1.2 g), trimethylsilyltrifluoromethanesulfonate (835 μl) was added, and the mixture was stirred at 50 ° C. for 1 hr. Triethylamine (1.40 ml) was added at room temperature, the reaction solution was filtered, the filtrate was diluted with methylene chloride, washed with water, dried and the solvent was distilled off. Subsequently, the residue was subjected to column chromatography using silica gel (150 g) (toluene-acetone-methanol 500: 300:
The compound 7-2 (2.45 by purifying in 8).
g) was obtained as a colorless oil.

[Α] _D ²⁷ -14.7 ° (c1.18, C
HCl ₃ ) IR (CHCl ₃ ): 3622, 1747, 1678c
m ^{−1 1} H-NMR (CDCl ₃ ) δ: 6.61 (1H, d,
J = 9.3 Hz), 5.11-5.10 (2H, m),
4.79 (1H, d, J = 8.5Hz), 4.26 (1
H, dd, J = 12.2, 4.6 Hz), 4.17 (1
H, dd, J = 12.2, 2.4 Hz), 4.10 (1
H, m), 3.90 (1H, m), 3.86-3.76.
(2H, m) 3.81 (2H, t, J = 6.3H
z), 3.75-3.58 (18H, m), 3.48.
(2H, t, J = 6.3 Hz), 2.09, 1.97
(Each 3H, s), 2.01 (6H, s).

(3) Synthesis of compound 7-3 To a methanol solution (40 ml) in which compound 7-2 (13.0 g) was dissolved, 28% sodium methoxide-methanol solution (0.3 ml) was added, and the mixture was allowed to stand at room temperature. Stir for 80 minutes. The reaction solution was used as a cation exchange resin (Dowex 50w
After neutralizing with x8 (H ⁺ )), the insoluble matter was filtered off, the filtrate was concentrated under reduced pressure (10.2 g), and the obtained residue (10.2 g) was added to N, N′-dimethylformamide. Dissolve in (50 ml), add benzaldehyde dimethyl acetal (10.9 ml) and d-camphorsulfonic acid (125 mg), and reduce the pressure at 55 ° C. for 3 hours (45 mm).
Hg) Stir. The reaction solution was treated with an anion exchange resin (AG-1).
(OH ^-)) was neutralized with, the insoluble material removed by filtration, the filtrate was concentrated under reduced pressure, followed by column chromatography of the residue on silica gel (15 g) (toluene - acetone - methanol 200: 300: 10) Compound 7-3 (7.57 g) was obtained as a colorless amorphous substance by purification by.

[Α] _D ²⁸ -55.4 ° (c1.04, C
HCl ₃ ) IR (CHCl ₃ ): 3352, 1666 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.52-7.47
(2H, m), 7.38-7.32 (3H, m), 7.
15 (1H, br.d, J = 6.3 Hz), 5.57
(1H, s), 4.75 (1H, d, J = 8.1H
z), 4.33 (1H, dd, J = 10.5, 4.9H)
z), 3.94-3.77 (5H, m), 3.75 (2)
H, t, J = 6.2 Hz), 3.72-3.57 (17)
H, m), 3.45 (1H, m, H-5), 3.44
(2H, t, J = 6.2Hz), 2.07 (3H,
s).

(4) Synthesis of compound 7-4 It was synthesized according to the description of J. Carbohydrate Chemistry, 10 (4), 549-560 (1991).

(5) Synthesis of Compound 7-5 Compound 7-3 (636 mg) and Compound 7-4 (697 mg) were added to methylene chloride (20 ml) containing Molecular Sieves 4A (10 g), and the mixture was stirred at room temperature for 2 hours. After that, dimethyl (methylthio) sulfonium triflate (1.16 g) was added at 0 ° C., and the mixture was stirred at the same temperature for 30 minutes. Methanol (2 ml) and triethylamine (1 ml) were added to the reaction solution, the mixture was filtered, the filtrate was diluted with methylene chloride, the organic layer was washed with water, dried and the solvent was distilled off. Subsequently, the residue obtained was treated with silica gel (70
The compound 7-5 (896 mg) was obtained as a colorless oil by purification by column chromatography (methylene chloride-methanol 50: 1) using g). According to ¹ H-NMR observation, the obtained compound was contaminated with about 6% of a compound in which fucose was presumed to have a β-glycoside bond, but the compound proceeded to the next step without further purification. It is.

IR (CHCl ₃ ): 1677 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.45-7.42
(2H, m), 7.39-7.24 (18H, m),
6.05 (1H, d, J = 8.1Hz), 5.50 (1
H, s), 5.17 (1H, d, J = 3.7 Hz),
4.92 (1H, d, J = 8.3 Hz), 4.91,
4.79, 4.71, 4.57 (each 1H, d,
J = 11.7 Hz), 4.78, 4.70 (each)
1H, d, J = 11.5 Hz), 4.33 (1H, d
d, J = 10.5, 4.9 Hz), 4.22 (1H, d
d, J = 9.5, 9.5 Hz), 4.11 (1H, q,
J = 6.3 Hz), 4.04 (1H, dd, J = 10.
3, 3.7 Hz), 3.94 (1H, dd, J = 10.
3,2.7 Hz), 3.78 (2H, t, J = 6.3H
z), 3.80-3.72 (2H, m), 3.70-
3.58 (20H, m), 3.56 (1H, m), 3.
52-3.45 (2H, m), 3.45 (2H, t, J
= 6.3 Hz), 1.75 (3H, s), 0.82 (3
H, d, J = 6.3 Hz).

(6) Synthesis of Compound 7-6 Compound 7-5 (5.20) was added to a tetrahydrofuran solution (60 ml) containing Molecular Sieves 3A (20 g).
g) was dissolved and stirred at room temperature for 2 hours, and sodium cyanoborohydride (4.66 g) was slowly added. After the sodium cyanoborohydride was completely dissolved, a hydrogen chloride-ether solution was added dropwise until gas generation stopped, and the mixture was stirred for 15 minutes. The reaction solution was filtered, the filtrate was diluted with methylene chloride, washed with 2N hydrochloric acid and 2N aqueous sodium hydroxide solution, and then dried, and the solvent was evaporated. The residue was purified by column chromatography (methanol) using polymer gel (900 cc) and column chromatography (methylene chloride-methanol 50: 1) using silica gel (600 g) to give compound 7-6 (4.02 g). ) Was obtained as a colorless amorphous.

[Α] _D ²⁸ -50.8 ° (c0.51, C
HCl ₃ ) IR (CHCl ₃ ): 3631, 3450, 1674c
m ^{−1 1} H-NMR (CDCl ₃ + D ₂ O) δ: 7.41−
7.25 (20H, m), 6.14 (1H, d, J =
7.8 Hz), 4.97 (1H, d, J = 3.7H)
z), 4.95, 4.61 (each 1H, d, J =
11.2Hz), 4.84 (1H, d, J = 8.3H)
z), 4.81, 4.79, 4.75, 4.67 (ea)
ch 1H, d, J = 11.7 Hz), 4.62, 4.
58 (each 1H, d, J = 12.2 Hz), 4.
13 (1H, q, J = 6.3 Hz), 4.06 (1H,
dd, J = 10.3, 3.7 Hz), 3.97 (1H,
m), 3.95 (1H, m), 3.84-1.57 (2
3H, m), 3.78 (2H, t, J = 6.3Hz),
3.52-3.41 (3H, m), 3.45 (2H, m
t, J = 6.3 Hz), 1.66 (3H, s), 1.1
4 (3H, J = 6.3 Hz).

(7) Synthesis of Compound 7-7 It was synthesized as described in J. Carbohydrate Chemistry, 8 (2) 265-283 (1989).

(8) Synthesis of compound 7-8 Compound 7-7 (400 mg) and compound 7-6 (1.07 g) were dissolved in a methylene chloride solution (6 ml) containing molecular sieves 4A (2 g), and the mixture was allowed to stand at room temperature. After stirring for 2 hours at 0 ° C., boron trifluoride diethyl ether complex (100 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 2 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography using silica gel (120 g) (ethylene chloride-methanol 30: 1) to obtain compound 7-8 (460 mg) as a colorless powder.

[Α] _D ²⁸ -34.0 ° (c0.63, C
HCl ₃ ) IR (CHCl ₃ ): 1744, 1688 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.97 (2H,
m), 7.50 (1H, m), 7.37 (2H, m),
7.36-7.22 (20H, m), 6.23 (1H,
m), 5.69 (1H, ddd, J = 9.3, 5.6,
2.7 Hz), 5.37 (1H, dd, J = 9.3,
2.7 Hz), 5.21 (1H, d, J = 3.7H)
z), 5.06 (1H, br.d), 5.04 (1H, br.d)
br. d), 4.97 (1H, dd, J = 10.0,
8.1 Hz), 4.94, 4.63 (each 1H,
d, J = 11.7 Hz), 4.90 (1H, ddd, J
= 12.0, 10.3, 4.7 Hz), 4.81 (1
H, d, J = 7.8 Hz), 4.81, 4.70 (ea
ch 1H, d, J = 12.9 Hz), 4.77, 4.
75, 4.57, 4.44 (each 1H, d, J =
12.2Hz), 4.74 (1H, d, J = 7.3H)
z), 4.67 (1H, dd, J = 10.0, 3.7H)
z), 4.30 (1H, dd, J = 12.4, 2.7H)
z), 4.24 (1H, dd, J = 11.0, 7.1H)
z), 4.20 (1H, m), 4.17 (1H, dd,
J = 11.0, 7.3 Hz), 4.10-4.05 (2
H, m), 4.04 (1H, m), 4.01 (1H,
m), 3.98 (1H, dd, J = 12.4, 5.6H
z), 3.94 (1H, dd, J = 7.3, 7.1H)
z), 3.91 (1H, dd, J = 10.0, 4.7H)
z), 3.87 (1H, dd, J = 10.3, 2.4H
z), 3.87-3.73 (5H, m), 3.79 (2)
H, t, J = 6.3 Hz), 3.75 (3H, s),
3.67-3.48 (21H, m), 3.46 (2H,
t, J = 6.3 Hz), 2.56 (1H, dd, J = 1)
2.4, 4.6 Hz), 2.22, 2.09, 2.0
7, 2.01, 1.97, 1.96, 1.89, 1.8
5 (each 3H, s), 1.71 (1H, dd, J
= 12.4, 12.0 Hz, 1.10 (3H, d, J
= 6.3 Hz).

(9) Synthesis of compound 7-9 Palladium-carbon (10%, 8%) was added to a solution of compound 7-8 (200 mg) in tetrahydrofuran (15 ml).
0 mg) was added, and the mixture was stirred under a hydrogen stream at room temperature for 24 hours. After the catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was purified by preparative thin layer chromatography (methylene chloride-methanol 8: 1) to give compound 7-9.
(118 mg) and compound 7-10 (25 mg) were obtained.

[0095] Compounds 7-9; colorless powder ^{_{[α] D 27 -53.1 ° (}} c0.66, CHCl 3) IR (KBr): 3450,1749,1665cm -1 1 H-NMR (CDCl 3 + D 2 O ) Δ: 8.04 (2
H, m), 7.59 (1H, m), 7.48 (2H,
m), 5.61 (1H, m), 5.33 (1H, dd,
J = 9.3, 2.7 Hz), 5.06 (1H, d, J =
2.7Hz), 5.05 (1H, d, J = 3.9H
z), 4.98 (1H, dd, J = 10.3, 8.1H)
z), 4.89 (1H, ddd, J = 12.0, 10.
7, 4.6 Hz), 4.75 (1H, d, J = 8.1H)
z), 4.66 (1H, d, J = 5.1Hz), 4.6
3 (1H, dd, J = 10.3, 3.4Hz), 4.4
3 (1H, dd, J = 12.4, 2.9 Hz), 4.4
2-4.38 (2H, m), 4.23 (1H, dd, J
= 11.0, 6.8 Hz), 4.15-3.90 (7
H, m), 3.85 (1H, m), 3.81 (2H,
t, J = 6.3 Hz), 3.77 (3H, s), 3.7
3-3.60 (25H, m), 3.48 (2H, t, J
= 6.3 Hz), 2.59 (1H, dd, J = 12.
7, 4.6 Hz), 2.24, 2.11, 2.06
2.04, 2.01, 1.85 (each 3H,
s), 2.14 (6H, s), 1.71 (1H, dd,
J = 12.7, 12.0 Hz), 1.27 (3H, d,
J = 6.6 Hz).

[0096] Compounds 7-10; colorless powder ^{_{[α] D 28 -56.5 ° (}} c0.34, CHCl 3) IR (KBr): 3452,1749,1663cm -1 1 H-NMR (CDCl 3 + D 2 O ) Δ: 8.04 (2
H, m), 7.59 (1H, m), 7.48 (2H,
m), 5.61 (1H, m), 5.33 (1H, dd,
J = 9.1, 2.7 Hz), 5.07-5.04 (2
H, m), 4.98 (1H, dd, J = 10.3, 8.
3Hz), 4.89 (1H, m), 4.74 (1H,
d, J = 8.3 Hz), 4.66 (1H, d, J = 4.
9 Hz), 4.63 (1H, dd, J = 10.3, 3.
4Hz), 4.42 (1H, dd, J = 12.5, 2.
9Hz), 4.39 (1H, m), 4.39 (1H, d
d, J = 11.0, 6.8 Hz), 4.23 (1H, d
d, J = 11.2, 6.8 Hz), 4.16-3.89.
(7H, m), 3.87-3.76 (3H, m), 3.
78 (3H, s), 3.75-3.57 (23H,
m), 3.53 (2H, q, J = 7.1Hz), 2.5
9 (1H, dd, J = 12.7, 4.4Hz), 2.2
4,2.14,2.14,2.11,2.06,2.0
5, 2.01, 1.86 (each 3H, s), 1.
72 (1H, dd, J = 12.7, 12.0Hz),
1.26 (3H, d, J = 6.6Hz), 1.21 (3
H, t, J = 7.1 Hz).

(10) Synthesis of compound 7-11 Compound 7-10 (14 mg) dissolved in methanol (2
ml) solution, 3% sodium methoxide-methanol solution (400 μl) was added, and the mixture was stirred at room temperature for 30 minutes. Cation exchange resin (Dowex 50w)
After neutralizing with H ⁺ ), the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. Then, a 0.1 N sodium hydroxide aqueous solution (2 ml) was added to the obtained residue, and the mixture was stirred at room temperature for 10 minutes. Then cation exchange resin (Dowex 50w
H ⁺ ) to neutralize, insoluble matter is removed by filtration, the filtrate is concentrated under reduced pressure, and purified by column chromatography (methanol) using polymer gel (20 cc) to give compound 7-11 (9 mg). Was obtained as a colorless powder.

[Α] _D ²⁷ -39.6 ° (c0.33, M
OH) IR (KBr): 3400, 1650 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 5.03 (1 H, d,
J = 3.9 Hz), 4.86-4.79 (1H, m),
4.52-4.49 (2H, m), 4.03 (1H, d
d, J = 9.8, 2.9 Hz), 4.00 (1H, d
d, J = 12.2, 3.4 Hz), 3.95-3.49.
(38H, m), 3.53 (2H, q, J = 7.1H
z), 3.45-3.40 (2H, m), 2.81 (1
H, dd, J = 12.2, 4.2 Hz), 2.01,
1.97 (each 3H, s), 1.83 (1H, d
d, J = 12.2, 12.2 Hz), 1.19 (3H,
t, J = 7.1 Hz), 1.16 (3H, d, J = 6.
6 Hz).

(11) Synthesis of compound 7-12 To a solution of compound 7-9 (220 mg) in methanol (5.0 ml) was added 28% sodium methoxide-
A methanol solution (200 μl) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was added to a cation exchange resin (Dowex 5
After being neutralized with ⁰ wH ⁺ , the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. Then, 0.1N aqueous sodium hydroxide solution (3.0 ml) was added to the obtained residue, and the mixture was stirred at room temperature for 10 minutes.
Stir for minutes. Then cation exchange resin (Dowex 5
Compound (7-12) (150 mg) was obtained by neutralizing with ⁰ wH ⁺ ), filtering off insoluble matter, concentrating the filtrate under reduced pressure, and purifying by column chromatography (methanol) using a polymer gel (200 cc). Obtained as a colorless powder.

[Α] _D ²⁷ -39.4 ° (c0.66, M
OH) IR (KBr): 3460, 1653 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 5.03 (1 H, d,
J = 3.9 Hz), 4.85-4.83 (1H, m),
4.52 (1H, d, J = 7.8Hz), 4.50 (1
H, d, J = 7.6 Hz), 4.03 (1H, dd, J
= 9.5, 2.9 Hz), 4.00 (1H, dd, J =
12.2, 3.7 Hz, 3.95-3.83 (8H,
m), 3.81 (2H, t, J = 6.1Hz), 3.7
9-3.60 (28H, m), 3.56 (1H, dd,
J = 9.5, 7.8 Hz), 3.51 (2H, t, J =
6.1 Hz), 3.50 (1H, dd, J = 9.0,
1.2 Hz), 3.45-3.41 (2H, m), 2.
80 (1H, dd, J = 12.4, 4.4 Hz), 2.
00, 1.97 (each 3H, s), 1.85 (1
H, dd, J = 13.3, 11.5 Hz), 1.16
(3H, d, J = 6.6 Hz).

(12) Synthesis of Sialyl Lewis X Modified Carboxymethyl Chitosan (Compound 7) Carboxymethyl chitosan (40 mg) and Compound 1 (2)
76 mg, 0.24 mmol) were dissolved in 0.5% aqueous sodium hydrogen carbonate solution (3 ml), sodium hydrogen carbonate (20 mg) was added, and the mixture was stirred at 60 ° C. for 160 hr. The reaction solution was added to 99.5% ethanol (35 ml) to precipitate a crude target product, and the precipitate was added in order of 95% ethanol (40 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml). It was washed and then dried under reduced pressure (58 mg).

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000) was prepared using a dialysis membrane (Spectra / Pore Co., Ltd .: molecular weight exclusion limit 12000-14000).
0 ml) was used as an external solution and dialyzed at room temperature for 12 hours. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate the target substance, and the precipitate was washed with 95% ethanol (40 ml), acetone (40 ml) and diethyl ether (40 ml) in this order, Then, it is dried under reduced pressure to give sialyl Lewis X-modified carboxymethyl chitosan (compound 7, 47 mg, ds: 0.17, SLe
X content: 33%) was obtained (ds was determined by the resorcinol-hydrochloric acid method, which is a quantitative method for sialic acid).

Example 8 (1) Synthesis of Compound 8-1 A solution of compound 7-3 (240 mg) in N, N′-dimethylformamide (3 ml) was dissolved in barium oxide (1).
27 mg), barium hydroxide octahydrate (24 mg) and benzyl bromide (90 μl) were added, and the mixture was added at room temperature to 12
Stir for hours. 28% with methanol (3 ml) in the reaction mixture
Sodium methoxide-methanol solution (150 μl)
Was added, and the mixture was stirred at room temperature for 20 minutes, diluted with ethylene chloride, washed with saturated brine, dried, and the solvent was evaporated. Subsequently, the obtained residue was treated with silica gel (30 g).
Compound 8-1 (233 mg) was obtained as a colorless amorphous substance by purification by column chromatography using (Ethanol chloride-methanol 50: 1).

[Α] _D ²⁷ -13.4 ° (c1.03, C
HCl ₃ ) IR (CHCl ₃ ): 3460, 3340, 1674c
^{m -1 1 H-NMR (CDCl} 3) δ: 7.51-7.47
(2H, m), 7.42-7.22 (8H, m), 6.
36 (1H, d, J = 8.1Hz), 5.57 (1H,
s), 4.95 (1H, d, J = 8.1Hz), 4.9.
0,4.66 (each 1H, d, J = 12.0H
z), 4.35 (1H, dd, J = 10.5, 5.1H)
z), 4.05 (1H, dd, J = 9.8, 9.5H)
z), 3.90 (1H, m), 3.80 (1H, m),
3.77 (2H, t, J = 6.3Hz), 3.44 (2
H, t, J = 6.3 Hz), 3.73-3.46 (22)
H, m), 1.95 (3H, s).

(2) Synthesis of Compound 8-2 Compound 8-1 (500 mg) was dissolved in a tetrahydrofuran solution (8 ml) containing Molecular Sieves 3A (3 g), and the mixture was stirred at room temperature for 2 hours and then cyanoborohydride. Sodium (650 mg) was added slowly. After sodium cyanoborohydride was completely dissolved,
The hydrogen chloride-ether solution was added dropwise until gas evolution stopped and stirred for 10 minutes. The reaction solution was filtered, the filtrate was diluted with methylene chloride, washed with 2N hydrochloric acid and 2N aqueous sodium hydroxide solution, then dried and the solvent was distilled off. The residue was subjected to column chromatography (methanol) using silica gel (300 cc) and silica gel (1
Compound 8 was purified by column chromatography (methylene chloride-methanol 50: 1) using 50 g).
-2 (395 mg) was obtained as a colorless amorphous.

[Α] _D ²⁴ -14.8 ° (c1.11, C
HCl ₃ ) IR (CHCl ₃ ): 3460, 3350, 1674c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 7.38-7.24
(10H, m), 6.36 (1H, d, J = 7.3H
z, NH), 4.81 (1H, d, J = 8.3 Hz),
4.77, 4.71 (each 1H, d, J = 11.1.
5 Hz), 4.61, 4.56 (each 1H, d,
J = 12.0 Hz), 3.90 (1 H, m), 3.80
-3.72 (5H, m), 3.77 (2H, t, J =
6.3 Hz), 3.71-1.57 (19H, m),
3.51 (1H, m), 3.45 (2H, t, J = 6.
3 Hz), 2.76 (1H, br.s, OH), 1.9
6 (3H, s).

(3) Synthesis of compound 8-3 Compound 7-7 (200 mg) and compound 8-2 (370 mg) were dissolved in a methylene chloride solution (5 ml) containing molecular sieves 4A (1.5 g), and the mixture was allowed to stand at room temperature. After stirring for 2 hours at 0 ° C., boron trifluoride diethyl ether complex (50 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 3 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography (ethylene chloride-methanol 30: 1) using silica gel (20 g) to give compound 8-3 (272 mg) as a colorless powder.

[Α] _D ^23.5 −21.8 ° (c0.38,
CHCl ₃ ) IR (CHCl ₃ ): 3300,1751,1662c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 7.96 (2H,
m), 7.53 (1H, m), 7.38 (2H, m),
7.34-7.18 (10H, m), 6.30 (1H,
br. d, J = 8.0 Hz), 5.60 (1H, dd
d, J = 9.3, 6.1, 2.9 Hz, 5.36 (1
H, dd, J = 9.3, 2.7 Hz), 5.04 (1
H, dd, J = 10.3, 8.1 Hz), 5.04 (1
H, br. d), 5.03 (1H, br.d), 4.8.
8 (1H, ddd, J = 12.0, 10.5, 4.7H
z), 4.85 (1H, d, J = 7.8Hz), 4.7
6,4.67 (each 1H, d, J = 11.5H
z), 4.71 (1H, d, J = 6.1 Hz), 4.6
6 (1H, dd, J = 10.3, 3.7Hz), 4.5
9,4.51 (each 1H, d, J = 12.0H
z), 4.32 (1H, dd, J = 12.4, 2.4H
z), 4.16 (1H, dd, J = 11.0, 6.6H)
z), 4.11 (1H, dd, J = 11.0, 7.1H)
z), 4.05 (1H, qlike), 4.04 (1
H, tlike), 3.97 (1H, dd, J = 12.
2, 6.1 Hz), 3.95-3.88 (4H, m),
3.84 (1H, tlike), 3.79 (2H, t,
J = 6.3 Hz), 3.75 (3H, s), 3.70-
3.56 (22H, m), 3.45 (2H, t, J =
6.3 Hz), 2.58 (1H, dd, J = 12.7,
4.7 Hz), 2.22, 2.11, 2.01, 1.9
6, 1.96, 1.85 (each 3H, s), 2.
07 (6H, s), 1.72 (1H, dd, J = 12.
7, 12.0 Hz).

(4) Synthesis of compound 8-4 A solution of compound 2-3 (310 mg) in tetrahydrofuran (15 ml) was dissolved in palladium-carbon (10%, 1%).
00 mg) and 1N hydrochloric acid (100 μl) were added, and the mixture was stirred under a hydrogen stream at room temperature for 3 hours. The catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was purified by preparative thin layer chromatography (methylene chloride-methanol 20: 1) to give compound 8-4 (234 mg) as a colorless powder. Obtained.

[Α] _D ²⁵ -4.4 ° (c0.41, CH
_{Cl 3) IR (KBr):} 3500,1747,1664cm -1 1 H-NMR (CD 3 OD) δ: 8.07 (2H,
m), 7.61 (1H, m), 7.49 (2H, m),
5.64 (1H, ddd, J = 9.3, 6.3, 2.7
Hz), 5.34 (1H, dd, J = 9.3, 2.7H)
z), 5.13 (1H, J = 3.4 Hz), 4.99
(1H, dd, J = 10.0, 8.1Hz), 4.97
(1H, m), 4.86 (1H, d, J = 8.1H
z), 4.75 (1H, dd, J = 10.0, 3.4H)
z), 4.51 (1H, d, J = 8.5 Hz), 4.4
1 (1H, dd, J = 12.5, 2.7Hz), 4.3
7 (1H, dd, J = 11.0, 5.9Hz), 4.2
3 (1H, dd, J = 11.2, 7.3Hz), 4.1
8 (1H, dd, J = 7.3, 5.9 Hz), 3.99
(1H, dd, J = 12.5, 6.3Hz), 3.96
(1H, tick), 3.91 (1H, m), 3.8
8 (1H, m), 3.80 (3H, s), 3.80 (2
H, t, J = 6.1 Hz), 3.82-3.70 (3
H, m), 3.68-3.55 (21H, m), 3.5
0 (2H, t, J = 6.1Hz), 3.39 (1H,
m), 2.58 (1H, dd, J = 12.5, 4.9H
z), 2.26, 2.14, 2.12, 2.07, 2.
03, 1.97, 1.95, 1.81 (each 3
H, s), 1.54 (1H, dd, J = 12.5, 1
2.5 Hz).

(5) Synthesis of Compound 8-5 Compound 8-4 (390 mg) dissolved in methanol (6
ml) solution, 3% sodium methoxide-methanol solution (600 μl) was added, and the mixture was stirred at room temperature for 30 minutes. Cation exchange resin (Dowex 50w)
After neutralizing with H ⁺ ), the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was then added to 1,4-dioxane (1 ml) and 0.1N aqueous sodium hydroxide solution (6.0
ml) was added, and the mixture was stirred at room temperature for 10 minutes. After that, the compound was neutralized with a cation exchange resin (Dowex 50wH ⁺ ), insoluble matter was filtered off, the filtrate was concentrated under reduced pressure, and purified by column chromatography (methanol) using a polymer gel (200 cc). 8-5 (27
8 mg) was obtained as a colorless powder.

[Α] _D ²⁴ -12.2 ° (c1.02, M
OH) IR (KBr): 3446, 1735, 1655 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 4.50 (1 H, d,
J = 8.3 Hz), 4.45 (1H, d, J = 7.8H)
z), 4.05 (1H, dd, J = 9.8, 2.9H)
z), 3.95-3.55 (36H, m), 3.81.
(2H, t, J = 6.1 Hz), 3.51 (2H, t,
J = 6.1 Hz), 3.50 (1 H, m), 3.40
(1H, m), 2.79 (1H, dd, J = 12.9,
4.2 Hz), 2.00, 1.98 (each 3H,
s), 1.87 (1H, tricke).

(6) Synthesis of Sialyl-N-acetyllactosamine Modified Carboxymethyl Chitosan (Compound 8) Carboxymethyl Chitosan (40 mg) and Compound 8-5
(240 mg) was dissolved in 0.5% aqueous sodium hydrogen carbonate solution (3 ml), and then sodium hydrogen carbonate (20 m
g) was added and the mixture was stirred at 60 ° C. for 160 hours. The reaction solution is 9
The crude product was precipitated by adding it to 9.5% ethanol (35 ml), and the precipitate was mixed with 95% ethanol (40 ml).
It was washed three times with acetone (40 ml) and diethyl ether (35 ml) in that order, and then dried under reduced pressure (42 mg). Then, the crude product was dissolved in 10 ml of water, and then purified water (100 ml)
(00 ml) was used as an external solution and dialyzed for 12 hours at room temperature. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate a target substance, and the precipitate was washed with 95% ethanol (40 ml), acetone (40 ml) and diethyl ether (40 ml) in this order. Then, it is dried under reduced pressure to give sialyl-N-acetyllactosamine-modified carboxymethyl chitosan (compound 8, 28 mg, d
s: 0.17, sialyl-N-acetyllactosamine content: 29%) was obtained (ds was determined by resorcinol-hydrochloric acid method, which is a quantitative method for sialic acid).

Example 9 (1) Synthesis of compound 9-1 Lithium azide (81 mg) was added to a dimethylformamide solution (8 ml) in which compound 1-3 (900 mg) was dissolved.
Was added and the mixture was stirred at 65 ° C. for 3 hours. The reaction solution was cooled to room temperature, diluted with ethylene chloride, washed with saturated saline, dried, and then the solvent was distilled off. Then, the obtained residue was purified by column chromatography (ethylene chloride-methanol 50: 1) using silica gel (70 g) to obtain compound 9-1 (810 mg) as a colorless oil.

[Α] _D ²¹ -16.0 ° (c1.07, C
HCl ₃ ) IR (CHCl ₃ ): 2108, 1745, 1690c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 5.39 (1 H, dd
d, J = 8.5, 5.4, 2.7 Hz), 5.32 (1
H, dd, J = 8.5, 2.0 Hz), 5.10 (1
H, d, J = 9.3 Hz), 4.86 (1H, ddd,
J = 12.2, 9.8, 4.6 Hz), 4.30 (1
H, dd, J = 12.5, 2.7 Hz), 4.09 (1
H, dd, J = 12.5, 5.4 Hz), 4.08-
4.02 (2H, m), 3.90 (1H, ddd, J =
10.7, 5.1, 3.4 Hz), 3.80 (3H,
s), 3.69-3.60 (20H, m), 3.46.
(1H, ddd, J = 10.7, 6.6, 3.4H
z), 3.39 (2H, t, J = 6.3 Hz), 2.6
2 (1H, dd, J = 12.9, 4.6 Hz), 2.1
4,2.14,2.04,2.03,1.88 (eac
h 3H, s), 1.98 (1H, dd, J = 12.
9, 12.2 Hz).

(2) Synthesis of Compound 9-2 Compound 9-1 (100 mg) dissolved in methanol (5
ml) solution, Lindlar catalyst (100 mg) and paratoluenesulfonic acid monohydrate methanol solution (25 mg)
Was added, and the mixture was stirred under a medium-pressure hydrogen stream (50 psi) at room temperature for 3 hours. The catalyst was filtered off from the reaction solution, the filtrate was concentrated under reduced pressure, and the residue was subjected to column chromatography using silica gel (20 g) (ethylene chloride-methanol-water).
7: 3: 1) to give compound 9-2.
(103 mg) was obtained as a colorless oil.

[Α] _D ²³ -12.4 ° (c1.01, C
HCl ₃ ) IR (CHCl ₃ ): 1745, 1688 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.78 (2H, A ₂
B ₂ , J = 8.1 Hz), 7.16 (2H, A ₂ B ₂ ,
J = 8.1 Hz), 5.50 (1H, d, J = 9.0H)
z), 5.39 (1H, ddd, J = 8.5, 5.8,
2.9 Hz, 5.32 (1H, dd, J = 8.5,
2.0 Hz), 4.89 (1H, ddd, J = 12.
5, 9.8, 4.6 Hz), 4.30 (1H, dd, J
= 12.5, 2.7 Hz), 4.12 (1H, dd, J
= 10.7, 2.0 Hz), 4.09 (1H, qlik
e) 4.09 (1H, dd, J = 12.5, 5.9H)
z), 3.87 (1H, ddd, J = 11.0, 4.
9,3.2Hz), 3.81 (2H, t, J = 5.0H
z), 3.82 (3H, s), 3.74-3.60 (1
8H, m), 3.52 (1H, ddd, J = 11.0,
6.5, 2.5 Hz), 3.23-3.13 (2H,
m), 2.64 (1H, dd, J = 12.5, 4.6H)
z), 2.35, 2.14, 2.13, 2.04, 2.
02, 1.88 (each 3H, s), 1.93 (1
H, dd, J = 12.5, 12.5 Hz).

(3) Preparation of carboxymethyl pullulan By the method described in Japanese Patent Application No. 5-38635 (pullulan (average molecular weight: 150,000) and chloric acid are reacted in a 1N aqueous sodium hydroxide solution) carboxymethyl Pullulan (degree of carboxymethylation: 0.6) was obtained.

(4) Synthesis of sialic acid-modified carboxymethyl pullulan (9) Carboxymethyl pullulan (50 mg) and compound 9-2
1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 610 mg) was added to a mixed solution of water (2 ml) in which (115 mg) was dissolved and N, N'-dimethylformamide (2 ml), 160 at ℃
After stirring for an hour, the reaction mixture was concentrated, 1N aqueous sodium hydroxide solution (5 ml) was added to the residue, and the mixture was further stirred for 12 hours. The reaction mixture was mixed with 99.5% ethanol (35 ml).
In addition to the to precipitate the crude product, 95% of the precipitate
Ethanol (40 ml x 3 times), acetone (40 ml)
The extract was washed with diethyl ether (40 ml) and dried under reduced pressure (63 mg).

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000)
0 ml) was used as an external solution and dialyzed at room temperature for 16 hours. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate the target substance, and the precipitate was sequentially washed with 95% ethanol (40 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml). After washing and then drying under reduced pressure, sialic acid-modified carboxymethyl pullulan (compound 9, 43 mg, ds: 0.12, sialic acid content: 13.5%) was obtained (ds was calculated by the resorcinol hydrochloric acid method). .

Example 10 (1) Synthesis of Compound 10-1 A solution of sodium azide (92.5 mg) in dimethylformamide solution (3 ml) in which compound 7-6 (750 mg) was dissolved
mg, 1.42 mmol) was added, and the mixture was stirred at 70 ° C. for 17 hours. The reaction solution was cooled to room temperature, diluted with ethylene chloride, washed with saturated saline, dried, and then the solvent was distilled off. Subsequently, the obtained residue was treated with silica gel (150
g) by column chromatography (ethylene chloride-methanol 100: 1) using
Compound 10-1 (674 mg) was obtained as a colorless amorphous.

[Α] _D ²⁶ -31.0 ° (c1.07, C
HCl ₃ ) IR (CHCl ₃ ): 3450, 2106, 1674c
m ^{−1 1} H-NMR (CDCl ₃ + D ₂ O) δ: 7.41−
7.26 (20H, m), 6.13 (1H, d, J =
7.6 Hz), 4.98 (1H, d, J = 3.4H)
z), 4.95, 4.61 (each 1H, d, J =
11.5Hz), 4.84 (1H, d, J = 8.5H
z), 4.81, 4.79, 4.75, 4.67 (ea)
ch 1H, d, J = 11.7 Hz), 4.62, 4.
58 (each 1H, d, J = 12.2 Hz), 4.
13 (1H, q, J = 6.6 Hz), 4.06 (1H,
dd, J = 10.2, 3.4 Hz), 3.97 (1H,
m), 3.95 (1H, m), 3.85-3.77 (2
H, m), 3.76-3.56 (23H, m), 3.5
2-3.43 (3H, m), 3.36 (2H, t, J =
5.0Hz), 1.66 (3H, s), 1.14 (3
H, J = 6.6 Hz).

(2) Synthesis of Compound 10-2 Compound 7-7 (350 mg) and Compound 10-1 (542 mg) were dissolved in a methylene chloride solution (10 ml) containing Molecular Sieves 4A (3 g), and the mixture was stirred at room temperature for 2 hours. After stirring, boron trifluoride diethyl ether complex (87 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 30 minutes. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography (ethylene chloride-methanol 20: 1) using silica gel (70 g) to give compound 10-2 (242 mg) as a colorless powder.

[Α] _D ²⁸ -27.9 ° (c0.55, C
HCl ₃ ) IR (KBr) 2108, 1745, 1685 cm ^{-1 1} H-NMR (CDCl ₃ ) δ: 7.97 (2H,
m), 7.50 (1H, m), 7.49-7.16 (2
2H, m), 6.25 (1H, br.s), 5.59
(1H, ddd, J = 9.3, 5.6, 2.7 Hz),
5.37 (1H, dd, J = 9.3, 2.7Hz),
5.22 (1H, d, J = 3.7Hz), 5.07 (1
H, d, J = 10.3 Hz), 5.04 (1 H, d, J
= 3.7 Hz), 4.97 (1H, dd, J = 10.
0, 8.1 Hz), 4.92, 4.63 (each 1
H, d, J = 11.7 Hz), 4.90 (1H, dd
d, J = 12.0, 10.5, 4.6 Hz), 4.81
(1H, d, J = 8.1Hz), 4.74 (1H,
d) 4.81, 4.77, 4.75, 4.70, 4.
58, 4.44 (each 1H, d, J = 12.0H
z), 4.67 (1H, dd, J = 10.0, 3.7H)
z), 4.29 (1H, dd, J = 12.4, 2.7H
z), 4.24 (1H, dd, J = 11.0, 6.8H)
z), 4.23 (1H, q, J = 6.6Hz), 4.1
6 (1H, dd, J = 11.0, 7.3 Hz), 4.0
8 (1H, m), 4.08 (1H, dd, J = 10.
0, 3.7 Hz), 4.04 (1H, m), 4.01
(1H, m), 3.98 (1H, dd, J = 12.4,
5.6 Hz), 3.90 (1 H, m), 3.87 (1
H, dd, J = 10.0, 2.7 Hz), 3.87-
3.79 (4H, m), 3.75 (3H, s), 3.6
5 (2H, t, J = 5.1Hz), 3.65-3.48
(21H, m), 3.37 (2H, t, J = 5.1H
z), 2.56 (1H, dd, J = 12.7, 4.6H)
z), 2.22, 2.08, 2.07, 2.01, 1.
97, 1.96, 1.89, 1.85 (each 3
H, s), 1.71 (1H, dd, J = 12.7, 1
2.0Hz, 1.10 (3H, d, J = 6.6H
z).

(3) Synthesis of compound 10-3 Palladium-carbon (10%, 15%) was added to a solution of compound 10-2 (200 mg) in methanol (15 ml).
0 mg) and 0.5 N hydrochloric acid (328 μl) were added, and the mixture was stirred at room temperature under a medium-pressure hydrogen stream (50 psi) for 12 hours.
After the catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was subjected to column chromatography using silica gel (45 g) (methylene chloride-methanol-water 65:35:10).
(Lower layer)) to give compound 10-3 (1
40 mg) was obtained as a colorless powder.

[Α] _D ²⁵ -58.3 ° (c0.52, C
HCl ₃ ) IR (KBr): 3420, 1749, 1663 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 8.05 (2H,
m), 7.63 (1H, m), 7.51 (2H, tli
ke), 5.60 (1H, ddd, J = 9.3, 5.
6,2.9 Hz), 5.37 (1H, dd, J = 9.
3,2.7 Hz), 5.17 (1H, d, J = 3.4H
z), 5.06 (1H, d, J = 3.9Hz), 4.9
8 (1H, dd, J = 10.0, 8.3Hz), 4.9
1 (1H, d, J = 8.3 Hz), 4.87 (1H,
m), 4.86-4.81 (1H, m), 4.60 (1
H, dd, J = 10.0, 3.4 Hz), 4.52 (1
H, dd, J = 10.5, 5.9 Hz), 4.45 (1
H, d, J = 8.5 Hz), 4.41 (1H, dd, J
= 12.7, 2.9 Hz), 4.15 (1H, dd, J
= 10.5, 8.5 Hz), 4.09-3.99 (4
H, m) 3.95 (1H, dd, J = 10.5,1
0.5 Hz), 4.16 (1H, dd, J = 12.0,
4.9 Hz), 3.85 (1 H, m), 3.84-3.
79 (5H, m), 3.74 (3H, s), 3.76-
3.62 (22H, m), 3.42 (1H, m), 3.
22 (1H, ddd, J = 13.7, 6.1, 4.6H
z), 3.15 (1H, ddd, J = 13.7, 10.
0, 5.1 Hz), 2.58 (1H, dd, J = 12.
4, 4.6 Hz), 2.26, 2.16, 2.08,
2.07, 2.04, 1.99, 1.97, 1.81
(Each 3H, s), 1.53 (1H, dd, J =
12.4, 12.2Hz), 1.27 (3H, d, J =
6.8 Hz).

(4) Synthesis of Sialyl Lewis X Modified Carboxymethyl Pullulan (Compound 10) Carboxymethyl Pullulan (50 mg) and Compound 10-
3 (184 mg, 0.124 mmol) dissolved in water (2 ml) and N, N'-dimethylformamide (2 m
l) mixed solution of 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 610 m
g) was added and the mixture was stirred at 40 ° C. for 160 hours, the reaction mixture was concentrated, and the residue was 1N aqueous sodium hydroxide solution (7 ml).
Was added and the mixture was further stirred for 12 hours. The reaction mixture was added to 99.
The crude product was precipitated by adding it to 5% ethanol (35 ml), and the precipitate was mixed with 95% ethanol (40 ml × 3).
Washed sequentially with acetone (40 ml) and diethyl ether (40 ml), and then dried under reduced pressure (8).
3 mg).

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000) was used using a dialysis membrane (Spectra / Pore Co .: molecular weight exclusion limit 12000 to 14000).
0 ml) was used as an external solution and dialyzed at room temperature for 16 hours. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate a target substance, and the precipitate was sequentially washed with 95% ethanol (40 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml). After washing and then drying under reduced pressure, sialyl Lewis X-modified carboxymethyl pullulan (compound 10, 57 mg, ds: 0.1
3, sialyl Lewis X content: 31%) was obtained (ds was determined by the resorcinol hydrochloric acid method which is a quantitative method for sialic acid).

[0129]Example 11 (1) Synthesis of compound 11-1 Dimethylform in which compound 8-2 (620 mg) was dissolved
In an amide solution (6 ml), lithium azide (83 mg,
1.70 mmol) and stirred at 70 ° C. for 12 hours
It was After cooling the reaction solution to room temperature, dilute it with methylene chloride.
And wash with saturated saline, then dry and evaporate the solvent.
It was Subsequently, the obtained residue was treated with silica gel (150 g).
Column chromatography used (methylene chloride-meta
Compound 1 by purifying with 70: 1)
1-1 (575 mg) is colorless amorphous Got as quality.

[Α] _D ²⁶ -14.3 (c1.02, CH
Cl ₃ ) IR (CHCl ₃ ): 2108, 1674 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.37-7.26
(10H, m), 6.33 (1H, br.s), 4.8
2 (1H, d, J = 8.3 Hz), 4.77, 4.71
(Each 1H, d, J = 11.5 Hz), 4.6
1,4.57 (each 1H, d, J = 12.2H
z), 3.89 (1H, m), 3.80-3.72 (4)
H, m), 3.71-1.57 (22H, m), 3.5
1 (1H, dt, J = 9.5, 4.9 Hz), 3.36
(2H, t, J = 5.1Hz), 2.15 (1H, d,
J = 2.2 Hz, OH), 1.96, 1.70 (eac
h 3H, s).

(2) Synthesis of compound 11-2 Compound 7-7 (200 mg) and compound 11-1 (350 mg) were dissolved in a methylene chloride solution (5 ml) containing molecular sieves 4A (2 g), and the mixture was allowed to stand at room temperature for 2 times. After stirring for an hour, boron trifluoride diethyl ether complex (50 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 3 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography using silica gel (45 g) (methylene chloride-methanol 15: 1) to obtain compound 11-2 (145 mg) as a colorless powder.

[Α] _D ^23.5 -20.9 ° (c1.05
CHCl ₃ ) IR (CHCl ₃ ): 2100, 1744, 1682c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 7.96 (2H, dl
ike), 7.53 (1H, tricke), 7.38
(2H, tick), 7.35-7.18 (10H,
m), 6.32 (1H, br.s), 5.60 (1H, br.s)
ddd, J = 9.3, 6.1, 2.7 Hz), 5.36
(1H, dd, J = 9.3, 2.7 Hz), 5.09-
5.00 (3H, m), 4.89 (1H, ddd, J =
12.0, 10.7, 4.6 Hz), 4.85 (1H,
d, J = 8.3 Hz), 4.77, 4.67 (each)
1H, d, J = 11.5Hz), 4.71 (1H,
d, J = 6.1 Hz), 4.66 (1H, dd, J = 1)
0.3, 3.7 Hz), 4.59, 4.51 (each
1H, d, J = 12.0Hz, 4.31 (1H, b
r. d), 4.16 (1H, dd, J = 11.0, 6.
8 Hz, 4.11 (1H, dd, J = 11.0, 7.
3 Hz), 4.05 (1H, qlike), 4.04-
4.03 (1H, m), 3.97 (1H, dd, J = 1
2.7, 6.1 Hz), 3.95-3.86 (4H,
m), 3.84 (1H, tricke), 3.78 (2
H, t, J = 6.3 Hz), 3.75 (3H, s),
3.70-3.56 (22H, m), 3.37 (2H,
t, J = 5.1 Hz), 2.58 (1H, dd, J = 1)
2.7, 4.6 Hz), 2.22, 2.10, 2.0
1, 1.85 (each 3H, s), 2.07, 1.
96 (6H, s), 1.72 (1H, dd, J = 12.
7,12.3 Hz).

(3) Synthesis of compound 11-3 Palladium-carbon (10%, 50%) was added to a solution of compound 11-2 (100 mg) in methanol (10 ml).
mg) and 1N hydrochloric acid (99 μl) were added, and the mixture was stirred under a hydrogen stream at room temperature for 12 hours. After the catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was purified by column chromatography using silica gel (20 g) (methylene chloride-methanol-water 65:35:10 (lower layer)) to give Compound 11. -3 (65 mg) was obtained as a colorless powder.

[Α] _D ²⁶ -3.4 ° (c1.04, CH
Cl ₃ ) IR (KBr): 3480, 3420, 1743, 16
88 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 8.07 (2H, dl
ike), 7.62 (1H, tricke), 7.48
(2H, tick), 5.63 (1H, ddd, J =
9.5, 5.9, 2.7 Hz), 5.36 (1H, d
d, J = 9.5, 2.4 Hz), 5.14 (1H, d,
J = 3.2 Hz), 5.00 (1H, dd, J = 10.
0, 8.3 Hz), 4.88 (1 H, m), 4.85
(1H, d, J = 8.3 Hz), 4.73 (1H, d
d, J = 10.3, 3.2 Hz), 4.40 (1H,
d, J = 8.5 Hz), 4.39 (1H, dd, J = 1)
2.7, 2.7 Hz), 4.36 (1H, dd, J = 1)
1.2, 5.8 Hz), 4.30 (1H, dd, J = 1)
1.2, 7.3 Hz), 4.17 (1H, tlik
e), 4.05 (1H, m), 3.99 (1H, dd,
J = 12.2, 5.9 Hz), 3.97 (1H, dd,
J = 10.3, 10.3 Hz), 3.93 (1H,
m), 3.89 (1H, dlike), 3.83-3.
52 (25H, m), 3.80 (3H, s), 3.41
(1H, m) 3.23 (1H, ddd, J = 13.
2, 7.1, 3.9 Hz), 3.11 (1H, ddd,
J = 13.2, 5.9, 3.4 Hz), 2.58 (1
H, dd, J = 12.4, 4.6 Hz), 2.31,
2.14, 2.12, 2.07, 2.03, 1.97,
1.97, 1.81 (each 3H, s), 1.54
(1H, dd, J = 12.5, 12.0Hz).

(4) Synthesis of sialyl-N-acetyllactosamine-modified carboxymethyl pullulan (Compound 11) Carboxymethyl pullulan (40 mg) and Compound 11-
3 (133 mg) dissolved in water (1.6 ml) and N,
To a mixed solution of N'-dimethylformamide (1.6 ml), 1-ethoxycarbonyl-2-ethoxy-1,2-
Add dihydroquinoline (EEDQ, 490 mg) and add 4
After stirring at 0 ° C. for 160 hours, the reaction solution was concentrated, 1N aqueous sodium hydroxide solution (6 ml) was added to the residue, and the mixture was further stirred for 12 hours. The reaction mixture was added to 99.5% ethanol (35 ml) to precipitate a crude target product, and the precipitate was added in order of 95% ethanol (40 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml). Washed and then dried under reduced pressure (48 mg).

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000) was used using a dialysis membrane (Spectra / Pore Co .: molecular weight exclusion limit 12000 to 14000).
0 ml) was used as an external solution and dialyzed at room temperature for 16 hours. The dialyzed solution was added to 99.5% ethanol (140 ml) to precipitate a target substance, and the precipitate was sequentially washed with 95% ethanol (40 ml × 3 times), acetone (40 ml) and diethyl ether (40 ml). Washed and then dried under reduced pressure to give sialyl-N-acetyllactosamine-modified carboxymethyl pullulan (Compound 11, 40m
g, ds: 0.13, sialyl-N-acetyllactosamine content: 27%) were obtained (ds was determined by resorcinol-hydrochloric acid method, which is a quantitative method for sialic acid).

Example 12 Carboxymethyl chitosan (50 mg) was added to distilled water (10 m).
lactose monohydrate (C ₁₂ H ₂₂ O ₁₁ : H ₂
O = 360.31) 313 mg was added, and then acetic acid was added to the reaction solution to adjust the pH to 6.6. Na in this reaction solution
72 mg of BH ₃ CN (95%, Mw = 62.84) was added, and the mixture was stirred at room temperature for 1 week. The reaction solution was dialyzed with a dialysis membrane (Spectra / Pore Co .: molecular weight cutoff of 12,000 to 1).
It was dialyzed for 2 days at 4 ° C. using purified water as an external solution. The dialyzed solution was further passed through a membrane filter (0.22 μm). This eluate was added to 280 ml of 99.5% ethanol, and the resulting precipitate was washed with 95% ethanol, acetone, and ether in this order, and dried under reduced pressure to give a compound (51 m
g) was obtained as a white amorphous. The degree of substitution of lactose was 0.15 as determined by the phenol-sulfuric acid method using galactose as a standard.

The lactose-introduced compound (2
(0 mg) was dissolved in 5 ml of a saturated aqueous solution of sodium hydrogen carbonate, 80 μl of acetic anhydride was added in 4 portions, and the mixture was stirred overnight at room temperature. The reaction solution was dialyzed (Spectra / Pore Co .:
Using a molecular weight cutoff of 12,000 to 14,000), the mixture was dialyzed with purified water as an external solution at 4 ° C for 2 days. The dialyzed solution was further passed through a membrane filter (0.22 μm). This eluate was added to 150 ml of 99.5% ethanol.
In addition, the resulting precipitate was washed with 95% ethanol, acetone, and ether in this order, and dried under reduced pressure to introduce lactose, and a compound (13 mg) in which the amino group of carboxymethyl chitosan was acetylated was white. Obtained as a crystalline material.

Example 13 Carboxymethyl chitosan (100 mg) was added to distilled water 10
Dissolve in malt and maltose monohydrate (C ₁₂ H ₂₂ O ₁₁ · H
641 mg of ₂ O = 360.32) was added, and then dilute acetic acid was added to the reaction solution to adjust the pH to 6.6. To this reaction solution was added NaBH ₃ CN (95%, Mw = 62.84) 140 m.
g was added, and the mixture was stirred at room temperature for 6 days. The reaction solution was dialyzed with a dialysis membrane (Spectra / Pore Co., Ltd .: molecular weight cutoff of 12,000).
0 to 14,000) and purified water as an external solution at 4 ° C.
And dialyzed for 2 days. The dialyzed solution was further passed through a membrane filter (0.22 μm). This eluate was added to 99.
Add to 300 ml of 5% ethanol and remove the resulting precipitate by 95%.
The compound in which maltose was introduced by washing with ethanol, acetone, and ether in this order and then drying under reduced pressure (1
20 mg) was obtained as a white amorphous. The degree of substitution of maltose was 0.26 as determined by the phenol-sulfuric acid method using glucose as a standard.

The compound (3 containing the maltose introduced above
0 mg) was dissolved in 3 ml of saturated aqueous sodium hydrogen carbonate solution, 120 μl of acetic anhydride was added in 4 portions, and the mixture was stirred overnight at room temperature. The reaction solution was dialyzed for 2 days at 4 ° C. using purified water as an external solution using a dialysis membrane (Spectra / Pore Co., Ltd .: molecular weight cutoff 12,000 to 14,000). The dialysis solution was further filtered with a membrane filter (0.22μ
m). This eluate was added to 99.5% ethanol 15
The resulting precipitate was washed with 95% ethanol, acetone, and ether in this order and dried under reduced pressure to introduce maltose, and the compound (30 mg) in which the amino group of carboxymethyl chitosan was atticelized was white. Obtained as an amorphous.

Example 14 Carboxymethyl chitosan (50 mg) was added to distilled water (10 m).
Dissolve in l, maltopentaose (C ₃₀ H ₅₂ O ₂₆ = 82
870 mg of 8.73) and then diluted acetic acid was added to the reaction solution to adjust the pH to 6.5. NaBH ₃ was added to this reaction solution.
72 mg of CN (95%, Mw = 62.84) was added, and the mixture was stirred at room temperature for 6 days. The reaction solution was dialyzed (Spectra /
Pore: molecular weight cut-off 12,000 to 14,000
0) was dialyzed for 2 days at 4 ° C. using purified water as an external solution. The dialysis solution is further filtered with a membrane filter (0.2
2 μm). This eluate was added to 250 ml of 99.5% ethanol, and the resulting precipitate was washed with 95% ethanol, acetone, and ether in this order, and dried under reduced pressure to obtain a compound having maltopentaose introduced (63 m).
g) was obtained as a white amorphous. When the phenol-sulfuric acid method using maltotetraose as a standard, it was determined that the substitution degree of maltopentaose was 0.12.

The above-mentioned compound (30 mg) into which maltopentaose was introduced was saturated with an aqueous solution of sodium hydrogen carbonate (3 m).
Dissolve in 1 l, add 120 μl of acetic anhydride in 4 portions,
Stirred overnight at room temperature. The reaction solution was dialyzed (Spectra /
Pore: molecular weight cut-off 12,000 to 14,000
0) was dialyzed for 2 days at 4 ° C. using purified water as an external solution. The dialysis solution is further filtered with a membrane filter (0.2
2 μm). The eluate was added to 100 ml of 99.5% ethanol, and the resulting precipitate was washed with 95% ethanol, acetone, and ether in this order, and dried under reduced pressure to introduce maltopentaose, thereby introducing the amino group of carboxymethyl chitosan. Compound (26
mg) was obtained as a white amorphous.

Example 15 A maltotriose-introduced carboxymethyl chitosan complex was obtained by the same method as described in Example 12, except that maltotriose was used instead of lactose monohydrate.

Example 16 A cellobiose-introduced carboxymethyl chitosan complex was obtained by the same method as described in Example 12, except that cellobiose was used instead of lactose monohydrate.

Example 17 A chitobiose-introduced carboxymethyl chitosan complex was obtained by the same method as that described in Example 12, except that chitobiose was used instead of lactose monohydrate.

Example 18 (1) Preparation of Carboxymethyl Chitosan-Peptide Complex Carboxymethyl chitosan (320 m) of Example 1 (5)
g) as a 0.5% aqueous sodium hydrogen carbonate solution (32 ml)
After the solution was dissolved in water, dimethylformamide (28 ml) was added to form a uniform polysaccharide solution. On the other hand, 3- (p-hydroxyphenyl) propionic acid (deaminotyrosine) (11m
g) was dissolved in 0.6 ml of dimethylformamide and then N-
Hydroxysuccinimide (7.6 mg) and N, N'-
Dicyclohexylcarbodiimide (12.0 mg) was added and reacted at room temperature for 2 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at 4 ° C. for 18 hours. The reaction solution was added to ethanol (256 ml), and the deposited precipitates were collected and dried under reduced pressure to obtain 280 mg of carboxymethyl chitosan-deaminotyrosine complex. The content of deaminotyrosine in this complex was determined from the ultraviolet (276 nm) absorbance analysis.
It was 0.43% (% by weight).

The above complex (120 mg) was dissolved in a 0.5% aqueous sodium hydrogencarbonate solution (12 ml), and dimethylformamide (10.5 ml) was added thereto to give a uniform polysaccharide solution, while the tert-butoxycarbonyl group ( Bo
c-group-protected peptide N-Boc-Gly.Gl
y-Gly-OH (34 mg) was dissolved in 0.9 ml of dimethylformamide, N-hydroxysuccinimide (13.8 mg) and N, N'-dicyclohexylcarbodiimide (22.2 mg) were added, and the mixture was reacted at room temperature for 4 hours. To give the active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at 4 ° C. for 18 hours. The reaction mixture was added to ethanol (80 ml), and the deposited precipitates were collected, dried under reduced pressure, and 111 mg of carboxymethyl chitosan-deaminotyrosine-Gly.G.
A ly.Gly-Boc complex was obtained.

(2) Preparation of 9- (galactosyl-β) nonanoic acid RULemieux, DRBundle, DA Beker et al., US Pat.
The compound of the following formula (X) was synthesized according to the method described in 238473 (1980).

[0149]

[Chemical 6]

A 1N aqueous sodium hydroxide solution (1.2 ml) was added to a mixed solution of methanol (5 ml) -tetrahydrofuran (4 ml) in which this compound (350 mg) was dissolved.
Was added, and the mixture was stirred at room temperature for 18 hours. The reaction solution was neutralized with a cation exchange resin (Amberlite IR-120B (H ⁺ )), the insoluble material was filtered off, and the filtrate was concentrated under reduced pressure to obtain the target product (327 mg) as a colorless solid.

M. p. 107.5-109 ° C Anal. Calcd for C ₁₅ H ₂₈ O ₈ / 3H ₂ O; C, 5
1.74; H, 8.11. Found: C, 51.76; H, 8.25. [Α] _D ²⁵ -10.3 ° (c1.0, MeOH) IR (KBr): 3420, 1730, 1740, 16
35 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 4.20 (1 H, d,
J = 8.0 Hz), 3.89 (1H, dt, J = 8.
5,7.0Hz), 3.83 (1H, d, J = 3.5H)
z), 3.69-3.78 (2H, m), 3.53 (1
H, dt, J = 8.5, 7.0 Hz), 3.46-3.
52 (2H, m), 3.45 (1H, dd, J = 9.
5,3.5Hz), 2.26 (2H, t, 7.0H
z), 1.66-1.55 (4H, m), 1.44-
1.30 (8H, m). FAB-MS m / z: 359
(M + Na ⁺ ).

(3) Preparation of galactose-introduced carboxymethyl chitosan-peptide complex Carboxymethyl chitosan-deaminotyrosine-Gly
-Gly-Gly-Boc complex (60 mg) 0.5
After being dissolved in an aqueous sodium hydrogen carbonate solution (6 ml), dimethylformamide (5.3 ml) was added to obtain a uniform polysaccharide solution. On the other hand, 9- (galactosyl-β) nonanoic acid (Gal-O—C ₈ H ₁₆ COOH) (60.6 mg) was dissolved in 1.2 ml of dimethylformamide, and then N-hydroxysuccinimide (21 mg) and N, N ′ were added. -Dicyclohexylcarbodiimide (36 mg) was added, and the mixture was reacted at room temperature for 18 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at room temperature for 3 days. The reaction solution is ethanol (48m
l) and the deposited precipitates were collected and dried under reduced pressure to obtain 6
5 mg galactose-introduced carboxymethyl chitosan-
A deaminotyrosine-Gly.Gly.Gly-Boc complex was obtained. Galactose-OC ₈ H ₁₆ C of this complex
O content was analyzed by phenol-sulfuric acid method (absorbance 490n
m) was 5.6% (weight%). Galactose was again introduced under the same conditions to give 49.
Obtained 6 mg (11%).

The above complex (47.6 mg) was dissolved in saturated aqueous sodium hydrogen carbonate solution (5 ml), acetic anhydride (0.2 ml) was added, and the mixture was N-acetylated at room temperature for 5 hours. After neutralizing the reaction solution, it was added to ethanol (20 ml), and the deposited precipitates were collected and dried under reduced pressure.
7 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly / Gly / Gl
A y-Boc complex was obtained.

(4) Introduction of Drug The above complex (20 mg) was dissolved in 0.5N hydrochloric acid (2 ml), and the Boc group was deprotected at 30 ° C. for 16 hours. After neutralizing the reaction solution, add it to ethanol (8 ml),
The deposited precipitates were collected and dried under reduced pressure, and 15.8 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly.Gly.Gly-NH
_Two complexes were obtained.

50.5 mg of methotrexate (MT
X) was dissolved in dimethylformamide (1 ml), then N,
N'-dicyclohexylcarbodiimide (20.5m
g) was added and the reaction was carried out at 4 ° C. for 17 hours.
-Hydroxysuccinimide (9.2mg) and pyridine (12.6ul) were added and reacted at room temperature for 5 hours to obtain MT.
The active ester of X was prepared.

On the other hand, the above-mentioned galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gl
y · Gly · Gly-NH ₂ complex (15mg) 0.
After dissolving in a 5% aqueous sodium hydrogen carbonate solution (3 ml), 340 ul of the reaction solution containing the active ester of MTX was added, and the mixture was reacted at room temperature for 3 hours. The obtained reaction solution was added to ethanol (20 ml), and the deposited precipitates were collected and dried under reduced pressure to give 12.2 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-.
Gly.Gly.Gly-MTX complex was obtained as a yellow powder. The MTX content of this complex was determined to be ultraviolet (308n
It was 13.5% (% by weight) from the absorbance analysis of m).

Example 19 (1) Preparation of Carboxymethyl Chitosan-Peptide Complex Carboxymethyl chitosan of Example 1 (5) (192 m
g) to a 0.5% aqueous sodium hydrogen carbonate solution (19.2 m
l) and then dimethylformamide (16.8 ml)
Was added to form a uniform polysaccharide solution. On the other hand, 3- (p-hydroxyphenyl) propionic acid (deaminotyrosine)
After dissolving (6.6 mg) in 0.36 ml of dimethylformamide, N-hydroxysuccinimide (4.56 m
g) and N, N'-dicyclohexylcarbodiimide (7.2 mg) were added and reacted at room temperature for 2 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at 4 ° C. for 18 hours.
The reaction solution was added to ethanol (150 ml), and the deposited precipitates were collected and dried under reduced pressure to obtain 177 mg of carboxymethyl chitosan-deaminotyrosine complex. The deaminotyrosine content of this complex was determined to be ultraviolet (276 nm).
It was 0.37% (% by weight) from the absorbance analysis.

The above complex (40 mg) was dissolved in a 0.5% aqueous sodium hydrogen carbonate solution (4 ml), and dimethylformamide (3.5 ml) was added to give a uniform polysaccharide solution. On the other hand, the peptide N-Boc- protected by the Boc group
GlyPheGlyGly-OH (18 mg) was dissolved in 0.3 ml of dimethylformamide, N-hydroxysuccinimide (4.6 mg) and N, N'-dicyclohexylcarbodiimide (7.4 mg) were added, and the mixture was allowed to stand at room temperature. And reacted for 4.5 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at 4 ° C. for 18 hours. The reaction solution was added to ethanol (32 ml), and the deposited precipitate was collected, dried under reduced pressure, and dried under reduced pressure to give 37.1 mg of carboxymethyl chitosan-deaminotyrosine-Gly.Gly.Phe.Gly-Bo.
A c-complex was obtained. The peptide content of this complex was determined to be 7.3% (% by weight) based on the ultraviolet (258 nm) absorbance analysis.
Met.

(2) Preparation of galactose-introduced carboxymethyl chitosan-peptide complex After dissolving the above complex (35 mg) in a 0.5% aqueous sodium hydrogen carbonate solution (5.25 ml), dimethylformamide (4.6 ml) was added. To give a uniform polysaccharide solution. On the other hand, 9- (galactosyl-β) nonanoic acid (Example (2)) (35.4 mg) was dissolved in 0.7 ml of dimethylformamide, and then N-hydroxysuccinimide (1
2.3 mg) and N, N'-dicyclohexylcarbodiimide (21 mg) were added and reacted at room temperature for 18 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at room temperature for 3 days. The reaction solution was added to ethanol (28 ml), and the deposited precipitates were collected and dried under reduced pressure to obtain 33.6 mg of galactose-introduced carboxymethyl chitosan-deaminotyrosine-Gly.Gly.Phe.Gly-Boc complex. . The galactose-OC ₈ H ₁₆ CO content of this complex was analyzed by the phenol-sulfuric acid method (absorbance 490 nm),
It was 7.2% (% by weight). The above complex (32.4m
g) was dissolved in saturated aqueous sodium hydrogen carbonate solution (4 ml), acetic anhydride (0.16 ml) was added, and the mixture was N-acetylated at room temperature for 5 hours. After neutralizing the reaction solution, it was added to ethanol (16 ml), and the deposited precipitates were collected and dried under reduced pressure to give 32.9 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly.
-Gly-Phe-Gly-Boc complex was obtained.

(3) Introduction of drug The above complex (32.9 mg) was added to 0.5N hydrochloric acid (3.5 m).
After dissolution in l), the Boc group was deprotected at 37 ° C. for 16 hours. After neutralizing the reaction solution, ethanol (16 ml)
Add to the inside and collect the deposited precipitate and dry under reduced pressure.
2.6 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-GlyGly.
A Phe.Gly-NH ₂ complex was obtained.

The above complex (20 mg) was dissolved in a 0.5% aqueous sodium hydrogen carbonate solution (4 ml), and then 400 ul of a reaction solution containing an active ester of MTX prepared in the same manner as in Example 18 was added, and the mixture was allowed to stand at room temperature. The reaction was carried out for 3 hours. The obtained reaction solution was added to ethanol (24 ml), and the deposited precipitates were collected and dried under reduced pressure to give 18.3 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly / Gly / Phe / Gly-. MT
The X-complex was obtained as a yellow powder. The MTX content of this complex was determined to be 8.3% by ultraviolet (308 nm) absorbance analysis.
(% By weight). The molar ratio of MTX to peptide in this complex was calculated to be 1.1.

Example 20 (1) Preparation of Carboxymethyl Chitosan-Peptide Complex Carboxymethyl chitosan of Example 1 (5) (160 m
g) as a 0.5% aqueous sodium hydrogen carbonate solution (16 ml)
After the solution was dissolved in water, dimethylformamide (14 ml) was added to obtain a uniform polysaccharide solution. On the other hand, 3- (p-hydroxyphenyl) propionic acid (deaminotyrosine) (16m
g) was dissolved in 0.8 ml of dimethylformamide and then N-
Hydroxysuccinimide (11 mg) and N, N'-dicyclohexylcarbodiimide (17.4 mg) were added and reacted at room temperature for 2 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at 4 ° C. for 18 hours. The reaction solution was added to ethanol (128 ml), and the deposited precipitates were collected and dried under reduced pressure to obtain 135 mg of carboxymethyl chitosan-deaminotyrosine complex. The content of deaminotyrosine in this complex was determined from the ultraviolet (276 nm) absorbance analysis.
It was 1.1% (% by weight).

The above complex (100 mg) was dissolved in a 0.5% aqueous sodium hydrogencarbonate solution (10 ml), and dimethylformamide (8.75 ml) was added to give a uniform polysaccharide solution. On the other hand, peptide N-Bo protected by Boc group
c-Phe.Phe.Gly-OH (47 mg) was added to 0.
After dissolving in 5 ml of dimethylformamide, N-hydroxysuccinimide (11.5 mg) and N, N'-dicyclohexylcarbodiimide (18.5 mg) were added and reacted at room temperature for 3 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution,
The reaction was carried out at 4 ° C for 18 hours. The reaction solution is ethanol (80
ml) and the deposited precipitates were collected and dried under reduced pressure to obtain 64.2 mg of carboxymethyl chitosan-deaminotyrosine-Gly.Phe.Phe-Boc complex. The peptide content of this complex was determined to be ultraviolet (258n
It was 13% (% by weight) from the absorbance analysis of m).

(2) Preparation of galactose-introduced carboxymethyl chitosan-peptide complex The above complex (20 mg) was dissolved in 0.5% sodium hydrogen carbonate aqueous solution (2 ml), and dimethylformamide (1.75 ml) was added to homogenize the complex. A simple polysaccharide solution was used. On the other hand, 9- (galactosyl-β) nonanoic acid (Example (2)) (20.2 mg) was dissolved in 0.4 ml of dimethylformamide, and then N-hydroxysuccinimide (7
mg) and N, N'-dicyclohexylcarbodiimide (12 mg) were added and reacted at room temperature for 19.5 hours to give an active ester. The whole amount of the reaction solution containing this active ester was added to the above-mentioned polysaccharide solution and reacted at room temperature for 3 days. The reaction solution was added to ethanol (16 ml), and the deposited precipitates were collected, dried under reduced pressure and dried with 21 mg of galactose-introduced carboxymethyl chitosan-deaminotyrosine-G.
A ly.Phe.Phe-Boc complex was obtained. Analyzing the content of galactose _-O-C 8 H ₁₆ CO of the complex by the phenol-sulfuric acid method (absorbance 490 nm) was at 9.5%
(% By weight).

The above complex (19 mg) was dissolved in a saturated aqueous sodium hydrogen carbonate solution (2 ml), and then acetic anhydride (0.0 mg) was added.
8 ml) was added and N-acetylated at room temperature for 5 hours.
After neutralizing the reaction solution, add it to ethanol (8 ml),
The deposited precipitates were collected and dried under reduced pressure to give 17.5 mg of galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly.Phe.Phe-Bo.
A c-complex was obtained.

(3) Introduction of drug The above complex (16.8 mg) was added to 0.5N hydrochloric acid (3 ml).
After dissolution in, the Boc group was deprotected at 37 ° C. for 24 hours. After neutralizing the reaction solution, it was added to ethanol (8 ml), and the deposited precipitates were collected and dried under reduced pressure to 11.5 m.
g-galactose-introduced N-acetylcarboxymethyl chitosan-deaminotyrosine-Gly / Phe / Phe-
An NH ₂ complex was obtained.

The above complex (11.5 mg) was dissolved in a 0.5% aqueous sodium hydrogencarbonate solution (3 ml), and the resulting mixture was used in Example 1.
300 ul of reaction solution containing the active ester of MTX prepared in the same manner as in 8 was added, and the mixture was reacted at room temperature for 3 hours.
The obtained reaction solution was added to ethanol (18 ml), and the deposited precipitates were collected and dried under reduced pressure, and 10.8 mg of galactose-introduced N-acetylcarboxymethyl chitosan-
Deaminotyrosine-Gly.Phe.Phe-MTX complex was obtained as a yellow powder. The MTX content of this complex is
When calculated from the ultraviolet absorption analysis (308 nm) 1
It was 2.1% (% by weight). The molar ratio of MTX to peptide in this complex was calculated to be 0.96.

Example 21 (1) Synthesis of compound 21-1 Synthesis was carried out according to the method described in Japanese Patent Application No. 1-180897.

(2) Synthesis of Compound 21-2 Compound 21-1 (20.0 g) was mixed with methanol (80 m
It was dissolved in 1), dibutyltin oxide (21.4 g) was added, and the mixture was heated under reflux for 2 hours. The solvent was evaporated under reduced pressure, benzene (160 ml), paramethoxybenzyl chloride (28 ml) and tetra-n-butylammonium bromide (23.0 g) were added to the residue, and the mixture was heated under reflux for 1.5 hr.

Then, after returning the reaction solution to room temperature, methanol (160 ml) and 28% sodium methoxide-
A methanol solution (80 ml) was added, and the mixture was stirred at room temperature for 1 hour. After the precipitate was filtered off from the reaction solution, the filtrate was diluted with ethyl acetate, washed with saturated saline and then dried, and the solvent was distilled off under reduced pressure. The residue is silica gel (900 g)
Compound 21-2 (18.8 g) was obtained as colorless crystals by purifying by column chromatography using (n-hexane-ethyl acetate-methanol 300: 400: 7).

[Α] _D ²⁸ -8.9 ° (c1.03, C
HCl ₃ ) IR (CHCl ₃ ): 3332 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 7.30 (2H, A ₂
B ₂ , J = 8.3 Hz), 6.90 (2H, A ₂ B ₂ ,
J = 8.3 Hz), 4.68 (2H, s), 4.26
(1H, d, J = 7.8Hz), 4.02 (1H,
m), 3.98 (1H, br.s), 3.96 (1H,
dd, J = 11.7, 6.3 Hz), 3.82 (1H,
m), 3.81 (3H, s), 3.74 (1H, dd,
J = 9.5, 7.8 Hz), 3.59 (1H, m),
3.49 (1H, tick), 3.42 (1H, d
d, J = 9.5, 3.4 Hz), 1.07-0.95
(2H, m), 0.02 (9H, s).

(3) Synthesis of Compound 21-3 Pyridine (4) in which Compound 21-2 (4.45 g) was dissolved
Acetic anhydride (20 ml) was added to the solution (0 ml), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed with 2% hydrochloric acid and saturated aqueous sodium hydrogen carbonate solution, and dried, and the solvent was evaporated under reduced pressure (5.47 g). Then, the obtained residue (5.
47 g) was dissolved in a methylene chloride (100 ml) solution, water (5 ml) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ, 7.56 g) were added, and the mixture was stirred at room temperature for 30 minutes. did. The reaction solution was diluted with methylene chloride, washed with saturated sodium hydrogen carbonate, then dried and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (n-hexane-ethyl acetate 3: 2) using silica gel (330 g) to give compound 21-3 (4.20 g) as a colorless powder.

¹ H-NMR (CDCl ₃ ) δ: 5.31
(1H, d, J = 3.4 Hz), 4.93 (1H, d
d, J = 10.0, 7.8 Hz), 4.43 (1H,
d, J = 7.8 Hz), 4.19-4.10 (2H,
m), 3.98 (1H, m), 3.84-3.78 (2
H, m), 3.55 (1H, m), 2.48 (1H, b)
r. d), 2.16, 2.11, 2.04 (each
3H, s), 0.98 (1H, ddd, J = 14.0,
10.5, 6.8Hz), 0.89 (1H, ddd, J
= 14.0, 10.0, 5.1 Hz), 0.00 (9
H, s).

(4) Synthesis of Compound 21-4 To a solution of Compound 21-3 (13.8 g) in methylene chloride (90 ml) was added pyridine (16 ml) and chloroacetyl chloride (5.40 ml), and the mixture was allowed to stand at room temperature. 30
Stir for minutes. After diluting the reaction solution with ethyl acetate, 2%
Washed with hydrochloric acid and saturated aqueous sodium hydrogen carbonate solution,
Then, it was dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography (n-hexane-ethyl acetate 3: 1) using silica gel (330 g) to give compound 21-4 (15.0 g) as a colorless powder.

[Α] _D ³³ −8.8 ° (c1.14, C
HCl ₃ ) IR (CHCl ₃ ): 1753 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 5.39 (1 H, d
d, J = 3.4, 1.0 Hz), 5.23 (1H, d
d, J = 10.5, 8.1 Hz), 5.08 (1H, d
d, J = 10.5, 3.4 Hz), 4.51 (1H,
d, J = 8.1 Hz), 4.22 (1H, dd, J = 1)
1.2, 6.3 Hz), 4.14 (1H, dd, J = 1)
1.2, 7.1 Hz), 4.00 (1 H, m), 3.9
7 (2H, s), 3.95 (1H, tricke), 3.
58 (1H, m), 2.16, 2.06, 2.05 (e
ach 3H, s), 0.99 (1H, ddd, J = 1
3.9, 10.7, 6.6 Hz), 0.92 (1H, d
dd, J = 13.9, 10.3, 5.1 Hz), 0.0
2 (9H, s).

(5) Synthesis of Compound 21-5 Compound 21-4 (3.55 g) was treated with methylene chloride (30 m).
l), dissolved in boron trifluoride dier ether complex (3.62 ml), and stirred at 0 ° C. for 5 hours. The reaction mixture was diluted with methylene chloride, washed with saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (n-hexane-ethyl acetate 3: 2) using silica gel (150 g) to give compound 21-5 (2.52 g) as a colorless powder. IR (CHCl ₃ ): 1747 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: It was confirmed that the 2- (trimethylsilyl) -ethyl group had been removed.

(6) Synthesis of Compound 21-6 Compound 21-5 (8.27 g) was treated with methylene chloride (50 m).
l) dissolved in trichloroacetonitrile (12.5m
l) and 1,8-diazabicyclo [5.4.0] -7-undecene (323 μl) were sequentially added, and the mixture was stirred at 0 ° C. for 30 minutes. The reaction mixture was concentrated under reduced pressure and then concentrated on silica gel (45
0g) and purified by column chromatography (n-hexane-ethyl acetate 3: 1) to give compound 21-6.
(9.10 g) Obtained as a colorless powder.

[Α] _D ²⁵ + 103.8 ° (c0.9
8, CHCl ₃ ) IR (CHCl ₃ ): 1753,1676 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.69 (1H,
s), 6.62 (1H, d, J = 3.4Hz), 5.5
6 (1H, m), 5.50 (1H, dd, J = 10.
7, 3.2 Hz), 5.41 (1H, dd, J = 10.
7,3.4 Hz), 4.45 (1H, tick),
4.18 (1H, dd, J = 11.2, 6.8Hz),
4.10 (1H, dd, J = 11.2, 6.6Hz),
4.01 (2H, s), 2.18, 2.04, 2.03
(Each 3H, s).

(7) Synthesis of Compound 21-7 Compound 21-6 (1.24 g) and Compound 10-1 (480 mg) were dissolved in a methylene chloride (8 ml) solution containing Molecular Sieves 4A (3 g), and the mixture was stirred at room temperature. After stirring for 2 hours, boron trifluoride diethyl ether complex (97 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 2 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using silica gel (120 g) (toluene-acetone-methanol 200:
100: 6) to give compound 21-7 (395
mg) was obtained as a colorless amorphous.

[Α] _D ³⁰ −27.9 ° (c0.97,
CHCl ₃ ) IR (CHCl ₃ ): 2108, 1753, 1676c
^{m -1 1 H-NMR (CDCl} 3) δ: 7.41-7.22
(20H, m), 6.01 (1H, d, J = 7.3H
z), 5.22 (1H, d, J = 3.2 Hz), 5.1
4 (1H, d, J = 3.7Hz), 5.01 (1H, d
d, J = 10.3, 8.1 Hz), 4.96, 4.7
5, 4.73, 4.40 (each 1H, d, J = 1)
2.0 Hz), 4.90 (1H, d, J = 7.1H)
z), 4.83 (2H, dlike), 4.79 (1
H, dd, J = 10.3, 3.4 Hz), 4.78,
4.69 (each 1H, d, J = 11.7 Hz),
4.55 (1H, d, J = 8.3Hz), 4.46 (1
H, q, J = 6.3 Hz), 4.17 (1H, dd, J
= 8.3, 8.1 Hz), 4.15-4.08 (2H,
m), 3.98 (1H, dd, J = 11.7, 5.9H)
z), 3.96 (1H, dd, J = 8.3, 8.1H)
z), 3.93 (2H, s), 3.91-3.85 (2
H, m), 3.81-3.76 (2H, m), 3.69.
-3.55 (23H, m), 3.48-3.43 (2
H, m), 3.37 (2H, t, J = 5.1Hz),
2.01, 2.00, 1.88, 1.77 (each
3H, s), 1.17 (3H, d, J = 6.3Hz).

(8) Synthesis of Compound 21-8 A solution of compound 21-7 (210 mg) in ethanol (4 ml) -ethylene chloride (1 ml) was added to thiourea (58 mg) and 2,6-lutidine (35.5 μm).
l) was added, and the mixture was stirred at 60 ° C. for 1 hr, the reaction mixture was diluted with methylene chloride, washed with saturated aqueous sodium hydrogen carbonate solution, dried, and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography using silica gel (40 g) (methylene chloride-methanol 40: 1) to obtain Compound 21-8 (162 mg) as a colorless amorphous substance.

[Α] _D ³⁰ −29.6 ° (c1.06
CHCl ₃ ) IR (CHCl ₃ ): 2108, 1747, 1676c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 7.41-7.24
(20H, m), 6.05 (1H, d, J = 7.3H
z), 5.19 (1H, d, J = 3.4 Hz), 5.1
4 (1H, d, J = 3.7 Hz), 4.95, 4.8
2, 4.70, 4.68 (each 1H, d, J = 1
1.7 Hz), 4.90 (1H, d, J = 7.3H)
z), 4.83, 4.78, 4.74, 4.42 (ea)
ch 1H, d, J = 12.0 Hz), 4.76 (1
H, tlike), 4.53 (1H, d, J = 8.1H
z), 4.46 (1H, q, J = 6.3 Hz), 4.1
7 (1H, dd, J = 8.3, 8.1Hz), 4.14
-4.10 (2H, m), 3.96 (1H, m), 3.
95 (1H, dd, J = 8.3, 8.1Hz), 3.9
3 (1H, m), 3.87 (1H, dt, J = 11.
2,4.4 Hz), 3.85 (1H, dd, J = 10.
7, 3.2 Hz), 3.79 (1H, dd, J = 10.
7, 3.2 Hz), 3.68-3.48 (26H,
m), 2.38 (1H, br.s), 2.08, 2.0
2, 1.92, 1.76 (each 3H, s), 1.
14 (3H, d, J = 6.3 Hz).

(9) Synthesis of Compound 21-9 To a solution of compound 21-8 (373 mg) in dimethylformamide (5 ml) was added sulfur trioxide pyridine complex (398 mg), and the mixture was stirred at 55 ° C. for 1 hour. After the reaction solution was concentrated, it was dissolved in methanol (5 ml), and a cation exchange resin (Dowex 50w Na ⁺ ) was added.
Stir for 0 minutes. The insolubles were removed by filtration, the filtrate was concentrated under reduced pressure, and the obtained residue was subjected to column chromatography using silica gel (30 g) (methylene chloride: methanol).
8: 1) to give compound 21-9 (25
2 mg) was obtained as a colorless amorphous.

[Α] _D ²⁹ -38.2 ° (c0.97,
CHCl ₃ ) IR (CHCl ₃ ): 3450, 2110, 1747,
1670 cm ^{-1 1} H-NMR (CDOD ₃ ) δ: 7.44-7.22
(20H, m), 5.54 (1H, d, J = 3.7H
z), 5.33 (1H, d, J = 3.7Hz), 5.0
0 (1H, dd, J = 10.0, 8.3Hz), 4.8
7, 4.68, 4.54 (each 1H, d, J = 1
1.5 Hz), 4.81, 4.79, 4.71, 4.5
5 (each 1H, d, = 12.0 Hz), 4.76
(1H, dlike), 4.76 (1H, m), 4.6
7 (1H, d, J = 8.3Hz), 4.45 (1H,
d, J = 8.1 Hz), 4.37 (1H, dd, J = 1)
0.0, 3.7 Hz), 4.17 (1H, dd, J = 1)
1.0, 8.1 Hz), 4.04-3.91 (7H,
m), 3.90 (1H, dd, J = 11.0, 6.1H
z), 3.82 (1H, m), 3.75 (1H, br.
d) 3.71-3.56 (22H, m), 3.46.
(1H, m), 3.38 (2H, tick), 2.0
6, 2.00, 1.99, 1.94 (each 3H,
s), 1.22 (3H, d, J = 6.6Hz).

(10) Synthesis of Compound 21-10 Compound 21-9 (160 mg) was dissolved in a 2% sodium methoxide-methanol solution (3.5 ml), and the mixture was stirred at room temperature for 30 minutes. After adding water (0.5 ml) and dry ice and stirring for 10 minutes, the reaction solution was concentrated, and then the residue was purified by column chromatography (methanol) using polymer gel (200 cc) to give compound 21-10. (130 mg) was obtained as a colorless powder.

[Α] _D ²⁹ -54.8 ° (c0.98,
CHCl ₃ ) IR (CHCl ₃ ): 3450, 2106, 1668c
m ^{-1 1} H-NMR (CD ₃ OD) δ: 7.42-7.22
(20H, m), 5.36 (1H, d, J = 3.9H
z), 4.89-4.81 (6H, m), 4.77 (1)
H, d, J = 11.7 Hz), 4.64 (1H, d, J
= 12.0 Hz), 4.57-4.54 (3H, m),
4.48 (1H, d, J = 7.8Hz), 4.41 (1
H, d, J = 8.3 Hz), 4.16 (1H, dd, J
= 9.3, 9.3 Hz), 4.13-4.06 (4H,
m), 4.00-3.91 (4H, m), 3.79 (1
H, m), 3.71-1.55 (24H, m), 3.5
3 (1H, m) 3.33 (2H, t, J = 5.1H
z), 2.01 (3H, s), 1.15 (3H, d, J
= 6.6 Hz).

(11) Synthesis of Compound 21-11 A solution of compound 21-10 (20 mg) in methanol (4 ml) was added with palladium-carbon (10%, 40 m).
g) and 1N hydrochloric acid (34 μl) were added, and the mixture was stirred under a hydrogen stream (1 atm) at room temperature for 2 hours. The catalyst was filtered off from the reaction solution, the filtrate was concentrated, and the residue was purified by column chromatography (methylene chloride: methanol: water: 5: 5: 1) using silica gel (1.2 g) to give compound 21- 11 (10 mg) was obtained as a colorless powder.

[Α] _D ²⁹ -56.6 ° (c0.33
MeOH) IR (KBr): 3418, 1675 cm ^{-1 1} H-NMR (CD ₃ OD) δ: 5.05 (1 H, d,
J = 3.9 Hz), 4.81 (1H, m), 4.57
(1H, d, J = 7.6 Hz), 4.44 (1H, d,
J = 8.3 Hz), 4.21 (1H, dd, J = 9.
5.3.2 Hz), 4.19 (1H, br.d), 4.
05 (1H, dd, J = 10.0, 8.3Hz), 4.
04-3.99 (4H, m), 3.93 (1H, dd,
J = 9.3, 9.3 Hz), 3.91 (1H, dd, J
= 12.0, 2.4 Hz), 3.84 (1H, dd, J
= 10.5, 3.4 Hz), 3.82 (1H, m),
3.80 (1H, dd, J = 9.3Hz, 9.3H
z), 3.79-3.62 (23H, m), 3.49.
(1H, m), 3.42 (1H, m), 3.22 (1
H, ddd, J = 13.5, 6.1, 4.4 Hz),
3.16 (1H, ddd, J = 13.5, 5.6, 4.
4Hz), 1.98 (3H, s), 1.16 (3H,
d, J = 6.6 Hz).

(12) Synthesis of Sulfated Lewis X Modified CM Pullulan (Compound 21) Carboxymethyl Pullulan (30 mg) and Compound 2
Water (1.3 ml) in which 1-11 (67.5 mg) was dissolved
And N, N'-dimethylformamide (1.3 ml)
1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (366 mg) and sodium hydrogen carbonate (6.5 mg) were added to the mixed solution of, and the mixture was stirred at 40 ° C. for 110 hours, and then sodium hydrogen carbonate (13 mg). Was further added, and the reaction solution was concentrated. The reaction mixture was added to 99.5% ethanol (35 ml) to precipitate a crude product, and then the precipitate was added in order of 95% ethanol (40 ml × 3 times), acetone (40 ml), diethyl ether (40 ml). It was washed and then dried under reduced pressure.

Then, the crude product was dissolved in 10 ml of water, and then purified water (1000) was used using a dialysis membrane (Spectra / Pore Co .: molecular weight exclusion limit 12000 to 14000).
(0 ml) as an external solution for 16 hours at room temperature, and the dialyzed internal solution is lyophilized under reduced pressure to give sulfated Lewis X-modified carboxymethyl pullulan (compound 21, 28 mg,
ds: 0.03) was obtained.

Example 22 (1) Synthesis of Compound 22-1 Compound 21-2 (2.78 g) dissolved in pyridine (2
Benzoyl chloride (20 ml) was added to the solution (0 ml), and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed with 2% hydrochloric acid and saturated aqueous sodium hydrogen carbonate solution, and then dried, and the solvent was evaporated under reduced pressure. Then, the obtained residue was purified by column chromatography (n-hexane-ethyl acetate 6: 1) using silica gel (330 g) to obtain compound 22-1 (4.92 g) as a colorless powder.

[Α] _D ³⁰ + 65.9 ° (c 1.10,
CHCl ₃ ) IR (CHCl ₃ ): 1724 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.27,8.14,
8.05 (each2H, dlike), 7.71-
7.65 (4H, m), 7.59-7.52 (6H,
m), 7.12 (1H, d, J = 8.8Hz), 6.6
8 (1H, d, J = 8.8Hz), 5.97 (1H, d
d, J = 3.4, 1.0 Hz), 5.58 (1H, d
d, J = 10.0, 8.1 Hz), 4.71 (1H,
d, J = 8.1 Hz), 4.71, 4.51 (each)
1H, d, J = 12.7 Hz), 4.70 (1H,
m), 4.53 (1H, dd, J = 10.9, 6.1H)
z), 4.16 (1H, tricke), 4.08 (1
H, m), 3.86 (1H, dd, J = 10.0, 3.
4 Hz), 3.80 (3H, s), 3.66 (1H,
m), 1.02 (1H, ddd, J = 13.9, 11.
0, 6.6 Hz), 0.95 (1H, ddd, J = 1
3.9, 10.5, 5.4 Hz), -0.01 (9H,
s).

(2) Synthesis of compound 22-2 A solution of compound 22-1 (4.20 g) in methylene chloride (60 ml) was mixed with water (3 ml) and 2,3-dichloro-5,6-dicyano-p. -Benzoquinone (DDQ,
4.02 g) was added and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with methylene chloride, washed with saturated sodium hydrogen carbonate, then dried and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (n-hexane-ethyl acetate 4: 1) using silica gel (150 g) to give compound 22-2 (4.20 g) as a colorless powder.

[Α] _D ²⁸ -1.2 ° (c1.00, C
HCl ₃ ) IR (CHCl ₃ ): 3520, 1726 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.18-8.16
(2H, m), 8.07-8.03 (4H, m), 7.
63-7.55 (3H, m), 7.51-7.42 (6
H, m), 5.77 (1H, d, J = 3.4 Hz),
5.35 (1H, dd, J = 10.0, 7.8Hz),
4.74 (1H, d, J = 7.8Hz), 4.60 (1
H, dd, J = 11.2, 7.1 Hz), 4.44 (1
H, dd, J = 11.2, 6.1 Hz), 4.15 (1
H, tick), 4.12 (1H, m), 4.04.
(1H, m), 3.65 (1H, m), 2.73 (1
H, m), 0.99 (1H, ddd, J = 13.9, 1
1.0, 6.6 Hz), 0.92 (1H, ddd, J =
13.9, 10.5, 5.3 Hz), -0.06 (9
H, s).

(3) Synthesis of Compound 22-3 To a methylene chloride (1.5 ml) solution in which compound 22-2 (415 mg) was dissolved, pyridine (300 μl) and chloroacetyl chloride (112 μl) were added, and the mixture was allowed to stand at room temperature for 3 minutes.
Stir for 0 minutes. After diluting the reaction solution with methylene chloride,
The extract was washed with 1% hydrochloric acid and saturated aqueous sodium hydrogen carbonate solution, and then dried, and the solvent was evaporated under reduced pressure (5.47).
g). The obtained residue was subjected to column chromatography using silica gel (30 g) (n-hexane-ethyl acetate).
Compound 22-3 (434) by purification with 6: 1).
mg) as a colorless oil.

[Α] _D ³⁰ + 30.6 ° (c1.06
CHCl ₃ ) IR (CHCl ₃ ): 1770, 1720 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.15 (2H, dl
ike), 8.01-7.99 (4H, m), 7.63
(1H, tricke), 7.60-7.55 (2H,
m), 7.51 (2H, tick), 7.46-7.
42 (4H, m), 5.83 (1H, dlike),
5.61 (1H, dd, J = 10.3, 8.1Hz),
5.43 (1H, dd, J = 10.3, 3.4Hz),
4.77 (1H, d, J = 8.1Hz), 4.67 (1
H, dd, J = 11.2, 6.6 Hz), 4.39 (1
H, dd, J = 11.2, 6.8 Hz), 4.25 (1
H, tick), 4.04 (1H, ddd, J = 1)
1.0, 9.8, 5.6 Hz), 3.91 (1H, d,
J = 15.3 Hz), 3.84 (1H, d, J = 15.
3Hz), 3.64 (1H, m), 0.96 (1H, d)
dd, J = 13.9, 11.0, 6.6 Hz), 0.9
0 (1H, ddd, J = 13.9, 10.5, 5.6H
z), -0.067 (9H, s).

(4) Synthesis of Compound 22-4 Compound 22-3 (402 mg) was treated with methylene chloride (5 m
l), dissolved in boron trifluoride diethyl ether complex (369 μl), and stirred at 0 ° C. for 5 hours. The reaction mixture was diluted with methylene chloride, washed with saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was evaporated under reduced pressure (354 m
g). The resulting residue (354 mg) was dissolved in methylene chloride (50 ml), trichloroacetonitrile (2 ml) and potassium carbonate (41 mg) were added,
The mixture was stirred at room temperature for 20 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and then column chromatography using silica gel (45 g) (n-hexane-ethyl acetate 5:
The compound 22-4 (152 mg) was obtained as a colorless powder by purification in 1).

[Α] _D ³⁰ + 103.8 ° (c0.9
5, CHCl ₃ ) IR (CHCl ₃ ): 1770,1728,1678c
m ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.63 (1H,
s), 8.13 (2H, dlike), 7.99-7.
96 (4H, m), 7.65 (1H, tick),
7.60-7.50 (4H, m), 7.46-7.40
(4H, m), 6.88 (1H, d, J = 3.7H
z), 6.01 (1H, m, H-4), 5.88 (1
H, dd, J = 10.7, 3.2 Hz), 5.78 (1
H, dd, J = 10.7, 3.7 Hz), 4.79 (1
H, tick), 4.60 (1H, dd, J = 11.
2,6.8 Hz), 4.40 (1H, dd, J = 11.1.
2, 6.1 Hz), 3.96 (1H, d, J = 15.4)
Hz), 3.91 (1H, d, J = 15.4Hz).

(5) Synthesis of Compound 22-5 In a dimethylformamide solution (50 ml) in which Compound 1-2 (13.8 g) was dissolved, sodium azide (3.9) was added.
0 g) was added and the mixture was stirred at 70 ° C. for 12 hours. The reaction solution was cooled to room temperature, ethyl acetate (150 ml) was added,
The precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using silica gel (330 g) (methylene chloride-methanol 25: 1) to give compound 22-5 (11.5 g) as a colorless oil.

IR (CHCl ₃ ): 2106 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 3.75-3.63
(20H, m), 3.62-3.60 (2H, m),
3.39 (2H, t, J = 5.1Hz).

(6) Synthesis of compound 22-6 Compound 22-4 (140 mg) in a methylene chloride (5 ml) solution containing molecular sieves 4A (1.5 g),
Compound 22-5 (191 mg) was dissolved and stirred at room temperature for 2 hours, boron trifluoride diethyl ether complex (51 μl) was added at 0 ° C., and the mixture was stirred at the same temperature for 5 hours. After filtering the reaction solution, the filtrate was washed with a saturated aqueous sodium hydrogen carbonate solution, dried and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography using silica gel (35 g) (methylene chloride-methanol 100: 1) to give compound 22-6 (134 mg) as a colorless amorphous substance.

[Α] _D ²⁷ + 16.5 ° (c1.06
CHCl ₃ ) IR (CHCl ₃ ): 2108, 1769, 1730c
m ^{-1 1} H-NMR (CDCl ₃ ) δ: 8.16-8.13
(2H, m), 8.02-7.98 (4H, m), 7.
45-7.40 (9H, m), 5.84 (1H, dd,
J = 3.4, 1.0 Hz), 5.61 (1H, dd, J
= 10.3, 8.1 Hz), 5.44 (1H, dd, J
= 10.3, 3.4 Hz), 4.89 (1H, d, J =
8.1 Hz), 4.66 (1H, dd, J = 11.2,
6.6 Hz), 4.38 (1H, dd, J = 11.2,
6.8 Hz), 4.27 (1H, tick), 4.0
1 (1H, m), 3.91, 3.86 (each 1
H, d, J = 15.4 Hz), 3.82 (1 H, m),
3.68-3.58 (14H, m), 3.54-3.5
1 (2H, m), 3.47-3.43 (2H, m),
3.40-3.31 (4H, m).

(7) Synthesis of Compound 22-7 To a solution of Compound 22-6 (230 mg) in ethanol (3 ml) -ethylene chloride (1 ml) was added thiourea (106 mg) and 2,6-lutidine (65 μl), After stirring at 60 ° C. for 7 hours, the reaction solution was diluted with methylene chloride, washed with 2% hydrochloric acid and saturated aqueous sodium hydrogen carbonate solution, dried, and the solvent was evaporated under reduced pressure. The obtained residue was subjected to column chromatography using silica gel (20 g) (n-hexane-ethyl acetate-methanol 20.
0: 300: 5) to give compound 22-7.
(160 mg) was obtained as a colorless amorphous.

[Α] _D ^26.5 −0.9 ° (c1.07,
CHCl ₃ ) IR (CHCl ₃ ): 2108, 1724 cm ^{−1 1} H-NMR (CDCl ₃ ) δ: 8.16, 8.08,
8.04 (each2H, dlike), 7.62,
7.59, 7.57 (each 2H, tick),
7.49, 7.46, 7.44 (each 2H, tl
ike), 5.78 (1H, d, J = 3.2 Hz),
5.37 (1H, dd, J = 9.8, 7.8Hz),
4.87 (1H, d, J = 7.8Hz), 4.60 (1
H, dd, J = 11.5, 6.8 Hz), 4.43 (1
H, dd, J = 11.5, 6.3 Hz), 4.19-
4.14 (2H, m), 4.00 (1H, m), 3.8
4 (1H, m), 3.67-3.58 (14H, m),
3.57-3.54 (2H, m), 3.53-3.50
(2H, m), 3.46-3.43 (2H, m), 3.
35 (2H, t, J = 5.1Hz), 2.88 (1H,
d, J = 6.0 Hz).

(8) Synthesis of Compound 22-8 A sulfur trioxide pyridine complex (75 mg) was added to a dimethylformamide (3 ml) solution in which the compound 22-7 (140 mg) was dissolved, and the mixture was stirred at 45 ° C. for 12 hours. After the reaction solution was concentrated, it was dissolved in methanol (3 ml), and a cation exchange resin (Dowex 50w Na ⁺ ) was added.
Stir for 0 minutes. The insoluble material was filtered off, the filtrate was concentrated under reduced pressure, and the obtained residue was subjected to column chromatography using silica gel (20 g) (methylene chloride: methanol).
Compound 22-8 (81 m
g) was obtained as a colorless amorphous.

[Α] _D ²⁵ + 38.3 ° (c0.70,
CHCl ₃ ) IR (CHCl ₃ ): 2108, 1724 cm ^{−1 1} H-NMR (CD ₃ OD) δ: 8.14-8.11
(4H, m), 8.00 (2H, dlike), 7.6
5, 7.60, 7.58 (each 2H, tlik
e), 7.52, 7.47, 7.42 (each 2)
H, tlike), 6.11 (1H, d, J = 3.2H
z), 5.57 (1H, dd, J = 10.3, 8.1H
z), 4.97 (1H, d, J = 8.1Hz), 4.9.
5 (1H, dd, J = 10.3, 3.4Hz), 4.4
9-4.45 (2H, m), 4.39 (1H, tlik
e) 3.96 (1H, dd, J = 11.7, 4.2H)
z), 3.77 (1H, dd, J = 11.7, 4.9H)
z), 3.67-3.55 (14H, m), 3.51-
3.48 (2H, m), 3.45-3.41 (2H,
m), 3.39-3.33 (4H, m).

(9) Synthesis of Compound 22-9 Compound 22-8 (70 mg) was dissolved in 3% sodium methoxide-methanol solution (2 ml), and the mixture was stirred at room temperature for 30 minutes. After adding water (0.2 ml) and dry ice and stirring for 10 minutes, the reaction solution was concentrated, and the residue was purified by column chromatography (methanol) using polymer gel (90 cc) to give compound 22- 9 (45 mg) was obtained as a colorless amorphous.

[Α] _D ^25.5 -7.4 ° (c0.31,
CHCl ₃ ) IR (CHCl ₃ ): 3450, 2108 cm ^{−1 1} H-NMR (CD ₃ OD) δ: 4.41 (1 H, d,
J = 7.8 Hz), 4.27 (1H, dd, J = 9.
8.2.9 Hz), 4.19 (1H, br.s), 4.
06 (1H, m), 3.81-3.73 (4H, m),
3.73-3.61 (20H, m), 3.59 (1H,
m) 3.43-3.39 (2H, m).

(10) Synthesis of Compound 22-10 Compound 22-9 (37 mg) dissolved in methanol (7
ml) solution, palladium-carbon (10%, 40 mg).
And 1N hydrochloric acid (175 μl) were added, and under hydrogen flow (1 at
m), and stirred at room temperature for 1 hour. After the catalyst was filtered off from the reaction solution, the filtrate was concentrated and purified by column chromatography (methanol) using polymer gel (90 cc) to obtain Compound 22-10 (31 mg) as a colorless powder.

[Α] _D ²⁷ -3.2 ° (c0.56, M
OH) ¹ H-NMR (CD ₃ OD) δ: 4.42 (1 H, d,
J = 7.8 Hz), 4.27 (1H, dd, J = 9.
8, 3.2 Hz), 4.20 (1H, br.d), 4.
07 (1H, m), 3.82-3.62 (23H,
m), 3.61-1.58 (2H, m), 3.22-
3.17 (2H, m).

(11) Synthesis of galactose-3-sulfate modified carboxymethyl pullulan (compound 22) Carboxymethyl pullulan (35 mg) and compound 22-
10 (51 mg) dissolved in water (1.5 ml) and N,
To a mixed solution of N'-dimethylformamide (1.5 ml), 1-ethoxycarbonyl-2-ethoxy-1,2-
Dihydroquinoline (EEDQ, 427 mg) and sodium hydrogen carbonate (7.5 mg) were added, and the mixture was stirred at 40 ° C. for 110 hr, sodium hydrogen carbonate (15 mg) was further added, and the reaction mixture was concentrated. The reaction mixture was added to 99.5% ethanol (35 ml) to precipitate a crude product, and then the precipitate was added in order of 95% ethanol (40 ml × 3 times), acetone (40 ml), diethyl ether (40 ml). It was washed and then dried under reduced pressure.

Then, the crude product was dissolved in 10 ml of water, and then purified water (10 liters) was used as an external liquid at room temperature using a dialysis membrane (manufactured by Spectra / Pore Co., Ltd .: molecular weight exclusion limit 12000 to 14000). It is dialyzed for 16 hours, and the dialysis solution is freeze-dried under reduced pressure to give galactose-3-
Sulfate-modified carboxymethyl pullulan (compounds 22, 32
mg, ds: 0.10) was obtained.

The compounds of the above Examples are shown in the table below.

[0214]

[Table 1]

The synthetic process of the above examples is shown below as a scheme.

[0216]

[Chemical 7]

[0219]

Embedded image

[0218]

[Chemical 9]

[0219]

[Chemical 10]

[0220]

[Chemical 11]

[0221]

[Chemical 12]

[0222]

[Chemical 13]

[0223]

Embedded image

[0224]

[Chemical 15]

[0225]

Embedded image

[0226]

[Chemical 17]

[0227]

[Chemical 18]

[0228]

[Chemical 19]

[0229]

Embedded image

[0230]

[Chemical 21]

Pharmacological test (1) Organ mobilization (I) 1 mg / in the jugular vein of SD male rats (about 200 g)
The ³ H-labeled compound according to the invention was administered at a dose of kg. One group consisted of three animals, and about 1, 2 and 3 minutes after administration, about 0.2 ml of blood was collected from the jugular vein, plasma after centrifugation was precisely weighed, and burned with a sample oxidizer.
Then, the radioactivity was measured with a liquid scintillation counter, and the plasma concentration was calculated.

Five minutes after the administration of the compound of the present invention, the blood was killed and the main organs were removed. Next, each organ was cut into an appropriate size and precisely weighed, the radioactivity was measured, and the concentration in each organ was calculated. The distribution clearance of each organ is calculated by measuring the AUC of the plasma concentration up to 5 minutes after the administration.
It was calculated by dividing by (area under the curve). The clearance of each organ when the compound according to the present invention is administered is shown in Table 2.
As shown in.

[0233]

[Table 2]

(2) Organ migration (II) ICR male mice (about 30 g) were coated with arachidonic acid dissolved in acetone (20 mg at 50 mg / ml).
Immediately after inflammation, the ¹²⁵ I-labeled compound of the present invention was administered to the tail vein at a dose of 1 μg / kg. Blood was collected at 0.5, 1, 2, 4, and 6 hours after administration and blood was removed, and then lung, spleen, kidney, and liver were extracted. The concentration of the compound of the present invention in each organ was determined by measuring radioactivity with a γ counter. Changes in plasma and organ concentrations A
UC (area under the curve) was determined by the trapezoidal method. The transferability of unmodified carboxymethyl pullulan and the compounds of Examples 9 and 10 into each organ is as shown in Table 3 below. In order to evaluate specific transferability, the ratio of AUC between each organ and plasma up to 6 hours was compared. As a result, the compound of Example 9 avoided the reticuloendothelial system tissues of the liver and spleen.

Table 3 (AUC O-6h, tissue) / (AUC 0-6h, plasma) Lung Spleen Kidney Liver Carboxymethyl pullulan 0.18 1.99 0.15 1.24 Compound of Example 9 0.11 0.23 0.10 0.25

(3) Inflammatory site migration (I) Inflammatory site migration can be evaluated by, for example, the inhibitory activity of infiltration of neutrophils into inflammation sites, and the infiltration of neutrophils into inflammation sites. Can be determined by measuring the activity of myeloperoxidase (hereinafter referred to as "MPO"), which is a marker enzyme for neutrophils (Bradley, PP, et al. (1982) J. Invest. Der
matol. 78 , 206-209). The measurement was specifically performed as follows.

IL-1β was intradermally administered to the right ear of male ICR mice (about 30 g) (2 ng / 20 μl physiological saline), and 2 hours later, the compound according to the present invention (Examples 9 and 1).
0) was administered into the tail vein at an appropriate concentration. After 4 hours, the edema-causing part of the right ear was punched and stored at -80 ° C until measurement. 1m of punched pinna
l of 5 mM EDTA and 0.5% Cetyltrimethyl
50 mM phosphate buffer containing ammonium bromide (pH
6.0) minced into 3 ml of the same buffer
Add, and use a Polytron homogenizer under ice cooling for 20,000 m.
Homogenized at in-1 for 3 minutes. Further, freeze-thawing was repeated 3 times, and after each thawing, ultrasonic treatment was performed for 10 seconds under ice cooling. After centrifugation at 3000 rpm for 20 minutes, the supernatant was transferred to a microtube and centrifuged again at 14000 rpm for 15 minutes to measure the MPO activity of the supernatant. The MPO activity was measured using a 96-well plate, and the supernatant was
l, 3,3 ', 5,5'-tetramethylbenzidine to 2
5 ml (dissolved in dimethyl sulfoxide gives a final concentration of 0.16 mM), 200 ml of H ₂ O ₂ (0.
After diluting with 08M phosphate buffer (pH 5.4) and adding in the order of final concentration of 0.24 mM and incubating at 37 ° C for 5 minutes, 25 ml of bovine catlase (final concentration 13.
6 mg / ml) was added to stop the reaction. MTP after reaction
660 with -32 microplate reader (Corona)
The absorbance at nm was measured. Human leukocyte-derived MPO was used to prepare the standard curve. The result is as shown in FIG.

(4) Inflammatory site migration (II) Arachidonic acid dissolved in acetone was applied to the right ear of an ICR male mouse (about 30 g) (20 mg of 50 mg / ml was applied).
Immediately after inflammation, the ³ H-labeled compound of the present invention (Examples 7 and 10) was administered to the tail vein at a dose of 1 mg / kg. 0.08, 0.5, 1, 2, 4, after administration
Blood was removed at 6, 8, 12, and 24 hours, and the right and left ears were extracted. For the concentration in both ears, ³
After recovering H as H ₂ O, radioactivity was measured with a liquid scintillation counter to determine the concentration of the compound according to the present invention. The AUC (area under the curve) of the concentration transition in both ears was determined by the trapezoidal method. The results are shown in Table 4. The compounds of Examples 7 and 10 had significantly increased AUC _0-24h to the inflamed right ear compared to unmodified polysaccharide.

Table 4 Specimen AUC 0-24h (%: dose / hr / g / tissue) Right ear (a) Left ear Plasma (b) (a / b) Sugar Modified Carboxymethyl Chitosan Compound of Example 1 46.5 8.2 61.4 0.76 Compound of Example 7 126.7 15.4 124.4 1.02 Sugar Modified Carboxymethyl Pullulan Compound of Example 9 276.8 33.6 588.9 0.47 Compound of Example 10 692.2 24.6 368.4 1.88

[Brief description of drawings]

FIG. 1 is a view showing the neutrophil infiltration inhibitory activity of a compound according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C08B 37/08 A 7433-4C Z 7433-4C 37/10 7433-4C // A61K 31/405 AAH 31/505 ADU 31/57 ABE 31/71 (72) Inventor Shiro Miyoshi 206-1 Anku, Mishima City, Shizuoka Prefecture Tamura Heights No. 5 (72) Inventor Syugenbara 1 Nishi-Kashiwadai, Kashiwa City, Chiba Prefecture 2-1- 1 City Palace Kashiwa 1018 (72) Inventor Satoshi Okuno 8-5-18 Waseda, Misato City, Saitama Prefecture (72) Inventor Hiroshi Hamana 2694 Yamazaki, Noda City, Chiba Prefecture View Parade No. Umego A-307 (72) Inventor I Kamiwasa 5-6-6 Matsugaoka, Funabashi, Chiba Prefecture (72) Inventor Teruomi Ito 6-89, Shin Matsudo, Matsudo City, Chiba Prefecture 104 (72) Inventor, Takayuki Kawaguchi Sugamo, Toshima-ku, Tokyo 1-15 2-A406

Claims (5)

[Claims] 1. A compound represented by the following general formula (I): ^{E-T 1 -T 2 -F (} I) ( In the formula, E is chosen glucose, fructose, mannose, fucose, neuraminic acid, uronic acid, galactosamine, glucosamine, ribose, deoxyribose, arabinose and xylose Monosaccharides, or N- or O-acyl derivatives of these monosaccharides, O-
Represents an alkyl derivative or an ester derivative, or represents an oligosaccharide consisting of 2 to 6 of these monosaccharides or derivatives of monosaccharides, and T ¹ represents —CH ₂ CH ₂ (OCH ₂ C
H ₂ ) m- (m represents an integer of 1 to 10),-(CH
₂ ) n- (n represents an integer of 2 to 16) or the following group (II): (In the above group, X is, -NHCOCH _3, -OH or -
NH ₂ represents a) ^{T 2} is, -NH -, - NHCO -, - CONH- or an -NHCONH-, F is chitosan, pullulan,
Represents a polysaccharide selected from dextran, mannoglucan, heparin, hyaluronic acid, and chondroitin sulfate or a derivative of these polysaccharides) 2. E is a monosaccharide or a derivative of a monosaccharide selected from glucose, mannose, galactose, fucose, neuraminic acid, galactosamine, glucosamine, sialic acid, N-acetylgalactosamine and N-acetylglucosamine, or a derivative thereof. The compound according to claim 1, which represents an oligosaccharide consisting of 2 to 4 sugar or monosaccharide derivatives, and F represents carboxymethyl chitosan or carboxymethyl pullulan. 3. The compound according to claim 1, which is a drug carrier. 4. The compound according to any one of claims 1 to 3, wherein the polysaccharide derivative carries a pharmaceutical compound. 5. The compound according to claim 4, wherein the pharmaceutical compound is selected from methotrexate, doxorubicin, dexamethasone, indomethacin.