JP2791001B2 - Glycosyl-protein derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a glycosyl-protein derivative useful as a carrier for selectively transporting a drug to a target tissue, particularly bone marrow or brain, and an intermediate thereof.

[0002]

2. Description of the Related Art Bone marrow tissues include red blood cells, lymphocytes, monocytes,
It is a tissue that produces granulocytes and is the most important tissue responsible for hematopoiesis and immunity in the body. Radiation therapy and anticancer drug administration in cancer patients cause bone marrow hypofunction,
It is an inevitable side effect at present, and as a result of myelosuppression, a severe decrease in white blood cells and platelets, particularly severe infections associated with a decrease in granulocytes, has become a serious problem.

[0003] Further, in order for a drug to be transported to brain tissue, it must pass through the blood-brain barrier, which hinders the transfer of the drug into the brain. For this reason, development of a means for delivering a drug into the brain has been demanded.

On the other hand, with the recent development of immunology, many bioactive proteins have been isolated and purified, and their pharmacological actions have been elucidated. Furthermore, with the development of genetic engineering technology, many cytokines such as erythropoietin, a hematopoietic hormone for erythrocytes, several types of colony stimulating factors (CSF), which are hematopoietic factors for leukocytes, and α-, β
-, Γ-interferon, interleukin 2, etc.
Mass production has become possible, and applications to pharmaceuticals have been attempted.

However, many cytokines are
After being administered in vivo, it is rapidly metabolized,
There is a problem that the onset of the drug effect in the target tissue cannot be sufficiently achieved.

In order to solve this problem, as carriers for transporting physiologically active substances and synthetic drugs to target sites, antibodies, glycoproteins, lipoproteins, lectins, hormones, liposomes, deoxyribonucleic acids, polysaccharides, synthetic polymers, A wide range of substances ranging from naturally occurring polymers, molecular assemblies and synthetic polymers, such as polyamino acids, have been proposed.

For example, Proc. Nath. Acad. Sci. USA 84 ,
1487-1491 (1987) reports a study in which several cytokines are modified with a synthetic polymer such as polyethylene glycol to increase the half-life in vivo without losing the activity of the cytokines themselves.

Japanese Patent Application Laid-Open No. 63-152393 discloses that a polyethylene glycol derivative having a sugar chain can be used for modifying cytokines. -Suggested for use to enhance delivery to tissues. However, there are no reports on bone marrow or brain tropism.

[0009]

DISCLOSURE OF THE INVENTION The present invention utilizes a sugar recognizing property on the surface of bone marrow or brain cells to deliver more drugs (such as cytokines and low-molecular-weight drugs) to the target bone marrow or brain tissue. The invention of a drug delivery carrier that enables a drug to delay the in vivo half-life of a drug, and develops a system (Targeting Drug Delivery System) for delivering a target drug to a target tissue. It is the purpose.

[0010] In particular, the present invention relates to patients having hematopoietic dysfunction such as aplastic anemia, abnormal bone marrow dysfunction such as immunodeficiency associated with various lymphocytic diseases, furthermore, myeloid leukemia,
Efficiently collect drugs used in the treatment of bone marrow tissue in patients with myeloid cancer such as myelomes, plasmacytomas, and multiple myeloma, or in patients with reduced bone marrow function due to side effects associated with cancer treatment Accordingly, it is an object of the present invention to develop a drug delivery carrier which can delay the half-life of the drug in vivo and is expected to have higher efficacy.

Another object of the present invention is to develop a drug delivery carrier directly into the brain for treating Alzheimer's disease and the like.
It allows treatment while retaining its activity and reducing the possibility of side effects.

[0012]

The present invention SUMMARY OF] has the formula: _{[R-O- (CH 2) 2} O (CH 2) 2 O (CH 2) 3 -CO- ] _m -Z (I) (wherein R represents a glycosyl group, Z represents a protein, and m represents 5 to 50).

The above-mentioned compound of the present invention can be used as a drug delivery carrier for cells, organs, organs, particularly bone marrow or brain tissues, which are intended for drugs. The drug carrier is (i)
Good biocompatibility, (ii) stability for a certain period of time after administration, and (iii) release of the drug by chemical or enzymatic reaction when the drug reaches the site of action is required. .

The sugar chain constituting the carrier of the above formula can be used as a target recognition site having the ability to specifically recognize a target cell organ, organ or the like. Examples of the glycosyl group represented by R include a xylopyranosyl group, a mannopyranosyl group, a fucopyranosyl group, a 2-galactopyranosyl group, a 2-acetamido-2-deoxy-fucopyranosyl group, a 2-acetamido-2-deoxy-mannopyranosyl group, Monosaccharides such as acetamido-2-deoxy-galactopyranosyl group; mannopyranosyl-mannopyranosyl group, (2-acetamido-2-deoxy-mannopyranosyl) -mannopyranosyl group, (2-acetamido-2-deoxy-glucopyranosyl) -mannopyranosyl Group, fucopyranosyl- (2-acetamido-2-deoxy-glucopyranosyl) group, galactopyranosyl- (2-acetamido-2-deoxy-glucopyranosyl) group, galactopyranosyl- (2-acetamido-2- Oxy - mannopyranosyl) group, galactopyranosyl - disaccharide or di like glucopyranosyl group (2-acetamido-2-deoxy - glucopyranosyl) - mannopyranosyl group, di (galactopyranosyl)
Trisaccharides such as -2-acetamido-2-deoxy-glucopyranosyl group can be mentioned. Particularly preferably, a mannopyranosyl group, a fucopyranosyl group, a 2-acetamido-2-deoxy-fucopyranosyl group, a mannopyranosyl-mannopyranosyl group, (2-acetamido-
2-deoxy-glucopyranosyl) -mannopyranosyl group, and galactopyranosyl-glucopyranosyl group.

In the compound of the formula (I), the dioxacarboxylic acid as a modifier for linking a sugar chain to a protein is a dioxaalkanoic acid via an ethylene group or a propylene group, preferably 5,8-dioxadecanoic acid. . The bond between the sugar chain and the dioxacarboxylic acid may be an α-bond or a β-bond. Z
In addition to proteins such as human serum albumin, proteins such as interleukin, erythropoietin, interferon, tissue plasminogen activator, ulcer necrosis factor (TNF) or colony stimulator which are physiologically active proteins in addition to proteins such as human serum albumin It may be a factor (CSF). Although these proteins themselves do not have organ specificity, they become target-identifying by chemical modification with a sugar chain having target-identifying ability.

To produce the carrier of formula (I), for example, hydrazine is reacted with an alkyl ester of glycosyl-dioxaalkanoic acid or an alkyl ester of 2-acetamido-2-deoxy-glycosyl-dioxaalkanoic acid, The obtained acid hydrazide is converted into an acid azide by a conventional method, and this is reacted with a protein to obtain a target product in which glycosyl-dioxaalkanoic acid is amide-bonded to a part of an amino group in the protein. The bonding number of the modifying group is 5 to 50 mol per protein molecule. The degree of modification can be selected by increasing or decreasing the molar ratio of glycosyl-dioxaalkanoic acid to protein, or by increasing or decreasing the concentration of the reaction solution of protein and glycosyl-dioxaalkanoic acid.

The solvent used in the reaction may be any solvent as long as it does not hinder the reaction, and examples thereof include a phosphate buffer, a Tris buffer, an acetate buffer and a borate buffer. The reaction is carried out between 0 ° C. and room temperature near neutrality. The reaction solution is purified by an ordinary protein purification method such as dialysis, ion exchange chromatography, or gel filtration to obtain the desired product. The number of introduced modified groups can be determined by hydrolyzing the modified protein with hydrochloric acid and then measuring by the Elson-Morgan method or the like.

The reaction between glycosyl-dioxaalkanoic acid and protein can also be produced by reacting in the presence of a condensing agent such as water-soluble carbodiimide. Also, other active derivatives of dioxaalkanoic acid may be used,
Examples of such active derivatives include amide compounds, active esters, active thioesters, and the like. These active derivatives can be appropriately selected by those skilled in the art to form an amide bond with the protein.

The present invention also relates to an intermediate for producing the drug delivery carrier of the formula (I), wherein the intermediate is a glycosyl-dioxacarboxylic acid of the formula (II) or an active derivative thereof. R—O— (CH ₂ ) ₂ O (CH ₂ ) ₂ O (CH ₂ ) ₃ COOH (II) wherein R is particularly a xylopyranosyl group, a fucopyranosyl group, a galactopyranosyl group, a 2-acetamido-2-deoxy -Fucopyranosyl group, 2-acetamido-2-deoxy-mannopyranosyl group, 2-acetamido-2-
Deoxy-galactopyranosyl group, (2-acetamido-2-deoxy-mannopyranosyl) -mannopyranosyl group, (2-acetamido-2-deoxy-glucopyranosyl) -mannopyranosyl group, fucopyranosyl- (2
-Acetamido-2-deoxy-glucopyranosyl)
Group, galactopyranosyl- (2-acetamido-2-deoxy-glucopyranosyl) group, galactopyranosyl-
(2-acetamido-2-deoxy-mannopyranosyl) group, galactopyranosyl-glucopyranosyl group, di (2-acetamido-2-deoxy-glucopyranosyl) -mannopyranosyl group or di (galactopyranosyl) -2-acetamido-2 -A glycosyl group such as a deoxy-glucopyranosyl group.

To prepare the intermediate of the formula (II), (A)
A glycosyl halide having a protected hydroxyl group is reacted with an alkyl ester of ω-hydroxydioxaalkanoic acid to give a glycosyl-α (or β) -dioxaalkanoic acid alkyl ester, which is obtained by deprotection,
(B) the alkyl 2-acetamido-2-deoxy-glycosyl-dioxaalkanoate is the corresponding 2
-Azido-2-deoxy-glycosyl-dioxaalkanoic acid alkyl ester obtained by acetylation. Further, it can be led to another active derivative if necessary.

In order to prepare the carrier of the formula (I), an intermediate of the formula: R—O— (CH ₂ ) ₂ O (CH ₂ ) ₂ O (CH ₂ ) ₃ —COOH (II) It can be produced by the method shown in the reaction formula.

[0022]

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[0023]

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[0024]

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[0025]

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[0026]

EFFECT OF THE INVENTION The glycosyl-protein derivative of the present invention binds a drug as shown in the test examples described below, and when administered to animals as shown in the test examples, the drug concentrates on bone marrow or brain tissue. Can be distributed.

[0027]

EXAMPLES Examples and test examples of the present invention will be described below.

EXAMPLE 1 2- (2-Benzyloxyethoxy) ethoxyethanol (402) Triethylene glycol (13.5 g, 0.338 mol) suspended in 300 ml of anhydrous dimethylformamide was dissolved in 300 ml of anhydrous dimethylformamide. 401) 50.0 g
(0.33 mol) was added dropwise. After stirring at room temperature for 1 hour, 56.0 g (0.33 mol) of benzyl bromide was added dropwise, and the mixture was further stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was dissolved in 200 ml of ethyl acetate and washed with 200 ml of water. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain an oily crude product. This was purified by silica gel column chromatography [silica gel 70-230 mesh 500 g, solvent system: hexane / ethyl acetate (1: 1)].
27.5 g (34%) of the title compound (402) were obtained.

¹ H-NMR (CDCl ₃ , δ ppm): 7.26 to 7.35 (5H,
m), 4.57 (2H, s), 3.73 (2H, t, J = 4.5Hz), 3.66 to 3.71 (6
H, m), 3.63 to 3.65 (2H, m), 3.62 (2H, t, J = 4.5Hz), 2.25
(1H, br) IR (neat) cm ^-1 : 3450 (OH), 1099 (COC)

Example 2 2- (2-Benzyloxyethoxy) ethoxyethyl bromide (403) Phosphorus tribromide was added to a solution of 36.0 g (0.15 mol) of compound (402) in 100 ml of anhydrous ether under ice-cooling. 0g
(0.052 mol) and stirred for 1 hour. After further stirring at room temperature for 1 hour, 100 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain a syrupy crude product. This was purified by silica gel column chromatography [silica gel 70-230 mesh, 250 g, solvent system: hexane / ethyl acetate (9: 1)] to obtain 13.6 g (36%) of the title compound (403).

¹ H-NMR (CDCl ₃ , δ ppm): 7.26 to 7.35 (7H,
m), 4.58 (2H, s), 3.81 (2H, t, J = 7.0Hz), 3.66 to 3.71 (6
H, m), 3.61 to 3.63 (2H, m), 3.47 (2H, t, J = 6.7Hz) IR (neat) cm ^-1 : 1112 (COC)

EXAMPLE 3 Methyl 4- [2- (2-benzyloxyethoxy) ethoxy] -2-methoxycarbonylbutanoate (40
4) To a suspension of 10.0 g (0.25 mol) of 60% sodium hydride in 300 ml of anhydrous dimethylformamide was added 33.0 g of dimethyl malonate, and the mixture was stirred at 40 ° C for 1 hour. To this was added 41.1 g (0.136 mol) of the compound (403) at a time, and the mixture was further stirred at 40 ° C. for 6 hours and left at room temperature overnight. The reaction mixture was neutralized with 10% hydrochloric acid, the solvent was distilled off under reduced pressure, and the obtained residue was partitioned between 100 ml of water and 200 ml of ethyl acetate. After separating the organic phase, it was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to obtain a syrupy crude product. This was subjected to silica gel column chromatography [silica gel 70-230 mesh 250 g, solvent system: hexane / ethyl acetate (20:
1)] to give 36.8 of the title compound (404).
g (77%).

¹ H-NMR (CDCl ₃ , δ ppm): 7.26 to 7.34 (5H,
m), 4.56 (2H, s), 3.72 (3H, s), 3.52 (2H, t, J = 5.9Hz), 2.
18 (2H, m) IR (neat) cm ^-1 : 1754 (C = O), 1735 (CO ₂ CH ₃ )

Example 4 Methyl 4- [2- (2-benzyloxyethoxy) ethoxy] butanoate (405) 23.2 g (65.5 mmol) of the compound (404) and 4.5 g (76.9 mmol) of sodium chloride were obtained. 4 ml of water
And a mixture of 80 ml of dimethyl sulfoxide and 150 ml
It heated and stirred at 160 degreeC for 4 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was partitioned between 100 ml of water and 100 ml of ethyl acetate. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting syrupy crude product was purified by silica gel column chromatography [silica gel 70-230 mesh 250 g, solvent system: hexane / ethyl acetate (5: 1)].
17.0 g (87%) of the title compound (405) were obtained.

¹ H-NMR (CDCl ₃ , δ ppm): 7.24 to 7.33 (5H,
m), 4.57 (2H, s), 3.66 (3H, s), 3.63 to 3.68 (6H, m), 3.5
7 to 3.58 (2H, m), 3.50 (2H, t, J = 6.2Hz), 2.42 (2H, t, J
= 7.3Hz), 1.90 (2H, m) IR (neat) cm ^-1 : 1738 (CO ₂ CH ₃ ), 1113 (COC)

Example 5 Methyl 4- [2- (2-hydroxyethoxy) ethoxy] butanoate (406) To a solution of 16.6 g (56.0 mmol) of compound (405) in 20 ml of methanol was added 2.0 g of 10% palladium on carbon.
Was added and hydrogenated at room temperature for 4 hours. After completion of the reaction, the catalyst was filtered off, and the filtrate was concentrated to dryness under reduced pressure to obtain 11.3 g (99%) of the title compound 406 as a colorless oil.

¹ H-NMR (CDCl ₃ , δ ppm): 3.73 (2H, t, J = 4.
5Hz), 3.68 (3H, s), 3.65 to 3.68 (2H, m), 3.57 to 3.63 (4
H, m), 3.51 (2H, t, J = 6.2Hz), 2.42 (2H, t, J = 7.3Hz), 1.
92 (2H, m), 1.70 (1H, br) IR (neat) cm ^-1 : 3450 (OH), 1738 (CO ₂ CH ₃ ), 1119 (COC) MS (EI) m / z: 207 (M + H ⁺ )

Example 6 2- [2- (3-methoxycarbonylpropyloxy)
Ethoxy] ethyl-2,3,4,6-tetra-O-benzyl-β-D-mannopyranoside (409) and 2-
[2- (3-Methoxycarbonylpropyloxy) ethoxy] ethyl-2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside (408) 577 mg (2.8 mmol) of compound (406), carbonic acid 500 mg (1.79 mmol) of silver and 500 dry light
After stirring a solution of 5 mg of methylene chloride at 0 ° C. for 30 minutes under a nitrogen stream, Koto, Morishima, Miyata, and Zen, Bu
ll. Chem. Soc. Jpn., 49 , 2639 (1976).
2,3,4,6-tetra-O-benzyl-α / β-mannopyranosyl p-nitrobenzoate was synthesized by blowing dry hydrogen chloride gas into a methylene chloride solution.
3 ml of a methylene chloride solution of 580 mg (0.84 mmol) of 3,4,6-tetra-O-benzyl-α-D-mannopyranosyl chloride (407) was slowly added dropwise. Thereafter, the mixture was stirred at an internal temperature of 0 to 5 ° C for 4 hours. After completion of the reaction, the reaction product was filtered using celite, and the insoluble material was thoroughly washed with methylene chloride. The filtrate and the washing solution were combined, and the solvent was distilled off under reduced pressure to obtain 1.1048 g of a crude product. The crude product was purified by flash chromatography [silica gel 230-4.
00 mesh, 60 parts, solvent system: ethyl acetate / hexane (2: 3)] to give the title compound (409).
520 mg (81.2%) and the title compound (408) 12
0 mg (18.8%) was obtained.

The compounds (409) [α] ^{_{D 24 - 44.6 ° (C O.67}} , CHCl 3) 1 H-NMR (CDCl 3, δppm): 7.18~7.50 (20H, m, aromatic
H), 4.85~4.97 (2H, AB -q, J = 12.5Hz, benzyl-CH 2), 4.54
~4.62 (2H, AB-q, J = 12Hz, benzyl-CH 2), 4.50~4.90 (2
H, AB-q, J = 12Hz, benzyl-CH ₂ ), 4.41-4.51 (2H, AB-q,
J = 12Hz, benzyl-CH ₂ ), 4.44 (1H, s, H-1), 4.02-4.08 (1
H, m, H-2) , 3.64~3.93 (7H, m), 3.63 (3H, s, CO 2 CH 3),
3.40~3.64 (8H, m), 2.35~2.40 (2H, m, CH 2 CO -), 1.80~
1.95 (2H, m, CH ₂ CH ₂ CO-) IR (CHCl ₃ ) cm ^-1 : 1726 (CO ₂ CH ₃ ) MS (FAB) m / z: 751 (M + Na ⁺ ), 727 (M-1 )

Compound (408) [α] _D ²⁴ + 13.9 ° (C O.70, CHCl ₃ ) ¹ H-NMR (CDCl ₃ , δ ppm): 7.18 to 7.50 (20H, m, aromatic
H), 4.91 (1H, d, J = 1.71 Hz, H-1), 4.68 to 4.75 (2H, AB-q,
J = 12.5Hz, benzyl-CH ₂ ), 4.604 (2H, s, benzyl-CH ₂ ), 4.
47~4.86 (2H, AB-q, J = 10.75Hz, benzyl-CH 2), 4.51~4.
65 (2H, AB-q, J = 12Hz, benzyl-CH ₂ ), 3.65-4.00 (8H,
m), 3.63 (3H, s, CO ₂ CH ₃ ), 3.44 to 3.60 (8H, m), 2.30 to 2.4
0 (2H, m, CH ₂ CO-), 1.80 to 1.90 (2H, m, CH ₂ CH ₂ CO-) IR (CHCl ₃ ) cm ^-1 : 1725 (CO ₂ CH ₃ ) MS (FAB) m / z : 727 (M-1)

Example 7 2- [2- (3-methoxycarbonylpropyloxy)
Ethoxy] ethyl-β-D-mannopyranoside (41
0) 500 mg (0.686 mmol) of compound (409) was dissolved in 10 ml of methanol, and 10% palladium-carbon 50
0 mg was added, and the mixture was stirred vigorously at room temperature under a hydrogen stream for 40 hours. After completion of the reaction, the catalyst was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure to give 232.4 mg (92%) of the title compound (410).
was gotten.

Compound (410) [α] _D ²³ -22.3 ° (C 2.18, MeOH) ¹ H-NMR (CD ₃ OD, δ ppm): 4.53 (1H, s, H-1), 3.30 to 4.00 (1
4H, m), 3.62 (3H , s, CO 2 CH 3), 3.10~3.25 (2H, m), 2.37
(2H, t, J = 7.3Hz, CH ₂ CO-), 1.83 (2H, t, J = 7.3Hz, CH ₂ C
_{H 2 CO-) IR (CHCl 3} ) cm -1: 3350 (OH), 1732 (CO 2 CH 3) MS (FAB) m / z: 391 (M + Na +)

Example 8 2- [2- (3-methoxycarbonylproxyoxy)
[Ethoxy] ethyl 2,3,4-tri-O-acetyl-
β-L-fucopyranoside (412) HM Flowers et al. [Carbohydr. Res., 4 , 189-195]
(1967)], 2,3,4-tri-O-acetyl-α-L-fucopyranosyl bromide (411) 16
To a solution of 6 mg (0.469 mmol) in 10 ml of benzene were added 100 mg (0.485 mmol) of the compound (406), 123 mg (0.485 mmol) of mercuric cyanide and 350 mg of powdered anhydrous calcium sulfate, and the mixture was placed in an argon stream. Stirred at room temperature for 24 hours. After filtering the reaction solution, 5 ml of ethyl acetate was added to the filtrate, and the mixture was washed with 5 ml of water. After drying over anhydrous magnesium sulfate and concentration under reduced pressure, the resulting syrup was purified by silica gel column chromatography (chloroform: methanol = 200: 1) to obtain 136 mg (58.6%) of the title compound (412).

Compound (412) [α] _D ²⁵ -2.9 ° (C 1.1, CHCl ₃ ) ¹ H-NMR (CDCl ₃ , δ ppm): 1.22 (3H, d, J = 6.4 Hz, H-6), 1.9
9, 2.06, 2.18 (9H, 3s, OAc), 2.42 (2H, t, J = 7.3Hz, C H
₂ , COOCH ₃ ), 3.68 (3H, s, OCH ₃ ), 4.76 (1H, d, J = 8.1 Hz,
H-1) IR (NaCl) cm ^-1 : 1750, 1700

Example 9 2- [2- (3-methoxycarbonylpropyloxy)
Ethoxy] ethyl 2,3,4-β-L-fucopyranoside (413) 180 mg (0.376 mmol) of compound (412) was dissolved in 2 ml of methanol, and 0.05 ml of 28% sodium methylate was added. Stir for 2 hours. The reaction solution was neutralized with Amberlite IR-120B (H ⁺ ), the ion exchange resin was collected by filtration, and the filtrate was concentrated under reduced pressure to obtain 109 mg (82.3%) of the title compound (413).

[Α] _D ²⁶ -2.1 ° (C 1.3, MeOH) ¹ H-NMR (CD ₃ OD, δ ppm): 1.23 (3H, d, J = 6.6 Hz, H-6), 2.3
7 (2H, t, J = 7.6Hz, C H 2 COOCH 3), 3.67 (3H, s, OCH 3), 4.
19 (1H, d, J = 7.3Hz, H-1) IR (NaCl) cm ^-1 : 3450, 1740, 1070

Example 10 2- [2- (3-methoxycarbonylpropyloxy)
[Ethoxy] ethyl 3,4,6-tri-O-acetyl-
2-azido-2-deoxy-α-D-mannopyranoside (414) and 2- [2- (3-methoxycarbonylpropyloxy) ethoxy] ethyl 3,4,6-tri-
O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside (415) 3,4,6-tri-O-acetyl-2-synthesized by the method of Carbohydr. Res., 136 , 153 (1985). Azido-2-deoxy-α-D-mannopyranosyl bromide 930 mg
(2.35 mmol) and methyl 4- [2- (2-hydroxyethoxy) ethoxy] butanoate (406) 4
To a solution of 85 mg (2.35 mmol) in 20 ml of toluene,
Silver silicate 700mg and molecular sieve 4 in powder form
A (500 mg) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction solution was filtered using celite, the filtrate was concentrated to dryness under reduced pressure, and the obtained residue was purified by a rover column (hexane: ethyl acetate = 1: 1) to give the title compound (414) 5.
25 mg (43%) and 143 mg of the title compound (415)
(11.7%) was obtained.

Compound (414) [α] _D ²⁴ + 58.2 ° (C O.57, CHCl ₃ ) ¹ H-NMR (CDCl ₃ , δ ppm): 5.39 (1H, dd, J _3,4 = 10 Hz, J _{2 , 3} =
4.0Hz, H-3), 5.32 (1H, t, J = 10Hz, H-4), 4.91 (1H, s, H
-1), 4.25 (1H, dd , J 5,6a = 4.5Hz, J 6a, 6b = 12Hz, H-6a),
4.02 (1H, m, H- 5), 2.40 (2H, t, J = 7.5Hz, -C H 2 CO -), 2.1
0, 2.09, 2.04 (each 3H, each s, 3 × OAc) IR (film) cm ^-1 : 2110, 1747, 1438, 1369, 1230, 1049

[0049] Compound (415) [α] ^{_{D 24 - 60.1 ° (C O.9}} , CHCl 3) 1 H-NMR (CDCl 3, δppm): 5.24 (1H, t, J 3,4 = 9.5Hz, H -Four),
4.99 (1H, dd, J _2,3 = 3.5Hz, H-3), 4.79 (1H, s, H-1), 4.2
_{5 (1H, dd, J 5,6a} = 5.5Hz, J 6a, 6b = 12Hz, H-6a), 4.00 (1H,
m, H-5), 2.40 (2H, t, J = 7.5Hz, -C H ₂ CO-), 2.10, 2.08,
2.03 (each 3H, each s, 3 × OAc) IR (film) cm ^-1 : 2112, 1743, 1371, 1236, 1055

Example 11 2- [2- (3-methoxycarbonylpropyloxy)
Ethoxy] ethyl 2-azido-2-deoxy-α-D
-Mannopyranoside (416) 1M sodium methoxide was added to a solution of 525 mg (1.01 mmol) of compound (415) in 10 ml of anhydrous methanol.
0.25 ml of a methanol solution was added and left at room temperature for 4 hours. Add ion exchange resin Amberlite IR-12 to the reaction solution.
After neutralizing by adding 0B (H ⁺ type), the resin was filtered off and washed with a small amount of methanol. The filtrate and washing solution were combined, and the solvent was distilled off under reduced pressure to give a syrup of the title compound (41).
6) 154 mg were obtained.

Example 12 2- [2- (3-methoxycarbonylpropyloxy)
Ethoxy] ethyl 2-acetamido-2-deoxy-α
-D-mannopyranoside (417) To a solution of 150 mg (0.381 mmol) of compound (416) in 4 ml of ethanol was added 380 mg of nickel chloride hexahydrate.
(1.6 mmol) was added to a solution of 0.1 ml of a solution of ethanol in 10 ml of ethanol, and then 43 mg of sodium borohydride was added.
To a solution of (1.143 mmol) in ethanol was added 4 ml with stirring. After stirring at room temperature for 30 minutes, the reaction solution was neutralized by adding acetic acid. Then, 0.5 ml of acetic anhydride was added thereto and left at room temperature for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform: methanol = 7: 1) to obtain 157 mg of the title compound (417).

[Α] _D ²⁶ + 26.4 ° (C 0.62, MeOH) ¹ H-NMR (CD ₃ OD, δ ppm): 4.72 (d, 1 H, J = 1 Hz, H-1), 4.31
(dd, 1H, J _1,2 = 1Hz, J _2,3 = 4.7Hz), 2.01 (s, 3H, NAc) IR (KBr) cm ^-1 : 3446, 1730, 1660, 1550, 1132, 1068

Example 13 Neoglycoprotein (419) To a solution of 85 mg (0.241 mmol) of compound (413) in 0.8 ml of ethanol was added 0.24 of hydrazine hydrate.
ml was added and the mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, 1 ml of water was added, and the mixture was further concentrated under reduced pressure to give 85 mg of 2- [2- (3-hydrazinocarbonylpropyloxy) ethoxy] ethyl β-L-fucopyranoside.
I got To this was added 3 ml of DMF to dissolve, and
After cooling, 0.14 ml of a 4N solution of hydrogen chloride in dioxane was added. At the same temperature, 35.5 mg of t-butyl nitrite (0.
241 mmol) in DMF (0.2 ml) and stirred for 30 minutes, followed by sulfamic acid (16.8 mg) in DMF (0.1 ml).
A 2 ml solution was added and stirred for 15 minutes to obtain a reaction solution containing the corresponding acid azide. 332 mg (4.82 × 1) of human serum albumin (HSA) cooled to 0 ° C.
0 ^-3 mmol) borate buffer (0.08M Na ₂ B ₄
O ₇ , 0.35 M KHCO ₃ ) 20 ml
Stirred for hours. After dialysis at 4 ° C. overnight, lyophilization gave 343 mg of a powder of fucose-modified albumin (419). It was found that the number of moles of sugar chains bound to HSA / mol by the phenol-sulfuric acid method was 28.

A list of neoglycoproteins and intermediates used is shown in Table 1. The content of the introduced sugars was determined by the phenol-sulfuric acid method for neutral sugars and the Elson-Morgan method for hexosamines after hydrolysis with 4N hydrochloric acid. The physical constants of these neoglycoproteins are shown in Table 2.

[0055]

[Table 1]

[0056]

[Table 2]

Test Example Neoglycoproteins 418, 419 and 420 were prepared as test samples, and HSA was prepared as a control sample. The sample was labeled with iodine by the following method, and a distribution experiment in animals was performed.

Test Method Each neoglycoprotein was iodolabeled using the chloramine T method. As a result, specific activity 18-30 MBq /
Iodine-labeled neoglycoprotein of 20 μg protein was obtained. TCA treatment was performed to confirm that radioactive iodine was bound to the protein. That is, after appropriately diluting with a 0.1 M phosphate buffer containing 0.5% bovine serum albumin, 300 μl of the diluted solution was placed in a test tube, and 15%
600 μl of a TCA (trichloroacetic acid) solution was added, and the mixture was stirred and centrifuged at 3,000 rpm for 10 minutes to measure the activity of radioactive iodine present in the supernatant and the precipitate. As a result, 95% or more of the total radioactivity was present in the precipitate, indicating that radioactive iodine was bound to the protein.

Using this iodolabeled neoglycoprotein, a distribution experiment in animals was performed. First, 4 μg of iodine-labeled neoglycoprotein was added to 0.1 M phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin.
It was dissolved in 1 ml and used as a test solution for administration. When the specific activity of the iodolabel was high, dilution was performed using unlabeled neoglycoprotein to obtain a test solution for administration.

This solution was used for SD male rats (body weight 240
To 300 g) from the femoral vein so as to be 0.1 ml per 100 g of body weight. After that, exactly one minute, two minutes,
Three minutes later, blood is collected from the jugular vein, and five minutes after administration, systemic blood is collected from the inferior aorta, and the blood is killed. Immediately thereafter, heart, lung, thymus, spleen, kidney, muscle, bone marrow, skin and liver were collected.

1 minute, 2 minutes, 3 minutes, 5 minutes after administration, serum and 5 minutes of whole blood, heart, lung, thymus, spleen, kidney, muscle,
Bone marrow, skin and liver were accurately weighed and their radioactivity was measured.

Further, TCA treatment was performed to confirm that radioactivity present in the test solution for administration and the serum 5 minutes after administration was bound to neoglycoprotein. The operation was performed in the same manner as described above, and it was confirmed that 90% or more of the total radioactivity was bound to neoglycoprotein.

Next, the obtained serum concentration (1 minute after administration,
From the results at 2 minutes, 3 minutes and 5 minutes), the area under serum concentration (AUC _0-5 ) from 0 minutes to 5 minutes after administration was calculated using a trapezoidal approximation method. AUC _0-5 is defined by the following equation. AUC _0-5 = ∫ ⁵ ₀ Cdt where C is the serum concentration at neoglycoprotein time (t).

Further, tissue distribution clearance was calculated from the ratio of _{neoglycoprotein} concentration in each tissue to AUC _0-5 . Table 3 shows the results.

This indicates that the glycosyl-protein derivative is useful as a drug carrier for targeting bone marrow tissue.

[0066]

[Table 3]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C07K 14/76 A61K 37/48 (72) Inventor Tetsu Okuno 8-5-18 Waseda, Misato City, Saitama Prefecture (72) Inventor Takashi Kato 1-7-1 Higashi, Kashiwa City, Chiba Pref. Grandle Ari 104 (56) References JP-A-58-15993 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/76 C07K 14/52 C07H 15/04 CA (STN) REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A compound represented by the formula: [R-X-] _m -Z, wherein R represents a glycosyl group and X is O- (CH ₂ ) _{2
O (CH 2) 2 O (} CH 2) represents a ₃ -CO, Z represents a protein, glycosyl m is represented by the representative) 10-50 - drug delivery carrier containing the protein derivative. 2. The glycosyl group is a xylopyranosyl group,
Mannopyranosyl group, fucopyranosyl group, galactopyranosyl group, 2-acetamido-2-deoxy-fucopyranosyl group, 2-acetamido-2-deoxy-mannopyranosyl group, 2-acetamido-2-deoxy-galactopyranosyl group, mannopyranosyl- Mannopyranosyl group, (2-acetamido-2-deoxy-mannopyranosyl) -mannopyranosyl group, (2-acetamido-2
-Deoxy-glucopyranosyl) -mannopyranosyl group, fucopyranosyl- (2-acetamido-2-deoxy-glucopyranosyl) group, galactopyranosyl- (2
-Acetamido-2-deoxy-glucopyranosyl)
Group, galactopyranosyl- (2-acetamido-2-deoxy-mannopyranosyl) group, galactopyranosyl-
The drug delivery carrier according to claim 1, which is a glucopyranosyl group, di (2-acetamido-2-deoxy-glucopyranosyl) -mannopyranosyl group, or di (galactopyranosyl) -2-acetamido-2-deoxy-glucopyranosyl group. . 3. The drug delivery carrier according to claim 1, wherein the protein is albumin, cytokines or an enzyme. 4. A carrier for delivering a drug to bone marrow, comprising the glycosyl-protein derivative according to claim 1. 5. A carrier for delivering a drug to the brain, which comprises the glycosyl-protein derivative according to any one of claims 1 to 3.