JP2001261706A - Sialic acid-branched cyclodextrin derivative and its intermediate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cyclodexrin derivative having a sialic acid at its side chain in a situation that chemical synthesis of a sialic acid derivative is still difficult, at the same time, there are many problems, for example, via what kind of bond should be branched in a side chain of a cyclodextrin, and accordingly a cyclodextrin derivative having a sialic acid at its side chain is not known yet. SOLUTION: A sialic acid-branched cyclodextrin derivative prepared by replacing hydrogen of the amino group of a 6-monoaminodextrin derivative with a sialic acid-containing acyl group expressed by structural formula (1) (wherein, n is an integer 0-5), the 6-monoaminodextrin derivative being prepared by replacing the primary hydroxy group of a cyclodextrin with an amino group. An intermediate is expressed by structural formula (2) (wherein, R is 9- fluorenylmethyl group).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cyclodextrin derivative having sialic acid in a side chain. Cyclodextrins are compounds well known as inclusion compounds. Since it can include an aromatic compound, it is widely used as a pharmaceutical or food additive. On the other hand, it has become clear that sugar chains play important functions such as cell recognition and adhesion on the cell surface. In particular, it is said that sialic acid plays an important role in virus and bacterial infection. For example, it has been clarified that influenza virus adheres and acts as a ligand for infection. Therefore, if sialic acid can be bound to cyclodextrin, it can be used as a tool for so-called drug delivery systems, especially target drug delivery systems, by using the sugar chain recognition mechanism by including drugs and using them to transport new drugs. It is thought that technology can be provided.

[0002]

2. Description of the Related Art However, the chemical synthesis of sialic acid derivatives is still a difficult problem, and at the same time, there are many problems such as what kind of bond should be branched to the side chain of cyclodextrin. Examples of cyclodextrin derivatives having in the side chain have not yet been seen.

[0003]

The present invention provides a cyclodextrin derivative having sialic acid in the side chain.

[0004]

Therefore, the present inventors have made intensive studies with reference to a method for producing a sialic acid derivative,
The present invention has been reached. That is, the present invention relates to a method for converting hydrogen of an amino group of a 6-monoaminocyclodextrin derivative in which a primary hydroxyl group of cyclodextrin is substituted with an amino group into

Embedded image (Wherein n represents an integer of 0 to 5), and a sialic acid-branched cyclodextrin derivative substituted with a sialic acid-containing acyl group represented by the general formula:

Embedded image (Wherein, R represents a 9-fluorenylmethyl group).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

First, as the aminocyclodextrin derivative in which the primary hydroxyl group of cyclodextrin as a raw material of the method of the present invention has been substituted with an amino group, known derivatives can be used. Examples of the cyclodextrin include α-cyclodextrin having six glucose units, seven β-cyclodextrin, eight γ-cyclodextrin, and the like. In addition, there is no problem even if the derivative is chemically or enzymatically synthesized. For example, there may be mentioned those having 5 or less glucose units, those having 9 or more glucose units, and derivatives having a monosaccharide other than glucose as a constituent sugar. One of such a series of cyclodextrins
Derivatives in which one of the graded hydroxyl groups is substituted with an amino group can be used.

A well-known method can also be used for the substitution with an amino group. In general, a suitable leaving group is introduced into a hydroxyl group, and this is azido-formed and then reduced.

It is needless to say that the hydroxyl group of the cyclodextrin derivative can be prepared by protecting the hydroxyl group by a well-known method and then deprotecting after acylation, but it is not always necessary to protect the hydroxyl group. When an amino group is acylated, if a well-known acylation method having a difference in reactivity between the amino group and the hydroxyl group is selected, the amino group can be used without protection of the hydroxyl group. For example, active esters such as succinimide ester and pentafluorophenyl ester, symmetric acid anhydride, dimethylthiophosphinic acid mixed acid anhydride method and the like can be mentioned.

Next, a method for producing the compound of the present invention will be described. This compound is a derivative obtained by sequentially condensing carboxypropyl glycoside derivative of sialic acid, 6-aminocaproic acid, and 6-monoaminocyclodextrin derivative. Actually, the method and order of condensation with the 6-monoaminocyclodextrin derivative are not limited at all. For example, a sialic acid derivative and 6-monoaminocaproic acid can be condensed and then condensed with a 6-monoaminocyclodextrin derivative, or conversely, a 6-aminoaminocyclodextrin derivative can be condensed with 6-aminocaproic acid. The carboxypropyl glycoside derivative of sialic acid can then be condensed.

The method of condensing the respective components is not limited at all, and a known method can be used. For example, an active ester method, a symmetric acid anhydride method, a mixed acid anhydride method, a so-called condensation reagent and the like can be used. Specifically, use of succinimide ester, pentafluorophenyl ester, succinic anhydride, dimethylthiophosphinic acid mixed acid anhydride, N,
N-dicyclohexylcarbodiimide (DCC) and the like can be mentioned.

6-Monoaminocaproic acid is condensed using a derivative having an amino group protected by a well-known method, followed by deprotection to proceed with the reaction. Specific examples of the protecting group include a benzyloxycarbonyl group, a tertiary butyloxycarbonyl group, and a 9-fluorenylmethyloxycarbonyl group. First, a derivative having a long-chain caproic acid oligomer can be prepared by reacting with 6-monoaminocyclodextrin and repeating deprotection and condensation. Further, condensation with carboxypropyl glycoside of sialic acid can lead to the compound derivative of the present invention. On the contrary, it is needless to say that the carboxyl group can be protected and condensed. In addition, a 6-monoaminocaproic acid derivative having an amino group protected and a free 6-monoaminocaproic acid or a derivative having a carboxyl group protected are condensed to prepare an oligomer in advance, and condensed with a sialic acid derivative and monoaminocyclodextrin. It goes without saying that you can do it.

The carboxyl group at the 1-position of the carboxylpropyl glycoside of sialic acid is used after being protected. A well-known protecting group can be used as the protecting group. For example, it is protected as an aliphatic ester or an aromatic ester. Specific examples include methyl ester, ethyl ester, allyl ester, tertiary butyl ester, benzyl ester, 9-fluorenyl methyl ester and the like. On the other hand, the hydroxyl group can be used without protection, but is usually protected by a well-known method. For example, it can be protected with an acyl group, an alkyl group, a silyl group and the like. Specific examples include an acetyl group, a benzoyl group, a benzyl group, and a tert-butyldimethylsilyl group. In particular, a derivative in which the carboxyl group at the 1-position is protected with 9-fluorenylmethyl ester and the hydroxyl group is protected with an acetyl group is preferred as an intermediate from the viewpoint of isolation and purification.

[0013]

EXAMPLE Methyl (4-pentenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5) prepared by an existing method
-Dideoxy-D-glycero-α-D-galact-2-nonulopyranoside) onate (400 mg, 0.71 mmol) in pyridine (70
ml), lithium iodide (1.0 g, 7.5 mmol) was added, and the mixture was stirred at 120 ° C. for 1 day under an argon atmosphere. The reaction solution was concentrated under reduced pressure, and the obtained residue was extracted with dichloromethane. The organic layer was washed with 2M hydrochloric acid and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane (3 ml), and 9-fluorenylmethanol (280 mg, 1.4 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (270 mg, 1.4
mmol), 4-dimethylaminopyridine (43 mg, 0.35 mmol)
Was added and stirred at room temperature for 1 day. The reaction solution was poured into saturated saline and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, the desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (Wakogel C-300) to give dichloromethane:
Elution with methanol = 70: 1 was performed using 9-fluorenylmethyl (4-pentenyl 5-acetamide-4,7,8,9-tetra-O
-Acetyl-3,5-dideoxy-D-glycero-α-D-galacto-
2-nonulopyranoside) onate (127 mg, 0.17 mmol, 25
%).

[0014]¹H-NMR (CDCl_Three) δ 1.60 (m, 2H, H-2 of
4-pentenyl), 1.88 (s, 3H, AcN), 1.97 (t, 1H, J
_{3ax, 3eq} = J_3ax, 4 = 12.7 Hz, H-3ax), 2.02, 2.03, 2.
11, 2.13 (4s, 12H, 4AcO), 2.06 (m, 2H, H-3 of 4-pen
tenyl), 2.53 (dd, 1H, J_{3ax, 3e q} = 12.7 Hz, J_{3eq, 4} 
= 4.6 Hz, H-3eq), 3.15, 3.72 (2m, 2H, H-1 of 4-pen
tenyl), 3.98 (dd, 1H, J_5,6 = 10.0 Hz, J_6,7 = 1.7 H
z, H-6), 4.06 (q, 1H, J _4,5 = J_5,6 = J_{5, NH} = 10.0 H
z, H-5), 4.08 (dd, 1H, J_8,9 = 5.3 Hz, J_{9,9 '} = 12.4
Hz, H-9), 4.24 (t, 1H, J_{CH, CH2} = 6.6 Hz, CH of F
m), 4.28 (dd, 1H, J_{8,9 '} = 2.7 Hz, J_{9,9 '} = 12.4 Hz,
H-9 '), 4.51 (m, 2H, CH_TwoO of Fm), 4.76 (m, 1H, H-
4), 4.94, 5.00 (2m, 2H, H-5 of 4-pentenyl), 5.21
(d, 1H, J_{5, NH}= 10.0 Hz, NH), 5.29-5.34 (m, 2H, H-
7, H-8), 5.77 (m, 1H, H-4 of 4-pentenyl), 7.29-7.7
9 (m, 8H, 2Ph).

¹³ C-NMR (CDCl ₃ ) δ 20.88,20.93,20.96,
21.21 (4AcO), 23.28 (AcN), 28.83 (C-2 of 4-penten
yl), 30.03 (C-3 of 4-pentenyl), 38.09 (C-3), 46.65
(CHof Fm), 49.46 (C-5), 62.32 (C-9), 64.17 (C-1 o
f 4-pentenyl), 67.31 (C-7), 67.42 (CH ₂ O of Fm), 6
8.74 (C-8), 69.13 (C-4), 72.41 (C-6), 98.53 (C-2),
114.60 (C-5 of 4-pentenyl), 120.03, 120.08, 124.5
6, 124.72, 127.02, 127.10, 124.68, 127.76, 141.06,
142.89, 142.97 (2Ph), 137.80 (C-4 of 4-pentenyl),
167.62, 169.66, 169.77, 169.89, 170.28, 170.48 (6
CO).

The obtained 9-fluorenylmethyl (4-pentenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-Dideoxy-D-glycero-α-D-galact-2-nonulopyranoside) onate (200 mg, 0.28 mmol) was dissolved in dichloromethane (3 ml), acetonitrile (3 ml), and water (3 ml). Sodium periodate (300 mg, 1.40 mmol) and a catalytic amount of ruthenium chloride hydrate were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with dichloromethane, and the organic layer was washed with 2M hydrochloric acid and dried over anhydrous sodium sulfate. The drying agent was filtered off, the filtrate was concentrated under reduced pressure, and the residue obtained was subjected to silica gel column chromatography, eluting with dichloromethane / methanol = 15: 1 to give 4-O- (9-
Fluorenylmethyl 5-acetamide-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto
2-Nonulopyranosylonate) -butanoic acid
(110 mg, 0.15 mmol, 53%).

¹ H-NMR (CDCl ₃ ) δ 1.88 (s, 3H, AcN),
2.02, 2.03, 2.11, 2.13 (4s, 12H, 4AcO), 2.46 (m, 1
H, H-3eq), 3.19, 3.71 (2m, 2H, H-4 of Bu), 4.55
(m, 2H, CH ₂ O of Fm), 4.74 (m, 1H, H-4), 7.27-7.78
(m, 8H, 2Ph).

¹³ C-NMR (CDCl ₃ ) δ 20.76, 20.82, 21.0
7, 23.07 (5Ac), 24.72 (C-3 of Bu), 30.31 (C-2 of B
u), 37.82 (C-3), 46.54 (CH of Fm), 49.32 (C-5), 6
2.42 (C-9), 63.52 (C-4 of Bu), 67.32 (CH ₂ O of Fm),
67.23, 68.64, 68.92, 72.35 (C-4, C-6, C-7, C-8), 9
8.42 (C-2), 120.12, 124.56, 124.67, 127.07, 127.1
5, 127.74, 127.81, 141.12, 142.96 (2Ph), 167.66, 1
69.91, 170.02, 170.35, 170.67, 170.73, 176.91 (7C
O).

Next, the obtained 4-O- (9-fluorenylmethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
Dideoxy-D-glycero-α-D-galact-2-nonulopyranosylonate) -butanoic acid (50 mg, 0.067
was dissolved in dichloromethane (1 ml), 1M N, N-diisopropylethylamine in N, N-dimethylformamide (0.075 ml, 0.075 mmol) was added, and the mixture was cooled to 0 ° C and 1M dimethylphosphino chloride. A solution of thioyl in dichloromethane was added (0.075 ml, 0.075 mmol), and the mixture was stirred for 1 hour while warming to room temperature. The residue obtained by concentrating the reaction solution under reduced pressure was dissolved in N, N-dimethylformamide (1 ml), and separately prepared 6- (6-aminohexane-1-amide) -hexanoic acid trifluoroacetate (72 mg,
0.20 mmol) and 1M N, N-diisopropylethylamine N,
An aqueous solution (1 ml) obtained by combining an N-dimethylformamide solution (0.15 ml, 0.15 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added to the reaction solution and extracted, and the organic layer was
After washing with hydrochloric acid, it was dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (Wakogel C-300), eluting with dichloromethane: methanol = 10: 1 to give 6- {6- [4 -O- (9-Fluorenylmethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galact-2-nonulopyranosyl [Nate) -butane-1-amide] -hexane-1-amide} -hexanoic acid (23 mg, 0.023 mmol, 35%) was obtained.

¹ H-NMR (CD ₃ OD-CDCl ₃ ) δ 1.27-2.33 (m,
20H, 10CH ₂ ), 1.86 (s, 3H, AcN), 2.03, 2.04, 2.13,
2.14 (4s, 12H, 4AcO), 2.55 (dd, 1H, J _{3ax, 3eq} = 12.
7 Hz, J _{3eq, 4} = 4.6 Hz, H-3eq), 3.17 (m, 5H, 2CH ₂ N,
H-4 of Bu), 3.66 (m, 1H, H-4 of Bu), 4.54 (m, 2H,
CH ₂ O of Fm), 4.76 (m, 1H, H-4), 5.28-5.36 (m, 2H,
H-7, H-8), 7.31-7.80 (m, 8H, 2Ph).

¹³ C-NMR (CD ₃ OD-CDCl ₃ ) δ 20.81, 20.83,
20.90, 21.21, 22.68 (5Ac), 24.65, 25.55, 26.08, 2
6.50, 29.09, 29.13, 33.20, 34.08, 36.33, 37.92, 3
9.32 (C-3, 12CH ₂ ), 46.77 (CH of Fm), 49.85 (C-5), 6
2.79 (C-9), 64.26 (C-4 ofBu), 67.75 (CH ₂ O of Fm),
67.61, 68.79, 69.52, 72.23 (C-4, C-6, C-7, C-8), 9
8.73 (C-2), 120.13, 120.16, 124.85, 127.21, 127.2
5, 127.92, 127.97, 141.18, 141.24, 143.04, 143.20
(2Ph), 167.61, 170.27, 170.73, 171.24, 171.53, 17
3.68, 174.16, 176.20 (8CO).

6- {6- [4-O- (9-fluorenylmethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α- D-galact-2-nonulopyranosylonato) -butan-1-amide] -hexane-1-amide} -hexanoic acid (23 mg, 0.024 mmol) was dissolved in dichloromethane (0.5 ml), and 1M N N, N-dimethylformamide solution of N, N-diisopropylethylamine (0.024 ml, 0.
024 mmol) and 1 M dimethylphosphinochioil chloride (0.03
0 ml, 0.030 mmol) at 0 ° C. and stirred for 1 hour while warming to room temperature. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in N, N-dimethylformamide (1 ml), and 1M N, N
-Diisopropylethylamine in N, N-dimethylformamide (0.050 ml, 0.050 mmol) and 6-aminocyclomaltoheptaose (134 mg, 0.118 mmol) in water (1
ml), and the mixture was stirred at room temperature for 1 day. The residue obtained by concentrating the reaction solution under reduced pressure was subjected to column chromatography (Diaion HP-20), and the fraction eluted with methanol was concentrated under reduced pressure. The residue was subjected to anion exchange column chromatography (Dowex 1, HCOO-form), and the fraction eluted with water was lyophilized to give 6- (6- {6- [4-O
-(9-Fluorenylmethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galact-2-nonulopyranosylonate) -Butane-1-amide] -hexane-1-amide} -hexane-1-amide) -cyclomaltoheptaose was obtained as a crude product (18 mg).

MALDI-TOF MASS: Calcd for C₉₁H₁₃₄N
_FourO₅₀; [M + Na]⁺ 2107.03. Found; [M + Na] ⁺2109.52

Subsequently, the above-mentioned 6- (6- {6- [4-O- (9-fluorenylmethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- Dideoxy-D-glycero-α-D-galact-2-nonulopyranosylonate) -butan-1-amide] -hexane-1-
Amide} -hexane-1-amido) -cyclomaltoheptaose (18 mg) was dissolved in water (1 ml), a 0.3 M aqueous potassium hydroxide solution (0.5 ml) was added, and the mixture was stirred at room temperature for 1 day. After the reaction solution was neutralized using Amberlite IR-120 (H + form), the resin was separated by filtration and washed with water. The filtrate and the washing were combined and concentrated under reduced pressure, and the obtained residue was subjected to column chromatography using a synthetic adsorbent DIAION HP-20. The fraction eluted with water is lyophilized, and the cyclodextrin derivative 6- (6- {6- [4-O
-(5-acetamido-3,5-dideoxy-D-glycero-α-D-galact-2-nonulopyranosyl ionic acid) -butane-
1-Amido] -hexane-1-amide} -hexane-1-amide) -cyclomaltoheptaose (10 mg, 0.006 mmol, 24%)
I got

MALDI-TOF MASS: Calcd for C ₆₉ H ₁₁₆ N
_{4 O 46;. [MH]} - 1736.64 Found; [MH] - 1735.39.

[0026]

The cyclodextrin derivative having sialic acid in the side chain is a new drug delivery system (DDS) carrier for pharmaceuticals that combines the functions of the sialic acid moiety such as virus adhesion ability and cyclodextrin inclusion ability. Available. Therefore, the compound of the present invention is of great industrial value as a new material that can contribute to the development and production of new medicines.

Claims (3)

[Claims] 1. The hydrogen of the amino group of 6-monoaminocyclodextrin is represented by the following structural formula: (Wherein, n represents an integer of 0 to 5). A branched sialic acid cyclodextrin derivative, which is substituted with a sialic acid-containing acyl group represented by the formula: 2. The sialic acid-branched cyclodextrin derivative according to claim 1, which is a β-cyclodextrin derivative. 3. A compound of the general formula The intermediate for producing a sialic acid-branched cyclodextrin derivative according to claim 1, wherein R is a 9-fluorenylmethyl group.