WO2004085487A1 - Novel cyclodextrin derivatives - Google Patents

Abstract

A cyclodextrin derivative having branches constituted of sugar chains and spacer arms, characterized in that (a) the sugar chains are located respectively at the ends of the spacer arms and contain one or more sugars selected from among galactose, glucose, mannose, and sialic acids, (b) each spacer arm is located between the sugar chain and the cyclodextrin and has a skeleton comprising at least two substituted or unsubstituted hexanoic acid units, and (c) the branches are present in a proportion of one branch per molecule of the glucose constituting the cyclodextrin ring.

Description

 Description New cyclodextrin derivative Technical field

 The present invention relates to a novel cyclodextrin derivative. More specifically, the present invention relates to a novel branched cyclodextrin derivative useful in the field of the pharmaceutical industry, a method for producing the same, and a use thereof. Background art

 Targeted drug delivery systems (targeting DDS) are useful for so-called missile therapy, which efficiently and specifically delivers drugs to targets to be acted on, and have been studied so far. A drug carrier for targeting DDS requires a portion having a function of specifically recognizing a target and a portion having a function of transporting a drug.

The use of sugar chains as the part responsible for target recognition has been studied. Sugar chains play an important role in cell recognition, and it is known that some cells have sugar-specific receptors. For example, substances having hepatocytes galactose residue, Kupffer cells _c these sugar residues have respective receptor that recognizes mannose residues and glucose residues on their surface, bind to the respective receptors It is specifically taken up by hepatocyte or Kupffer cells by endcytosis. Influenza virus has a receptor (hemagglutinin) on its surface that recognizes a sugar chain having sialic acid at its terminal, and specifically binds to (a cell having) such a sugar chain.

Carbohydrate recognition has proven to be useful in targeting DDS. For example, when unmodified superoxide dismutase (SOD) is administered to mice, it hardly accumulates in the liver, whereas when SOD is modified with mannose or galactose, non-liver parenchymal cells and Specifically delivered to liver parenchymal cells, At 10 minutes after administration, about 60 to 80% of the administered dose is detected in the liver (Reference 1).

 On the other hand, various compounds (inclusion compounds') that take in the drug and form a complex compound are being studied as the part that transports the drug.

 Cyclodextrin (hereinafter sometimes abbreviated as “CD”) is one such clathrate compound, and is a non-reducing, donut-shaped form in which D-dalcoviranose is α-1,4 bonded. Cyclic oligosaccharides with hydrophilic outside and hydrophobic inside. Those consisting of 6, 7, and 8 darcoviranoes molecules are called α- and β-γ-CD, respectively. CD is used for chemical stabilization of substances, improvement of solubility, pulverization of liquid substances, etc.It is also used for various purposes in the pharmaceutical industry, but especially for targeting DDS As a drug carrier, there are advantages such as being able to stereoselectively include a pharmaceutical compound as a guest molecule in its hydrophobic cavity, being nontoxic to living organisms, and having low antigenicity. Have. Therefore, targeting DDS using sugar-protein interaction using CD modified with sugar chains has been studied.

 For example,

Man,

 Man

^Man Man one GicNAc ₂ one

 No

 Man―an '

(In the formula, “M an” represents mannose, and “G 1 c NA c j represents N-acetyl-D-darcosamine. The same applies to the following.)

CD having a sugar chain (hereinafter referred to as “M6CD”), and Man

 X

 Man

^Man Man Man GlcNAc ₂

 No

 A CD with a Man-Man-sugar chain (hereinafter referred to as “M7CD”) was synthesized, and the inclusion of the drug and recognition by immobilized concanapalin A (ConA) were examined. It had inclusion ability and protein recognition ability. However, CDs containing these branched bran chains require the preparation of asparagine, a natural oligosaccharide chain from chicken eggs (egg white albumin), so it is difficult to produce them in large quantities, making them practical as drug carriers. Is practically impossible.

 For this reason, CDs having sugar chains produced by synthesis without using sugar chains derived from natural products are also being studied (see Reference 2, Table 2). In recent years, sugar cluster-type CDs having a plurality of sugar chains have been synthesized, and β-CD (heptaGa1-β-1CD) having seven galactose chains has been described as having one galactose chain (monosaccharide). Compared with G a1 -j8-CD), both drug inclusion ability and lectin binding ability were improved. Further, when a CD having a series of two galactose chains with different arm lengths was synthesized and examined, it was found that the longer the arm, the better the lectin binding ability.

 However, none of these synthetic CDs were comparable to CDs having natural sugar chains in both drug inclusion ability and binding ability to lectin. Reference 1:

 Mitsuru Hashida, “Drug Delivery System”, Tokyo Kagaku Doujin (1995) Reference 2:

Kenjiro Hattori and Toshiyuki Inatsu, "Synthesis of Glycocluster Cluster Cyclodextrin and Double Recognition of Lectin Proteins and Drugs", Journal of Synthetic Organic Chemistry, Vol.59, No.8 (2001) pp.742-754 Disclosure of the invention

 The present invention provides a novel CD derivative and a method for producing the same, which have high target protein recognition ability and drug binding ability comparable to M6CD and M7CD having a sugar chain derived from a natural product, and can be easily produced in large quantities, And to provide the use of these compounds as carrier for targeted drug delivery.

 The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, developed a novel cyclodextrin derivative having a specific structure, and completed the present invention. The cyclodextrin derivative of the present invention is a cyclodextrin derivative having a branch consisting of a sugar chain and a spacer arm,

 a) the sugar chain is located at the end of the spacer arm and contains one or more sugars selected from galactose, glucose, mannose and sialic acid;

 b) a spacer arm positioned between the sugar chain and the cyclodextrin, having a skeleton containing at least two substituted or unsubstituted hexanoic acid units,

 c) There should be one branch per glucose molecule constituting the cyclodextrin ring.

It is characterized.

Scan Bae Saamu preferably has a backbone containing hexanoic acid unit to 2-5 amino, more preferably Darukono _{- [NH (CH 2) 5} CO] n - NH - or (CH 2) 3 - _{S - (CH 2) 2 -} CO [NH (CH 2) 5 CO] n - NH - ( wherein, n represents an integer of 2-5) Ru Oh a group represented by.

 The sugar chain is preferably a galactosyl group, a darcosyl group, a mannosyl group, or a sialic acid group (a sialyl group).

Particularly preferably, the derivative of the present invention comprises hexakis (6-galactosyl darcona amide (hexanamide) J-cyclodextrin, heptakis (6-galactosinolenoregonamide) (hexanoic acid). A (Mid) _n ) _j3-cyclodextrin, cytakis (6-galactosyl darconoamide (hexanoic acid amide) _η ) — γ-cyclodextrin (where η represents an integer from 2 to 5) The same applies to the following);

Hexakis (6-darcosyldarconoamide (hexanamide) _η ) -a-cyclodextrin, heptakis (6-darcosylgluconoamide (hexanamide) _n ) _j3—cyclodextrin, otatax (6-darcosyldarconamide, hexamide) _n ) _γ-cyclodextrin;

Hexakis (6 — mannosinolepropynolethiocinoleamide (hexanoic acid amide) _η ) 1 α-cyclodextrin, heptakis (6 — mannosylpropylthioethylamide) (Hexanic acid amide) _η ) -β-cyclodextrin, cytakis (6-mannosylpropinorechoet / reamid)-(hexanic acid amide) J-γ-cyclodextrin Hexakis (6-propylthioethylamyl sialate-amide (amide hexanoate) J-<< cyclodextrin, heptakis (6-hydroxypropylthioethyl sialate-amide) de) _n) _ β - consequent Rodez kiss Application Benefits down, and Okutakisu (6-sialic acid propylthiouracil E Chiru § Mi hexanoate § mi de to dough () J one gamma -. a Shikurodekisu Application Benefits emissions also present The present invention provides a method for producing the above-mentioned cyclodextrin derivative, wherein the method of the present invention comprises a sugar chain containing one or more sugars selected from galactose, glucose, mannose and sialic acid, or a functional group comprising the sugar chain. The presence of a condensing agent by combining a sugar chain unit containing a side chain having a functional group with a cyclodextrin unit containing a side chain having a functional group and a dextrin or a cyclodextrin having a functional group; It is characterized in that the reaction is performed under or in the absence.

Specifically, a sugar chain unit and a cyclodextrin unit are reacted in the presence or absence of a condensing agent; a bran chain and a cyclodextrin unit are reacted in the presence or absence of a condensing agent. The reaction is carried out in the presence; or the sugar chain unit and the cyclodextrin having a functional group can be reacted in the presence or absence of a condensing agent. Further, in the method of the present invention, the side chain having a functional group constitutes a spacer arm of the CD derivative of the present invention.

 The present invention also provides a target-directed drug carrier containing the cyclodextrin derivative of the present invention as described above.

 Further, the present invention also provides a targeting drug comprising the cyclodextrin derivative of the present invention and a drug.

 According to the present invention, there is provided a novel CD derivative compound exhibiting a very high sugar chain recognition interaction and an inclusion interaction with a drug, which are very high, comparable to M 6 CD bound to a natural sugar chain, and a method for producing the same. Further, according to the present invention, a drug carrier having a very high target directivity comparable to that of M6CD and an inclusion interaction with a drug, and a very high drug recognition efficiency by efficiently recognizing a sugar chain containing a drug are included. Drugs having targeting properties are also provided.

 Since the CD derivative of the present invention has high safety, extremely high targetability and extremely high drug inclusion ability, it can efficiently include a predetermined drug and reliably and specifically release it without releasing the drug on the way. It can be transported to the target site, where the drug can be specifically incorporated into target cells via sugar-specific receptors. Therefore, the CD derivative of the present invention has very high utility in targeting DDS.

 Moreover, the compound of the present invention can be synthesized by an organic synthesis reaction, and does not require the preparation of a difficult natural sugar chain which requires a great deal of time and effort. Therefore, the CD derivative of the present invention can be easily supplied in large quantities, and is highly practical. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing an outline of a procedure for synthesizing the cyclodextrin derivative heptakis (6-Ga1-cap2) -β-CD of the present invention in Example 1. Fig. 1A shows the synthesis of a sugar chain unit, Fig. 1B shows the synthesis of a CD unit, and Fig. 1C shows the outline of the assembly of each unit.

FIG. 2 is a diagram comparing the protein recognition ability and the drug inclusion ability of the conventional mouth dextrin derivative and the derivative of the present invention. Figure 2 AF It is a figure explaining a compound.

FIG. 2A: Heptakis (6-Gal-cap2) -β-CO (compound "I") of the cyclodextrin derivative of the present invention of Example 1;

Figure 2B: Cyclodextrin derivative heptakis of the comparative example (6-Ga1-cap1) -β-CO (compound “a”);

Figure 2 C: Heptakis (Gal) — / 3-CD (compound “b”);

Figure 2D: Bis (Gal—cap5) — / 3-CD (compound “c”); FIG. 2E: M 6 CD (compound “d”); and

Figure 2F: M7CD (compound "e").

 · Figure 2G shows the X-axis of the compound of Figures 2A-F and -CD (compound “f”) as the logarithm of the inclusion association constant Ka with the drug, and the y-axis as the sugar chain with lectin. This is a two-dimensional map in which each value is plotted as the logarithm of the recognition association constant Ka. BEST MODE FOR CARRYING OUT THE INVENTION

 Cyclodextrin derivative

 The components constituting the cyclodextrin derivative of the present invention are basically a sugar chain, a spacer arm, and cyclodextrin.

 Cyclodextrin

The cyclodextrin used for synthesizing the cyclodextrin derivative of the present invention is not particularly limited as long as it has the basic skeleton of cyclodextrin. Generally, α-CD, β-CD and γ-CD are mentioned. These CDs differ in the number of darcoviranoses that make up the ring (α: 6; / 3: 7; γ: 8), and therefore have different cavities (α: 0.45 nm; β 0.70 nm; γ: 0.85 nm). For example, α-CD is large enough to contain a benzene ring, and can include trichlorne, perclene, and the like. Also, j3-CD has a size enough to contain a naphthalene ring, and γ-CD has a size enough to contain two anthracene-naphthalene rings. Therefore, those skilled in the art can appropriately select a CD having an optimum cavity diameter in consideration of the molecular size of the drug to be included. Further, from the viewpoint of the inclusion ability of the drug, it is preferable that the number of sugar chain branches, that is, the number of glucoviranose molecules constituting CD is large. From the viewpoint of availability, a-CD, β-CD and γ-CD are preferred. The sugar chain for modifying the CD of the present invention is not particularly limited as long as it is specifically recognized by a specific target. The number of sugars may be at least one, and may be two or more. In the present specification, the term “sugar chain” includes not only a case where two or more sugars are linked but also a case where there is one sugar.

 The sugar constituting the sugar chain may be unmodified or modified. Preferably, it is selected from galactose, glucose, mannose and sialic acid; amino sugars such as galactosamine, dalcosamine and mannosamine. From the viewpoint of ease of synthesis, galactose, glucose, mannose and sialic acid are preferred. When the sugar chain contains two or more sugars, the sugars may be of the same kind or different kinds.

 In addition, the sugar chain may be linear or branched.

 From the viewpoints of availability and ease of synthesis, the sugar chain is preferably 1 to 6 sugar chains, more preferably 1 to 4 sugar chains, and still more preferably 1 to 4 sugar chains. It is a linear sugar chain, and the most preferred sugar chain is a monosaccharide, that is, a galactosyl group, a darcosyl group, a mannosyl group, and a sialic acid group.

 Spacer arm

The spacer arm used in the CD derivative of the present invention is located between the sugar chain and the CD and connects both, and generally, the C4 of the sugar at the reducing end of the sugar chain is used. Refers to the portion between the carbon atom and the carbon atom at the C 6 position of the glucose molecule that makes up the CD. The spacer arm only needs to have a skeleton containing at least two substituted or unsubstituted hexanoic acid units [— (CH ₂ ) ₅ —CO—].

The spacer arm preferably has 2 to 5 hexanoic acid units [one (CH ₂ ) ₅ —C _n ] _n or an aminoamino acid unit [one NH— (CH ₂ ) ₅ -C O-] _n (wherein, n represents an integer of 2 to 5). Scan Bae Saamu is for ease of synthesis, particularly preferably Darco Bruno - [NH (CH _{2) 5} C_〇] _n - NH - or _{(CH 2) 3 - S -} (CH

_{2) 2} - CO _{[NH (CH 2) 5 CO]} n - NH - ( wherein, n is a group represented by represents) the integer of 2-5.

 The backbone of the spacer arm may be substituted or unsubstituted, for example, hexanoic acid (synonymous with caproic acid), aminoethanol, aminoethanethiol, polyethylene glycol (synonymous with polyoxyethylene), It is composed of a group derived from polypropylene glycol cornole, octaethylenedali cornole, bisepoxyanolylene, diaminoalkylene, dicarboxyalkylene, succinic anhydride and the like, or a combination thereof.

 The length of the spacer arm is related to the size of the cell surface protein, etc. of the target to which the drug is delivered, but the sugar chains facing each other in a form in which the spacer arm bound to CD opens outward in solution. It is preferable that the distance between them is close to the diameter of the target protein (if the target protein is a dimer, the distance between the binding sites at two sites). Therefore, when selecting a long sugar chain, the spacer arm can be relatively short.

 The spacer arm may contain atoms other than carbon in the skeleton. In that case, nitrogen, zeolite, oxygen and the like are preferable.

 Substituents that may be present on the spacer arm skeleton include alkyl, aryl, phenylene, amide, amino, ketone, hydroxyl, thiol, and carboxyl groups. Examples of the group include, but the size, type and number of the substituent are not particularly limited as long as the flexibility of the spacer arm portion is not hindered.

 Branch

 The branch consists of a sugar chain and a spacer arm. The number of branches is the same as the number of glucose molecules constituting the CD ring, that is, one branch per glucose molecule constituting the CD ring.

The location of the branch on the CD is not particularly limited, and may be on the primary side (ie, the C 6 position of the dalcopyranose ring) or on the secondary side (C 3rd). Generally, the primary side is preferable from the viewpoint of high reactivity and ease of synthesis.

Accordingly, specific examples of particularly preferred CD derivatives of the present invention include hexakis (6-galactosyldarconoamide (hexanic acid amide) J_α-cyclodextrin, heptakis (6-galactosylgluconoamide) (Hexanoic acid amide) _η ) — j3 — Cyclodextrin, Cytakis (6 — Galactosyldarconoamide (hexanic acid amide) J — γ — Cyclodextrin (where η represents an integer of 2 to 5. The same applies to the following);

Hexakis (6 — darcosyl darconoamide (hexanic acid amide))) α-Cyclodextrin, heptakis (6 — guanolecosinoregnoleconoamide (hexanoic acid amide) J) 1/3 -cyclodextrin, octakis (6-darcosyldarconamide) (hexanoic acid amide) J- _γ -cyclodextrin;

Hexakis (6—Mannosylpropinorethioethyl) amide (Hexanamide) J _α—Cyclodextrin, Heptakis (6—Mannosylpropylthioethyl-amide (Hexanamide) ) _η ) — β —cyclodextrin, cytakis (6 —mannosylpropylpyruchoethyl) -amide— (hexanoic acid amide) J-γ—cyclodextrin;

Hexakis (6-propylthioethyl sialate-amide (hexanoic acid amide) J-α-Cyclodextrin, heptakis (6-propyl thiothiol sialate-Amido (hexanoic acid amide) J 1] 3—cyclodextrin and octoctakis (6-propyl thiothiolamide amide— (hexanamide) J-γ—cyclodextrin

Is mentioned. These have the following formula: cyclo

(Wherein, r is, represents the 6, 7 or 8; X is galactosyl - glucono _{- (NH (CH 2) 5} CO) n NH -, Darukoshiru - Darukono _{- (NH (CH 2) 5} CO) n NH -, mannosyl _{- (CH 2) a S (} CH 2) 2 CO (NH (CH 2) 5 CO) n NH - or sialic acid _{- (CH 2) 3 S (} CH 2) 2 CO (NH (CH 2 ) ₅ CO) _n represents NH −; n represents an integer of 2 to 5.)

Most preferred for ease of synthesis is the compound of the above formula where n = 2, ie, hexakis (6-galactosylgluconoamide (hexanoic acid amide) ₂ ) —α-cyclodextrin Lin, heptakis (6-galactosyldarconoamide (hexanic acid amide) ₂ ) 1; 3-cyclodextrin, cystakis (6-galactosylgluconoamide (hexanic acid amide) ₂ ) mono-γ-cyclodextrin;

Hexakis (6-darcosyldarconoamide (hexanic acid amide) ₂ ) — α-cyclodextrin, heptakis (6-darcosyldarconoamide (hexanic acid amide) ₂ ) 1-cyclodextrin, otataxi (6-glucosylgluconoamide (hexanamide)) 1 γ-cyclodextrin;

Hexakis (6-mannosinolepropizolethiotinoleamide (hexanoic acid amide) ₂ ) —one cyclodextrin, heptakis (6-mannosylpropylthioethyl-amide— hexanoic acid Ami de) ₂₎ - 3- consequent Rodekisu bird down, Okutakisu (6 mannosyl prop Honoré Chio E chill Ichia Mi hexanoate § mi de to dough () ₂₎ Single _gamma - Shikurodekisu DOO phosphorus;

Hexakis (propylthioethyl 6-sialate monoamide (hexanoic acid) Amide) ₂ ) — 1-cyclodextrin, heptakis (6-thiopropyl thiolate) -amide (hexanic acid amide) ₂ ) _; 8-cyclodextrin, octakis (6-sialic acid) Propylthioethyl-amide (hexanoic acid amide) ₂ ) One γ-cyclodextrin.

 Compounds with η = 2 to 5 are most preferred from the viewpoint of target protein recognition ability. Production method .

 The method for producing a CD derivative of the present invention comprises a sugar chain containing at least one sugar selected from galactose, glucose, mannose and sialic acid, or a sugar chain containing this sugar chain and a side chain having a functional group. A unit having a functional group-containing side chain and a CD, or a CD having a functional group, in the presence or absence of a condensing agent.

 Specifically, in the method of the present invention, (1) the sugar chain unit is reacted with the cyclodextrin in the presence or absence of a condensing agent; (2) the sugar Reacting the chain with the CD unit in the presence or absence of a condensing agent; or (3) reacting the bran chain unit with CD having a functional group in the presence or absence of a condensing agent Can be reacted.

 The bran chain unit has a sugar chain composed of one or more sugars selected from glucose, galactose, mannose and sialic acid, and a side chain having a functional group.

 The sugar chain contained in the sugar chain unit is as described for the CD derivative of the present invention. Ratatose (lactose) is used to make the non-reducing terminal of the sugar chain galactose, and maltose (maltose) is used as the material of the sugar chain to make glucose. The reducing end is oxidized to form the lactone ring. It is easy to synthesize by forming and bonding to the side chain using the reactivity of this lactone ring. In this case, glucose on the reducing end side of the raw material disaccharide becomes a dalcono group to form a part of the side chain of the sugar chain unit, and eventually becomes a part of the spacer arm.

When the non-reducing terminal of the sugar chain is mannose or sialic acid, the sugar chain unit can be synthesized from each sugar by a known method. For example, dissolve mannose in aryl alcohol and add acid catalyst The mixture is refluxed at 97 ° C. under a nitrogen stream to produce arylmannoside, which can be used as a sugar chain unit. To further extend the side chain, for example, mercaptopopionic acid can be added to arylmannoside.

 The side chain having a functional group will be described later.

 CD is also as described for the CD derivative of the present invention. In the reactions of the above types (1) and (2), a CD unit produced by introducing a side chain having a functional group into each darcoviranose molecule constituting the CD ring is used. In the above-mentioned type (3), a reaction is used in which a functional group is directly introduced into each dalcovaranose molecule constituting the CD ring. A method for introducing a side chain or a functional group having a functional group into CD is known.

 The side chain having a functional group of the sugar chain unit and / or the side chain having a functional group of the CD unit may be converted into a sugar chain of the CD derivative of the present invention as a result of the above-mentioned reactions (1) to (3). Build a surarm. Therefore, whether these side chains are the side chains contained in the sugar chain unit or the side chains present in the CD, the side chain is basically the same as the spacer arm of the CD derivative of the present invention described above. It has a similar skeleton.

 When performing the reaction of the type (1) above, the side chain contained in the sugar chain unit and the side chain present on the CD each constitute a part of the spacer arm, and after the synthesis, the spacer arm is formed. Make up the whole. When the reaction of the type (2) is performed, the side chain contained in the CD unit is used. When the reaction of the type (3) is performed, the side chain contained in the bran chain unit is used. Each has a length and a structure sufficient to constitute the entire spacer arm.

Therefore, those skilled in the art will understand whether the side chain having a functional group is the side chain included in the sugar chain unit or the side chain included in the CD unit, and the description of the spacer arm already described. Based on the above, an appropriate length and structure of a side chain and a combination of side chains in each case can be appropriately selected. Specifically, these side chains, whether included in the bran chain unit or the CD unit, are based on aliphatic hydrocarbon skeletons that make up all or part of the spacer arm. Having. The side chain preferably has a skeleton containing a substituted or unsubstituted hexanoic acid unit [— (CH ₂ ) ₅ —C.〇I] _n , and more preferably an aminohexanoic acid unit [1-NH— (CH ₂ ) ₅ —CO—] _n (wherein, n represents an integer of 2 to 5). N in the above formula is 1 to 5 in the case of the type (1), and 2 to 5 in the case of the type (2) or (3). Further, these side chains have a functional group at the terminal for reaction.

 The functional group present on the side chain and / or the functional group of the CD having a functional group includes an ether group, a thioether group, an amino group, a carboxyl group, an azide group, a p-toluenesulfonyl group, an epoxide group, an unsaturated group, and a thiol. Group, an acetooxy group, a phenoxy group, or a halogen group of iodine or bromine or chlorine.

Examples of the condensing agent that can be used in the reaction of the types (1) to (3) include dicyclohexyl carpoimide (hereinafter referred to as “DCC”) and water-soluble carpoimide (hereinafter “WSC”). And the like, and condensing agents known in the art. One skilled in the art can select an appropriate condensing agent depending on the selected functional group and the type of reaction solvent. For example, in the reactions of the above types (1) and (3), the functional group at the terminal of the side chain of the sugar chain unit is a hydroxyl group, and the functional group on the side chain of the CD unit or on the CD is amide. When a carboxyl group is reacted with an amino group, as in the case of a heterocyclic group, a new condensing agent developed by Kunisnima et al. (Kunishima et al., Tetrahedron (1999) 55., pp. 13159-13170) 4 It is preferable to use-(4,6-dimethyoxy-1,3,5-triazine-2-yl) -4-methylmorpholinidium chloride (hereinafter referred to as "DMT-MM") in terms of efficiency. DMT-MM reacts with carboxylic acids to form active esters, and then reacts with amines to form amide bonds. It has been reported that the reaction proceeds in various solvents such as ethanol, methanol, upper-propanol, and water, and that the reaction proceeds quantitatively. The reaction of the type (1) can also be carried out in the absence of a condensing agent. For example, it is possible to use a lactone ring obtained by oxidizing glucose at the reducing end of a sugar chain to lactobonic acid and then cyclizing the lactone ring. Further, a reaction between a thiol group and a halogen group on each side chain between the sugar chain unit and the CD unit and a reaction between a thiol group and a double bond group are possible.

 In the type (2) reaction, even in the absence of a condensing agent, a sugar chain can be directly reacted with a functional group present on a side chain of a CD unit to bond the sugar chain. For example, the reactivity of sialic acid with the carboxylic acid can be used to react with the functional group on the side chain of the CD unit in the presence of a condensing agent. It is sufficient to use an object or acid-rich lid. When an amino sugar such as galactosamine, dalcosamine or mannosamine is used, these can be reacted with a carboxylic acid group introduced on the side chain.

 The reaction of the type (3) can also be carried out in the absence of a condensing agent. For example, a thiol group can be introduced as a functional group into a sugar chain unit, a halogen group such as iodine can be introduced into CD, and both can be reacted in an atmosphere of argon.

 The production of the derivatives of the present invention can all be performed at room temperature. As the reaction solvent, dimethylformamide (DMF), water, methanol, N-methylpyrrolidinone (NMP) can be used depending on the solubility of the ligated compound. Those skilled in the art can appropriately design a side chain, select a condensing agent, and select a reaction solvent.

 For example, in the cases of (1) and (3) above, it is possible to use a molar equivalent of triethylamine equivalent to the carboxylic acid as the reaction solvent and a 5-fold molar equivalent of DMT-MM as the condensing agent. it can.

Specifically, for example, it can be synthesized as follows. First, a desired sugar chain is condensed with 6-aminohexanoic acid to form a sugar chain unit in which the sugar chain and hexanoic acid are amide-bonded. On the other hand, CD (for example, -CD) in which the primary hydroxyl group is substituted with an amino group is replaced with 6-aminohexanoic acid To produce a CD (eg, heptakis (6-amino)-] 3-CD) having the same number of 6-aminohexaneamide branches as the number of dalcoviranose molecules constituting the CD. The above sugar chain unit and the branched CD are combined and combined by a condensation reaction to prepare a CD derivative of the present invention.

 Structure confirmation and purification of the synthesized CD can be performed by ordinary methods known in the art.

 Targeted drug carriers

 The targeting drug carrier of the present invention is characterized by containing the cyclodextrin derivative of the present invention. The target of the targeting drug carrier of the present invention differs depending on the sugar chain present in the cyclodextrin derivative. Targets are, for example, hepatocytes, Kupffer cells, influenza viruses and the like.

 The targeting drug carrier may include water, ethanol, ethylene glycol, propylene glycol, phosphate, inorganic salts such as NaCl, and the like.

 Targeting drugs

 The targeting drug of the present invention contains the cyclodextrin derivative of the present invention and a drug. In the targeting drug of the present invention, the drug is included in a cyclodextrin molecule.

 Drugs to be included in the cyclodextrin derivative of the present invention include, but are not limited to, anticancer agents, therapeutic agents for liver diseases, drugs acting on the immune system, antiviral agents, and the like. A drug to be acted on the target can be appropriately selected, and a cyclodextrin derivative having a cavity diameter suitable for the selected drug can be produced and used.

 The method of including a guest molecule such as a drug with the cyclodextrin derivative of the present invention may be any method known in the art. For example, the guest molecule is included by contacting the cyclodextrin derivative (host) of the present invention with the guest molecule in an aqueous solution, and the solution is dried by a method such as freeze drying or spray drying. A dried product can be obtained.

Such drug inclusion complex of sugar-modified CD is dissolved in physiological saline etc. at the time of use. Can be injected intravenously. It can also be formulated into other administration forms, for example, enteric preparations. Such a formulation method is known. Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

 In the following synthesis and product confirmation experiments, the following equipment was used as needed:

 Laser ionization time-of-flight mass spectrometer (MA LDI-TOF-MS: V oyager DEPro-T (Applied iosystems)) Superconducting FT-NMR system: J NM_Lambda 500 (manufactured by JEOL) Double focusing mass Analyzer: SX102A (JEOL)

 Analytical HPLC: L-600 (manufactured by Hitachi), UV-975 (manufactured by JASCO) Preparative HPLC: HLC-870 (manufactured by Tosoichi) Example 1. Heptakis (6-galatatosylglucono) -Amid -Hexanoic acid amide -Hexanoic acid amide--CD [Heptakis (6-Galapap2) -β-CD]

 Heptakis (6-Gal-cap2) -β-CO was synthesized by the following procedure. The outline of the synthesis scheme is shown in FIG.

 1) Synthesis of sugar chain

Lactonolactone was synthesized according to the report of Kobayashi et al. (Kobayashi et al., Polym. J. (1998) 30, pp.653-658). Briefly, lactose is added to a methanol solution of iodine, and a methanol solution of 4% KOH is added dropwise at 45 ° C to form a potassium carboxylate having an oxidized reducing end. Cooled and crystallized. Potassium is removed by ion exchange resin (Amberlite IR-120B, available from Rohm and Haas Co., Ltd., Organo Co., Ltd.), and dehydrated with ethanol / methanol using an evaporator. The ring was opened to obtain lactonolactone. 2) Preparation of sugar chain unit

 This rata tonolactone was azeotroped with ethanol / ethanol using an evaporator, and two equivalents of 6-aminohexanoic acid were combined with 60% dimethylformamide (60 ° C) under a nitrogen stream. (Hereinafter referred to as “DMF”) and reacted overnight (about 12 hours). This was subjected to an ion exchange resin (CM-Sephadex, C25, manufactured by Pharmacia, available from SI GMA) to remove unreacted 6-aminohexanoic acid, and to remove galactosyldarcono-amide-hexane. An acid (hereinafter sometimes referred to as “Ga 1 -cap 1 -OH”) was obtained.

 G a1 -cap1 -OH dissolves in methanol, while lactobionic acid (a ring-opened product of lactonolactone) in the raw material does not dissolve. Utilizing this, purification was performed by methanol extraction. Further, the remaining lactobionic acid was precipitated with a mixed solvent of methanol and ethyl acetate (1: 1.5 (V / V)) to obtain Gal-capl-OH as a dissolved product. The yield was 68%. The Rf value by thin layer chromatography (TLC) was 0.47 (developing solvent butanol: ethanol: water = 5: 4: 3), confirming that the sample was a spot spot.

 3) Synthesis of condensing agent

 DMT-MM was synthesized according to the method of Kunishima et al. (1999) for use as a condensing agent for the amide bond formation reaction. Briefly, a beaker is charged with 1.1 equivalents of 2-chloro-2,2,6-dimethoxy-1,3,5-triazine (hereinafter referred to as ΓC DMT J) and the THF 60 m Then, 1 equivalent of N-methylmorpholine (hereinafter referred to as “NMM”) was added, and the mixture was stirred at room temperature for 30 minutes. The resulting white precipitate was filtered and washed with THF to give the product. The yield was 95%.

As a confirmation of the synthesis, when FAB-MS was measured, it coincided with the value described in Kunishima et al. (1999), indicating that the target compound was synthesized. 4) Heptakis (6-amino-hexanoic acid amide)-3-CD

 To synthesize heptakis (6-amino-hexanoic acid amide) -β-CD as a CD having a side chain, first synthesize heptakis (6-amino) -j8-CD, Finally, the product was de-Bocylated as ptakis (6-Boc-hexanoic acid amide) -β-CD to obtain the desired compound.

 First, heptakis (6-amino) -β-CD was synthesized according to the report of Peter R. Ashton et al. (J. Org. Chem. (1996) 61, pp. 903-908). Specifically, 2 g of heptakis (6-azito)--CD and 6.36 g of triphenylphosphine (hereinafter, referred to as "TPP") were added to DMF, and the mixture was added at room temperature. After stirring for 24 hours, 8.4 ml of 25% ammonia water was added, and the mixture was further stirred for 18 hours.

 This solution was concentrated and dried by a rotary evaporator. Thereafter, an appropriate amount of ethanol was added to this solid to suspend and wash, and the washing was removed by filtration. The filtrate was dissolved in 0.1 M hydrochloric acid, insoluble matter was removed by filtration, and the filtrate was concentrated using a rotary evaporator and dried. The yield was 42%.

The synthesis was confirmed by ¹³ C-NMR and TOF-MS. ^{In 13} C-NMR, it was confirmed that the signal of the carbon at the C 6 position was shifted. The hydrochloride was neutralized with dilute NaOH solution and analyzed for free amine products. In TOF-MS, a peak was observed at mZz: 1127 [M] ⁺ with respect to the molecular weight of 112, confirming that the desired compound was synthesized.

 After adding 1 equivalent of the above prepared heptakis (6-amino) -β-CD and 8 equivalents of Boc hexanoic acid to DMF and dissolving them, add 40 equivalents of the condensing agent DMT-MM and add room temperature. For 24 hours. After the reaction, the mixture was concentrated by an evaporator, and water was added to precipitate a product. After removing the filtrate, the filtrate was dissolved in methanol and dried. Purification by HPLC yielded heptakis (6-Boc-hexanoic acid amide)-/ 3-CD.

Next, the purified heptakis (6-Boc-hexanoic acid amide) -β- TFA was added to the CD, and the mixture was stirred under ice-cooling for 2 hours to perform a de-Boc reaction. Yield 4 1. /. Met.

 When T OF -MS was measured, the molecular weight of the product was 19

/ Z: 1 9 2 0 [ M + H] +, 1 9 4 2 [M + N a] +, 1 9 5 8 Now that you observe a peak only [M + K] +, the desired compound is synthesized Was confirmed.

 5) Synthesis of Heptakis (6-Gal-cap2) _- CD

In H ₂ 〇 mixed solvent, 8 equivalents of Heputaki scan prepared as described above - NM P (6 - Amino - hexanoic acid § Mi de) - 0 - CD, 1 equivalent of G al - cap 1 - OH, 8 After adding and dissolving equivalent amounts of triethylamine (hereinafter referred to as “TEA”), 40 equivalents of a condensing agent DMT-MM were added and reacted at room temperature for 48 hours. After the reaction, the mixture was concentrated by an evaporator and dropped into ice-cooled acetone to precipitate. Thereafter, filtration was performed, and after removing the filtrate, the filtrate was dissolved in water.

 For purification, first, CM-Sephadex (C25, manufactured by Pharmacia, S

After removing the unreacted amino group-containing CD derivative using IMA, gel filtration using a TOPEARL HW-40F column (4.2 cm x 46 cm; manufactured by Tosoh) (eluate) Water). Finally, isolation was confirmed by analytical HPLC. The yield was 30%.

 Comparative Example 1. Heptakis (6-galactosyldarcono-amide-hexanoic acid amide) -β-CO [Heptakis (6-Gal-capl) -β

-C D]

NM P - to H ₂ 0 mixed solvent, one equivalent of Cheb Takis prepared in the same manner as in Example 1 (6 - Amino) - jS - CD in the same manner as in Example 1 prepared 8 equivalents of G al - CAPL - OH, After adding and dissolving 8 equivalents of {Τ} respectively, 40 equivalents of a condensing agent DMT-MM were added and reacted at room temperature for 48 hours. After the reaction, the mixture was concentrated with an evaporator and dropped into ice-cooled acetone to precipitate. Thereafter, filtration was performed, and after removing the filtrate, the filtrate was dissolved in water.

For purification, first, an unreacted amino group-containing CD derivative was extracted using CM-Sephadex (C25, manufactured by Pharmacia, available from SIGMA). After the removal, the mixture was subjected to gel filtration (eluent: water) using a Toyopearl HW-40F column (4.2 cmX 46 cm; manufactured by Tosoh Corporation). Finally, the isolation was confirmed by analysis HP. The yield was 31%.

 Test Example 1. Evaluation of lectin recognition ability

 The target protein recognition ability was evaluated by surface plasmon resonance (SPR) using peanut lectin (PNA) as the target protein. IASsys (manufactured by Thermo) was used as an SPR optical biosensor, and the obtained data was analyzed using the attached software FASTfit.

 1) Immobilization of PNA

Immobilization of PNA (tetramer) is described in Imata (Graduate School of Engineering, Tokyo Polytechnic University, Graduate School of Engineering, Doctoral Course in Industrial Chemistry (1997)) and Yasuda (Graduate School of Engineering, Tokyo Polytechnic University, Graduate School of Engineering, Master's Thesis in Industrial Chemistry) (1999)). First, as a linker agent for reacting with the Amino group of the lectin protein to amino silane based on SPR optical by-O sensor particulate Beck preparative surface, bis (Suruhosukushini Mijiru) scan base rate ^{(BS 3) (1 ηιΜ -} BS ^3/1 0 mm- phosphate buffered saline (PBS), was p H 6. 5) reacted in advance. This was repeated several times until the response became difficult to change, and an unreacted active aminosilane group was inactivated by adding an acetic anhydride-acetic acid solution (mixing volume ratio 1: 1), and blocking was performed.

After blocking, replace with pH 5.3, 10 mM-acetate buffer solution, and then 200 μL of PNA solution (1 mg PNAZ 10 mM-acetate buffer solution, pH 5.3) Was added to the cuvette and reacted. Next, a 1 M-ethanolanol aqueous solution and pH 8.5 were added to inactivate the blocking of the succinic acid amide ester group. Response (R) = 1 ng Zmm ² at 600 arcsec and one aminosilane group per nm ² is present. Since the amount of change in the response of the PNA was about 176 s, the immobilization rate was calculated to be about 1.6%.

 2) Measurement

10 mM-acetate buffer solution containing l mM-MgCl ₂ , 100 mM-NaCl, ImM-CaCI, pH 5.3, (0.2-1) X Ten A solution containing 100 μL of each CD to be tested, which was changed to a concentration of 6 points in the range of ⁵ M, was placed in a cuvette on which PNA was immobilized, and the interaction analyzer (SPR optical The interaction between each CD and immobilized PNA was measured using a biosensor. The measurement temperature was 25.0 ° C, and the required time was 5 to 30 minutes until the response reached saturation.

 3) Results

 The CD derivative heptakis (6-Gal-cap2) -β-CD of the present invention with respect to PNA measured as described above and the CD derivative heptakis (6-Gal-capl) -β-CD of the comparative example Table 1 shows the analysis results of the association behavior. For reference, similar measured values for other CD derivatives described in the known literature are also shown. Table 1: Aggregation behavior for lectin protein

 : From Reference 2, Table 7

 Abe (From Tokyo Polytechnic University Graduate School, Graduate School of Engineering, Industrial Chemistry Master's Thesis (2000))

: From Reference 2, Table 5 (however, C on A is used as protein) Assuming that Ka of the CD derivative of the comparative example having a spacer arm for one hexanoic acid unit is 1, K of the CD derivative of the present invention having a spacer arm for two hexanoic acid units a has a 54-fold higher association ability, and the derivative of the present invention has a very high recognition ability even when compared with the relatively high recognition ability of conventional CD derivatives. found.

 Test example 2. Measurement of inclusion ability of drug

 Using the antibiotic doxorubicin (Doxorubicin; “DXR”) as a drug, the inclusion ability of the derivative of the present invention was examined in the same manner as in Test Example 1.

 1) DXR immobilization

 DXR is immobilized on the optical biosensor cuvette surface in the same manner as the lectin immobilization described above, except that a buffer with a different pH was used as a buffer solution for dissolving the drug. went.

The Aminoshiran group cuvette preparative surface as a Menori Lanka one agent is reacted with Amino group of DXR, 1 mM - were PBS, reacted with ^{p H 6. 5 - BS 3/} 1 0 mM. This is repeated several times until the response of the optical biosensor does not change, and an unreacted aminosilane group is inactivated by adding an acetic anhydride-acetic acid solution (mixing volume ratio 1: 1), and blocking is performed. Was.

After blocking, the solution was replaced with a 10 mM-acetate buffer solution of pH 5.3, and a DXR solution (2 mg DXR / 10 mM-acetate buffer solution, pH 5.3) was added and reacted. Then, taking into account the effect of the background on the cuvette, the succinic amide estenole group was blocked with a 1 M-ethanolamine aqueous solution, pH 8.5. Since variable I inhibit the amount of response of DXR was about 5 5 arcsec, and shall not exist aminosilane group one per 1 nm ² with R = 6 0 0 arcsec Noto-out 1 ng / mm ² Thus, the immobilization rate was calculated to be about 9.6% of the aminosilane groups.

 2) Measurement

Using the DXR-immobilized cuvette prepared above, the interaction between DXR and each CD was measured in the same manner as in Test Example 1. However, concentration of CD tested ranged from ^{(0. 2~1) X 1 0- 4} M. 3) Results

 The CD derivative heptakis (6-Gal-cap2) -β-CD of the present invention with respect to DXR measured as described above and the CD derivative heptakis (6-Gal-capl) -β-CD of the comparative example Table 2 shows the analysis results of the association behavior. For reference, similar measured values for other CD derivatives described in the known literature are also shown. Table 2: Association behavior for DXR

 : From Reference 2, Table 8

 Abe (From Tokyo Polytechnic University Graduate School, Graduate School of Engineering, Industrial Chemistry Master's Thesis (2000))

 : From Reference 2, Table 6 (however, the value for cholic acid instead of DXR) Compared with the Ka of the CD derivative of the comparative example, the association ability of the CD derivative of the present invention was increased about 16-fold. . In addition, since other known CD derivatives are inferior to the CD derivatives of the comparative examples, the CD derivatives of the present invention have extremely excellent drug inclusion ability as compared with the conventional CD derivatives having a synthetic sugar chain. It was found to have.

The data for M6CD and M7CD listed in Table 2 were obtained using cholic acid, which is easily included in CD, as a drug. M6CD and M7CD differ from the CD derivative and the like of the present invention in that a hydrophobic field is easily formed due to the presence of the Fmoc group. Taking these differences into account, the drug (DXR) inclusion capacity of M6CD and M7CD is estimated to be on the order of one-tenth of these data. As a result, the CD derivative of the present invention is interpreted to be equivalent to the CD having these natural sugar chains also in drug inclusion ability.

 Test Example 3. Evaluation of Double Recognition of Various Sugar Chain Branched CDs by Two-Dimensional Map In order to evaluate the drug inclusion ability and lectin binding ability of known cyclodextrin tris derivatives and the compound of the present invention, Tables 1 and 2 were used. Based on the indicated values, the X axis is the logarithm of the inclusion association constant Ka with the drug, and the y axis is the logarithm of the sugar chain recognition association constant Ka with lectin.] 3-CD, known Two-dimensional maps were prepared by plotting some sugar-chain-modified jS-CD and the CD derivative of the present invention (FIG. 2G).

 Unmodified β-CO (Γ f) has a lg Ka of about 3 for the drug, but has 0 for lectin because it has no sugar. Conventionally known CD a ”to“ c ”having a galactose chain; FIGS. 2B to D) show 1 og Ka force S 3 to about 5 for the drug, and log 1 and 4 for the lectin of 4 to 6 It is plotted in a small area. The M6CD (“d”; FIG. 2E) and the M7CD (“e”; FIG. 2F) located at the top right of the map have 6 and 7 mannose residues, respectively. It has a natural sugar chain consisting of N-acetyl-D-darcosamine 2 residues, and has the highest association ability with lectins and drugs that has been examined so far.

Heptakis (6-Gal-cap2) -j3-CD ("1"; FIG. 2) synthesized in Example 1 has the same ability to associate with lectin as M6CD and M7CD, The meeting ability is interpreted to be similar. Therefore, according to the present invention, a CD derivative having extremely high protein recognition ability and drug inclusion ability comparable to CD having a natural sugar chain by synthesis can be obtained. This application is a Japanese patent application filed on March 27, 3-8 7192, and the contents of the specification and claims of Japanese Patent Application No. 2003-87192 are all included in the specification of this application. You.

Claims

The scope of the claims 1. A cyclodextrin derivative having a branch consisting of a sugar chain and a spacer arm,  a) the sugar chain is located at the end of the spacer arm and contains one or more sugars selected from galactose, glucose, mannose and sialic acid;  b) the spacer arm is located between the sugar chains and the cyclodextrin, and has a skeleton containing at least two substituted or unsubstituted hexanoic acid units;  c) There should be one branch per glucose molecule constituting the cyclodextrin ring.  A cyclodextrin derivative characterized by the following.  2. The cyclodextrin derivative according to claim 1, wherein the spacer arm has a skeleton containing 2 to 5 aminohexanoic acid units. 3. The spacer arm is Darcono-[NH (CH ₂ ) ₅ CO] _n -NH-or (CH ₂ ) a-S-(CH ₂ ) ₂ -CO [NH (CH ₂ ) ₅ CO] _n- The cyclodextrin derivative according to claim 2, which is a group represented by NH-(wherein, n represents an integer of 2 to 5). 4. The cyclodextrin derivative according to any one of claims 1 to 3, wherein the sugar chain is a galactosyl group, a darcosyl group, a mannosyl group, or a sialic acid group. 5. Hexakis (6-galactosyldarconoamide (hexanic acid amide) „) — α-cyclodextrin, heptakis (6-galactosyldarconoamide (hexanic acid amide) _η ) β-cyclodextrin, octakis (6-galactosyldarconamide (hexanic acid amide) _η ) _γ-cyclodextrin (where η represents an integer of 2 to 5). The same applies to the following); Hexakis (6-Darcosylgnoconamide (Hexamic acid amide) _η ) — a—Cyclodextrin, Heptakis (6-Darcosine) Darconamide (hexanamide) _n ) — —cyclodextrin, octakis (61-glucosylgnoconamide (hexanoic acid amide) _η ) — γ-cyclodextrin; Hexakis (6-mannosinolepropynolethiotinoleamide (hexamic acid amide) _η ) —Hycyclodextrin, heptakis (6-mannosylpropylthioethylamide (hexanoic acid amide)) 1) | 3-cyclodextrin, octakis (6-mannosylpropyl thioethyl-amide (hexanoic acid amide) _η ) — γ-cyclodextrin; Hexakis (6 -propinolethiotinol sialic acid amide (amide hexanoic acid) _η )-α-cyclodextrin, heptakis (6-thiopropyl sialate-amide ( Hexanoic acid amide) _η ) — / 3-cyclodextrin, and octakis (Propyl 6-sialate thioethyl monoamide (hexanic acid amide) J-γ — Cyclodextrin  5. The cyclodextrin derivative according to claim 4, which is any of the following. 6. The method for producing cyclodextrin according to any one of claims 1 to 5, wherein the sugar chain comprises one or more sugars selected from galactose, glucose, mannose and sialic acid, or a sugar chain comprising the sugar chain. A condensing agent comprising: a sugar chain unit containing a sugar chain and a side chain having a functional group; and a cyclodextrin unit containing a side chain having a functional group and cyclodextrin or a cyclodextrin having a functional group. A reaction in the presence or absence of a compound.  7. The method according to claim 6, wherein the sugar chain unit is reacted with the cyclodextrin unit in the presence or absence of a condensing agent.  8. The method according to claim 6, wherein the sugar chain is reacted with the cyclodextrin in the presence or absence of a condensing agent.  9. The method according to claim 6, wherein the sugar chain cut is reacted with cyclodextrin having a functional group in the presence or absence of a condensing agent. 10. The side chain and the functional group of Ζ or cyclodextrin are Claims 6 to 6, wherein the group is selected from a ter group, a thioether group, an amino group, a carboxyl group, an azide group, a p-toluenesnoleophyl group, an epoxide group, an unsaturated group, a thiol group, and halogen groups of iodine, bromine and chlorine. 10. The method according to any one of 9 above. 11. The side chain having a functional group of a sugar chain unit and / or the side chain having a functional group of a cyclodextrin unit has a skeleton containing a substituted or unsubstituted hexanoic acid unit. The method according to any one of claims 1 to 10.  12. The side chain having a functional group of a sugar chain unit and / or the side chain having a functional group of a cyclodextrin unit has a skeleton containing 1 to 5 aminoaminohexanoic acid units. 6. The method according to any one of items 6 to 11.  13. A targeting drug carrier comprising the cyclodextrin derivative according to any one of claims 1 to 5.  14. A targeting drug comprising the cyclodextrin derivative according to any one of claims 1 to 5, and a drug.