JP5144957B2 - Multi-branched cyclodextrin compounds, methods for their production, and drug delivery agents for targeted drug delivery systems - Google Patents

Description

  The present invention relates to a multi-branched cyclodextrin compound used as a drug delivery agent for a targeted drug delivery system (TDDS), and a method for producing the same, and more particularly, to the 6-position primary of glucopyranose constituting cyclodextrin. The present invention relates to a multibranched cyclodextrin compound in which a hydroxyl group is substituted with a group recognized by a target protein, a method for producing the multibranched cyclodextrin compound that does not require complicated steps, and a drug delivery agent comprising the multibranched cyclodextrin compound .

  A cyclodextrin typified by β-cyclodextrin represented by the following formula (hereinafter sometimes simply referred to as “CD”) is widely used in drug delivery systems because it has a drug inclusion ability.

  A primary hydroxyl group exists at the 6-position of glucopyranose constituting CD. For example, when there are 6 glucopyranoses constituting CD, there are 6 primary hydroxyl groups in CD. A CD compound (hereinafter referred to as “multi-branched CD compound”) in which a plurality of the primary hydroxyl groups present in CD, each represented by the following general formula a, are substituted with sugar chain arms formed by binding sugar chains, respectively. Production) is being attempted.

In the general formula a, the substituent A represents a sugar chain arm.
However, conventionally, a multi-branched CD compound in which all of the primary hydroxyl groups present in a plurality of CDs (for example, all seven in the case of β-CD) are substituted with a sugar chain arm is used for isolation of a production intermediate. Production was difficult due to difficulty, steric hindrance and the like. In addition, the incompletely substituted multi-branched CD compound in which the reactive substituent of the intermediate remains without any sugar chain arm being substituted on all the primary hydroxyl groups may have toxicity.
Previously, a mannose-containing natural sugar chain arm separated and purified from egg white obalmin was bound to β-CD (for example, see Non-Patent Document 1). The obtained multi-branched CD compound has a very high associating ability with both medicine and lectin, so-called double recognition ability, but it is very difficult to separate and purify mannose-containing natural sugar chains. It was not suitable for. It was desired to construct a spacer arm designed to be suitable for mass synthesis by mimicking a natural sugar chain and leaving only some monosaccharide moieties important for lectin recognition.
In particular, for the construction of a mannose- or galactose-containing spacer arm, a production method suitable for large-scale synthesis with a complicated process has been desired (see, for example, Patent Document 1, Non-Patent Documents 2 and 3). Moreover, in the conventional manufacturing method, since the hyperbranched CD compound was manufactured by using protective groups, such as an acetyl group, the process was complicated.
Furthermore, conventionally, evaluation of the manufactured CD compound as a drug delivery agent has been performed through the process of animal experiments and clinical trials, but excellent drug delivery can be achieved by determining the association constant with the drug and target protein. A method for exploring agents was desired.
WO2004 / 085487A1 Kenjiro Hattori and Toshiyuki Inazu, “Synthesis of sugar chain cluster cyclodextrin and double recognition of lectin proteins and pharmaceuticals”, Journal of Synthetic Organic Chemistry (2001) 59: 742-754. H. Abe, A .; Kenmoku, N .; Yamaguchi, K .; Hattori, J. et al. Inclusion Phenom. Macrocyclic Chem. 52, 39-47 (2002) K. Hattori, A .; Kenmoku, T .; Mizuguchi, D.M. Ikeda, M .; Mizuno, T .; Inazu, J. et al. Inclusion Phenom. Macrocyclic Chemistry, 56, 9-16 (2006)

  An object of the present invention is to solve the above-described problems, a multi-branched CD compound in which the primary hydroxyl group at the 6-position of glucopyranose constituting CD is substituted with a group recognized by the target protein, An object of the present invention is to provide a method for producing the multi-branched CD compound suitable for mass production without requiring an operation, and a drug delivery agent comprising the multi-branched cyclodextrin compound.

The above problems have been achieved by the following means.
That is, the present invention
(1) A multibranched cyclodextrin compound in which the primary hydroxyl group at the 6-position of glucopyranose constituting cyclodextrin is substituted with a substituent represented by the following general formula 1,

In the general formula 1, α-glycosyl represents a glycosyl group that is α-bonded via an oxygen atom at the anomeric position, and C represents a bonding position with the 6-position carbon atom of glucopyranose constituting the cyclodextrin. N represents an integer of 0 to 2, and when n is 0, it represents a single bond connecting a carbonyl carbon atom and an amino nitrogen atom.
(2) The multi-branched cyclodextrin compound according to (1), wherein the α-glycosyl is α-D-mannosyl, α-D-galactosyl, α-L-fucosyl or α-D-glucosyl,
(3) The multi-branched cyclodextrin compound according to (1) or (2), wherein the cyclodextrin is β-cyclodextrin,
(4) Heptakis-6- (1-α-glycosyl-oxypropylthioethylamide) -β-cyclodextrin, heptakis-6- (1-α-glycosyl-oxypropylthioethylamidohexanoylamide) -β-cyclo Any one of (1) to (3), which is dextrin or heptakis-6- (1-α-glycosyl-oxypropylthioethylamidohexanoylamidohexanoylamide) -β-cyclodextrin A hyperbranched cyclodextrin compound according to
(5) Cyclodextrin having an amino group or an amino oligocaproic acid amide group at the 6-position primary hydroxyl group of each glucopyranose molecule constituting the cyclodextrin ring, and 1-α-glycosyloxypropylthio fatty acid The method for producing a multi-branched cyclodextrin compound according to any one of (1) to (4), and (6) any one of (1) to (4), wherein Provided are targeted drug delivery agents comprising the described multi-branched cyclodextrin compounds.

The multi-branched cyclodextrin compound of the present invention can include and associate a drug with a CD moiety contained in the compound, while the primary hydroxyl group at the 6-position of glucopyranose constituting the CD is associated with a target protein. The target orientation for the target protein increased exponentially due to the sugar cluster effect (see, for example, YC Lee and RT Accc Chem. Res, 1995, 28). Play.
The multi-branched cyclodextrin compound of the present invention can have a spacer arm composed of aminocaproic acid for adjusting the distance between recognition sites in order to enhance the recognition ability with a target protein, and the spacer arm can be produced in large quantities. Suitable for.
The method for producing a multi-branched cyclodextrin compound of the present invention is obtained by converting a monosaccharide as a starting material into mannose, glucose, galactose, fucose, sialic acid without going through complicated protection / deprotection steps using a protecting group such as an acetyl group. By selecting from the above, a drug delivery agent for TDDS can be produced for diseases such as cancer via macrophages for mannose and glucose, liver cancer for galactose, colon cancer for fucose, and influenza for sialic acid.
Since the target-directed drug delivery agent of the present invention comprises the multi-branched CD compound, it can be suitably used for TDDS against diseases such as cancer, liver cancer, colon cancer, influenza and the like via macrophages.

First, the multibranched cyclodextrin compound of the present invention will be described.
In the multi-branched cyclodextrin compound of the present invention, substantially all of the 6-position primary hydroxyl groups of glucopyranose constituting cyclodextrin (hereinafter sometimes simply referred to as “CD”) are represented by the following general formula 1. It is substituted with a substituent.

In the general formula 1, α-glycosyl represents a glycosyl group that is α-bonded through an oxygen atom at the anomeric position (position 1), and C represents a bonding position with the 6-position carbon atom of glucopyranose constituting cyclodextrin. . N represents an integer of 0-2.
In the present invention, the α-glycosyl is preferably α-D-mannosyl, α-D-galactosyl, α-L-fucosyl or α-D-glucosyl.
In general formula 1, the moiety represented by the following formula is a repeating unit derived from aminocaproic acid as a spacer arm as described later.

  Therefore, in the present specification, hereinafter, the portion represented by the above formula may be simply represented by “cap”. Therefore, the general formula 1 can also be expressed as follows.

In the general formula 1, α-glycosyl, C, and n are as described above.
In this specification, when n is 2, it is simply referred to as cap2, and when n is 1, it may be simply referred to as cap1.
When n is 0, cap represents a single bond connecting the carbonyl carbon atom and the amino nitrogen atom in the formula.
The CD is preferably β-CD. In that case, the multi-branched CD compound of the present invention is preferably represented by the following general formula a.

The substituent A in the general formula a represents the substituent represented by the general formula 1.
In the present specification, the β-CD site of the multi-branched CD compound of the present invention represented by the general formula a is simply represented by the following general formula b.

In the general formula b, the substituent A represents the substituent represented by the general formula 1.
In this case, the multi-branched CD compound of the present invention can be represented by the following general formula c.

In the general formula c, α-glycosyl, cap and n are as described above.
Examples of the hyperbranched cyclodextrin compound of the present invention include heptakis-6- (1-α-glycosyl-oxypropylthioethylamide) -β-cyclodextrin, heptakis-6- (1-α-glycosyl-oxypropylthioethylamide). Hexanoylamide) -β-cyclodextrin or heptakis-6- (1-α-glycosyl-oxypropylthioethylamidehexanoylamidehexanoylamide) -β-cyclodextrin is preferred.
Although the following exemplary compounds 1-9 are shown as a specific example of the hyperbranched cyclodextrin compound of this invention, this invention is not limited to these.

Next, the CD used as a raw material in the present invention will be described.
The CD used as a raw material in the present invention is not particularly limited as long as it has a cyclodextrin basic skeleton, and examples thereof include α-CD, β-CD, and γ-CD. These CDs differ in the number of glucopyranoses constituting the ring (α: 6; β: 7; γ: 8), and therefore the cavity diameters are also different (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 trichlene, parklene, and the like. Further, β-CD is a size capable of containing a naphthalene ring, and γ-CD is a size capable of containing anthracene or two 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 drug inclusion ability, it is preferable that the number of sugar chain branches, that is, the number of CD-constituting glucopyranose molecules is large.

Next, a method for producing the multi-branched CD compound of the present invention will be described.
The method for producing the multi-branched CD compound of the present invention comprises:
(1) A sugar arm unit containing a sugar and a CD having a functional group at all the primary hydroxyl groups at the 6-positions of each glucopyranose molecule constituting a CD ring in the presence or absence of a condensing agent (2) In the presence or absence of a condensing agent, the sugar arm unit and the CD having a spacer arm on all the primary hydroxyl groups at the 6-position of each glucopyranose molecule constituting the CD ring It is made to react.
The sugar arm unit has one or more sugars or sugar chains.
The sugar constituting the sugar arm unit is not particularly limited as long as it is specifically recognized by a specific target protein. The sugar may be a monosaccharide or may be one in which two or more sugars are linked to form a sugar chain. The sugar may be unmodified or modified. The sugar or sugar chain is preferably selected from mannose, galactose, fucose, glucose, sialic acid, galactosamine, glucosamine, mannosamine and the like. From the viewpoint of ease of synthesis, mannose, galactose, fucose, glucose or sialic acid is more preferred, and mannose, galactose or fucose is more preferred. When the sugar chain contains two or more sugars, the sugars may be the same type or different types. In addition, the sugar chain may be linear or branched.

From the viewpoint of easy availability and synthesis, it is preferably a saccharide having 1 to 6 sugars or a sugar chain, more preferably a sugar having 1 to 4 sugars or a sugar chain, and more preferably 1 to 1 sugar. Four straight-chain sugars or sugar chains, particularly preferably monosaccharides, that is, a mannosyl group, a galactosyl group, a glucosyl group, a fucosyl group, and the like according to the purpose of the TDDS disease.
Next, the construction of the sugar arm unit will be described.
The sugar arm unit can be constructed as shown in the following reaction scheme when the glycoside is mannoside.

A sugar arm unit can be constructed by oxypropenation of a sugar or sugar chain with allyl alcohol in the presence of an acid catalyst, and a photoaddition reaction using a mercapto fatty acid having 2 to 7 carbon atoms. As the mercapto fatty acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid and the like are preferable.
Specifically, an allyl glycoside can be synthesized by dissolving a sugar or sugar chain in allyl alcohol, adding an acid catalyst, and refluxing at 97 ° C. in a nitrogen stream. A sugar arm unit can be constructed by subjecting the obtained allylglycoside to a photoaddition reaction with a mercapto fatty acid such as 3-mercaptopropionic acid.

The reaction between the allyl glycoside and the mercapto fatty acid proceeds by irradiating ultraviolet rays in an inert gas atmosphere such as nitrogen or argon.
The ultraviolet rays to be irradiated are used in the range of 200 nm to 400 nm, preferably 340 to 380 nm. The irradiation time is 1 to 20 hours, preferably 3 to 7 hours. It can be appropriately changed depending on the reaction scale and the reaction apparatus. As the reaction solvent, DMF, methanol, water or the like is used.
The product can be purified by adsorbing the product with an ion exchange resin and then desorbing the product with aqueous ammonia. As the ion exchange resin used for the adsorption, DEAE-Sephadex and Dowex1-X8 are used.

Next, the CD having the functional group will be described.
In the present invention, the CD having a functional group is preferably formed by substituting all the primary hydroxyl groups at the 6-position of each glucopyranose molecule constituting the CD ring with a functional group. The introduction of the functional group into CD can be performed by any method.
The functional group of CD having a functional group at the 6-position primary hydroxyl group of each glucopyranose molecule constituting the CD ring is amino group, ether group, thioether group, carboxyl group, azide group, p-toluenesulfonyl group, epoxide Groups, unsaturated groups, thiol groups, acetoxy groups, phenoxy groups, iodine, bromine and chlorine halogen groups, and the like, with amino groups being more preferred.

  As a specific example, heptakis-6-amino-β-cyclodextrin in which all the primary hydroxyl groups at the 6-position of each glucopyranose molecule constituting the CD ring are substituted with amino groups (formula 1 below) (hereinafter simply referred to as “peramino acid”). Is sometimes referred to as “modified CD”) by any method in the art to chlorinate (so-called perchlorination) all the primary hydroxyl groups at the 6-position of each glucopyranose molecule, perazid and then peramination. Is obtained.

Next, a CD having spacer arms in all the primary hydroxyl groups at the 6-position of each glucopyranose molecule constituting the CD ring will be described.
The CD having the spacer arm is one or more (preferably 1 or 2) as a spacer arm on a CD having a functional group at all the primary hydroxyl groups at the 6-position of each glucopyranose molecule constituting the CD ring. It can be constructed by subjecting aminocaproic acid to a condensation reaction.
The condensation reaction is represented by the following scheme.

Specifically, in a reaction solvent such as a mixed solvent of methanol / water, amino group is protected with tetra-butyloxycarbonyl (Boc) group against heptakis-6-amino-β-CD (formula 1). It is preferable that 8-40 equivalents of aminocaproic acid be subjected to a condensation reaction at room temperature (20 ° C.) for 2 hours to 150 hours (preferably 2 hours to 24 hours), followed by an arbitrary deprotection (debocification) reaction. The selection of the condensing agent and the selection of the reaction solvent can all be appropriately carried out by those skilled in the art. -4-Methylmorpholinium chloride is preferred, and the reaction solvent is preferably a methanol / water mixed solvent in which a basic compound such as triethylamine (TEA) or N-methylmorpholine (NMM) is present.
By repeating the condensation reaction and deprotection reaction n times, a CD having n aminocaproic acids as spacer arms in all the primary hydroxyl groups at the 6-positions of each glucopyranose molecule constituting the CD ring is constructed. Can do. As described later, n is preferably 1 or 2.
Hereinafter, β-CD represented by Formula 2 in the case of n = 1 may be simply referred to as “heptakis-6-amino-cap1-β-CD”. In Formula 3 in the case of n = 2, The represented β-CD may be simply referred to as “heptakis-6-amino-cap2-β-CD”.
In the production method of the present invention, it is possible to mimic natural sugar chains that are difficult to separate and purify and are difficult to obtain without requiring complicated operations by the spacer arm. Here, “mimics a natural sugar chain” means that only a specific sugar most involved in recognition by a target protein such as a lectin is included in the sugar chain arm unit among natural sugar chains and does not strongly relate to the recognition. The part is mimicked as a spacer arm to enable mass synthesis.
In the spacer arm, n is preferably 1 or 2 from the viewpoint of having a length and structure sufficient to mimic a natural sugar chain. The spacer arm has a functional group for the condensation reaction at the terminal.

In the production method of the present invention, the peraminated CD (Formula 1), the heptakis-6-amino-cap1-β-CD (Formula 2) or the heptakis-6-amino-cap2-β-CD (Formula 3) ) And the above sugar arm unit are combined together by a condensation reaction to produce the desired multi-branched CD compound.
The condensation reaction is represented by the following scheme when the glycoside of the sugar arm unit is a mannoside.

Specifically, the peraminated CD (Formula 1), the heptakis-6-amino-cap1-β-CD (Formula 2) or the heptakis-6-amino-cap2-β-CD (Formula 3) In contrast, 8 to 40 equivalents of a sugar arm unit (formula 4) was dissolved in a reaction solvent, and 4- (4,6-dimethoxy-) was added in the presence of a basic compound such as triethylamine (TEA) or N-methylmorpholine (NMM). The reaction of 1,3,5-triazin-2-yl) -4-methylmorpholinium chloride and peraminated CD is preferably carried out at room temperature (20 ° C.) for 2 hours to 150 hours, preferably for 2 hours to 24 hours. It is more preferable.
As the reaction solvent, dimethylformamide (DMF), water, methanol, isopropyl alcohol, t-butyl alcohol, N-methylpyrrolidinone (NMP) or the like can be used depending on the solubility of the compound. The selection of the condensing agent and the selection of the reaction solvent can all be appropriately performed by those skilled in the art.
The condensing agent that can be used in the above condensation reaction is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (hereinafter referred to as “DMT-MM”). And any condensing agent in the art such as dicyclohexylcarbodiimide (hereinafter referred to as “DCC”) and water-soluble carbodiimide (hereinafter referred to as “WSC”). A person skilled in the art can select an appropriate condensing agent according to the type of the selected functional group and reaction solvent. For example, in the above condensation reaction, when the functional group at the end of the side chain of the sugar arm unit is a carboxyl group and the functional group on CD is an amino group, the carboxyl group and the amino group are reacted, Use of the condensing agent DMT-MM is preferable from the viewpoint of efficiency.
DMT-MM reacts with a carboxylic acid to produce an active ester and then reacts with an amine to produce an amide bond. There are reports that the reaction also proceeds in various solvents such as ethanol, methanol, i-propanol, water and the like, and the reaction proceeds quantitatively, and has attracted attention (for example, M. Kunishima, C. Kawachi, J Morita, K. Terao, F. Iwasaki, S. Tani, Tetrahedron, 55, 13159-13170 (1999)).
For example, a molar equivalent of triethylamine equivalent to carboxylic acid can be used as a reaction solvent, and a 5-fold molar equivalent of DMT-MM can be used as a condensing agent.
The product is preferably purified and isolated by GPC chromatogram after removal of unreacted material by DEAE-Sepahdex ion exchange resin.
As the gel used for GPC, Sephadex, TOSO-PW gel, Bio gel (all are trade names) and the like are used, but Bio gel is preferable from the viewpoint of separation and purification ability.
The condensation reaction can be performed in the absence of a condensing agent. For example, a thiol group can be introduced as a functional group into a sugar arm unit, and a halogen group such as iodine can be introduced into CD, and both can be reacted in an alkaline atmosphere.
The confirmation of the structure of the produced multi-branched CD compound can be performed by any method in the art.

Next, the targeted drug delivery agent of the present invention will be described.
The targeted drug delivery agent of the present invention is a drug delivery agent for TDDS comprising the multi-branched cyclodextrin compound.
Here, the target-directed drug delivery system (TDDS) is to selectively and locally administer a drug to target cells by precisely controlling the pharmacokinetics of the drug by devising the dosage form of the drug. Say.
The targeted drug delivery agent of the present invention is used for cancer diseases via macrophages when the α-glycosyl group in general formula 1 is a mannosyl or glucosyl compound.
In the case of a multi-branched CD compound in which the α-glycosyl group in the general formula 1 is galactosyl, the surface of the liver parenchymal cells can be targeted and used for liver cancer diseases.
In the case of a multi-branched CD compound in which the α-glycosyl group is fucosyl, colon surface cells can be targeted and used for colorectal cancer diseases. In the case of a multi-branched CD compound in which the α-glycosyl group is a sialic acid group, it is used for influenza virus diseases.

Next, evaluation of association with drugs and target proteins will be described.
The method for evaluating the double recognition ability of the multi-branched CD compound includes the inclusion association constant for the drug of the multi-branched CD compound obtained by the surface plasmon resonance method, and the recognition of the multi-branched CD compound for the target protein. It is preferable to use a two-dimensional plot consisting of association constants.
Here, “double recognition” means that the multi-branched CD compound of the present invention associates with a target protein through its sugar chain moiety, and includes and associates a drug with the CD moiety.
Examples of the drug to be included include an anticancer drug doxorubicin (DXR) and the like.
Examples of target proteins include lectins such as concanavalin A, receptor proteins present on the cell surface, and the like.
A multibranched CD compound that is promising as the target-directed drug delivery agent by obtaining each association constant using a SPR optical biosensor on which a target protein such as DXR or a target protein such as lectin is immobilized, and plotting it two-dimensionally Can be screened.
In the method for evaluating the dual recognition ability described above, the association constant between the drug and the corresponding lectin protein or the corresponding cell surface receptor protein is determined without animal experiments or clinical experiments, and the logarithm of each logarithm is obtained. The double recognition is evaluated by plotting on the axis and the vertical axis, and the higher the value is on the upper right, the better the drug delivery agent for TDDS, the better the drug inclusion ability and the ability to associate with the target protein.

Next, the principle of the SPR optical biosensor will be described.
The surface plasmon resonance (SPR) method using an SPR optical biosensor (for example, IAsys) can measure the intermolecular interaction of biopolymers.
Both the measurement of the association of the multi-branched CD compound of the present invention with the target protein and the measurement of drug inclusion association can be performed by the SPR method.
Specifically, a ligand (for example, concanavalin A) is immobilized on a cuvette, and an analyte (for example, the multibranched CD compound of the present invention) dissolved in a buffer solution is injected into the cuvette. Then, the incident angle when the evanescent wave and the surface plasmon wave generated when the laser is incident on the prism at an incident angle equal to or smaller than the total reflection angle causes surface plasmon resonance (SPR) is measured. Depending on the mass in the 600 nm evanescent field, the angle of incidence that causes SPR changes. The amount of change can be observed, and the amount of change at this time is called a response (R). This incident angle is proportional to the mass of the associated molecule. That is, the association dissociation interaction can be observed from these mass changes.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these.

Reference Example 1 Production of heptakis-6-amino-β-CD (formula 1) (1) Production of heptakis-6-chloro-β-CD β-CD 3.0274 g was placed in a two-necked flask, and ethanol 4 After dehydration by azeotropic distillation, vacuum drying (40 ° C.) was performed for 20 hours. After vacuum drying, the inside of the flask was substituted with N ₂ and taken out to obtain 2.75 g (2.42 × 10 ⁻³ mol) of β-CD. To this, 23 ml of DMF previously dehydrated with CaH ₂ was added and dissolved, and placed under a N ₂ stream. Further, 2.9 ml (37 × 10 ⁻³ mol) of methanesulfonyl chloride was gently added dropwise. Thereafter, the reaction was carried out at an oil bath temperature of 80 ° C. for 4 hours and 30 minutes. After cooling to room temperature, in order to stop the reaction, 5 ml of ethanol was added and stirred for 30 minutes, neutralized with about 12 ml of sodium methoxide 28% methanol solution (pH 7-8), and stirred for 16 hours and 20 minutes.
This was concentrated to dryness with an evaporator, transferred to a Buchner funnel, and the product was washed with methanol and water. After 24 hours and 20 minutes of vacuum drying (40 ° C.), a yield of 2.5886 g and a yield of 85% were obtained. The product was confirmed by TLC and laser ionization time-of-flight mass spectrometer (MALDI-TOF MS). TLC showed _Rf value 0.83 using developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde.

(2) Production of heptakis-6-azido-β-CD In an eggplant flask, 1.2630 g (9.9930 × 10 ⁻⁴ mol) of heptakis-6-chloro-β-CD was added to DMAc (dimethylacetamide): water = 60 ml: It melt | dissolved in 8 ml and 1.4232 g (218.9 * 10 < ^-4 > mol) sodium azide was added. After reaction at an oil bath temperature of 110 ° C. for 24 hours, the reaction solution was stirred for 1 hour until the temperature of the reaction solution decreased to room temperature. The solution was added dropwise to about 700 ml of water with a pipette to precipitate the product and remove the impurity filtrate. The filtrate was transferred to a Buchner funnel and washed with water. After 13 hours of vacuum drying (40 ° C.), a yield of 1.1562 g and a yield of 88% were obtained. The product was confirmed by TLC and MALDI-TOF MS. TLC showed Rf value 0.77 using developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde.

(3) Production of heptakis-6-amino-β-CD (formula 1) Hexakis-6-azido-β-CD 1.0015 g (7.6446 × 10 ⁻⁴ mol), triphenylphosphine in an eggplant flask 3. 1962 g (121.86 × 10 ⁻⁴ mol) was dissolved in 73 ml of DMAc, reacted at room temperature for 1 hour, 30 ml of 25% aqueous ammonia (4000 × 10 ⁻⁴ mol) was added, and the mixture was stirred for 22 hours and 30 minutes. After the reaction, the mixture was concentrated by an evaporator, and 500 ml of ethanol was added to precipitate the product to remove impurities as a filtrate. The filtrate was transferred to a Buchner funnel and washed with ethanol. After 19 hours and 50 minutes of vacuum drying (40 ° C.), a yield of 0.8321 g and a yield of 96% were obtained. The product was confirmed by TLC and MALDI-TOF MS. TLC used developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde and ninhydrin.

Reference Example 2 Production of heptakis-6-amino-cap1-β-CD (formula 2) In order to introduce aminocaproic acid into heptakis-6-amino-β-CD (formula 1) (1.71 g), Using aminocaproic acid (10 equivalents, 3.51 g) in which the amino group was protected with a Boc group, NMM (10 equivalents, 1.67 ml) in a methanol / water mixed solvent (1: 1 v / v, 20 ml: 20 ml). Then, DMT-MM (10 equivalents, 4.2 g) was added all at once and reacted at room temperature for 48 hours. After the reaction, the mixture was concentrated with an evaporator, and the product was reprecipitated with pure water. After removing the filtrate, it was dissolved in methanol, and the filtrate was dried to obtain heptakis-6-Boc-amino-cap1-β-CD.
About 4 ml of 4M-HCl / dioxane solution was added to heptakis-6-Boc-amino-cap1-β-CD, and the mixture was stirred for 3 hours under ice-cooling to perform de-Bocation, and heptakis-6-amino-cap1 -Β-CD (formula 2) was obtained. The yield was 2.17 g, and the yield was 74.6%. MALDI-TOF MS was used to confirm the product.
MALDI-TOF MS: Calcd. 1920.55 [M] ⁺ , 1943.54 [M + Na] ⁺ , 1959.65 [M + K] ⁺ ; Found m / z 1919.50, 1956.81

Reference Example 3 Production of heptakis-6-amino-cap2-β-CD (formula 3) Amino group for further introducing aminocaproic acid into the heptakis-6-amino-cap1-β-CD (1.0 g) Was added with NMM (20 eq, 1.15 ml) in methanol solvent (100 ml) using aminocaproic acid (20 eq, 2.4 g) protected with Boc group, followed by DMT-MM (20 eq, 2 g). .88 g) was added at once and reacted at room temperature for 48 hours. After the reaction, the mixture was concentrated with an evaporator, and the product was reprecipitated with pure water. After removing the filtrate, it was dissolved in methanol, and the filtrate was dried to obtain heptakis-6-Boc-amino-cap2-β-CD.
About 4 ml of 4M-HCl / dioxane solution was added to heptakis-6-Boc-amino-cap2-β-CD, and the mixture was stirred for 3 hours under ice-cooling to perform de-Bocation, and heptakis-6-amino-cap2 -Β-CD (formula 3) was obtained. The yield was 1.46 g, and the yield was 103.4%. MALDI-TOF MS was used to confirm the product.
MALDI-TOF MS: Calcd. 2712.3 [M] ⁺ , 2735.29 [M + Na] ⁺ , 2751.4 [M + K] ⁺ ; Found m / z 2711.45, 2732.78, 2748.80

Example 1 Production of Illustrative Compound 1 “Heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide) -β-CD” (1) Production of 1-α-D-mannosyl-oxypropene 1.0073 g (5.5942 × 10 ⁻³ mol) of mannose was placed in the flask, dehydrated by azeotropic distillation with ethanol four times, and vacuum dried (40 ° C.) for 139 hours and 17 minutes. After vacuum drying, the inside of the flask was purged with N ₂ and taken out, and 10 ml of allyl alcohol; 150 × 10 ⁻³ mol was added and dissolved, and placed under a N ₂ stream. To this, 0.6560 g of Dowex 50W-X8 was added as an acid catalyst, and the mixture was refluxed at an oil bath temperature of 125 ° C. for 2 hours. Then, Dowex 50W-X8 was removed by suction filtration, transferred to a two-necked flask (concentrated to dryness with methanol when the remainder in the suction bottle was transferred to a two-necked flask). Further, methanol was added and the operation of concentration to dryness was performed three times. After 44 hours and 24 minutes of lyophilization, the product was obtained in a yield of 1.2602 g and a yield of 100%. The product was confirmed by TLC and MALDI-TOF MS. TLC showed Rf value of 0.75 using developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde.

(2) Production of 1-α-D-mannosyl-oxypropylthioethylcarboxylic acid (formula 4) In a two-necked flask, 1.2938 g (5.879 × 10 ⁻³ ) of 1-α-D-mannosyl-oxypropene was obtained. mol) was dissolved in 5 ml of methanol and placed in an argon stream for 20 minutes. Thereafter, 0.5 ml (6 × 10 ⁻³ mol) of 3-mercaptopropionic acid was added, the argon stream was stopped, the lid was attached, and irradiation with ultraviolet rays (wavelength 365 nm) was performed for 5 hours while stirring.
After concentration to dryness and freeze-drying, purification was performed with a weak anion exchange resin DEAE-Sephadex and a strong anion exchange resin Dowex1-X8.
First, batch processing was performed for 10 hours with DEAE-Sephadex (30 g), the carboxylic acid group of the product was adsorbed, and suction filtration was performed. Further, the filtrate was concentrated to dryness, freeze-dried, divided into three parts, and subjected to column treatment with DEAE-Sephadex. The filtrate was concentrated to dryness, 15 ml of water and 11.35 g of Dowex1-X8 were added and batch-treated for 16 hours and 15 minutes, followed by suction filtration. About 18 g of Dowex1-X8 was added to the filtrate (from pH 4 to pH 6.5), and after 2 hours of batch treatment, suction filtration was performed. DEAE-Sephadex and Dowex1-X8 with adsorbed product were each batch-treated with 1M ammonia water (containing 5 moles or more of the resin exchange capacity) for 30 minutes to 1 hour to remove the product. Separation, suction filtration, and washing with 1M aqueous ammonia. This operation was performed 3 to 6 times, and the filtrate was concentrated. The mixed resin was filtered again and lyophilized. A yield of 1.6744 g and a yield of 87% were obtained.
The product was confirmed by TLC, MALDI-TOF MS and NMR. TLC showed Rf value 0.47 using developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde and bromocresol green.

(3) Production of Exemplified Compound 1 “Heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide) -β-CD” Heptakis-6-amino-β-CD (Formula 1) 44. 3 g and 591 mg of the 1-α-D-mannosyl-oxypropylthioethylcarboxylic acid (formula 4) (40 equivalents to heptakis-6-amino-β-CD) in 2 ml of water and 2 ml of methanol (1: 1 ), 510.2 g (40 equivalents to heptakis-6-amino-β-CD) of the condensing agent DMT-MM was added, and the mixture was reacted at room temperature for 24 hours.
After confirming the product by MALDI-TOF MS, the solution was added little by little using a Pasteur pipette to 10 times the amount of acetone to precipitate the product.
After overnight, the acetone was removed and the solid was dissolved in water, transferred to a sample bottle and lyophilized.
This crude product was subjected to column purification with Sephadex G-25, yielding 29.2 g of heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide) -β-CD (Exemplary Compound 1). A white solid was obtained in a yield of 23%.
Confirmation was performed by ¹ H NMR, MALDI-TOF MS, and TLC.
¹ H NMR (500 MHz, heavy water, 80 ° C.) δ (ppm): 2.28 (14H, s) 2.98 (14H, s) 3.04 (14H, s) 3.22 (14H, s) 5 .23 (7H, s) 5.48 (7H, s)
MALDI-TOF MS: Calcd. 3309.52 [M + Na] ⁺ , 3325.63 [M + K] ⁺ ; Found 3309.24 [M + Na] ⁺ , 3324.89 [M + K] ⁺
TLC Rf value: 0.77 Developing solvent: 1-butanol: water: DMF: ethanol: acetic acid = 10: 10: 3: 1: 1 Color solution: anisaldehyde

Example 2 Production of Illustrative Compound 2 “Heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide-cap1) -β-CD” Said heptakis-6-amino-cap1-β-CD 5.1 g And 59.8 g of the 1-α-D-mannosyl-oxypropylthioethylcarboxylic acid (formula 4) (30 equivalents to the heptakis-6-amino-cap1-β-CD) were dissolved in 3 ml of methanol. After adding 20.0 μl of desalting agent NMM (30 equivalents relative to the heptakis-6-amino-cap1-β-CD), 49.5 g of the condensing agent DMT-MM (said heptakis-6-amino-cap1 30 equivalents to -β-CD) and stirred at an oil bath temperature of 30 ° C. for 24 hours for reaction.
After confirming the product by MALDI-TOF MS, the solution was added little by little using a Pasteur pipette to 10 times the amount of acetone to precipitate the product.
After leaving overnight, the acetone was removed and the solid was dissolved in water, transferred to a sample bottle and lyophilized.
The crude product was purified by column with Bio Gel P-4, yield 2.97 g, 13% yield, colorless and clear, candy-like heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide -Cap1) -β-CD (Exemplary Compound 2) was obtained.
Confirmation was performed by ¹ H NMR, MALDI-TOF MS, and TLC.
¹ H NMR (500 MHz, heavy water, 70 ° C.) δ (ppm): 1.59 to 1.65 (14H, m) 1.78 to 1.85 (14H, m) 1.85 to 1.91 (14H, m) 2.17 to 2.22 (14H, m) 2.54 to 2.58 (14H, t, J = 8.25 Hz) 2.80 to 2.83 (14H, t, J = 7.03 Hz) 2.94 to 2.97 (14H, t, J = 7.15 Hz) 3.09 to 3.12 (14H, t, J = 6.85 Hz) 3.47 to 3.50 (14H, t, J = 6.55 Hz) 5.16 (7H, s) 5.35 (7H, s)
MALDI-TOF MS: Calcd. 4102.07 [M + Na] ⁺ ; Found 4101.43 [M + Na] ⁺
TLC Rf value: 0.88 Developing solvent: 1-butanol: DMF: water = 1: 1: 1
Coloring solution: anisaldehyde

Example 3 Production of Exemplary Compound 3 “Heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide-cap2) -β-CD” Heptakis in 6 ml of methanol in the presence of 53 μl of dehydrochlorinating agent NMM 6-amino-cap2-β-CD (43.2 mg) was reacted at room temperature with 164.6 mg of 1-α-D-mannosyl-oxypropylthioethylcarboxylic acid (formula 4) and DMT-MM (140.5 mg) at room temperature for 8 hours. Reprecipitation with acetone and suction filtration were performed. After washing with acetone, the product in the filter paper was dissolved with methanol and concentrated to dryness. After lyophilization, purification was performed by GPC using Bio-Gel P-4 (φ3 cm × 64.5 cm, elution rate 0.06 ml / min, sample size 34 mg / ml (water)), yield 22.8 mg, Heptakis-6- (1-α-D-mannosyl-oxypropylthioethylamide-cap2) -β-CD (Exemplary Compound 3) was obtained with a yield of 29%. Further, TLC Rf value: 0.78 Developing solvent: 1-butanol: DMF: water = 1: 1: 1 Color solution: anisaldehyde), MALDI-TOF MS: ([M + Na] ⁺ ; Calcd. 4893.72, Found 4896.11, [M + K] ⁺ ; Calcd. 4909.83, Found 4912.21), ¹ H NMR confirmed the product.
¹ H NMR: (500 MHz, heavy water, 70 ° C.) δ (ppm): 1.60 to 1.66 (28H, m) 1.78 to 1.85 (28H, m) 1.85 to 1.91 (28H) , M) 2.15 to 2.21 (14H, m) 2.50 to 2.53 (28H, t, J = 7.33 Hz) 2.80 to 2.82 (14H, t, J = 6.43 Hz) 2.94-2.96 (14H, t, J = 6.70 Hz) 3.08-3.11 (14H, t, J = 6.70 Hz) 3.47-3.50 (28H, t, J = 7.03 Hz) 5.15 (7H, s) 5.35 (7H, s)

Example 4 Production of Exemplary Compound 4 “Heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide) -β-CD” 1-α-D-mannosyl-oxypropylthio described above in Example 1 1-α-L-fucosyl-oxypropylthioethyl carboxylic acid was produced by the same procedure as in the production of ethyl carboxylic acid (Formula 4).
Calcd: 333.35 [M + Na] ⁺ , 349.46 [M + K] ⁺ ; Found: 332.21 [M + Na] ⁺ , 348.20 [M + K] ⁺
Next, a novel compound heptakis-6- (1-α-L-) obtained by condensation reaction of the 1-α-L-fucosyl-oxypropylthioethylcarboxylic acid and the heptakis-6-amino-β-CD. Fucosyl-oxypropylthioethylamide) -β-CD (Exemplary Compound 4) was produced.
7. 81.2 mg of the 1-α-L-fucosyl-oxypropylthioethyl carboxylic acid, 2.66 × 10 ⁻³ mol (30 equivalents), 102.0 mg of the heptakis-6-amino-β-CD, 8. 86 × 10 ⁻⁵ mol was placed in an Erlenmeyer flask and dissolved in 2.0 ml of methanol and 2.0 ml of water. Next, 736.9 mg of DMT-MM as a condensing agent was added and dissolved, and the mixture was stirred at room temperature for about 24 hours to be reacted.
Thereafter, acetone was added to the reaction solution to cause precipitation, followed by lyophilization. 286.4 mg of the reaction product was obtained. Purification was performed by GPC with Sephadex G25.
The product was confirmed by TLC, MALDI-TOF-MS, and ¹ H NMR.
The developing solvent of TLC was 1-butanol: water: DMF: methanol: acetic acid = 10: 10: 3: 1: 1, and the color reagent was anisaldehyde to obtain a single spot having an Rf value of 0.65. .
MALDI-TOF MS: Calcd. : 3197.53 [M + Na] ⁺ , 3213.63 [M + K] ⁺ ; Found: 3197.68 [M + Na] ⁺ , 3213.63 [M + K] ^+
1 H NMR (500 MHz, heavy water, 25 ° C.) δ (ppm): 1.10 to 1.13 (21H, d) 1.35 (14H, s) 2.43 (14H, s) 2.50 (14H, s) 2.70 (14H, s) 4.71 (7H, s) 4.98 (7H, s)

Example 5 Production of Exemplified Compound 5 “Heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide-cap1) -β-CD” Heptakis-6-amino-cap1- produced in Reference Example 2 β-CD (formula 2) is subjected to a condensation reaction of the 1-α-L-fucosyl-oxypropylthioethyl carboxylic acid to give a novel compound heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide). cap1) -β-CD (Exemplary Compound 5) was produced. To an Erlenmeyer flask were added heptakis-6-amino-cap1-β-CD (0.1 g) and the 1-α-L-fucosyl-oxypropylthioethylcarboxylic acid (30 equivalents, 0.49 g), and methanol (50 ml). Then, NMM (30 equivalents, 175 μl) and DMT-MM (30 equivalents, 0.44 g) were added all at once and reacted for 24 hours. Then, heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide-cap1) -β-CD (Exemplary Compound 5) was obtained after acetone reprecipitation, suction filtration, and lyophilization. The crude yield was 0.211 g, and the crude yield was 101.35%. Purification was then performed by column chromatography with Sephadex G25 and Bio-Gel P4. The purification yield was 9.4 mg, and the purification yield was 2.1%. The product was confirmed by MALDI-TOF MS, TLC, and ¹ H NMR.
¹ H NMR (500 MHz, heavy water, 70 ° C.) δ (ppm): 1.51 to 1.63 (14H, m) 1.80 to 1.83 (14H, m) 1.88 to 1.91 (14H, m) 2.19-2.20 (14H, m) 2.53-2.55 (14H, m) 2.80-2.83 (14H, t) 2.95-2.98 (14H, t) 3.09 to 3.12 (14H, t) 3.48 to 3.50 (14H, m) 5.16 to 5.17 (7H, d) 5.35 to 5.34 (7H, d)
MALDI-TOF MS: Calcd. 3966.63 [M] ⁺ , 3989.96 [M + Na] ⁺ , 4006.06 [M + K] ⁺ Found m / z 3991.29, 4007.90
TLC: Rf value 0.86 Developing solvent 1-butanol: DMF: water = 1: 1: 1 Color solution: anisaldehyde

Example 6 Production of Exemplified Compound 6 “Heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide-cap2) -β-CD” Heptakis-6-amino-cap2- produced in Reference Example 3 β-CD (formula 3) is subjected to a condensation reaction of the sugar chain unit 1-α-L-fucosyl-oxypropylthioethyl carboxylic acid to give a novel compound heptakis-6- (1-α-L-fucosyl-oxy). Propylthioethylamide-cap2) -β-CD (Exemplary Compound 6) was produced. Into an Erlenmeyer flask, heptakis-6-amino-cap2-β-CD (formula 3) (30 equivalents, 78.1 mg) and 1-α-L-fucosyl-oxypropylthioethylcarboxylic acid (30 equivalents, 0.27 g) ) And dissolved in methanol (20 ml), NMM (30 equivalents, 96 μl) and DMT-MM (30 equivalents, 0.24 g) were added all at once and reacted for 24 hours. And heptakis-6- (1-α-L-fucosyl-oxypropylthioethylamide-cap2) -β-CD was obtained after acetone reprecipitation, suction filtration, and freeze-drying. The crude yield was 76.9 mg, and the crude yield was 56.12%. Thereafter, purification was performed by column chromatography using Sephadex G25. The purification yield was 7.6 mg, and the purification yield was 12%. The product was confirmed by MALDI-TOF MS, TLC, and ¹ H NMR.
¹ H NMR (500 MHz, heavy water, 70 ° C.) δ (ppm): 1.06 to 1.11 (28H, m) 1.16 to 1.19 (28H, m) 1.33 to 1.43 (28H, m) 1.44 to 1.45 (14H, m) 2.04 to 2.07 (28H, t) 2.34 to 2.37 (14H, t) 2.50 to 2.53 (14H, t) 2.63 to 2.65 (14H, t) 3.01 to 3.04 (28H, t) 4.71 (5H, d) 4.80 (2H, d) 4.88 (7H, s)
MALDI-TOF MS: Calcd. 478.73 [M + Na] ⁺ , 4797.84 [M + K] ⁺ Found m / z 4785.03, 4801.62
TLC: Rf value 0.81 Developing solvent 1-butanol: DMF: water = 1: 1: 1 Color solution: anisaldehyde

Example 7 Production of Exemplified Compound 7 “Heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide) -β-CD” 1-α-D-mannosyl-oxypropylthio described above in Example 1 1-α-D-galactosyl-oxypropylthioethyl carboxylic acid was produced by the same procedure as that for the production of ethyl carboxylic acid (Formula 4).
Calcd. : 349.35 [M + Na] ⁺ , 365.46 [M + K] ⁺ ; Found: 349.02, 365.00
Next, 1-α-D-galactosyl-oxypropylthioethylcarboxylic acid (1.59 g), heptakis-6-amino-β-CD (formula 1) (0.105 g), DMT-MM (1. 23 g) was stirred in methanol at room temperature for 44 hours, reprecipitated with acetone, and then purified by GPC to obtain heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide) -β-CD in a yield of 0. Obtained at 0.08%. The product was confirmed by MALDI-TOF MS and TLC. (Developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde, Rf value 0.06)
GPC conditions: φ3 cm × 119 cm, elution rate 0.20 / min, concentration 10.6 mg / ml (water)
MALDI-TOF MS: Calcd. : 3309.52 [M + Na] ⁺ , Found: 3309.82 [M + Na] ⁺

Example 8 Production of Exemplary Compound 8 “Heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide-cap1) -β-CD” 1-α-D-galactosyl-oxypropylthioethylcarboxylic acid ( 184 mg), the heptakis-6-amino-cap1-β-CD (37 mg), DMT-MM (155 mg), and NMM (62 μl) were stirred in methanol at room temperature for 3 hours, reprecipitated with acetone, and purified by GPC. To give heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide-cap1) -β-CD (Exemplary Compound 8) in a yield of 25%. The product was confirmed by MALDI-TOF MS, TLC, and ¹ H NMR.
TLC: (developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde, Rf value 0.06).
GPC conditions: φ3 cm × 63 cm, elution rate 0.10 / min MALDI-TOF MS: Calcd. : 4101.62 [M + Na] ⁺ , 4117.72 [M + K] ⁺ , Found: 4103.64 [M + Na] ⁺ , 4118.96 [M + K] ^+
1 H NMR (500 MHz, heavy water, 80 ° C.) δ (ppm): 1.71 to 1.72 (14H, m) 1.89 to 1.92 (14H, m) 1.97 to 2.00 (14H, m) 2.26 to 2.29 (14H, m) 2.62 to 2.64 (14H, t, J = 8.55 Hz) 2.89 to 2.92 (14H, t, J = 7.00 Hz) 3.03 to 3.06 (14H, t, J = 6.50 Hz) 3.18 to 3.21 (14H, t, J = 7.05 Hz) 3.57 to 3.58 (14H, t, J = 7.90 Hz) 5.37 (7H, s) 5.43-5.44 (7H, s)

Example 9 Production of Exemplary Compound 9 “Heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide-cap2) -β-CD” 1-α-D-galactosyl-oxypropylthioethylcarboxylic acid ( 183 mg), heptakis-6-amino-cap2-β-CD (51 mg), DMT-MM (156 mg) and NMM (60 μl) are stirred in methanol at room temperature for 3 hours, reprecipitated with acetone, and purified by GPC. Application heptakis-6- (1-α-D-galactosyl-oxypropylthioethylamide-cap2) -β-CD was obtained in a yield of 4%. The product was confirmed by MALDI-TOF MS, TLC, and ¹ H NMR.
TLC: (developing solvent 1-butanol: ethanol: water = 5: 4: 3, color reagent anisaldehyde, Rf value 0.06).
GPC conditions: φ3 cm × 63 cm, elution rate 0.08 / min MALDI-TOF MS: Calcd. : 4893.91 [M + Na] ⁺ , 4910.02 [M + K] ⁺ , Found: 4897.98 [M + Na] ⁺ , 491.82 [M + K] ^+
1 H NMR (500 MHz, heavy water, 80 ° C.) δ (ppm): 1.70 to 1.74 (28H, m) 1.87 to 1.92 (28H, m) 1.97 to 1.99 (28H, m) 2.26 to 2.28 (14H, m) 2.59 to 2.61 (14H, m) 2.88 to 2.91 (28H, t, J = 6.98 Hz) 3.02 to 3. 05 (14H, t, J = 6.40 Hz) 3.17 to 3.20 (14H, t, J = 6.88 Hz) 3.55 to 3.58 (14H, t, J = 6.85 Hz) 37 (7H, s) 5.43 (7H, s)

Example 10 Evaluation test of recognition ability of exemplified compounds 1 to 3 for target protein (concanavalin A) and DXR Measurement of recognition and association ability for concanavalin A <immobilization of concanavalin A>
Reagents used for immobilization were obtained as follows.
-10 mM phosphate buffer solution (pH 7.7): 10 mM potassium dihydrogen phosphate solution and 10 mM disodium hydrogen phosphate solution were prepared and mixed to obtain pH 7.7.
-1 mM BS ³ solution: bis (sulfosuccinimidyl) suberate (BS ³ ) 2.9 mg was dissolved in 5 ml of the above 10 mM phosphate buffer solution (pH 7.7).
-10 mM acetate buffer solution (pH 5.3): A 10 mM acetic acid solution and a 10 mM sodium acetate solution were prepared and mixed so as to have a pH of 5.3.
Concanavalin A fixing solution: obtained by dissolving 2.5 mg of concanavalin A in 1 ml of the above 10 mM acetate buffer solution (pH 5.3).
1M ethanolamine solution: 210 μl of ethanolamine was dissolved in 5 ml of the above 10 mM acetate buffer solution (pH 5.3).

Concanavalin A was immobilized by the following methods (1) to (10) using the IAsys.
As for each injection volume, 200 μl was injected once for Concanavalin A, and 200 μl was injected three times for the other solutions.
(1) First, 10 mM phosphate buffer solution (pH 7.7) was added to the SPR optical biosensor cuvette several times at intervals of 0.5 to 2 hours to stabilize the response.
(2) As a linker agent for reacting the aminosilane group on the cuvette surface with the amino group of the lectin protein, the 1 mM BS ³ solution was injected and reacted in advance, and left for about 15 minutes until the response was balanced.
(3) A 10 mM phosphate buffer solution (pH 7.7) was injected into the cuvette and waited until the response was stabilized.
(4) The above (2) and (3) were repeated a plurality of times (for example, 4 times) until the response hardly changed.
(5) In order to inactivate and block the unreacted aminosilane group, an acetic anhydride-acetic acid solution (mixing volume ratio 1: 1) was injected into the cuvette.
(6) For washing, the 10 mM phosphate buffer solution (pH 7.7) was injected, and then 10 mM acetate buffer solution (pH 5.3) was injected to replace the solvent.
(7) The concanavalin A fixing solution was injected, reacted, and allowed to stand until equilibrium was reached.
(8) A 10 mM acetate buffer solution (pH 5.3) was injected and waited for stabilization.
(9) In order to inactivate and block the unreacted BS ^3- terminal succinamide ester group, the 1M ethanolamine solution was injected and allowed to stand for about 5 minutes.
(10) A 10 mM acetate buffer solution (pH 5.3) was injected and washed.
The cuvette obtained above was stored by adding a 0.1% aqueous sodium azide solution.
The response (R) corresponding to the immobilized amount was measured at the time of the injection of (7) and was 2000 arc sec.

<Calculation of immobilization rate>
The immobilization rate was calculated as follows.
Here, the immobilization rate is a value expressed as a percentage of the number of immobilization products immobilized on all aminosilane groups on the cuvette surface.
In the aminosilane cuvette used, the response (R) 600 arc sec corresponds to an immobilization amount of 1 ng / mm ^2, and thus the immobilization amount is R / 600 [ng / mm ² ].
The immobilized amount was converted into the number of moles of immobilizate, considering Avogadro's number, further, considering that aminosilane groups on the cuvette surface exists one in 100 Å ^2, the percentage of immobilization,
6.02R × 10 ¹⁴ / 600M [number of immobilized products / 1 × 10 ¹⁴ Å ² ] / one aminosilane group / 100Å ² = 602 R / 600M [number of immobilized products / one aminosilane group]
The required immobilization rate is expressed as 602R / 6M [%] as a percentage.
Here, R represents the immobilized response [arc sec], and M represents the molecular weight of the immobilized product.
Concanavalin A, which becomes a dimer at pH 5.3, has a molecular weight of about 53,000. Therefore, the immobilization rate of concanavalin A was calculated to be 3.79% as follows.
602 × 2000/6 × 53000 = 3.79%

<Association Evaluation of the Exemplified Compounds 1-3 for Concanavalin A>
Hexakis-6- (1-α-D-mannosyl-oxypropylthioethyl) dissolved in 10 mM acetate buffer solution (pH 5.3; containing 1 mM MnCl ₂ , 1 mM CaCl ₂ , 100 mM NaCl) in a cuvette in which concanavalin A is immobilized. When 200 μl of Carboxamide) -β-CD (Exemplary Compound 1) (1.9 × 10 ⁻⁶ M) was injected, the saturation curve shown in FIG. 1 was obtained. FIG. 1 is a diagram showing SPR measurement results representing a recognition association between the exemplified compound 1 and concanavalin A.
The measurement temperature was 25.0 ° C., and the required time was 5 to 15 minutes until the response reached saturation.
This confirmed that Exemplified Compound 1 was associated with Concanavalin A.

Furthermore, in order to determine the association constant for concanavalin A, the concentration was changed to a concentration of 6 points in a range of 0.1 × 10 ^{−5 to} 1.3 × 10 ⁻⁵ M in a 10 mM acetate buffer solution (pH 5.3). SPR measurement was performed by injecting 200 μL of the solution containing the exemplified compound 1 into a cuvette in which concanavalin A was immobilized. The results obtained are shown in FIG. FIG. 2a is a linear plot showing the kinetic association behavior for concanavalin A.
From the linear plot shown in FIG. 2a, the association constant of the exemplified compound 1 with respect to concanavalin A was 3.6 × 10 ⁵ M ⁻¹ .
Regarding the exemplified compounds 2 and 3, the association constant for concanavalin A was measured in the same manner as for exemplified compound 1, and the association constant for concanavalin A of the exemplified compounds 2 and 3 was 4.1 × 10 ⁶ M ⁻¹ , respectively. 1.4 × 10 ¹⁰ M ⁻¹ .

Test 2. Measurement of Inclusion Ability of Drug The inclusion ability of the CD compound produced according to the present invention was measured in the same manner as in Test Example 1 using doxorubicin (hereinafter referred to as “DXR”) as an anticancer antibiotic. evaluated.
1) Immobilization of DXR Immobilization of DXR on the optical biosensor cuvette surface was performed in the same manner as the immobilization of lectin described above, except that a buffer solution having a different pH was used as a buffer solution for dissolving the drug. It was.
As a linker agent for reacting the aminosilane group on the cuvette surface with the amino group of DXR, 1 mM BS ³ solution / 10 mM phosphate buffer solution, pH 6.5 was reacted. This was repeated several times until the response of the optical biosensor did not change, and an unreacted aminosilane group was inactivated by adding an acetic anhydride-acetic acid solution (mixing volume ratio 1: 1) to block.
After blocking, the solution was replaced with 10 mM-acetate buffer solution at pH 5.3, and DXR solution (2 mg DXR / 10 mM-acetate buffer solution, pH 5.3) was added and reacted. Then, in consideration of the influence of the background on the cuvette, the succinic acid amide ester group was blocked with 1M ethanolamine aqueous solution, pH 8.5.

The DXR immobilization rate can be calculated in the same manner as the aforementioned concanavalin A immobilization rate.
Since the change amount of the response of DXR was 186.1 arc sec, when R = 600 arc sec, it is assumed that one aminosilane group exists per 1 nm ² at 1 ng / mm ² and the immobilization rate is Calculated to 32.1%.
2) Measurement Using the DXR-immobilized cuvette prepared above, the kinetic association behavior of Exemplified Compound 1 and DXR was measured in the same manner as in Test 1. However, the concentration of the exemplified compound 1 was in the range of 0.5 × 10 ^{−5 to} 4.0 × 10 ⁻⁵ M. FIG. 2b shows the results obtained. FIG. 2b is a linear plot showing the kinetic association behavior for DXR.
Similarly, from the linear plot shown in FIG. 2b obtained by the measurement of the SPR sensor to which the anticancer drug DXR is immobilized, the inclusion association constant of Example Compound 1 for DXR is 1.5 × 10 ⁵ M ^−1. Met.
Regarding the exemplified compounds 2 and 3, the association constant for DXR was measured in the same manner as for the exemplified compound 1, and the association constant for DXR of the exemplified compounds 2 and 3 was 1.2 × 10 ⁶ M ⁻¹ , 3 0.0 × 10 ¹⁰ M ⁻¹ .

Test 3. Evaluation of double recognition of Exemplified Compounds 1 to 3 by two-dimensional plots The recognition association constants of Exemplified Compounds 1 to 3 for concanavalin A and inclusion association constants for DXR, respectively, converted to logarithm Was plotted on a two-dimensional map.
FIG. 3 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 1 to 3 with DXR and Concanavalin A.
In FIG. 3, unmodified β-CD as a comparative example and the following drug delivery agents I and II for TDDS manufactured by the present inventors were similarly plotted on a two-dimensional map.

FIG. 3 shows the correlation between the structure of the modified CD compound and the double recognition ability as described below.
In FIG. 3, as a comparative example, unmodified β-CD (“III”) has a log Ka of about 3.5 for the drug, but is 0 for the lectin because it has no sugar.
The broken line connecting I and II shown in FIG. 3 indicates that the longer the spacer arm, the better the double recognition ability.
As is clear from FIG. 3, Exemplified Compounds 1, 2, and 3 of the present invention show high dual recognition ability comparable to the other drug delivery agents I, II for TDDS obtained so far. From 2 to 2, the double recognition ability increased exponentially from 2 to 3, and it was proved that Exemplified Compound 3 is particularly promising as a drug delivery agent for TDDS.

Example 11 Evaluation of recognition and association ability of Exemplified Compounds 4 to 6 for AAL and DXR As a model substance of colon surface cells in which fucose residues exhibit specific association properties, aurea aurantia lectin (hereinafter referred to as AAL), which is a receptor model for colon surface cells. Was used to evaluate the sugar chain recognition interaction. AAL is known to bind strongly to α-L-fucose-containing sugar chains.
Test 1. Measurement of recognition and associating ability for AAL <Example of <Example 10> except that AAL 2.12 mg / ml solution (phosphate buffer solution, pH 7.2 + 0.02% NaN ₃ ) was used as the AAL fixing solution instead of the concanavalin A fixing solution. Immobilization of AAL was carried out by the same procedure as in Concanavalin A immobilization (immobilization rate: 1.84%), and the association constant for AAL was measured by the same procedure as in Example 10.
Test 2. Measurement of DXR Inclusion Ability The kinetic association behavior with DXR was measured using the DXR-immobilized cuvette prepared in Example 10.
FIG. 4 shows the results obtained in tests 1 and 2. FIG. 4a is a linear plot showing the kinetic association behavior for AAL. FIG. 4b is a linear plot showing the kinetic association behavior for DXR.
As is clear from FIGS. 4a and b, as a result of the measurement of association with AAL and DXR, it was 2.0 × 10 ⁸ M ⁻¹ and 2.1 × 10 ⁸ M ⁻¹ for exemplary compound 4, and for exemplary compound 5 2.8 × ¹⁰ 8 ^{M -1,} and 1.5 × ¹⁰ 8 ^{M -1,} and the exemplified compound 6, 3.4 × ¹⁰ 9 ^{M -1,} and became 1.3 × ¹⁰ 8 ^{M -1.}
Next, two-dimensional plots of association constants of Exemplified Compounds 4 to 6 with DXR and AAL were prepared to evaluate double recognition.
FIG. 5 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 4 to 6 with DXR and AAL.
As is clear from FIG. 5, it can be seen that the ability of the exemplary compounds 4 to 6 to associate with the lectin slightly improved in the order of the exemplary compounds 4 to 5, and 5 to 6. However, the ability to associate with drugs was similar.
Compared to the drug delivery agent II for TDDS shown in FIG. 5, the association constant was about 100 times. This is because the fucose group has a hydrophobic methyl group, and therefore, when DXR is included, the sea anemone effect (K. Hattori, A. Kenmoku, T. Mizuguchi, D. Ikeda, M. Mizuno, T .. Inazu, J. Inclusion Phenom. Macrocyclic Chemistry, 56, 9-16 (2006).).
From the above results, it was found that Exemplified Compounds 4 to 6 can be applied as TDDS delivery agents.

Example 12 Evaluation of recognition and associating ability of exemplified compounds 7 to 9 for target protein (PNA) and DXR PNA which is a model lectin of asialoglycoprotein receptor presenting specific recognition to β-galactose residues present on the surface of hepatocytes The association behavior with respect to the anti-cancer drug DXR was evaluated.
A PNA-immobilized cuvette was prepared in the same manner as in <Immobilization of concanavalin A> in Example 10 except that a PNA fixing solution was used instead of the concanavalin A fixing solution.
Furthermore, in order to obtain the association constant for PNA, each of the exemplary compounds 7 to 9 was infused with various concentrations of the solution into a cuvette on which PNA was immobilized, SPR measurement was performed, and kinetic association for PNA A linear plot showing the behavior was created.
Similarly, a linear plot showing the kinetic association behavior with DXR was created using the DXR-immobilized cuvette prepared in Example 10.
Table 1 shows association constants for PNA and DXR for each of Exemplified Compounds 7 to 9 obtained by the above procedure.

As is clear from Table 1, the ability to associate with lectins and drugs with increasing distance between galactoside and CD was observed. Moreover, the improvement of the association ability accompanying the increase in the distance between galactoside and CD showed a certain increase rate, and the improvement of the association ability from Exemplified Compounds 7 to 8 and 8 to 9 was about 10 times, respectively. From this, it can be seen that when the galactoside-CD is extended by about 0.7 nm with an alkyl chain spacer arm containing an amide bond, the association with the lectin and the drug is improved by about 10 times.
It can also be seen that the ability to associate with drugs was improved by increasing the hydrophobicity accompanying the extension of the spacer arm.
FIG. 6 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 7 to 9 with DXR and PNA.
As is clear from FIG. 6, it can be seen that the associating ability with respect to both the lectin and the drug is improved in the order of the exemplified compounds 7 to 8 and 8 to 9.

Example 13 Cellular uptake test of target-directed drug delivery agent comprising the multi-branched cyclodextrin compound of the present invention by flow cytometry A normal glycoprotein present in blood has a sialic acid end, a galactosyl group at the side chain end. A complex having a complex multi-antenna type oligosaccharide chain structure and a complex hierarchical structure is formed. As they become old and their lifetimes expire, the sialic acid located at the forefront of the antenna sugar side chain is cut, resulting in an asialoglycoprotein in which the pre-terminal galactosyl group is exposed. An asialoglycoprotein receptor exists on the surface of liver parenchymal cells, and has a function of taking in and recognizing intracellular changes while distinguishing changes in its terminal structure.
The in vitro incorporation of Exemplified Compound 7 into HepG2 cells (asialoglycoprotein receptor-expressing cells), which are human hepatoma cells, was evaluated by flow cytometry.
That is, HepG2 cells (2 × 10 ⁵ cells / well) cultured in a constant well of a 35 mm dish for 24 hours are reacted with a hydroxyl group of CD with tetramethylrhodamine isothiocyanate (TRITC) to form a thiourethane bond to fluoresce. 40 mM of labeled β-CD and Exemplified Compound 7 were added to each cell, and cultured for 1 hour in a medium without fetal calf serum, followed by flow cytometry (trade name FACSCalibur; manufactured by Becton Dickinson). A 10 mM isotonic phosphate buffer (pH 7.4) was used for washing and the like.
FIG. 7 is a histogram of cell number versus fluorescence intensity by flow cytometry showing the results obtained for intracellular uptake.
In FIG. 7, the peak a is a histogram of HepG2 cells to which nothing is added as a control, the peak b is a histogram of HepG2 cells to which unmodified β-CD has been added as a comparative example, and the peak c is the number of peaks of the present invention. It is a histogram of HepG2 cell which added the branched cyclodextrin compound (exemplary compound 7).
The fluorescence intensity of peak b of unmodified β-CD is not significantly different from control a to which nothing is added. On the other hand, the peak c to which the multibranched cyclodextrin compound of the present invention is added has an increased fluorescence intensity. Thereby, the hyperbranched cyclodextrin compound of the present invention was taken up into HepG2 cells, and it can be said that the fluorescence intensity increased due to the fluorescence by TRITC which is the label.
From this result, it can be seen that the hyperbranched cyclodextrin compound of the present invention can deliver a drug in a directed manner to target cells.

Example 14 Evaluation of intracellular behavior by confocal laser microscope The intracellular behavior of a targeted drug delivery agent comprising a multi-branched cyclodextrin compound of the present invention incorporated into HepG2 cells was evaluated by a confocal laser microscope.
That is, β-CD fluorescently labeled with TRITC and HexG 2 cells exemplarily compound 7 were added to HepG2 cells (2 × 10 ⁵ cells / well) cultured in a constant well of a 35 mm dish for 24 hours, respectively, and no fetal bovine serum was added. After culturing for 1 hour in the above medium, it was fixed with methanol and observed with a confocal laser microscope (trade name LSM-410; manufactured by Carl Zeiss). As a control, observation was also made with a confocal laser differential interference microscope.
Unmodified β-CD fluorescently labeled with TRITC was not taken up into HepG2 cells at all, and no fluorescence was observed in HepG2 cells. In contrast, the target-directed drug delivery agent comprising the hyperbranched cyclodextrin compound of the present invention (Exemplary Compound 7) is taken up by HepG2 cells, and the fluorescence derived from TRITC labeled with the hyperbranched cyclodextrin compound of the present invention is HepG2. It originated from each organ of the whole cell.
From this result, it can be seen that the multi-branched cyclodextrin compound of the present invention can deliver a drug directed to a target cell.

FIG. 1 is a diagram showing SPR measurement results representing a recognition association between Exemplified Compound 1 of the present invention and concanavalin A. FIG. 2a is a linear plot showing the kinetic association behavior of Exemplified Compound 1 to Concanavalin A. FIG. 2b is a linear plot showing the kinetic association behavior of Example Compound 1 against DXR. FIG. 3 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 1 to 3 with DXR and Concanavalin A. FIG. 4a is a linear plot showing the kinetic association behavior of Example Compound 4 to AAL. FIG. 4b is a linear plot showing the kinetic association behavior of Example Compound 4 with respect to DXR. FIG. 5 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 4 to 6 with DXR and AAL. FIG. 6 is a diagram showing a two-dimensional plot of association constants of Exemplified Compounds 7 to 9 with DXR and PNA. FIG. 7 shows a histogram by flow cytometry showing intracellular uptake of a targeted drug delivery agent comprising the hyperbranched CD compound (Exemplary Compound 7) of the present invention.

Claims (3)

Heptakis-6- (1-α-glycosyl-oxypropylthioethylamide) -β-cyclodextrin, heptakis-6- (1-α-glycosyl-oxypropylthioethylamidohexanoylamide) -β-cyclodextrin , and A multi-branched cyclodextrin compound selected from the group consisting of heptakis-6- (1-α-glycosyl-oxypropylthioethylamidohexanoylamidohexanoylamide) -β-cyclodextrin. Condensing a cyclodextrin having an amino group or an amino oligocaproic acid amide group with a 1-α-glycosyl-oxypropylthio fatty acid on the 6-position primary hydroxyl group of each glucopyranose molecule constituting the cyclodextrin ring The method for producing a multi-branched cyclodextrin compound according to claim 1 . A target-directed drug delivery agent comprising the multi-branched cyclodextrin compound according to claim 1 .

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