KR101622851B1 - A pH responsive cyclodextrin derivative, a preparation method therof and a pH responsive conjugate of the cyclodextrin derivative and a drug - Google Patents

Abstract

The present invention relates to a pH-responsive cyclodextrin derivative, a preparation method thereof, and a pH-responsive conjugate of the pH-responsive cyclodextrin derivative and a drug, and more specifically, to a cyclodextrin derivative having a novel structure, a preparation method thereof, and a pH-responsive conjugate of the pH-responsive cyclodextrin derivative and a drug. The cyclodextrin derivative has a novel structure formed by a covalent bond between a maleic anhydride derivative and a cyclodextrin or a cyclodextrin derivative, in which a residue of the maleic anhydride derivative bonds to a primary hydroxyl group of cyclodextrin or a terminal functional group, such as NH_2, N_3, or amino acid, of a cyclodextrin derivative to produce a pH-responsive cyclodextrin derivative.

Description

[0001] The present invention relates to a pH-responsive cyclodextrin derivative, a process for preparing the same, and a pH-responsive conjugate of a cyclodextrin derivative and a pH-responsive conjugate,

The present invention relates to a pH-responsive cyclodextrin derivative, a process for preparing the same, and a pH-responsive complex of the cyclodextrin derivative and the drug.

For efficient and safe delivery of drugs to specific targets, smart drug delivery techniques have evolved with the development of a variety of signal-responsive materials (Fleige E et al., Adv Drug Delivery Rev., 2012 , 64, 866-884). Rapid changes in the physico-chemical properties of smart materials in response to physical signals such as temperature and light, as well as chemical signals such as pH, glutathione and enzymes, can lead to drug encapsulation and release under certain conditions.

The pH has often been chosen as a controlling trigger because the pH value can represent a constant physiological condition of a number of target sites in the body. Changes in pH through the gastrointestinal tract have been used for site-specific drug delivery to strong acidic stomachs or weakly alkaline intestines. Increased glucose degradation in cancer tissues lowers the pH to 5-6, and similar pH drops are also observed in inflammatory tissues and abscesses. A weak acidic pH value is known as an induction signal for the release of anti-cancer drugs or anti-inflammatory drugs. In addition, the initial endosomal pH values of 5 to 6 may be used for endo-escape of macromolecular drugs such as pDNA, siRNA, or proteins that are difficult to penetrate cell membranes and must be delivered into cells through the endocytic pathway It is often used as a signal.

However, since changes in pH in the human body are not very large except for a few exceptions, smart materials with rapid degradation in response to small pH changes are still required for more practical drug delivery systems.

Conventionally, in order to introduce a pH-responsive substance into a cyclodextrin-based drug delivery vehicle, it takes a self-assembly form having weak binding force with a polymer having a pH-responsive property, And has a disadvantage of lowering intracellular permeability.

Under these circumstances, the present inventors have found that a cyclodextrin derivative of a novel structure formed by the covalent bond of a cyclodextrin or a derivative thereof and a maleic anhydride derivative, which is a cyclodextrin derivative of a cyclodextrin with NH ₂ , N ₃ or an amino acid of a cyclic dextrin derivative Is bonded to a drug having an amine group at a physiological pH condition, but the cyclodextrin derivative having a pH of 6.5 or less, which is an acidic pH condition, specifically, a weak acidic And thus the compound can be used as a pH-responsive drug delivery system. Thus, the present invention has been completed.

An object of the present invention is to provide a pH-responsive cyclodextrin derivative.

It is another object of the present invention to provide a process for preparing the pH-responsive cyclodextrin derivative.

Another object of the present invention is to provide a pH-responsive conjugate wherein a drug is bound to the pH-responsive cyclodextrin derivative.

It is still another object of the present invention to provide a method for producing the pH responsive conjugate.

It is still another object of the present invention to provide a pH-responsive drug delivery composition comprising the pH-responsive conjugate.

It is still another object of the present invention to provide an inclusion complex in which a substance containing an adamantane moiety is contained in the pH responsive binding body.

In order to solve the above-mentioned problems, the present invention relates to a method for producing a maleic anhydride derivative, wherein at least one glucose 6-carbon of a cyclodextrin or a derivative thereof has carboxylic acid moieties of a maleic anhydride derivative represented by the following formula 1 linked by an ester or amide bond, And a cyclodextrin derivative.

[Chemical Formula 1]

In this formula,

R 'is hydrogen or C _{1 -6} alkyl;

m is an integer of 1 to 10;

The cyclodextrin derivative having a pH-responsive property is a cyclodextrin derivative of a novel structure formed by the covalent bond of a cyclodextrin or a derivative thereof and a maleic anhydride derivative, and is a cyclodextrin derivative of a cyclodextrin having a primary hydroxyl group or a cyclodextrin derivative of NH ₂ , N ₃ or a compound of the medusa type in which a maleic anhydride derivative residue is bonded to a terminal functional group such as an amino acid. That is, the cyclodextrin derivative having the pH-responsive property of the present invention has a binding force through the covalent bond between the terminal functional group such as NH ₂ , N ₃ or amino acid of the primary hydroxyl group or the cyclodextrin derivative of the cyclodextrin and the maleic anhydride derivative residue And at the same time, the size of the molecule itself is small, so that the intracellular permeation increases without causing toxicity.

The cyclodextrin derivatives having pH-responsiveness of the present invention are those having a maleic anhydride derivative of the above formula (1) bonded thereto and bound to a drug having an amine group at a normal physiological pH, Can be used as a pH-responsive drug delivery system because it has the property of breaking the bond with a drug having an amine group under a weak acidic condition of pH 5 to 6.5.

In the present invention, the maleic anhydride derivative of Formula 1 may be bonded to the primary hydroxyl group of the cyclodextrin, or may be a maleic anhydride derivative of Formula 1 and an ester or ester of the maleic anhydride derivative of Formula 1, which is present in various cyclodextrin derivatives known in the art. Or may be bonded to various functional groups capable of forming amide bonds. Examples of various functional groups capable of forming such an ester or amide bond include, but are not limited to, NH ₂ , N _3, and amino acids. Examples of the amino acid include, but are not limited to, cysteine or phenylalanine.

That is, the cyclodextrin derivative having pH-responsiveness of the present invention has a structural characteristic that the maleic anhydride derivative of Formula 1 is bonded to cyclodextrin or a derivative thereof, so that a bond with a drug having an amine group is formed according to pH conditions It is possible to exhibit properties with pH-responsiveness that can be broken.

In a preferred aspect, the present invention is α-, β- or γ- cyclodextrins or cyclodextrin glucose 6 carbon of a hydroxyl group NH _2, N _3, or one or more of glucose 6 of the cyclodextrin derivative substituted by amino acid The carboxylic acid moiety of the maleic anhydride derivative represented by the following formula (1) is bonded by an ester or amide bond to give a cyclodextrin derivative having pH-responsiveness.

[Chemical Formula 1]

In this formula,

R 'is hydrogen or C _{1 -6} alkyl;

m is an integer of 1 to 10;

Preferably, R 'may be hydrogen or methyl.

Preferably, the cyclodextrin derivative in which the hydroxyl group of the glucose 6-carbon of?,? - or? -Cyclodextrin or cyclodextrin is replaced by NH ₂ , N ₃ or amino acid is a compound represented by the following formula .

(2)

In this formula,

R is OH, NH _2, N _3, or amino acid;

n is an integer of 1 to 3;

Preferably, the maleic anhydride derivative of formula (1) at the 6th carbon of glucose may be linked to the cyclodextrin by ester or amide bond in any one of formulas (3a) to (3e).

[Chemical Formula 3]

(3b)

[Chemical Formula 3c]

(3d)

[Formula 3e]

In this formula,

R 'is hydrogen or C _{1 -6} alkyl;

m is an integer of 1 to 10;

The term "cyclodextrin " used in the present invention means a cyclic functional oligosaccharide prepared by starch as a raw material by an enzymatic reaction.

There are three kinds of cyclodextrins present in the natural world, namely,? -,? - and? -Cyclodextrin, depending on the number of glucose units. The three kinds of cyclodextrins present in the natural system have numerous hydroxyl groups, but among them, 6 to 8 hydroxyl groups having excellent reactivity, which are bonded to specific carbon (generally, glucose 6-position carbon) Can be substituted.

Cyclodextrins also have structural features with voids, or cavities, in the interior, which can entrap other external molecules depending on the size of the cavity.

Typically, β-CD is a cyclic oligosaccharide composed of seven α-D-glucopyranoside units linked at positions 1 and 4, and includes an inner surface that is less hydrophilic and an outer surface that is more hydrophilic. Because of these structural features, β-CD can form inclusion complexes with various molecules through host-guest interactions.

The pH-responsive [beta] -CD derivatives of the present invention of the present invention can be used not only as a multifunctional drug delivery system for the rapid response at the pH of the initial endosome in intracellular drug delivery but also at a certain pH, It can be used as a multifunctional drug delivery system for targeting cancerous tissues, inflammatory tissues or abscesses with an environment.

The present invention also provides a method for producing a cyclodextrin derivative having pH responsiveness as an embodiment.

The cyclodextrin derivative having pH responsiveness of the present invention can be prepared by coupling the maleic anhydride derivative of Formula 1 to the compound represented by Formula 2, that is, cyclodextrin or a derivative thereof. At this time, the cyclodextrin or a derivative thereof and the maleic anhydride derivative of the formula (1) may be bonded by an ester or amide bond.

In the present invention, the compound represented by the above formula (2), that is, the cyclodextrin or a derivative thereof, can be commercially available or can be directly prepared by a known method.

As a preferred embodiment, there is provided a process for producing a cyclodextrin derivative having the pH responsiveness, which comprises the following steps.

reacting a cyclodextrin derivative in which the hydroxyl group of the glucose 6 carbon of? -,? - or? -cyclodextrin or cyclodextrin is substituted by NH ₂ , N ₃ or an amino acid and a maleic anhydride derivative of the following formula 1 Step 1):

[Chemical Formula 1]

In this formula,

R 'is hydrogen or C _{1 -6} alkyl;

m is an integer of 1 to 10;

Preferably, the cyclodextrin derivative in which the hydroxyl group of the glucose 6-carbon of?,? - or? -Cyclodextrin or cyclodextrin is replaced by NH ₂ , N ₃ or amino acid is a compound represented by the following formula .

(2)

In this formula,

R is OH, NH _2, N _3, or amino acid;

n is an integer of 1 to 3;

In the present invention, the reaction of step 1) may be carried out using diisopropylcarbodiimide (DIC). That is, in the present invention, a cyclodextrin derivative having pH-responsiveness can be prepared through a single step reaction of the step 1) by a carbodiimide coupling method using DIC. Although the coupling reaction of the compound represented by the formula (1) using thionyl chloride or oxalyl chloride has been previously known, the coupling reaction of the compound represented by the formula (2), that is, the cyclodextrin or its derivative and the compound represented by the formula There is a disadvantage that significant side reaction occurs. In the present invention, it is preferable to use a carbodiimide coupling method using DIC to prepare a cyclodextrin derivative having a pH response at a high yield.

In the present invention, the reaction of step 1) may be carried out in the presence of 4-dimethylaminopyridine (DMAP) and p-toluenesulphonic acid (PTSA) as a catalyst. Preferably, the DMAP and PTSA can be used together as a catalyst. When both DMAP and PTSA are added as a catalyst, ester formation can be promoted without formation of by-products containing N -acylurea in a polar solvent.

In the present invention, the reaction solvent in step 1) may be at least one selected from the group consisting of dimethylformamide (DMF), pyridine and methylene chloride (MC).

In the present invention, the reaction temperature of step 1) may preferably be 10 to 50 占 폚, more preferably 15 to 30 占 폚, and most preferably 25 占 폚. If the reaction temperature is lower than 10 ° C, unreacted materials are present and the yield of the product is lowered. If the reaction temperature is higher than 50 ° C, there is a disadvantage in that a side reaction is present and the yield is lowered.

In the present invention, the reaction time of the step 1) may be preferably 6 to 24 hours, more preferably 8 to 14 hours, and most preferably 12 hours. If the reaction time is shorter than 6 hours, there is a disadvantage in that the yield of the product is lowered due to the presence of unreacted materials. If the reaction time is longer than 24 hours, there is a disadvantage in that the yield is lowered due to side reactions.

According to another preferred embodiment of the present invention, there is provided a process for preparing a cyclodextrin derivative having the above pH responsiveness, comprising the steps of:

Reacting a compound represented by the formula (1) with prop-2-yn-1-ol to prepare a compound represented by the formula (1a);

Reacting a compound represented by the formula (2) and a compound represented by the formula (1a) (step 1-1):

[Chemical Formula 1]

[Formula 1a]

(2)

In this formula,

R 'is hydrogen or C _{1 -6} alkyl;

m is an integer from 1 to 10;

R is OH, or N < _{3 &} gt ;;

n is an integer of 1 to 3;

In the present invention, the step a) may be carried out in the presence of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and DMAP (4-dimethylaminopyridine).

In the present invention, the reaction solvent in step a) may be tetrahydrofuran (THF) or the like, but is not limited thereto.

In the present invention, the step 1-1) may be carried out in the presence of CuI and N, N-diisopropylethylamine (DIPEA).

In the present invention, the reaction solvent in step 1-1) may be dimethylformamide (DMF) or the like, but is not limited thereto.

Also, the present invention provides a conjugate wherein a cyclodextrin derivative having a pH-responsive property is coupled with a drug including an amine group.

In the present invention, the coupling between the cyclodextrin derivative having a pH-responsive property and a drug comprising an amine group can be formed by binding a maleic anhydride moiety of a cyclodextrin derivative having pH-responsiveness through an open ring reaction with an amine under alkaline conditions have.

In the present invention, the drug containing the amine group may be a drug including a primary amine group. Specifically, the drug containing the amine group may be an alkylamine-based drug such as butylamine; (Cefuroxime), cephodrine, cefadroxyl, cephaloglycin, cephapirin, cefroxadine, cefaclor, cefuroxime, Cefdinir, Cefotin, Cefoton, Cefuzonam, Cefotetan, Cefoxitin, Carbacephem, Cefotiam, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefotaxime, Cefovecin, Cefpodoxime, , Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Ceftizoxime, Ceftriaxone, Cefclidine, and the like. It is also known that Ceflestone, Cefloprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Take the (Ceftobiprole) and cephalosporins (cephalosporin) based drugs, such as chef Marty alkylene (Cefmatilen); Doxorubicin, or a mixture thereof.

In the present invention, the molar ratio of the cyclodextrin derivative having a pH-responsive property to the drug comprising an amine group may be 1: 1 to 8.

In the present invention, the conjugate is characterized in that the bond between the cyclodextrin derivative having the pH-responsive property and the drug comprising an amine group is decomposed under acidic conditions. As described above, the acidic condition may be a weak acidic condition of pH 6.5 or less, specifically, a pH of 5 to 6.5.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic diagram of pH-responsive drug binding and degradation of the conjugates of the present invention.

As shown in FIG. 1, the conjugate of the present invention has stable drug binding at pH 7.4, which is a general physiological pH condition, but when the pH value is lowered, the drug is decomposed. Thus, the conjugate of the present invention can be used as a target specific drug delivery vehicle for releasing a drug under acidic conditions.

Specifically, FIG. 2 shows a schematic conceptual diagram of pH-responsive drug binding and degradation of the conjugate of the present invention when seprradine is used as the drug.

2, it is possible to bind cephalosin, a cephalosporin antibiotic, as a drug targeted for weakly acidic inflammatory tissues, to a pH-responsive? -CD derivative such as medusa, and at pH values of weak acid It can be seen that cepladin is degraded to specifically release the drug, cephradine, in the inflammatory tissue of the desired target site.

In the present invention, due to the above-mentioned pH response, the conjugate can be used as a multifunctional drug delivery system for rapid response at pH of the initial endosome in intracellular drug delivery, as well as a specific pH, It can be used as a multifunctional drug delivery system for targeting cancer, inflammatory tissue or abscesses with a pH environment.

As a chemical moiety for the pH responsive smart material used in the present invention, maleic acid amide derivatives have rapid degradability at certain pH ranges. Unlike other amides which can be decomposed only under extreme pH conditions, maleic acid amide derivatives can be resolved within a physiologically acceptable pH range. The responsive pH range and rate of degradation can be finely tuned by changing the alkyl substituent on the cis-double bond. In addition, the maleic acid amide derivative can be synthesized through a single ring opening reaction between the amine and the corresponding maleic anhydride derivative under weakly alkaline conditions. The ability to readily synthesize maleic acid amide derivatives under mild conditions has the advantage over other pH-responsive linkages that require a fairly complex synthesis step in the binding of the drug or the backbone of the carrier backbone .

In one embodiment of the present invention, two maleic anhydride derivatives wherein the alkyl substituent is different are based on 1-methyl-2- (2'-carboxyethyl) maleic anhydride (MCM) or carboxylate dimethyl maleic anhydride Cyclodextrin derivatives with pH - responsive properties for use as drug delivery systems were synthesized. Since the acid amide derivatives obtained on the basis of MCM are stable at normal physiological pH 7.4 but rapidly degradable at pH 5.5, MCM derivatives may be one of the best candidate substances for binding with drugs targeting weak acidic environments . In one embodiment of the present invention, a drug delivery vehicle having an MCM moiety on its surface for easy coupling with an amine-based drug or cross-linker has been designed.

As described above, in one embodiment of the present invention, β-cyclodextrin (β-CD), which is a cyclic oligosaccharide having seven glucose molecules, was selected as the backbone of the drug delivery system. β-CD has high biocompatibility. In addition, a large number of functional groups can be easily introduced into the? -CD backbone due to the seven primary hydroxyl groups and the fourteen secondary hydroxyl groups. The structure of the medusa form can be synthesized by reaction with seven primary hydroxyl groups. The position-selective functionalization of? -CD makes it possible to achieve uniform chemical properties of the functional moiety to which it is attached.

The present invention also provides a process for producing a combination comprising the steps of:

Reacting the drug comprising the cyclodextrin derivative and the amine group having the pH responsiveness (Step 2).

In the present invention, the reaction molar ratio of the cyclodextrin derivative having pH responsiveness to the drug containing an amine group in the step 2) may be 1: 1 to 8.

In the present invention, the reaction of step 2) may be carried out using at least one selected from the group consisting of triethylamine, diethylamine and diisopropylethylamine as organic bases.

Also, the present invention provides a pH-responsive drug delivery composition comprising a complex in which a cyclodextrin derivative having a pH-responsive property is coupled with a drug including an amine group.

In the present invention, the pH-responsive drug delivery composition is characterized by releasing the drug under acidic conditions. Specifically, the pH-responsive drug delivery composition is capable of releasing the drug under weakly acidic conditions of pH 6.5 or less, specifically pH 5 to 6.5.

The present invention also provides an inclusion complex in which a drug containing an amine group is bound to a cyclodextrin derivative having a pH-responsive property and an additional drug or ligand as a guest substance is contained by host-guest inclusion do.

In the present invention, the additional drug or ligand used as the guest material is a hydrophobic molecule which can be embedded in the cyclodextrin by noncovalent interaction, and the kind thereof is not limited.

In the present invention, the guest material may be at least one selected from the group consisting of a targeting ligand, a cell penetrating moiety, a polymer and a nanoparticle, but is not limited thereto. Preferably, it may be a material comprising an adamantane moiety.

In the present invention, the inclusion complex may further exhibit functionality that can be provided by a substance encapsulated by the inclusion, in addition to the functionality that can be provided by the drug bound to the combination, and thus is multifunctional.

In the present invention, since the material containing the adamantane residue can strongly non-covalently interact with the inside of the toroidal form of the hydrophobic small molecule adamantane cyclodextrin as described above, the host-guest Can be contained in the internal space of the cyclodextrin portion of the conjugate by inclusion.

In other words, since the interior of the toroidal form of β-CD can strongly interact with hydrophobic small molecules such as adamantane through non-covalent interactions, the target ligand or cell permeability May be used for the introduction of additional functionality such as cell penetrating moieties. Polymers or nanoparticles having an adamantane moiety can be easily modified to a -CD derivatives by host-guest interaction.

The adamantane, a cycloalkane consisting of four linked cyclohexane rings, is the most representative guest molecule of β-CD. Thus, in the present invention, functional moieties such as a target ligand or a cytotoxic peptide can be introduced into? -CD-MCM by binding the adamantane moiety to the functional moiety and simply mixing with? -CD-MCM.

That is, the pH-responsive [beta] -CD derivatives of the present invention of the present invention can bind to drug molecules via pH-responsive MCM linkers by simple agitation under mild alkaline conditions, and other functional moieties having an adamantane moiety are bonded to the [beta] Lt; RTI ID = 0.0 > CD. ≪ / RTI > In addition, the surface of other drug delivery vehicles, such as polymers or nanoparticles, can be non-covalently modified with a? -CD derivative in the form of a pH-responsive medusa. The resulting carrier can rapidly release the bound drug under mildly acidic conditions.

In one embodiment of the present invention, the FITC-labeled adamantane derivative was synthesized and the inclusion complex was traced with FCS to confirm that the β-CD-MCM of the present invention can form an inclusion complex as described above (FIG. 7 ). 7, the binding constant between? -CD-MCM-CP and FITC-hex-ADM was calculated to be 1.49 × 10 ⁴ M ^-1 . In general, since the binding constant K of β-CD and adamantane is 1 × 10 ⁴ M ^-1 to 10 × 10 ⁴ M ^-1 , the results show that the interaction between β-CD-MCM-CP and adamantane derivatives Lt; RTI ID = 0.0 > β-CD < / RTI > and adamantane host-guest interaction.

In one embodiment of the present invention, the host-guest fold between β-CD-MCM-CP and adamantane derivatives was further confirmed by ROESY spectra (FIG. 8). The strong cross peak of β-CD-MCM-CP / FITC-hex-ADM was similar to β-CD / FITC-hex-ADM. The binding of the six MCM molecules confronting the primary hydroxyl group in the medusa form was not significantly affected by the inclusion. Based on these results, it was found that any functional moiety could be introduced into the beta -CD-MCM after formation of the inclusion complex after drug binding.

The present invention relates to novel structured cyclodextrin derivatives formed by the covalent bonding of cyclodextrin or derivatives thereof and maleic anhydride derivatives, wherein a terminal hydroxyl group such as NH ₂ , N ₃ or amino acid of the primary hydroxyl group or cyclodextrin derivative of cyclodextrin Wherein the pH-responsive cyclodextrin derivative is combined with a drug having an amine group at a physiological pH at an ordinary pH, but the pH-responsive cyclodextrin derivative may be an acidic can be used as a pH-responsive drug delivery system because the drug has a property of breaking the bond with a drug having an amine group at a pH of 6.5 or less, specifically, a pH of 5 to 6.5 under a weak acidic condition.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic diagram of pH-responsive drug binding and degradation of the conjugates of the present invention.
2 shows a schematic conceptual diagram of pH-responsive drug binding and degradation of the conjugate of the present invention when cepradine is used as the drug.
3 shows the results of ¹ H NMR spectra (a) and MALDI-TOF (b) analysis of? -CD-MCM.
FIG. 4 shows the results of confirming the release of BA from? -CD-MCM-BA at an acidic pH as measured by ¹ H NMR.
5 is a graph showing the pH-responsive release of CP from? -CD-MCM-CP at pH 5.5 (?) And pH 7.4 (?) As measured by HPLC.
Figure 6 shows the ¹ H NMR spectrum (a) and the ESI mass spectrum (b) of the released CP to identify CP released from? -CD-MCM-CP.
FIG. 7 is a graph showing a change in average diffusion time measured by FCS to confirm formation of a complex between? -CD-MCM-CP and FITC-hex-ADM.
Figure 8 shows NMR 2D ROESY spectra of the conjugates between? -CD and FITC-hex-ADM (a) and between? -CD-MCM-CP and FITC-hex-ADM (b).
FIG. 9 shows the cytotoxicity results of NIH3T3 cells of β-CD (), β-CD-MCM (), β-CD-MCM-CP (), CP (), and PEI .

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example  1: β- Cyclodextrin and  One- methyl -2- (2'- Carboxyethyl ) Maleic acid  The anhydride bond (? CD - MCM ) Synthesis of

material

Cephradine (CP) was obtained from Han Hwa Pharm. Co., ltd., South Korea. β- cyclodextrin (β-CD), 6- amino hexanoic acid, di-bit-butyl dicarbonate ((Boc) 2O), n-butylamine (BA), triethylamine (TEA), diisopropylethylamine (DIPEA), diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine (DMAP), p -toluenesulfonic acid (PTSA), trifluoroacetic acid (TFA), sodium hydride (HBTU), fluorene isothiocyanate (HBTU), and N, N ' , N' , N' -tetramethyl- O- (1 H -benzotriazol-1-yl) uronium hexafluorophosphate FITC) isomer I, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Triethyl-2-phosphonopropionate, dimethyl-2-oxoglutarate, and 1-adamantanamine hydrochloride were purchased from TCI (Japan). Ammonium chloride (NH ₄ Cl), magnesium sulfate (MgSO4), potassium hydroxide (KOH), sodium chloride (NaCl), sodium hydroxide (NaOH), sodium bicarbonate (NaHCO _3), tetrahydrofuran (THF), dimethylformamide ( (DMF), hexane, ethyl acetate (EA), methanol (MeOH), ethanol, dichloromethane (DCM), hydrochloric acid (HCl), acetonitrile (ACN) and pyridine were purchased from Daejung, South Korea. Anhydrous TEA, THF, and DMF were obtained via distillation of reagent grade material. Other reagents were used without further purification.

Step 1: 1- methyl -2- (2'- Carboxyethyl ) Maleic acid  anhydride( MCM , Compound 2)

1-methyl-2- (2'-carboxyethyl) maleic anhydride (MCM, Compound 2) was synthesized according to the following reaction formula.

Synthesis of Compound 1

Compound 1 was synthesized according to the previously reported method (Naganawa A et al., Tetrahedron, 1994, 50, 8969-8982). Briefly, Compound 1 was synthesized according to the following procedure.

First, NaH (0.37 g, 9.2 mmol) was slowly added to a solution of triethyl-2-phosphonopropionate (1.64 g, 6.89 mmol) in anhydrous THF (30 mL) at 0 <0> C under a nitrogen atmosphere. After the release of hydrogen gas ceased, dimethyl-2-oxoglutarate (1.00 g, 5.74 mmol) was added to the solution. The reaction mixture was further stirred while maintaining the temperature at 0 &lt; 0 &gt; C. After completion of the reaction was confirmed by TLC, a saturated aqueous NH ₄ Cl solution was added dropwise. THF was removed by rotary evaporation and the resulting mixture of solid and water was extracted several times with EA. After the combined organic phases, washed with de-ionized water (DIW) and brine, dried over MgSO ₄ was then concentrated to a diffraction evaporated. The residue was eluted with EA / hexanes Purification by silica gel chromatography provided Compound 1 as a colorless oil (yield: 91%). ^{1 H NMR (300 MHz, CDCl} 3): δ 1.25-1.30 (3H, t, C H 3 CH 2 O), 1.98 (3H, s, C H 3 C), 2.46-2.49 (2H, m, CC H _{2 CH 2), 2.62-2.65 (2H} , m, CH 2 C H 2 CO), 3.66 (3H, s, CO 2 C H 3), 3.73 (3H, s, C H 3 O 2 CC), 4.16- 4.22 (2H, q, CH ₃ C H ₂ O).

Synthesis of Compound 2

Compound 2 was synthesized according to the previously reported method (Naganawa A et al., Tetrahedron, 1994, 50, 8969-8982). Briefly, Compound 2 was synthesized according to the following procedure.

First, Compound 1 in a 2 M KOH solution in ethanol was refluxed for 1 hour. Deionized water was added and the hot reaction mixture was cooled to ambient temperature. After removing the ethanol by evaporation, the aqueous phase was washed several times with DCM and acidified to pH 2 using concentrated HCl. The water phase was then extracted several times with EA. Dry the organic phase over MgSO ₄ and concentrated under reduced pressure to give a white solid of 1-methyl-2- (2'-carboxyethyl) maleic anhydride (MCM) (2) (yield: 74%). ¹ H NMR (300 MHz, CDCl ₃ ):? 2.12 (3H, s, CC H ₃ ), 2.77 (4H, s, CC H ₂ C H ₂ CO).

Step 2: Cyclodextrin and  One- methyl -2- (2'- Carboxyethyl ) Maleic acid  The anhydride bond (? CD - MCM ) (Compound 3)

(Β-CD-MCM) (Compound 3) of β-cyclodextrin and 1-methyl-2- (2'-carboxyethyl) maleic anhydride was synthesized according to the following reaction formula.

To a solution of compound 2 (0.20 g, 1.09 mmol), beta -cyclodextrin (0.10 g, 0.092 mmol), DMAP (0.046 g, 0.37 mmol) and PTSA (0.070 g, 0.37 mmol) in DMF (10 mL) 0.33 g, 2.6 mmol). The reaction mixture was stirred overnight at ambient temperature. The crude product was purified by dialysis with a regenerated cellulose membrane (MWCO 1,000, SPECTRUM®) in phosphate buffer (pH 9.0), HCl solution (pH 3.0), and DIW, respectively. Then, freeze-drying was performed. Β-CD-MCM (Compound 3) was obtained as an off-white powder (yield: 85%). ¹ H NMR (300 MHz, 1.1 wt% NaOD in D ₂ O):? 1.65 (3H, s, CC H ₃ ), 1.98-2.10 (2H, m, CC H ₂ CH ₂ ), 2.21-2.33 _{m, CH 2 C H 2 CO} ), 3.26-3.36 (2H, m, 2 ', 4'-C H -β-CD), 3.67-3.80 (4H, m, 3', 5'-C H, 6 '-C H _{2 -} ? - CD), 4.94 (1H, s, 1'-C H - ? - CD) (FIG. MALDI-TOF (m / z): 1490, 1656, 1882, 1988, 2155, 2321 [M + Na] ⁺ (FIG.

Example  2: β- CD - MCM And drug complexes

Β-CD-MCM-BA (Compound 4) and β-CD-MCM-CP (Compound 5) were synthesized as a complex of β-CD-MCM and a drug according to the following reaction formula.

Synthesis of Compound 4

To a solution of compound 3 (0.050 g, 0.024 mmol) in ACN (5 mL) was added excess TEA at room temperature and stirred until completely clear. n -Butylamine (BA) (0.24 mL, 0.242 mmol) was added dropwise to the reaction mixture. After stirring overnight, the reaction mixture was evaporated to remove the solvent. 1 M NaOH solution (12 equiv.) Was carefully added to the aqueous solution of compound 4 to exchange the excess n -butylammonium cation with the sodium cation. The crude aqueous solution was evaporated and dried in vacuo to give β-CD-MCM-BA (4) as a pale brown powder (yield> 90%). ^{1 H NMR (300 MHz, D} 2 O of 1.1 wt% NaOD): δ 0.89-0.94 (3H, t, C H 3 CH 2), 1.32-1.39 (2H, m, CH 3 C H 2 CH 2), 1.46-1.54 (2H, m, CH ₂ C H ₂ CH ₂ ), 1.87 (3H, s, CC H ₃ ), 2.22-2.31 (2H, m, CH ₂ C H ₃ CO), 2.49-2.56 _{m, CC H 2 CH 2)} , 3.18-3.23 (2H, m, CH 2 C H 2 NH), 3.54-3.63 (2H, m, 2 ', 4'-C H -β-CD), 3.65-3.92 (4H, m, 3 ', 5'-C H , 6'-C H _{2 -} ? - CD), 5.05 (1H, s, 1H, s, 1'-C H- ?

Synthesis of Compound 5

CD-MCM-CP (Compound 5) was prepared by using the same method as the preparation of Compound 4, except that a solution of cephradine (CP) (6 equivalents) in ACN was used instead of n- Were synthesized. After purification, compound 5 was obtained as a yellow solid (yield:> 90%). ¹ H NMR (300 MHz, 1.1 wt% NaOD in D ₂ O):? 1.83 (3H, s, C H ₃ -Separosporin), 1.86 (3H, s, CC H ₃ ), 2.18-2.24 _{m, CC H 2 H 2)} , 2.45-2.48 (2H, m, CH 2 C H 2 CO), 2.62-2.71 (4H, m, 3 ', 6'-C H 2 - cyclohexadiene), 2.99- 3.08 (1H, m, SC H 2 cephalosporin C- to Sepharose), 3.12-3.18 (1H, m, SC H 2 cephalosporin C- to Sepharose), 3.39-3.49 (2H, m, 2 ', 4'-C H -β-CD), 3.77-3.83 (4H , m, 3 ', 5'-C H, 6'-C H 2 -β-CD), 4.73 (1H, s, 1'-CC H - cyclohexadiene ), 4.87 (1H, s, NC H S), 4.90 (1H, s, 1'-C H -β-CD), 5.71-5.73 (3H, m, NHC H CO, 4 ', 5'-C H -cyclohexadiene), 5.87 (1H, s, 2'-C H - cyclohexadiene).

Production Example 1: Synthesis of FITC-hex-ADM

FITC-hex-ADM (Compound 9) was synthesized according to the following reaction formula.

Synthesis of Compound 6

A solution of butyl dicarbonate (2.00 g, 9.15 mmol) - : (1 1) dissolved in a common sheet and, in D of the THF-tert: a 6-amino hexanoic acid (1.00 g, 7.26 mmol) THF saturated NaHCO ₃ aqueous solution Was added dropwise to the solution at room temperature. After completion of the reaction was confirmed by TLC, the THF was removed by rotary evaporation and the resulting solution was acidified to pH 1-2. The mixture was extracted several times with DCM. After washing the organic phase with the compound, and deionized brine, dried over MgSO ₄ and concentrated by rotary evaporation. N- Boc-aminohexanoic acid (Compound 6), which is a pure compound, was obtained as an opaque crystal (yield: 81%). ¹ H NMR (300 MHz, 1.1 wt% NaOD in D ₂ O): δ 1.10-1.28 (2H, m, CH ₂ C H ₂ CH ₂ ), 1.32-1.56 (13H, m, CH ₂ C H ₂ CH _{2 C H 2 CH 2, t -Boc} ), 2.10-2.15 (2H, t, CH 2 C H 2 CO 2 H), 2.99-3.03 (2H, t, NHC H 2 CH 2).

Synthesis of Compound 7

Compound 7 was prepared according to the previously reported method (Humblet V et al., J Med Chem, 2009, 52, 544-550). Briefly, Compound 7 was synthesized according to the following procedure.

A solution of compound 6 (0.10 g, 0.43 mmol) and 1-adamantanamine hydrochloride (0.080 g, 0.43 mmol) in dry THF (9 mL) was treated with diisopropylethylamine (0.12 mL, 0.86 mmol) and HBTU 0.16 g, 0.43 mmol). After stirring at room temperature for 16 hours, the reaction mixture was heated to 60 &lt; 0 &gt; C for 90 minutes. DCM and brine were then added and the organic phase was washed twice with 1 M HCl aqueous solution (10 mL), washed twice with 5% aqueous NaHCO ₃ (10 mL), then washed with 2 mL of brine (10 mL) washed once and dried over Na ₂ SO _4. The residue was purified by silica gel chromatography eluting with EA / hexane to give Compound 7 as a white solid (Yield: 54%). ^{1 H NMR (300 MHz, CDCl} 3): δ 1.27-1.35 (2H, m, CH 2 C H 2 CH 2), 1.42-1.49 (11H, s, m, t-Boc, C H 2 CH 2 CO) , 1.55-1.59 (2H, m, NHCH 2 C H 2), 1.61-1.65 (6H, br, CC H 2 CH-ADM), 1.89-1.97 (6H, br, CHC H 2 CH-ADM), 2.03- _{2.08 (5H, m, CH 2} C H CH 2 -ADM, CH 2 C H 2 CO), 3.08-3.14 (2H, m, NHC H 2 CH 2).

Synthesis of Compound 8

Excess TFA (10 mL) was added to compound 7 (0.75 g, 2.1 mmol) in DCM (30 mL) at 0 &lt; 0 &gt; C. After stirring the solution for 2 hours, concentrated, dried over and then, MgSO ₄ and washed with saturated NaHCO ₃ and brine. Compound 8 was obtained as a white solid (yield: 89%) without further purification. ¹ H NMR (300 MHz, 1.2 wt% DCI in D ₂ O): δ 1.16-1.26 (2H, m, CH ₂ C H ₂ CH ₂ ), 1.40-1.56 (10H, m, C H ₂ CH ₂ C H _{2, CC H 2 CH-ADM} ), 1.81 (6H, br, CHC H 2 CH-ADM), 1.89 (3H, br, CH 2 C H CH 2 -ADM), 2.04-2.09 (2H, t, CH 2 C H ₂ CO), 2.80-2.85 (2H, t, NH ₂ C H ₂ CH ₂ ).

Synthesis of Compound 9

Addition of FITC Isomer 1 (0.04 mg, 0.113 mmol) to a solution of MeOH (5 mL) solution of compound 8 (0.030 g, 0.11 mmol), followed by the addition of saturated NaHCO ₃ (1 mL). After stirring overnight, the reaction mixture was subjected to reversed-phase high performance liquid chromatography (RP-HPLC) with a linear gradient over 30 minutes at 50-100% ACN / DIW containing 0.1% TFA at a flow rate of 6 mL / min. Lt; / RTI &gt; Compound 9 was obtained as an orange solid (yield: 32%). ^{1 H NMR (300 MHz, MeOD} ): δ 1.40-1.45 (2H, m, CH 2 C H 2 CH 2), 1.61-1.66 (2H, m, C H 2 CH 2 CO), 1.69-1.73 (8H, _{s, m, CC H 2 CH} -ADM, NHCH 2 C H 2), 2.01 (9H, br, CHC H 2 CH-ADM, CH 2 C H CH 2 -ADM), 2.13-2.16 (2H, t, CH _{2 C H 2 CO), 3.65} (2H, br, NHC H 2 CH 2), 6.55 (2H, s, 2'-C H -xanthene), 6.56-6.58 (2H, dd, 4'-C H -xanthene ), 7.17-7.19 (2H, d, 5'-C H -xanthene), 7.20-7.22 (1H, d, 4'-C H -isobenzofuranone), 7.62-7.64 (1H, d, 5'-C H - isobenzofuranone), 7.82 (1H, s, 7 &apos; -C H -isobenzofuranone).

Experimental Example 1: Measurement of pH-responsive drug release

Release of BA from? -CD-MCM-BA

The pH-responsive release of BA from? -CD-MCM-BA (4) was confirmed by ¹ H NMR. β-CDMCM-BA was dissolved in 1.2 wt% DCl at a concentration of 3 mg / mL. After incubation for 8 hours at 37 ° C with stirring, ¹ H NMR was measured with a Bruker Avance DPX-300 (Germany) (Figure 4).

Acid solution showed peaks at (D ₂ O of 1.2 wt% DCl) from, is 3.18-3.23 ppm (denoted by x) methylene protons next to the nitrogen-β-BA CDMCM undamaged amides, methylene of free BA The peak corresponding to the peak (indicated by *) shifted to 2.55-2.62 ppm (FIG. 5). Through the chemical shifts, it is possible to confirm the decomposition of MCM acid amide and the release of bound BA.

Release of CP from? -CD-MCM-CP

Each of the solutions was incubated at 37 占 폚 with incubation at 37 占 폚, and the solution was incubated at 37 占 폚 for 1 hour at room temperature. The? -CD-MCM-CP (5) was dissolved in a pH 7.4 phosphate buffer solution (100 mM) or a pH 5.5 acetic acid buffer solution (100 mM) . YOUNGLIN HPLC (9000 HPLC, South Korea) equipped with a UV detector and a reversed phase column (Agilent Eclipse XDB-C18 4.6 × 150 mm, 5 μm) was used for HPLC-based measurements. At various time points, each sample was collected and diluted 10-fold with the same buffer. After filtration through a 0.2 μm PVDF syringe filter, the sample was injected into the HPLC system. A mixture of methanol: pH 9.0 phosphate buffer (50 mM) (3: 7 v / v) was used as the eluent and the flow rate was set at 0.5 mL / min. The release of CP was measured by UV absorbance at 245 and 270 nm wavelengths (Figure 5).

The cumulative release of CP from? -CD-MCM-CP at pH 5.5 and pH 7.4 was measured by HPLC to determine the decomposition kinetics in more detail. The results are shown in Fig.

From Figure 5, it can be seen that at pH 7.4, even after 5 hours, less than 20% CP is released from? -CD-MCM-CP. On the other hand, at pH 5.5, a rapid initial overdose of CP was observed, with over 80% CP released within 0.5 hours. The release was nearly complete by about 95% within one hour. Sudden differences in emission kinetics were observed between pH 5.5 and pH 7.4. In addition, the stability of CP during binding and release was confirmed in this experimental example. ¹ H NMR spectrum of the emitted CP from the β-CD-MCM-CP at pH 5.5 was the same as the ¹ H NMR spectrum of the original CP (Fig. 6a). Also, the mass spectra of the two CPs obtained by electrospray ionization (ESI) were the same (350.1 [M + H] ⁺ and 699.1 [M + M] ⁺ , Fig. Based on these data, it can be seen that the CP is fairly stable during bonding under mild basic conditions and during emission under mildly acidic conditions.

Experimental Example 2: Confirmation of complex formation of compounds 5 and 9 by fluorescence correlation spectroscopy (FCS) measurement

A self-made fluorescence confocal microscope setup was used to measure the autocorrelation of the fluorescence probe, FITC. Coupled to a single-mode fiber (Φ = 3.5 μm, P1-488-PM-FC, Thorlabs) for beam clean-up, a 488-nm continuous blue laser diode (TECBL-20GC-488, World Star Tech) The sample was irradiated with light through a water-immersion objective (NA = 1.20, 60x, f = 3 mm, Olympus) immobilized in the fabricating microscope body. The fluorescence signal was measured with excitation light by a dichroic mirror (ZT488rdc, Chroma) and further cleared with a radiation filter (HQ525 / 50 m, Chroma). Using a 1: 2 multimode fiber optocoupler (Φ = 62.5 μm, FCMM625-50A-FC) as a pin hole to remove the background from the focus volume and use two avalanche photodiodes (SPCM-AQR- 14-FC, Perkin Elmer). The correlation was measured with a correlator card (FLEX02-01D, Correlator.com) and added as a self-made analysis program coded in LabVIEW 2009 Respectively.

The fluorescence correlation spectroscopy (FCS) setup was calibrated as a phosphor to measure the axial and lateral area of the confocal volume using Alexa 488 with a diffusion coefficient of D = 4.35 x 10 ^-6 cm 2 / s (Petrasek Z et al Biophys J, 2008, 94, 1437-1448). The calibration followed the previously reported procedure (Kim SA et al., Nat Methods, 2007, 4, 963-973). The calibration showed that the 1 / e ² area of the side was 232 nm with an aspect ratio of 7.18, indicating that the measured focus volume was 0.501 fL.

For the determination of complex formation, a solution of Compound 9 (10 nM) in pH 9.0 phosphate buffer (50 mM) was prepared with varying concentrations of Compound 5. Bovine serum albumin was added to the mixture to a final concentration of 0.1 mg / mL to prevent nonspecific binding between Compound 5 and Compound 9. Each correlation curve was measured six times for 10 minutes (once for each concentration of compound 5) to suppress thermal noise and background signals.

The results are shown in Fig.

As described above, fluorescence correlation spectroscopy (FCS) was used to analyze the change in the diffusion coefficient of phosphors attached to a guest or a host, in order to confirm the formation of a complex between a drug complex of? -CD-base and an adamantane derivative Respectively. FITC was conjugated to an adamantane derivative as a probe (FITC-hex-ADM). The average diffusion time of the phosphor was measured while varying the concentration of the host molecule β-CD-MCM-CP. As the concentration of β-CD-MCM-CP increased, the mean diffusion time also decreased. That is, the diffusion coefficient of the phosphor was significantly decreased due to formation of the inclusion complex. Through this, molecular weight increase and complex formation can be confirmed (FIG. 7). The binding constants were calculated from the S-type curve between the diffusion time and the concentration of β-CD-MCM (FIG. 7). 7, the binding constant between? -CD-MCM-CP and FITC-hex-ADM was calculated to be 1.49 × 10 ⁴ M ^-1 . In general, since the binding constant K of β-CD and adamantane is 1 × 10 ⁴ M ^-1 to 10 × 10 ⁴ M ^-1 , the results show that the interaction between β-CD-MCM-CP and adamantane derivatives (Harries D et al., J Am Chem Soc, 2005, 127, 2184-2190). &Lt; / RTI &gt; Based on the value of the coupling constant, the inclusion percentage could be calculated and is shown in FIG.

Experimental Example  3: Rotational coordinate system Overhauser  Confirmation of formation of conjugate by effect spectroscopy

The complex formation of β-CD with compound 9 and β-CD-MCM-CP (compound 5) with compound 9 was confirmed by rotating frame overhauser effect spectroscopy (ROESY).

First, a complex of β-CD and Compound 9 and a complex of β-CD-MCM-CP (Compound 5) and Compound 9 were dissolved in D ₂ O-system pH 9.0 phosphate buffer (50 mM) By mole ratio. NMR spectra were recorded on an Agilent Technologies 400-MR DD2 (USA) spectrometer at 25 &lt; 0 &gt; C. Rotational Coordinate Overhaul Effect Spectroscopy (ROESY) experiments were performed using standard protocols (mixing time: 300 ms; T1 experiment) included in the spectrometer library.

8A and 8B show the ROESY spectra of a mixture of β-CD / FITC-hex-ADM and β-CD-MCM-CP / FITC-hex-ADM, respectively. The α, β and γ protons of adamantane in FITC-hex-ADM exhibit strong cross-peaks due to interaction with H3, H5, and H6 proton in the cavity of the original β-CD And showed weaker cross peaks due to interaction with H2 and H4 proton on the exterior of the β-CD cavity (FIG. 8A). Similarly, a cross peak between the proton of the adamantane derivative and? -CD-MCM-CP was also observed (FIG. 8b). The spectra showed a spatial proximity of the maximum limit of 5 Å in the proton of the β-CD derivative and adamantane moiety. Both data show complex formation between β-CD-MCM-CP and FITC-hex-ADM.

The host-guest fold between β-CD-MCM-CP and adamantane derivatives was further confirmed by ROESY spectra (FIG. 8). The strong cross peak of β-CD-MCM-CP / FITC-hex-ADM was similar to β-CD / FITC-hex-ADM. The binding of the six MCM molecules confronting the primary hydroxyl group in the medusa form was not significantly affected by the inclusion. Based on these results, it can be seen that any functional moiety can be introduced into the beta -CD-MCM after formation of the inclusion complex after drug binding. At this time, other drug delivery vehicles, such as polymers or nanoparticles with an adamantane moiety, can be easily modified through host-guest interaction of the binding partner with pH responsive binding of the present invention having a drug or other functional group.

Experimental Example 4: Cytotoxicity analysis

Cell viability was measured by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich (USA)) assay. NIH-3T3 cells (mouse embryonic fibroblast cell line) were inoculated into 90 μl of DMEM (Dulbecco's modified eagle's medium; WelGene (USA)) containing 96-well plates of 10% FBS (fetal bovine serum; WelGene × 10 ³ cells / well, and incubated at 37 ° C. for 24 hours. To determine cytotoxicity, 10 [mu] l of each sample solution at various concentrations was added to the medium and then incubated at 37 [deg.] C for 48 hours. For analysis, the cells were washed with DPBS (Dulbecco's phosphate-buffered saline; WelGene (USA)) and then 20 μl of filtered MTT solution (2 mg / mL in DPBS) was added. After incubation for 2 hours at 37 [deg.] C, the medium was removed from the wells and 150 [mu] l DMSO was added to dissolve the insoluble formalin particles. Absorbance was measured at 570 nm using a microplate reader (Molecular Devices Co., Menlo Park, Calif.). Relative cell viability (%) was defined as the percentage of viable cells versus the same percentage of control (cells treated with DPBS solution).

As described above, the short-term biocompatibility of the β-CD-MCM drug delivery system was confirmed by MTT assay on NIH3T3 cells. The results are shown in Fig.

A branched polyethylenimine ( b- PEI) 25 kDa, a known high molecular weight drug delivery with high cytotoxicity, was used as a positive control. Up to 100 μM, β-CD, β-CD-MCM, and β-CD-MCM-CP showed almost no cytotoxicity until 48 hours.

Example  3: β- Cyclodextrin  Amide derivative and 1- methyl -2- (2'- Carboxyethyl ) A conjugate of maleic anhydride (? CD - NH - MCM ) Synthesis of

Compound 10 was synthesized according to the following scheme using previously reported methods (NATURE PROTOCOLS, 2008, 3 (4), 691-697).

Example  4: β- Cyclodextrin Triazole  Derivatives and 1- methyl -2- (2'- Carboxyethyl ) Maleic acid  The anhydride bond (? CD - Triazole - MCM ) Synthesis of

Compound 11 was synthesized according to the following scheme using previously reported methods (Eur. J. Org. Chem., 2012, 4087-4105).

Example  5: β- Cyclodextrin  Phenylalanine derivatives and 1- methyl -2- (2'- Carboxyethyl ) Maleic acid  The anhydride bond (? CD - Phenylalaine - MCM ) Synthesis of

Compound 12 was synthesized according to the following scheme using previously reported methods (J. Org. Chem., 1996, 61, 903-908).

Example  6: β- Cyclodextrin  Cysteine derivative and 1- methyl -2- (2'- Carboxyethyl ) Maleic acid  The anhydride bond (? CD - Cysteine - MCM ) Synthesis of

Compound 13 was synthesized according to the following scheme using previously reported methods (J. Org. Chem., 1996, 61, 903-908).

Claims (22)

a hydroxyl group of at least one glucose 6 carbon of? -,? - or? -cyclodextrin;
Or a hydroxyl group of one or more glucose 6 of cyclodextrin carbon NH _2, N _3, or one or more of the glucose 6-carbon of the amino acid functional groups present in the cyclodextrin derivatives substituted with,
A cyclodextrin derivative having a pH-responsive property, which is connected to a carboxylic acid moiety of a maleic anhydride derivative represented by the following formula (1) by an ester or amide bond,
Wherein the functional group is at least one of a hydroxyl group, NH ₂ , N ₃ or an amino acid.
[Chemical Formula 1]

In this formula,
R ' is hydrogen or _C1-6alkyl ;
m is an integer of 1 to 10;
The method of claim 1, wherein the α-, β- or γ-cyclodextrin;
Or a cyclodextrin derivative in which the hydroxyl group of at least one glucose 6-carbon of the cyclodextrin is replaced by NH ₂ , N ₃ or an amino acid,
A cyclodextrin derivative having pH responsiveness, which is a compound represented by the following formula (2):
(2)

In this formula,
R is OH, NH _2, N _3, or amino acid;
n is an integer of 1 to 3;
The cyclodextrin derivative according to claim 1, wherein the amino acid is cysteine or phenylalanine.
The maleic anhydride derivative according to claim 1, wherein the maleic anhydride derivative of formula (1) is linked to cyclodextrin in the form of one of the following formulas (3a) to (3e) by an ester or amide bond. derivative:
[Chemical Formula 3]

(3b)

[Chemical Formula 3c]

(3d)

[Formula 3e]

In this formula,
R ' is hydrogen or _C1-6alkyl ;
m is an integer of 1 to 10;
A method for producing a cyclodextrin derivative having the pH responsiveness of claim 1, comprising the steps of:
? -,? - or? -cyclodextrin;
Or a cyclodextrin derivative in which the hydroxyl group of at least one glucose 6-carbon of cyclodextrin is replaced by NH ₂ , N ₃ or an amino acid,
Reacting a maleic anhydride derivative of formula (1) (step 1):
[Chemical Formula 1]

In this formula,
R ' is hydrogen or _C1-6alkyl ;
m is an integer of 1 to 10;
6. The composition of claim 5, wherein said alpha, beta, or gamma -cyclodextrin;
Or a cyclodextrin derivative in which the hydroxyl group of at least one glucose 6-carbon of cyclodextrin is replaced by NH ₂ , N ₃ or amino acid,
A compound represented by the following formula (2):
(2)

In this formula,
R is OH, NH _2, N _3, or amino acid;
n is an integer of 1 to 3;
6. The process according to claim 5, wherein the reaction of step 1) is carried out using diisopropylcarbodiimide (DIC).
6. The process according to claim 5, wherein the reaction of step 1) is carried out in the presence of 4-dimethylaminopyridine (DMAP) and p-toluenesulphonic acid (PTSA) Gt;
A conjugate wherein the cyclodextrin derivative having the pH responsiveness of claim 1 is combined with a drug including an amine group.
10. The pharmaceutical composition according to claim 9, wherein the drug comprising an amine group is selected from the group consisting of butylamine, cephradine, cefadroxyl, cephaloglycin, cephapirin, Cefroxadine, Cefaclor, Cefuroxime, Cefuzonam, Cefotetan, Cefoxitin, Carbacephem, Cefotiam, Cefcotin, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, Cefcaprofen, , Cefotaxime, Cefovecin, Cefpodoxime, Cefteram, Ceftibuten, Ceftiofur, Ceftiolene, Cephexin, Ceftiofur, Ceftizoxime, Ceftriaxone, Cefclidine, Cefepime, Cefluprenam, Cefoselis, A conjugate that is Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Cefmatilen, doxorubicin or a mixture thereof.
The conjugate according to claim 9, wherein the molar ratio of the cyclodextrin derivative having a pH-responsive property to the drug comprising an amine group is 1: 1 to 8.
10. The conjugate according to claim 9, wherein the bond between the cyclodextrin derivative having the pH-responsive property and the amine group-containing drug is degraded under acidic conditions.
13. The combination according to claim 12, wherein the acidic condition is a weak acidic condition of pH 5 to 6.5.
A method for manufacturing a combination according to claim 9 comprising the steps of:
Reacting a drug comprising the cyclodextrin derivative and the amine group having the pH responsiveness of claim 1 (step 2).
15. The method according to claim 14, wherein the reaction of step 2) is carried out using at least one selected from the group consisting of triethylamine, diethylamine and diisopropylethylamine as organic bases.
A composition for pH-responsive drug delivery comprising the conjugate of any one of claims 9 to 13.
The pH-responsive drug delivery composition of claim 16, wherein the drug is released under acidic conditions.
The pH-responsive drug delivery composition according to claim 16, wherein the acidic condition is a weak acidic condition of pH 5 to 6.5.
18. The composition of claim 16, which is used to target cancer, inflammatory tissue or abscess.
An inclusion complex in which an additional drug or ligand as a guest substance is contained by a host-guest inclusion in a complex formed by coupling a drug including an amine group to a cyclodextrin derivative having the pH responsiveness of claim 1.
21. The inclusion complex of claim 20, wherein the guest material is at least one selected from the group consisting of a targeting ligand, a cell penetrating moiety, a polymer and a nanoparticle.
22. The inclusion complex of claim 21, wherein the guest material comprises an adamantane moiety.

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