KR102051593B1 - Dual-responsive amphiphiles, Manufacturing method thereof and Drug delivery using the same - Google Patents

Abstract

The present invention relates to dual-responsive amphiphiles, a manufacturing method thereof, and a drug carrier using the same. More specifically, the present invention relates to dual-responsive amphiphiles which can be used in the manufacture of a drug carrier that maximizes the therapeutic efficacy of drugs by effectively releasing a large amount of drugs selectively in a short time while the drug carriers easily disintegrate for diseases of high active oxygen concentration and thiol compounds.

Description

Dual-responsive amphiphilic compound, preparation method thereof and drug delivery using the same {Dual-responsive amphiphiles, Manufacturing method etc. and Drug delivery using the same}

The present invention relates to thiol compounds (eg, glutathione (GSH), 1,4-dithiothreitol (DTT), cysteine, etc.) and reactive oxygen species (ROS): superoxide anion, hydrogenperoxide, hydroxy radical, etc.) The present invention relates to a double-responsive amphiphilic compound that reacts to a drug carrier using the same.

Drugs used to treat diseases, such as anticancer drugs or inflammatory diseases, have the disadvantage of not maximizing the therapeutic effect due to side effects due to toxicity and low solubility. To overcome these drawbacks, new drug formulations are being actively developed.

Drug delivery agents such as nanoparticles and micelles are being developed to minimize side effects due to toxicity of drugs and to improve solubility. In particular, the polymer micelle having a hydrophilic group and a hydrophobic group can form a thermodynamically stable and uniform spherical structure, and can improve the solubility of the drug by encapsulating a hydrophobic drug in the hydrophobic portion of the micelle. The micelle is designed to enclose a hydrophilic group to protect it from various mechanisms in the human body, and to enclose hydrophobic drugs in the hydrophobic group. Vascular vessels in most cancerous or inflammatory disease tissues are looser than normal tissues, and micelles having an appropriate size can easily accumulate around cancerous or inflammatory disease tissues by an EPR (Enhanced Permeability and Retention) effect.

Disulfide bonds, on the other hand, are present in numerous proteins in the human body, which can reversibly redox depending on the thiol concentration of human tissues. Thiol concentrations vary depending on the pathological conditions of the human body. For example, the concentration of thiol in cytosol is about 10 mM, about 10 uM in plasma, and around tens to hundreds of times more than that in normal tissues around cancer cells. It is reported. The concentration of active oxygen, such as hydrogen peroxide, is about 100 uM in any diseased area or cancer cell, which is 100 times that of normal cells.

Various researches and technologies using the characteristics of the disease occurrence site are being developed, and the demand and demand for efficient new drug carriers are increasing.

Republic of Korea Publication No. 10-2008-0009196 (published 2008.01.25)

Accordingly, the present inventors have completed a new high molecular compound having high sensitivity to both a thiol compound and active oxygen. That is, the present invention is to provide a novel double-responsive amphiphilic polymer compound, a drug carrier using the same and a method for preparing the same.

The present invention for solving the above problems includes a compound represented by the following formula 8 as a double-reaction amphiphilic compound.

[Formula 8]

In the formula (8), n is a natural number of 10 to 500, m is a natural number of 2 to 100.

In a preferred embodiment of the present invention, the double reactive amphiphilic compound is a thiol (-SH) reaction type and an active oxygen reaction type.

Another object of the present invention relates to a method for preparing the double-reactive amphiphilic compound, wherein the compound represented by the following formula 1 and the compound represented by the following formula 2 are reacted to synthesize a compound represented by the following formula 3 step; Performing a reaction of removing the amine protecting group from the compound represented by Formula 3 to synthesize a compound represented by Formula 4 below; Performing a reaction with a compound represented by Formula 4 and a methionine compound represented by Formula 5 to synthesize a compound represented by Formula 6; Performing a reaction of removing the thiol protecting group from the compound represented by Chemical Formula 6 to synthesize a compound represented by Chemical Formula 7; And five steps of synthesizing the compound represented by the following Chemical Formula 8 by oxidizing the compound represented by the chemical formula 7 between thiol groups.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

In Formulas 1 to 8, R ¹ is a thiol protecting group, R ² is an amine protecting group, n is a natural number of 10 to 500, and m is a natural number of 2 to 100.

As a preferred embodiment of the present invention, the reaction of step 1 may be performed for 15 to 36 hours at a temperature of 15 ° C to 35 ° C and atmospheric pressure under a reaction catalyst and an organic solvent.

In a preferred embodiment of the present invention, the reaction of step 1 may be performed by adding a coupling agent.

As a preferred embodiment of the present invention, one step of the reaction catalyst may be used (DMAP (4- (dimethylamino) pyridine).

As a preferred embodiment of the present invention, the coupling agent may include one or more selected from dicyclohexylcarbodiimide (DCC) and EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide).

In one preferred embodiment of the present invention, the organic solvent in one step may include one or more selected from methylene chloride, dimethylformamide and dimethylacetamide.

In a preferred embodiment of the present invention, the reaction of removing the amine protecting group in two steps may be performed with the compound represented by the formula (3) and the nitrogen-based compound in an organic solvent.

As a preferred embodiment of the present invention, the nitrogen compound of step 2 may include at least one selected from piperidine, pyrrolidine, pyridine and derivatives thereof.

In a preferred embodiment of the present invention, the organic solvent of the second step may include at least one selected from DMF (dimethylformamide) and methylene chloride.

In a preferred embodiment of the present invention, the amine protecting group of the second step is a methoxymethyl (MOM) group, methoxyethyl (MEM) group, 1-ethoxyethyl (EE) group, benzyloxymethyl group, (β-trimethyl Silylethoxy) methyl group, tetrahydropyranyl group, 2,2,2-trichloroethoxycarbonyl (troc) group, benzyloxycarbonyl (Cbz) group, tert-butoxycarbonyl (t-Boc) group , 9-fluoroenylmethoxycarbonyl (Fmoc) group, 2,2,2-trichloroethoxymethyl group, trimethylsilyl group, triethylsilyl (TES) group, tripropylsilyl group, dimethylethylsilyl group, (3 Tea-butyl) dimethylsilyl (TBS) group, diethylmethylsilyl group, dimethylphenylsilyl group or diphenylmethylsilyl group.

As a preferred embodiment of the present invention, the thiol group protecting group of the four steps, butoxycarbonyl (Boc) group, carbenzoxy (Cbz) group, 2-methoxycarbonyl-1-methylvinyl group, trityl (Trt), benzyl (Bzl), benzyloxycarbonyl (Z), formyl or chloroacetyl group.

As a preferred embodiment of the present invention, step 5 may dissolve the compound represented by Formula 7 in an organic solvent and then perform a thiol period oxidation reaction by stirring for 50 to 100 hours at room temperature and atmospheric pressure.

In a preferred embodiment of the present invention, the organic solvent of step 5 may include at least one selected from dimethylsulfoxide and tetrahydrofuran.

Another object of the present invention relates to a drug carrier using a dual reactive amphiphilic compound prepared by the method described above.

In one preferred embodiment of the present invention, the drug carrier of the present invention is a micelle type drug carrier, and may include the double-reactive amphiphilic compound and a hydrophobic drug.

In a preferred embodiment of the present invention, the drug carrier of the present invention may form the micelle of the double-responsive amphiphilic compound, and the hydrophobic drug may be supported inside the micelle.

In one preferred embodiment of the present invention, the drug may be an anticancer agent or an anti-inflammatory agent.

The term "room temperature" used in the present invention means "15 ~ 35 ℃, preferably" 18 ~ 30 ℃ ", more preferably" 20 ~ 25 ℃ ".

The double-reactive amphiphilic polymer compound of the present invention is highly sensitive to both thiol compounds and active oxygen, and when applied as a drug carrier, the drug carriers sensitive to thiols and free radicals accumulate in cancer or inflammatory disease tissues. In addition, in the environment of locally high thiols or free radicals, drug delivery disruption can be used to maximize the selective drug release at the disease site to improve treatment efficiency.

1 is a double reactivity measurement result of the double-reactive amphiphilic compound carried out in Experimental Example 1, Molecular weight change of the double-reaction amphiphilic compound by gel chromatography.
2 is a result of measuring the change in size and distribution of the solution phase dimer carried out in Experimental Example 1 by the light scattering method.
3 is a result of drug release properties of the drug carrier prepared in Preparation Example 1, carried out in Experimental Example 2.
Figure 4 is a result showing the cell death characteristics according to the drug release properties in connection with the drug carrier prepared in Preparation Example 1, carried out in Experimental Example 3.

Hereinafter, the present invention will be described in more detail.

The double reactive amphiphilic compound of the present invention is a dimer (DIMER), has two water-soluble groups and two fat-soluble groups, and is a compound represented by the following formula (8).

[Formula 8]

In the formula (8), n is a natural number of 10 to 500, preferably n is a natural number of 12 to 350, more preferably n is a natural number of 12 to 100. In this case, when n is less than 10, the molecular weight of the fat-soluble group should be designed to be low, which may cause a problem in the possibility of use as a drug carrier, and when n exceeds 500, there may be a problem in that the solubility in water-soluble solvents is too low. .

Further, m in the formula (8) is a natural number of 2 to 100, preferably m is a natural number of 3 to 60, more preferably a natural number of 4 to 30. At this time, if m is less than 2, there may be a problem in that micelles (micelles) are not formed well because the length of the fat-soluble group (fat-soluble portion) is short, and if m is more than 100, there is a problem in low solubility in water-soluble and fat-soluble solvents. Can be.

It is preferable to control the molecular weight of the water-soluble group and the oil-soluble group of the formula (8), and to adjust the n and m values appropriately so that the molecular weight of the water-soluble group is larger than the molecular weight of the oil-soluble group, it is preferable in terms of solubility in water-soluble solvents, micelle formation, and the like. Do.

In the double-reactive amphiphilic compound of the present invention, the lipophilic group and the active oxygen of the compound react to oxidize thioether (-S-) to sulfooxide (sulfoxide O = S = O) or sulfone (sulfone, S = O). It is a dual reactive amphiphilic compound which is converted and the disulfide bond (-SS-) of the compound is reduced by reacting with a compound having a thiol (-SH) to break the disulfide bond.

Such a double reactive amphiphilic compound of the present invention can be prepared by the following method.

The present invention is a step of synthesizing the compound represented by the formula (3) by reacting the compound represented by the formula (1) and the compound represented by the formula (2); Performing a reaction of removing the amine protecting group from the compound represented by Formula 3 to synthesize a compound represented by Formula 4 below; Performing a reaction with a compound represented by Formula 4 and a methionine compound represented by Formula 5 to synthesize a compound represented by Formula 6; Performing a reaction of removing the thiol protecting group from the compound represented by Chemical Formula 6 to synthesize a compound represented by Chemical Formula 7; And 5 steps of synthesizing the compound represented by Chemical Formula 8 by oxidizing the compound represented by Chemical Formula 7 between thiol groups.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

In Chemical Formulas 1 to 7, R ¹ is a thiol protecting group, and R ² is an amine protecting group.

The thiol protecting group is a butoxycarbonyl (Boc) group, a carbenzoxy (Cbz) group, 2-methoxycarbonyl-1-methylvinyl group, trityl (Trt) group, benzyl (Bzl) group, benzyloxy It may include a carbonyl (Z) group, formyl group, or chloroacetyl group, and preferably a butoxycarbonyl (Boc) group, carbenzoxy (Cbz) group, trityl (Trt) group or benzyl (Bzl) ) May be included.

Examples of the amine protecting group include methoxymethyl (MOM) group, methoxyethyl (MEM) group, 1-ethoxyethyl (EE) group, benzyloxymethyl group, (β-trimethylsilylethoxy) methyl group, tetrahydropyranyl group, 2,2,2-trichloroethoxycarbonyl (troc) group, benzyloxycarbonyl (Cbz) group, t-butoxycarbonyl (t-Boc) group, 9-fluoroenylmethoxycarbonyl (Fmoc) Group, 2,2,2-trichloroethoxymethyl group, trimethylsilyl group, triethylsilyl (TES) group, tripropylsilyl group, dimethylethylsilyl group, (t-butyl) dimethylsilyl (TBS) group, diethyl It may include a methylsilyl group, dimethylphenylsilyl group or diphenylmethylsilyl group, preferably 2,2,2-trichloroethoxycarbonyl (troc) group, benzyloxycarbonyl (Cbz) group, t- Butoxycarbonyl (t-Boc) group, 9-fluoroenylmethoxycarbonyl (Fmoc) group, 2,2,2-trichloroethoxymethyl group, trimethylsilyl group or triethylsilyl (TES) group have.

In addition, n in the general formulas (1) to (7) is a natural number of 10 to 500, preferably n is a natural number of 12 to 350, more preferably n is a natural number of 12 to 100. And, m in the formula (1) to (7) is a natural number of 2 to 100, preferably m is a natural number of 3 to 60, more preferably a natural number of 4 to 30.

In the production method of the present invention, step 1 is a step of preparing a compound represented by the formula (3) into which a water-soluble group is introduced, and the reaction of step 1 is carried out under a reaction catalyst and an organic solvent, preferably a reaction catalyst, a coupling agent, and an organic solvent. Under a temperature of 15 ° C. to 35 ° C. and atmospheric pressure for 15 to 36 hours, preferably at a temperature of 20 ° C. to 30 ° C. and for 18 to 30 hours. In this case, when the reaction temperature is less than 15 ℃ reaction rate is slow may be a long process time, when the reaction temperature exceeds 35 ℃ side reaction may occur. In addition, when the reaction time is less than 15 hours, the yield of the compound represented by Chemical Formula 3 may be too low, uneconomical exceeding 36 hours, rather, the side reaction may proceed and the yield may be lowered.

And, in the one-step reaction, if the reaction pressure is not at atmospheric pressure, the process may be complicated because a process for depressurization or pressurization should be added.

In addition, the reaction catalyst of step 1 may be used MAP (4- (dimethylamino) pyridine), the coupling agent (coupling agent) (dicyclohexylcarbodiimide) and EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) It may include one or more selected from.

In addition, the amount of the reaction catalyst and the coupling agent may be used by mixing the reaction catalyst and the coupling agent in a weight ratio of 1: 0.01 to 1, preferably, the reaction catalyst and the coupling agent in a weight ratio of 1: 0.25-0.7.

In addition, the organic solvent in one step may include one or more selected from methylene chloride, dimethylformamide and dimethylacetamide.

After completion of the reaction in one step, the solvent, the reaction catalyst, the unreacted coupling agent, and the like are removed, followed by precipitation in ether to obtain a compound represented by Chemical Formula 3 as a reaction product.

Next, in the preparation method of the present invention, step 2 is a step of removing the amine protecting group represented by R ² of Formula 3 to prepare a compound represented by Formula 4, and the compound represented by Formula 3 and nitrogen under an organic solvent. The reaction with the compound may be carried out to desorb the amine protecting group.

The nitrogen-based compound of step 2 may include at least one selected from piperidine, pyrrolidine and derivatives thereof, preferably at least one selected from piperidine and derivatives thereof. It may include.

The organic solvent in the second step may include one or more selected from dimethylformamide (DMF) and methylene chloride.

The amount of the reactant used in step 2 is 1 g of the compound represented by Formula 3, 2 to 30 ml of the organic solvent and 0.10 to 1 ml of the nitrogen-based compound, preferably 1 g of the compound represented by Formula 3, the organic solvent 4 After mixing ~ 20 ml and 0.2 ~ 0.8 ml of the nitrogen-based compound, it is preferable to perform a reaction to remove the amine protecting group.

In addition, the nitrogen-based compound of the second step may be carried out for 5 hours to 20 hours under a temperature and atmospheric pressure conditions of 20 ℃ ~ 30 ℃, preferably 9 hours ~ 16 under the temperature and atmospheric pressure conditions of 20 ℃ ~ 30 ℃ Can be done for hours. In this case, when the reaction temperature is less than 20 ℃ reaction rate is slow may be a long process time, when the reaction temperature exceeds 30 ℃ side reaction may occur. In addition, when the reaction time is less than 5 hours, the amine group protecting group may not be sufficiently removed from the compound represented by Formula 4, and it is uneconomical that the reaction time exceeds 20 hours. In addition, when the reaction pressure is not at atmospheric pressure, a process for depressurizing or pressurizing should be added, which may complicate the process.

After the completion of the two-step reaction, the solvent and the unreacted nitrogen-based compound are removed and then precipitated in ether to obtain a compound represented by Chemical Formula 4 as a reaction product.

Next, step 3 is a step of synthesizing the compound represented by the formula (6) in which the fat-soluble group is introduced by reacting the compound represented by the formula (4) from which the amine protecting group is removed with the methionine compound represented by the formula (5).

The reaction in the three steps is based on 100 parts by weight of the compound represented by the formula (4), 20 to 1100 parts by weight of the methionine carboxyanhydride compound represented by the formula (5), preferably 25 to 750 parts by weight of the methionine compound, more preferably 40 to 350 parts by weight is added to the organic solvent, stirred and dissolved. In this case, when the amount of the methionine compound is less than 20 parts by weight, there may be a problem in that the molecular weight of the oil-soluble part of the synthesized double-reactive amphiphilic compound is too low, and when it exceeds 1100 parts by weight, the molecular weight of the fat-soluble part may be too high. have. And, the melt can be carried out by reacting for 15 to 36 hours at a temperature of 15 ℃ to 35 ℃ and atmospheric conditions, preferably 18 to 30 hours at a temperature of 20 ℃ to 30 ℃ and atmospheric conditions.

After completion of the reaction in three steps, the solvent and the like are removed, followed by precipitation in ether to obtain a compound represented by Chemical Formula 6 as a reaction product.

Next, the fourth step is a step of synthesizing the compound represented by the formula (7) by performing a reaction to remove the thiol protecting group of the compound represented by the formula (6).

The step 4 may be performed by adding the compound represented by Chemical Formula 6 to an organic solvent, then adding a reaction catalyst, and performing the reaction for 10 hours to 30 hours under a temperature of 20 ° C. to 30 ° C. and atmospheric pressure. When the reaction temperature is less than 20 ° C may be a slow reaction rate, the process time may be long, and when the reaction temperature exceeds 30 ° C side reaction may occur. In addition, when the reaction time is less than 10 hours, the reaction may not be made sufficiently, and when the reaction time exceeds 30 hours, a side reaction may occur. In addition, if the reaction pressure is not at atmospheric pressure, the process may be complicated because a process for depressurizing or pressurizing must be added.

In addition, the reaction catalyst of step 4 may include at least one selected from trifluoroacetic acid (TFA) and hydogen bromide.

In addition, the organic solvent of step 4 may include one or more selected from methylene chloride and dimethylformamide.

In addition, the amount of the reactant used in step 4 per 1 g of the compound represented by the formula (6), 3 to 10 ml of the organic solvent and 1 to 7 ml of the reaction catalyst, preferably per 1 g of the compound represented by the formula (6), After mixing 4 to 8 ml of the organic solvent and 1.5 to 5 ml of the reaction catalyst, it is preferable to perform the reaction of desorbing the thiol protecting group.

After completion of the reaction in 4 steps, the solvent and the like are removed, followed by precipitation in ether to obtain a compound represented by Chemical Formula 7 as a reaction product.

Next, step 5 is a process for preparing the double-reactive amphiphilic compound of the present invention, which is a compound represented by the formula (8), after dissolving the compound represented by the formula (7) in which the thiol protecting group is removed in an organic solvent, at room temperature and atmospheric pressure Under 50 to 100 hours, preferably 60 to 85 hours, more preferably 65 to 80 hours by stirring the oxidation reaction between the thiol group can be obtained to the final reaction product.

The organic solvent of step 5 may include at least one selected from dimethylsulfoxide and tetrahadrofuran.

The double-reactive amphiphilic compound of the present invention prepared by the above-described method is a thiol (-SH) reaction and an active oxygen reaction type, and is highly sensitive to the thiol compound and active oxygen and thus applied as a drug carrier (for example, , Drug-carriers such as micelle-type drug carriers), the drug carriers easily disintegrate for diseases of high thiol compounds and active oxygen concentrations, and effectively release a large amount of drugs to the disease site (or target cell) in a short time. A drug carrier can be prepared that can maximize the therapeutic efficacy of.

Drugs enclosed in drug delivery and delivered may be anticancer agents and / or anti-inflammatory agents.

As a specific example, the anticancer agent may be paclitaxel, doxorubicin, cis-platin, dodetaxel, domotaxel, tamoxifen, camtothecin, anasterozole , Carboplatin, topotecan, topotecan, verootecan, belotecan, irinotecan, irinotecan, gleevec, and vincristine.

In addition, the anti-inflammatory agents such as salicylates (salicylates), ibuprofen (ibuprofen), naproxen (naproxen), phenopropene (fenoprofen), indomethacin (indometha cin), phenyltazone (phenyltazone), mesotrexate (methotrexate), cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, It may include one or more selected from cortisone (cortisone) and corticosteroid (corticoste roid).

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are not intended to limit the scope of the present invention, which should be construed as to help the understanding of the present invention.

[ Example ]

Example  1: dual reaction type Amphipathic  Preparation of the compound

(1) Preparation of the compound represented by Chemical Formula 3-1

2.0 g of the compound represented by the following formula (1-1) and 0.7 g of the compound represented by the formula (2-1) are dissolved in methylene chloride as a solvent, and 0.25 g of dicyclohexyl carbodiimide (DCC) as a reaction catalyst and 4- as a coupling agent are dissolved. 0.01 g of (dimethylamino) pyridine (DMAP) was added to remove the solvent and the reaction catalyst after stirring for about 24 hours at about 21 to 23 ° C. and atmospheric pressure, and a precipitate was formed in ether to give a precipitate represented by the following Chemical Formula 3-1. Yielded the compound (mPEG-Cys (Ttr) -Fmoc).

[Formula 1-1]

[Formula 2-1]

[Formula 3-1]

N in Formula 1-1 and 3-1 is about 44-46. In Formulas 2-1 and 3-1, R ¹ is a trityl (Trt, trityl) group, and R ² is a 9-fluoroenylmethoxycarbonyl (Fmoc) group.

(2) Preparation of the compound represented by Chemical Formula 4-1.

2.0 g of the compound represented by Chemical Formula 3-1 was dissolved in 10 mL of dimethylformamide (DMF), 0.5 mL of piperidine was added, and stirred and reacted at about 21 to 23 ° C. for 12 hours. Next, the reaction product was precipitated in ether to obtain a compound represented by Chemical Formula 4-1 (mPEG-Cys (Trt) -NH ₂ ) from which an amine protecting group was removed.

[Formula 4-1]

N in Formula 4-1 is about 44 to 46, and R ¹ in Formula 4-1 is a trityl (trityl) group.

(3) Preparation of the compound represented by Chemical Formula 6-1

1.50 g of the compound represented by Formula 4-1 was dissolved in 1.25 g of a methionine N-carboxyanhydride compound represented by the following Formula 5-1 in dimethylformamide, and stirred for 24 hours at room temperature and nitrogen atmosphere. And reacted. Next, it was precipitated in ether to obtain a compound represented by Chemical Formula 6-1 (mPEG-Cys (Trt) -PMT).

[Formula 5-1]

[Formula 6-1]

In Formula 6-1, m is about 8 to 10, n is about 44 to 46, and R ¹ is a trityl (trityl) group.

(4) Preparation of the compound represented by Chemical Formula 7-1

2.0 g of the compound represented by Chemical Formula 6-1 was dissolved in 10 mL of methylene chloride, and then 10 mL of trifluoroacetic acid was added thereto, followed by stirring and reaction for 3 hours under atmospheric pressure, and represented by Chemical Formula 7-1. To give the compound (mPEG-Cys-PMT).

[Formula 7-1]

In Formula 7-1, n is about 44 to 46 and m is about 8 to 10.

(5) Preparation of the compound represented by Formula 8-1.

1,7 g of the compound represented by Chemical Formula 7-1 was dissolved in 10 mL of dimethylsulfoxide, and stirred for 72 hours under atmospheric pressure to form a disulfide bond (-SS-) through an oxidation reaction between thiols of cysteine. The compound represented by the following Chemical Formula 8-1 (Gemini mPEG-Cys-PMT) was obtained.

[Formula 8-1]

In Formula 8-1, n is about 44 to 46 and m is about 8 to 10.

Experimental Example  1: dual reaction type Amphipathic  Dual Reactivity Determination of Compounds

0.1 g of the double-reactive amphiphilic compound, a dimer prepared in Example 1, was dissolved in 10 mL of distilled water, and then DTT (1,4-dithiothreitol) and / or H ₂ O ₂ were added, followed by stirring for 24 hours. . At this time, the concentration of DTT and H ₂ O ₂ added in the solution was titrated to 10 mM and 0.1 mM (= 100 uM), respectively.

In addition, the reduction of disulfide bond (-SS-) under DTT and the oxidation of methionine to thioether to sulfoxide (sulfoxide, O = S = O) or sulfone (sulfone, S = O) under H ₂ O ₂ were determined. The double reactivity of the compound prepared in Example 1 was measured by checking the molecular weight of the double-reactive amphiphilic compound by gel chromatography and the size and distribution of the solution phase dimer by the light scattering method. Figure 1 (change in molecular weight by gel chromatography) and Figure 2 (change in size and distribution by light scattering method).

(1) Regarding the graph of molecular weight change measured by gel chromatography of FIG. 1, the higher the molecular weight of the polymer of the compound, the faster the retention time of the peak. The red graph of FIG. 1 (original) is measured for a solution in which only a double reactive amphiphilic compound is added without adding both DTT and H ₂ O ₂ .

Referring to FIG. 1, when only 10 mM DTT was treated (blue graph), the disulfide bond (-SS-) of the dimer was broken to form a monomeric structure, resulting in a decrease in molecular weight, resulting in a later retention time than the original. The results were shown.

In addition, when only 0.1 uM H ₂ O _{2 was} treated (yellow graph), thioether (-S-) of methionine was oxidized to form sulfoxide (O = S = O) or sulfone (Sulfone, S = O). By forming, the molecular weight was increased and the retention time was the fastest. In addition, when 10 mM DTT was treated after treatment with 0.1 uM H ₂ O ₂ (green graph), the disulfide bond was broken and the molecular weight was reduced, resulting in a retention time slower than the yellow graph.

(2) Figure 2 shows the addition of DTT and H ₂ O ₂ of the double-reactive amphiphilic compound forming micelles in solution It is the result of measuring size and distribution change.

Looking at Figure 2, when compared with the original DTT and H ₂ O ₂ not treated (red graph), when only 10 mM DTT treatment (blue graph), it can be seen that the size of the micelle was reduced as the disulfide bond is broken. When only 100 uM H ₂ O _{2 was} treated (yellow graph), thioether (-S-) of methionine was oxidized to change to sulfoxide (sulfoxide O = S = O) or sulfone (sulfone, S = O). It can be seen that the change in size through, and when the two materials are treated simultaneously with the DTT and H ₂ O ₂ (green graph) it can be seen that the micelle size change occurs.

Production Example  1: Preparation of drug carrier

0.1 g and 0.02 g doxorubicin, the dimer prepared in Example 1, were dissolved in 1 mL of dimethylformamide, and then 10 mL of distilled water was added to encapsulate the drug and unsealed doxorubicin was removed by dialysis. Thus, a drug carrier in which doxorubicin is encapsulated in a micelle composed of a double reactive amphiphilic compound was prepared.

Experimental Example  2: measurement of drug release properties of drug carriers

The drug carrier of Preparation Example 1 was placed in a dialisian bag and the inside and outside of the bag were 10 mM DTT and / or 0.1 uM H ₂ O _2. The drug released to the outside by setting the concentration was measured by the UV spectrometer to measure the drug release characteristics, the results are shown in FIG.

Looking at Figure 3, DTT and H ₂ O ₂ Is not added at all (PBS, red graph) compared to H ₂ O ₂ The treatment (blue graph) shows that the rate of drug release increases due to the oxidation of methionine to the thioether and its transition to sulforoxide or sulfone. Or DTT treated, also, DTT Treatment (yellow graph) shows that the rate of drug release is further increased as the disulfide bonds of the compound are broken.

And DTT and H ₂ O ₂ All processed (triangle + crimson graph) is H ₂ O ₂ It can be seen that the release rate is faster than the single (blue graph) treatment.

Experimental Example  3: Measurement of Drug Release Characteristics of Drug Carrier by In Vitro Experiment

KB cells, one of the cancer cells, were cultured in a 96-well plate for 24 hours, followed by 10 mM GSH (Glutathione) or 100 uM H ₂ O _2. After washing uptake (uptake) in KB cells for 3 hours (washing), the drug carrier of Preparation Example 1 was treated with KB cells for 24 hours, according to the drug release according to the concentration of doxorubicin enclosed in the drug carrier The killing characteristics of cancer cells were examined by CCK-8 assay, and the results are shown in FIG. 4.

In FIG. 4, free-DOX refers to pure DOX not enclosed in micelles, and Dox-micelles are not treated with GSH and H ₂ O ₂ .

Drugs encapsulated in drug carriers during 10 mM GSH treatment resulted in increased drug release than undissolved (Dox-micelles) breakdown of disulfide bonds under GSH, thereby promoting KB cell killing and also 100 uM H ₂ O ₂ Methionine in the drug carrier during treatment It was confirmed that thioether was oxidized to induce destabilization of drug carriers through transition to sulfooxide or sulfone, thereby increasing drug release and promoting death of KB cells.

In addition, it was confirmed that the drug release rate was increased in the simultaneous treatment of the above substances, and cell death was increased more than when the single substance was treated.

Through the above examples, it was confirmed that the dual-reactive amphiphilic compound of the present invention has excellent thiol reactivity and reactivity to active oxygen, and when a dual-reactive amphiphilic compound having such characteristics is applied as a drug carrier, a specific disease It was confirmed that the drug can be effectively and quickly delivered to the site or cell site to maximize the effect of drug treatment.

Claims (10)

A step of synthesizing the compound represented by the formula (3) by reacting the compound represented by the formula (1) and the compound represented by the formula (2);
Performing a reaction of removing the amine protecting group from the compound represented by Formula 3 to synthesize a compound represented by Formula 4 below;
Performing a reaction with a compound represented by Formula 4 and a methionine compound represented by Formula 5 to synthesize a compound represented by Formula 6;
Performing a reaction of removing the thiol protecting group from the compound represented by Formula 6 to synthesize a compound represented by Formula 7 below; And
5 steps of synthesizing the compound represented by Formula 8 by oxidizing the compound represented by Formula 7 between thiol groups;
Method for producing a double-reactive amphiphilic compound comprising a;
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

In Formulas 1 to 8, R ¹ is a thiol protecting group, R ² is an amine protecting group, n is a natural number of 10 to 500, and m is a natural number of 2 to 100.
The method of claim 1, wherein the amine protecting group is a methoxymethyl (MOM) group, methoxyethyl (MEM) group, 1-ethoxyethyl (EE) group, benzyloxymethyl group, (β-trimethylsilylethoxy) methyl group, Tetrahydropyranyl, 2,2,2-trichloroethoxycarbonyl (troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (t-Boc), 9-fluoroenylme Oxycarbonyl (Fmoc) group, 2,2,2-trichloroethoxymethyl group, trimethylsilyl group, triethylsilyl (TES) group, tripropylsilyl group, dimethylethylsilyl group, (tert-butyl) dimethylsilyl A method for producing a double reactive amphiphilic compound, comprising a (TBS) group, diethylmethylsilyl group, dimethylphenylsilyl group or diphenylmethylsilyl group.
According to claim 1, wherein the thiol protecting group is a butoxycarbonyl (Boc) group, carbenzoxy (Cbz) group, 2-methoxycarbonyl-1-methylvinyl group, trityl (Trt) group, benzyl ( Bzl), a benzyloxycarbonyl (Z) group, a formyl group or a chloroacetyl group comprising a method for producing a double-reactive amphiphilic compound.
The method of claim 1, wherein the reaction in one step is performed for 15 to 36 hours at a temperature of 15 ° C to 35 ° C and atmospheric pressure under a reaction catalyst and an organic solvent.
The reaction of claim 1, wherein the reaction for removing the amine protecting group in the second step is carried out with the compound represented by the formula (3) and the nitrogen-based compound in an organic solvent,
The nitrogen-based compound is a method of producing a double-reactive amphiphilic compound characterized in that it comprises at least one selected from piperidine, pyrrolidine, pyridine and derivatives thereof.
According to claim 1, Step 5 of the double-reactive amphiphilic compound characterized in that the thiol-period oxidation reaction by dissolving the compound represented by the formula (7) in an organic solvent, stirred for 50 to 100 hours under atmospheric pressure Manufacturing method.
A double reactive amphiphilic compound comprising a compound represented by Formula 8;
[Formula 8]

In the formula (8), n is a natural number of 10 to 500, m is a natural number of 2 to 100.
The double-reactive amphiphilic compound according to claim 7, wherein the compound represented by Chemical Formula 8 is thiol (-SH) and an active oxygen reaction type.
As a drug carrier of the micelle type,
A dual reactive amphiphilic compound of claim 7 or 8 and a hydrophobic drug,
The dual-reactive amphiphilic compound forms a micelle, wherein the hydrophobic drug is supported inside the micelle.
The drug delivery carrier according to claim 9, wherein the drug is an anticancer agent or an anti-inflammatory agent.

Images