KR102097492B1 - Ring opening metathesis polymers with cis-alpha-beta unsaturated anhydride structures for pH-responsive grafting and degradation and use thereof - Google Patents

Abstract

The present invention relates to a novel ring-opening metathesis polymer, and more specifically, a pH-reactive ring-opening metathesis polymer prepared by ring-opening metathesis polymerization of an acid anhydride having a cis-α, β-double bond in the presence of a group's catalyst, and a drug delivery system thereof Provides such uses.

Description

Ring opening metathesis polymers with cis-alpha-beta unsaturated anhydride structures for pH-responsive grafting and degradation and use thereof}

The present invention relates to a ring-opening metathesis polymerization polymer, and more specifically, a ring-opening metathesis polymerization polymer and a drug delivery system, a tissue engineering body having a cis-alpha-beta double bond anhydride structure capable of pH responsive grafting and decomposition control. It relates to its use in implants and cosmetics in vivo.

In order to selectively release drugs to be delivered from organs of the gastrointestinal tract, skin, cancer or inflammatory tissues or intracellular organelles such as endosomes and lysosomes that exhibit a unique pH microenvironment, it is pH reactive in various drug delivery systems. Selective degradation of polymers is used (Felber et al ., Adv. Drug Deliv. Rev. 6 4: 979-992, 2012); Gao et al ., Mol. Pharm. 7: 1913-1920, 2010). Various pH-reactive degradable chemical bonds, including imine, hydrazone, acetal, and orthoester, are introduced into the polymer backbone or side chains to obtain pH reactivity suitable for each application (Tao et al ., Polym. Chem ., 9: 878-884, 2018; Wu et al ., RSC Adv ., 8: 5640-5646, 2018; Cao et al ., Polym. Chem ., 9: 169-177, 2018; Li et al ., Angew. Chem. Int. Edit. , 52: 13699-13702, 2013). In some studies, the pH-reactive degradation properties of acetal or imine bonds could be intentionally controlled by changing adjacent chemical structures (Tao et al ., Polym. Chem-UK , 9: 878-884, 2018; Mao et al ., ACS Appl. Mater.Inter., 8: 17109-17117, 2016; Cao et al ., Polym. Chem-UK , 9: 169-177, 2018; Sonawane et al ., Eur. J. Pharm. Sci . , 99: 45-65, 2017). However, when a drug is introduced through a degradable chemical bond to a polymer, it is most likely that the bond is decomposed under many reaction conditions, and thus, the drug is most often introduced through a complex reaction route including protection and deprotection. (Polyak et al ., Polym. Adv. Technol ., 24: 777-790, 2013). In addition, since many degradable bonds are often hydrolyzed in aqueous conditions, introduction of drugs in aqueous solutions is very difficult (Liu et al ., J. Mater. Chem ., 21: 6677-6682, 2011 ).

Among the pH-reactive bonds, β-carboxylic acid amides with cis-α, β-double bonds have unique degradation properties. Unlike other amide bonds that are easily hydrolyzed only at extreme pH conditions lower than 1 or higher than 13, β-carboxylic acid amides with cis-α, β-double bonds, i.e. maleic acid amide derivatives, are slightly acidic at pH around 3-7 Can be hydrolyzed. In addition, since the intramolecular attack of β-carboxylic acid on the amide carbonyl group is a major factor in promoting the hydrolysis of amide bonds under weakly acidic conditions, the reaction rate and reaction pH conditions of the decomposition regulate the substituents on cis-α, β-double bonds. This makes it easy to adjust. In addition, maleic acid amide derivatives can be readily synthesized through a single step reaction between the corresponding anhydride and amine, even in aqueous solution conditions. Due to these properties, maleic acid amide derivatives have been used as one of the key functions of smart biomaterials that degrade in response to slight changes in pH.

However, despite their attractive properties, the strategy for introducing maleic acid amide derivative functionality into polymers is quite limited. In all the previous research results, only a method of introducing a maleic acid amide derivative and the corresponding anhydride into the polymer skeleton after first forming the polymer skeleton was used (Rozema et al ., Proc. Natl. Acad. Sci. USA , 104: 12982-12987, 2007; Coulembier et al ., Prog. Polym. Sci ., 31: 723-747, 2006; Han et al ., Makromol. Chem., Macromol. Symp. Ser ., 33: 301 -309, 1990). Monomers containing maleic acid amide / anhydride moieties are rarely directly polymerized due to the reactivity of the double bond or anhydride functional groups of the monomers under traditional radical, cationic, anionic or other staged growth polymerization conditions.

Accordingly, there is an urgent need to develop a method for efficiently synthesizing a polymer having a residue capable of being rapidly degraded even in weakly acidic conditions reachable in vivo, such as maleic acid amide / anhydride derivatives.

The present invention is to solve a number of problems, including the above problems, the purpose of providing a novel ring-opening metathesis polymer capable of dissolving the drug or various bioactive substances or the skeleton is decomposed by pH-dependent decomposition. do. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, a ring-opening metathesis polymer having a structure of any one of the following Chemical Formulas 1 and 2 or a mixture of the two is provided:

(화학식 1)

(Formula 1)

(화학식 2)

(Formula 2)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 아릴에스터, 알킬에스터, 알킬아릴에스터, 알킬아민, 아릴아민, 알킬아미드 또는 아릴아미드이다)(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, aryl groups having 6 to 10 carbons, Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthioether having 7 to 10 carbon atoms, aryl containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur Ester, alkyl ester, alkyl aryl ester, alkylamine, arylamine, alkylamide or arylamide)

According to another aspect of the present invention, there is provided a compound-linked polymer conjugate having a structure of any one of the following Chemical Formulas 3 and 4 or a structure in which two are mixed:

(화학식 3)

(Formula 3)

(화학식 4)

(Formula 4)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 각각 독립적으로 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 탄소수 1 내지 10의 아릴에스터, 탄소수 1 내지 10의 알킬에스터, 탄소수 7 내지 11의 알킬아릴에스터, 탄소수 1 내지 10의 알킬아민, 탄소수 6 내지 10의 아릴아민, 탄소수 1 내지 10의 알킬아미드, 탄소수 6 내지 10의 아릴아미드 또는 구아니딘이며, 상기 R은 1차 또는 2차 아민기를 갖는 화합물이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are each independently alkyl having 1 to 10 carbons, alkyl ester, or 6 to 10 carbons. Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthio having 7 to 10 carbon atoms, containing a hetero atom selected from the group consisting of an aryl group, oxygen, nitrogen and sulfur Ether, an aryl ester having 1 to 10 carbons, an alkyl ester having 1 to 10 carbons, an alkyl aryl ester having 7 to 11 carbons, an alkylamine having 1 to 10 carbons, an arylamine having 6 to 10 carbons, an alkyl having 1 to 10 carbons Imide, and aryl amides, or guanidine group having 6 to 10 carbon atoms, wherein R is a compound having a primary or secondary amine).

According to another aspect of the present invention, the method for producing the ring-opening metathesis polymer comprising the step of ring-opening metathesis polymerization of an acid anhydride having a cis-α, β-double bond having the structure of Formula 5 in the presence of a group's catalyst This is provided:

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다). (In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

According to another aspect of the present invention, an acid anhydride having a cis-α, β-double bond having the structure of Chemical Formula 6 (7-oxabicyclo [2.2.1] hepta-2,5-diene-2,3 -Ring-opening metathesis polymerization of dicarboxylic acid anhydride) in the presence of a group's catalyst; And adding ferrous chloride to the cyclodegraded polymer to cut the cycloether bonds of the polymer to provide a method for preparing the ring-opening metathesis polymer:

(화학식 6).

(Formula 6).

According to another aspect of the present invention, a ring-opening metathesis polymer comprising a structure of any one of the following Chemical Formulas 1 and 2 or a structure in which both are mixed with a compound having a primary or secondary amine group in an aqueous solution under basic conditions A method of preparing a compound-grafted ring-opening metathesis polymer comprising reacting is provided:

(화학식 1)

(Formula 1)

(화학식 2)

(Formula 2)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 아릴에스터, 알킬에스터, 알킬아릴에스터, 알킬아민, 아릴아민, 알킬아미드 또는 아릴아미드이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, aryl groups having 6 to 10 carbons, Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthioether having 7 to 10 carbon atoms, aryl containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur Esters, alkyl esters, alkyl aryl esters, alkylamines, arylamines, alkylamides or arylamides).

According to another aspect of the present invention, there is provided a copolymer in which the ring-opening metathesis polymer and the second polymer are arranged in blocks, randomly or alternately.

According to another aspect of the present invention, there is provided a compound-grafted copolymer in which a compound having a primary or secondary amine in the acid anhydride group of the copolymer is connected by an amide bond.

According to another aspect of the invention, the step of adding a group catalyst to the end of the second polymer; Ring-opening metathesis polymerization of the second polymer to which the group's catalyst is added together with a maleic anhydride derivative having a cis-α, β-double bond having the structure of Formula 5; And removing the group's catalyst is provided:

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

According to another aspect of the invention, the step of polymerizing the second polymer; Ring opening metathesis polymerization of cis-α, β-maleic anhydride derivatives having a double bond having the structure of Chemical Formula 5; And linking the ring-opening metathesis polymer produced by the ring-opening metathesis polymerization with the second polymer.

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다.(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom composed of O, N, and S as a ring-member element.

According to one embodiment of the present invention made as described above, it is possible to implement a ring-opening metathesis polymer capable of releasing drugs, bioactive substances, crosslinking agents, etc. connected to the side chain by pH-dependent decomposition of the side chain. The ring-opening metathesis polymer can be used in the manufacture of drug carriers, tissue scaffolds, etc., which have reduced side effects for diseases with different pH conditions in the microenvironment, such as cancer, skin, intracellular environment, and inflammation.

However, these effects are exemplary, and the scope of the present invention is not limited thereby.

1 is a schematic diagram showing the synthesis, drug grafting, and pH-responsive drug release of a ring-opening metathesis polymer having an α, β-unsaturated acid anhydride group.
FIG. 2 is a reaction diagram schematically showing a polymer having various ring structures and α, β-unsaturated acid anhydride groups according to an embodiment of the present invention, and a drug grafting of the polymer and a polymer prepared by ring opening metathesis thereof. The synthetic process (a), the formation of the polymer by the ring-opening metathesis reaction (b), the allyl ester decomposition process of the polymer (c), and the drug grafting process of the ring-opening metathesis polymer (d) are shown.
Figure 3 shows the passage of time under various pH conditions (3.0, 5.5, 6.5 and 7.4) of n-propylamine-grafted polymers 6 (a), 7 (b) and 8 (c) according to one embodiment of the invention A graph showing the result of measuring the degree of amine release according to and a reaction formula (d) showing the process of grafting by acid amide bond between the acid anhydride group and n-propylamine of the polymer according to an embodiment of the present invention are shown.
FIG. 4 shows a variety of pH 5.8 (a) of alendronate-grafted polymers 9 (1 point dashed line ■), 10 (short dashed line ■), 11 (long dashed line ▲) and free alendronate (solid line ▼) according to an embodiment of the present invention. , pH 6.5 (b) and pH 7.4 (c) are a series of graphs showing the results of measuring cell viability after treatment with MDA-MB-231 cells by concentration.
Figure 5 is a reaction (top) and the ring-opening of the monomers having an oxoborodidine structure used in the preparation of the polymer according to an embodiment of the present invention to form the ring-opened monomers 12 and 13 by treating excess [Ru] It is a reaction diagram schematically showing the reaction (bottom) of forming the monomer 14 by cutting the 5-membered cyclic ether bond by adding ferric chloride (FeCl ₃ ) to the monomer 13.
FIG. 6 is a series of ¹ H-NMR spectra for polymer 3 of polymerization degrees 16 (a), 24 (b) and 42 (c), prepared according to one embodiment of the present invention.
7 is a ¹³ C-NMR spectrum for polymer 3 (DP = 42) prepared according to an embodiment of the present invention.
8 is a ¹ H-NMR spectrum (a) and ¹³ C-NMR spectrum (b) for monomer 12 prepared according to an embodiment of the present invention.
9 is a chromatogram showing the results of analyzing the polymer 3 (DP = 16, 24, and 42) prepared according to an embodiment of the present invention by gel permeation chromatography.
10 is a ¹ H-NMR spectrum (a) and ¹³ C-NMR spectrum for monomer 1 prepared according to an embodiment of the present invention.
FIG. 11 is a series of ¹ H-NMR spectra for polymers 4 of polymerization degrees 16 (a), 24 (b) and 42 (c), prepared according to one embodiment of the present invention.
12 is a series of ¹³ C-NMR spectra for Polymer 4 of Polymerization Degree 42 prepared according to one embodiment of the present invention.
13 is a ¹ H-NMR spectrum (a) and ¹³ C-NMR spectrum (b) for the monomer 13 prepared according to an embodiment of the present invention.
14 is a chromatogram showing the results of polymer 4 (DP = 16, 26, and 42) prepared according to an embodiment of the present invention analyzed by gel permeation chromatography.
15 is a ¹ H-NMR spectrum (a) and ¹³ C-NMR spectrum (b) for the monomer 14 prepared according to an embodiment of the present invention.
FIG. 16 is a series of ¹ H-NMR spectra for polymer 5 of polymerization degrees 16 (a), 26 (b) and 42 (c) prepared according to one embodiment of the present invention.
FIG. 17 is a series of ¹³ C-NMR spectra for polymer 5 of polymerization degree 42 prepared according to one embodiment of the present invention.
18 is a chromatogram showing the results of polymer 5 (DP = 16, 26, and 42) prepared according to an embodiment of the present invention analyzed by gel permeation chromatography.
19 is a ¹ H-NMR spectrum for each of the n-propylamine-grafted polymers 6 (a), 7 (b) and 8 (c) of polymerization degree 42 prepared according to an embodiment of the present invention.
20 is a chromatogram showing the results of polymer 6, 26 and 48 polymer 6 (a), 7 (b) and 8 (c) prepared according to an embodiment of the present invention analyzed by gel permeation chromatography.
Figure 21 shows the results of analyzing the n-propylamine-grafted monomers 12 (a), 13 (b) and 14 (c) prepared according to one embodiment of the present invention by density function theory (DFT) calculation.
FIG. 22 is a pH-dependent decomposition n-propylamine-grafted polymer showing that pH-dependent decomposition occurred and regenerated into polymers 3 (a), 4 (b) and 5 (b) according to an embodiment of the present invention. It is a series of ¹ H-NMR spectra.
23 shows each ¹ H for alendronate-grafted polymers 9 (a, DP = 42), 10 (b, DP = 48), and 11 (c, DP = 48) prepared according to one embodiment of the present invention. -NMR spectrum.
24 is a chromatogram showing the results of analyzing the alendronate-grafted polymers 9 (a), 10 (b) and 11 (c) having various degrees of polymerization prepared according to an embodiment of the present invention by gel permeation chromatography.
Figure 25 shows the passage of time at various pH conditions (3.0, 5.5, 6.5 and 7.4) of alendronate-grafted polymers 9 (a), 10 (b) and 11 (c) prepared according to one embodiment of the present invention. It is a series of graphs showing the results of measuring the degree of drug release.
FIG. 26 shows MDA-MB-231 cells in two pH conditions (5.8 and 7.4) at a concentration of 20 μg / mL of non-grafted polymers (polymers 3, 4 and 5) prepared according to one embodiment of the present invention. It is a graph showing the results of measuring the relative cell viability over time after treatment in.
27 is a graph showing the results of measuring relative cell viability over time after treatment with alendronate-grafted polymer (polymer 11) prepared according to an embodiment of the present invention and alendronate at a corresponding concentration at pH 5.8. to be.

Definition of Terms:

The term "ring opening metathesis polymerization" (hereinafter referred to as "ROMP") as used in this document is a type of olefin metathesis polymerization that produces industrially important polymers. The driving force of the reaction is norbornene or cyclo. Reduction of ring modification in cyclic olefins such as pentene For the ROMP reaction, a ruthenium chloride (RuCl ₃ ) -based alkylidene catalyst is used.

Detailed description of the invention:

According to one aspect of the present invention, a ring-opening metathesis polymer having a structure of any one of the following Chemical Formulas 1 and 2 or a mixture of the two is provided:

(화학식 1)

(Formula 1)

(화학식 2)

(Formula 2)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 아릴에스터, 알킬에스터, 알킬아릴에스터, 알킬아민, 아릴아민, 알킬아미드 또는 아릴아미드이다). (In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, aryl groups having 6 to 10 carbons, Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthioether having 7 to 10 carbon atoms, aryl containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur Esters, alkyl esters, alkyl aryl esters, alkylamines, arylamines, alkylamides or arylamides).

In the ring-opening metathesis polymer, the linker is a heteroalkyl having 1 to 10 carbon atoms, a heteroalkyl ether having 1 to 10 carbon atoms, an alkylene linker having 1 to 10 carbon atoms, an alkyl ether having 1 to 10 carbon atoms, 1 to 10 carbon atoms. It may be an alkylene oxide linker, an alkyl thioether having 1 to 10 carbon atoms, an alkyl amine having 1 to 10 carbon atoms, an ester having 1 to 10 carbon atoms, a thioester having 1 to 10 carbon atoms, or an alkyl amide having 1 to 10 carbon atoms.

In the ring-opening metathesis polymer, more preferably, n and m may be each independently an integer of 15 to 50, but are not limited thereto.

In addition, although not shown in Chemical Formulas 1 and 2, the terminal unit of the ring-opening metathesis polymer may be protected by a phenyl group (Ph).

In the above production method, the ring-opening metathesis polymer having the structure of Formula 1 uses a maleic anhydride derivative having a cis-α, β-double bond as a unit, and the ring-opening metathesis reaction of these maleic anhydride derivatives The resulting maleic anhydride derivative can be prepared by the reaction described in Scheme 1 below:

(Scheme 1).

In more detail, the reaction formula is reacted with but-2-ynedioic acid and cyclodiene such as cyclopentadiene, cyclohexadiene, and cycloheptadiene to form a bicyclodiene compound such as bicycloheptadien-2,3-dicarboxylic acid, and reacted with acetic anhydride to make final reaction. As a result, a maleic anhydride derivative having a cis-α, β-double bond having the structure of Formula 5 is synthesized.

According to another aspect of the present invention, there is provided a compound-linked polymer conjugate containing the polymer as an active ingredient.

According to one aspect of the present invention, there is provided a compound-linked polymer conjugate having a structure of any one of the following Chemical Formulas 3 and 4 or a structure in which two are mixed:

(화학식 3)

(Formula 3)

(화학식 4)

(Formula 4)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 각각 독립적으로 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 탄소수 1 내지 10의 아릴에스터, 탄소수 1 내지 10의 알킬에스터, 탄소수 7 내지 11의 알킬아릴에스터, 탄소수 1 내지 10의 알킬아민, 탄소수 6 내지 10의 아릴아민, 탄소수 1 내지 10의 알킬아미드, 탄소수 6 내지 10의 아릴아미드 또는 구아니딘이며, 상기 R은 1차 또는 2차 아민기를 갖는 화합물이다). (In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are each independently alkyl having 1 to 10 carbons, alkyl ester, or 6 to 10 carbons. Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthio having 7 to 10 carbon atoms, containing a hetero atom selected from the group consisting of an aryl group, oxygen, nitrogen and sulfur Ether, an aryl ester having 1 to 10 carbons, an alkyl ester having 1 to 10 carbons, an alkyl aryl ester having 7 to 11 carbons, an alkylamine having 1 to 10 carbons, an arylamine having 6 to 10 carbons, an alkyl having 1 to 10 carbons Imide, and aryl amides, or guanidine group having 6 to 10 carbon atoms, wherein R is a compound having a primary or secondary amine).

In the compound-linked polymer conjugate, the linker is a heteroalkyl of 1 to 10 carbon atoms, a heteroalkyl ether of 1 to 10 carbon atoms, an alkylene linker of 1 to 10 carbon atoms, an alkyl ether of 1 to 10 carbon atoms, 1 to 10 carbon atoms It may be an alkylene oxide linker of, an alkyl thioether having 1 to 10 carbon atoms, an alkyl amine having 1 to 10 carbon atoms, an ester having 1 to 10 carbon atoms, a thioester having 1 to 10 carbon atoms, an alkyl amide having 1 to 10 carbon atoms.

In the compound-linked polymer conjugate, the compound to be conjugated may be a drug, a bioactive substance, a crosslinking agent, or an amination derivative thereof, and the drug may be an anti-cancer agent, an antibiotic, or a therapeutic agent for osteoporosis. In addition, the bioactive material may be an enzyme, hormone, or peptide containing a primary or secondary amine group. The crosslinking agent may be a material having a primary or secondary amine group at both ends of the aryl or alkyl chain. In addition, in the case of a compound that does not have a diamine amine group, it can be bonded after introducing an amine group within a range that does not impair the properties of the compound. Accordingly, the scope of the present invention also includes amination derivatives of these substances.

The anticancer agent is doxorubicin, daunorubicin, epirubicin, idarubicin, cyclophosphamide, ifosfamide, allendronate, docetaxel, vinblastine, capecitabine, melphalan, 5-fluorouracil, temozolomide , Dacarbazine, Nitrogen Mustard, Polynin Acid, Dacarbazine, Procarbazine, Bleomycin, Trophycetron, Oxaliplatin, Bicalutamide, Flutamide, Nilutamide, Octreotide, Actinomycin D, My Tomycin C, carmustine, lomustine, methotrexate, thioguanine, hydroxyurea, azathioprine, cytarabine, pentostatin, mercaptopurine, lenalidomide, thalidomide, temozolomide, vinorelbine, amsacrine , Mitoxantrone, cladribine, vincristine, imatinib, azacitidine, bortezomib, gemcitabine, fludarabine, pemetrexed, octreotide, leu It may be a laurel or anti-cancer antibody, and the anti-cancer antibody may be cetuximab, bevacizumab, trastuzumab, rituximab, alemtuzumab, daclizumab, gemtuzumab, iritumomab, or edrepcolomab. have.

The therapeutic agent for osteoporosis may be cyclosporine, Odanakatip, thiazolidinedione, teriparatide, allendronate, denosumab, or calcitonin.

The antibiotics include ceftolozane, ceftaroline fosamil, eravacycline, ceftazidime, imipenem, brilacidin, and amibactam ), Plazomicin, penicillin, rifamycins, ciprofloxacin, trovafloxacin, garenoxacin, gatifloxacin, Gemifloxacin, hydrochlorothiazide, furosemide, sulfafurazole, sulfamethoxazole, sulfadimethoxine, sulfadimidine , Sulfamoxole, lincomycin, clindamycin, tetracycline, streptomycin, kanamycin, amikacin, tobramycin, D Bekacin ( dibekacin, gentamicin, sisomicin, netilmicin, neomycin, hygromycin, sephamycin, bacillomycin, bacinomycin (echinocandin), mycosubtilin, daptomycin, iturin, surfactin, azidamfenicol, doxycycline, eperezolid, Linezolid, radezolid, ranbezolid, sutezolid, thiampenicol, florfenicol, valnemulin, and seslo Cethromycin, solithromomycin, pristinamycin, tigecycline, methicillin, vancomycin, ampicillin, cefdinir, cefdinir, cefdinir, cefdinir, cefdinir, cefdinir, cefdinir, cefdinir, cefdinir Cefcapene, cefepime, ceftolozane, ceftovi Roll (ceftobiprole), oxacephem, cefdaloxime, ceftizoxime, cefmenoxime, cefpiramide, cefpiroxime, ceftodoxime ceftibuten, ceftitoren, cefaclor, cefotetan, cefadroxil, cefapirin, tiarcillin, piperacillin, oxalate Oxacillin, nafcillin, dicloxacillin, flucloxacillin, teicoplanin, ramoplanin, bacitracin, cyclo Serine (cycloserine), benzylpenicillin (bezylpenicillin), ticarcillin, piperacillin, ertapenem, meropenem, doripenem, cefrozil , Azteronam, imipenem, cefadroxil, cefapirin, cefachlor can be clor, clamamba, vaborbactam, aztrenam, amoxicillin, teixobactin, tyrothricin, or polymixin have.

According to another aspect of the present invention, the method for producing the ring-opening metathesis polymer comprising the step of ring-opening metathesis polymerization of an acid anhydride having a cis-α, β-double bond having the structure of Formula 5 in the presence of a group's catalyst This is provided:

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

In the above method, an acid anhydride having a cis-α, β-double bond having the structure of Chemical Formula 5 may be prepared by the Diels-Alder reaction described in Scheme 1 below:

(Scheme 1).

Detailed description of the reaction formula is the same as the description of the ring-opening metathesis polymer according to an embodiment of the present invention.

According to another aspect of the present invention, an acid anhydride having a cis-α, β-double bond having the structure of Chemical Formula 6 (7-oxabicyclo [2.2.1] hepta-2,5-diene-2,3 -Ring-opening metathesis polymerization of dicarboxylic acid anhydride) in the presence of a group's catalyst; And

A method for preparing the ring-opening metathesis polymer is provided, comprising adding a ferric chloride to the ring-opening metathesis polymer to cleave the cycloether bonds of the polymer:

(화학식 6).

(Formula 6).

According to another aspect of the present invention, a ring-opening metathesis polymer having a structure of any one of the following Chemical Formulas 1 and 2 or a mixed structure of two is reacted with a compound having a primary or secondary amine group in an aqueous solution under basic conditions. A method of preparing the compound-grafted ring-opening metathesis polymer comprising the steps of:

(화학식 1)

(Formula 1)

(화학식 2)

(Formula 2)

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이고, n 및 m은 각각 독립적으로 1 내지 300인 정수로서, 화학식 3 및 4가 혼합된 경우에 한하여 n 및 m은 모두 1일 수 있으며, A 및 A'은 탄소수 1 내지 10개의 알킬, 알킬에스터, 탄소수 6 내지 10의 아릴기, 산소, 질소 및 황으로 구성된 군으로부터 선택되는 헤테로 원자가 포함된 탄소수 2 내지 10의 헤테로알킬, 탄소수 4 내지 10의 헤테로아릴, 탄소수 7 내지 12의 아릴티오에스터, 탄소수 7 내지 10의 아릴티오에테르, 아릴에스터, 알킬에스터, 알킬아릴에스터, 알킬아민, 아릴아민, 알킬아미드 또는 아릴아미드이다). (In the above formula, X is absent, O, NH, S, guanidine (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, aryl groups having 6 to 10 carbons, Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthioether having 7 to 10 carbon atoms, aryl containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur Esters, alkyl esters, alkyl aryl esters, alkylamines, arylamines, alkylamides or arylamides).

In addition, although not shown in Chemical Formulas 1 and 2, the terminal unit of the ring-opening metathesis polymer may be protected by a phenyl group (Ph).

In the above method, the compound may be a drug, a bioactive substance, a crosslinking agent or an amination derivative thereof, and the drug may be an anti-cancer agent, an antibiotic or an agent for treating osteoporosis, and the anticancer agent, an antibiotic, and an agent for treating osteoporosis as described above same. Likewise, the bioactive material and crosslinking agent are also as described above.

Serial numbers that follow monomers and polymers unless otherwise noted in this document refer to the compound numbers shown in Figures 2, 3, and 5.

According to another aspect of the present invention, there is provided a copolymer in which the ring-opening metathesis polymer and the second polymer are arranged in blocks, randomly or alternately.

In the block copolymer, the second polymer may be a ring-opening metathesis polymer polymerized by ring-opening metathesis polymerization, and the ring-opening metathesis polymer may be a hydrophilic ring-opening metathesis polymer having a hydrophilic side chain, and the second The polymer may be a polyalkyloxazoline or a hydrophilic polymer, and the hydrophilic polymer may include polyethylene glycol (PEG), polylactic glycolic acid (PLGA), polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polyglycolic acid ( PGA), a bipolar polymer, or a polymer containing these as side chains. The bipolar polymer may be polymethacrylphosphoryl choline (PMPC), or polysulfonetian methacrylate (PSB), or polycarboxybeta methacrylate (PCB).

Examples of the synthesis of the block copolymer are as described in Schemes 2 or 3 below:

(Scheme 2),

(Scheme 3).

In addition, although not shown in Schemes 2 and 3, the terminal unit of the ring-opening metathesis polymer may be protected by a phenyl group (Ph).

The scheme 2 will be described in more detail as follows:

First, a block copolymer is prepared by synthesizing PEG [poly (ethylene glycol)] having a vinyl group through a ring-opening reaction, and then conjugating it with a polymer having a maleic anhydride derivative based on ROMP. The block copolymer thus prepared may be substituted with a maleic acid amide derivative by reacting a maleic anhydride derivative, which is a unit, with various types of amines. In this case, it is possible to manufacture a block copolymer having a biocompatible hydrophilic polymer PEG block and a maleic acid amide derivative having a very sensitive decomposition responsiveness in a desired pH region at the same time.

Next, Scheme 3 will be described in more detail as follows:

This method is a method of initiating ROMP polymerization using a PEG macroinitiator such as rutenium vynylsulfonyl methoxylpolyethylene glycol. That is, a macroinitiator is synthesized using a Grubbs catalyst in a PEG having a vinyl group at the terminal, and a PEG-b-maleic anhydride derivative block copolymer is synthesized through a ring-opening metathesis (ROMP) reaction with a maleic anhydride derivative monomer. . The block copolymer thus produced has a biocompatibility by linking a drug having a primary or secondary amine to an acid anhydride group of a maleic anhydride derivative by an amide bond, and is stable under physiological pH conditions but releases the drug under weakly acidic conditions. It is very useful as a drug delivery system with excellent pH responsiveness.

The block copolymer can be prepared by the reaction described in Scheme 4 below:

(Scheme 4).

On the other hand, although not shown in Scheme 4, the terminal unit of the ring-opening metathesis polymer may be protected by a phenyl group (Ph).

Referring to Scheme 4 in more detail as follows:

First, a maleic anhydride polymer block is synthesized using ROMP, and then a residue capable of electron transfer radical polymerization (ATRP) is introduced at the end. From this Br end group, a polymethacrylphosphocholine (PMPC) block polymer can be synthesized through ATRP using a methacrylphosphocholine (MPC) monomer. Of course, it is also possible to proceed with the ROMP reaction after first synthesizing the PMPC block, as in the above-described manufacturing process of the maleic anhydride derivative and the block copolymer of PEG.

According to another aspect of the present invention, there is provided a compound-grafted copolymer in which a compound having a primary or secondary amine in the acid anhydride group of the copolymer is connected by an amide bond.

In the compound-grafted copolymer, the compound is as described above.

According to another aspect of the present invention, there is provided a method for preparing a block copolymer comprising: adding a group's catalyst to the end of the second polymer; Ring-opening metathesis polymerization of the second polymer to which the group's catalyst is added together with a maleic anhydride derivative having a cis-α, β-double bond having the structure of Formula 5; And removing the Groups catalyst:

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

According to another aspect of the present invention, there is provided a method for preparing the block copolymer comprising the following steps: polymerizing a second polymer; Ring opening metathesis polymerization of cis-α, β-maleic anhydride derivatives having a double bond having the structure of Chemical Formula 5; And connecting the ring-opening metathesis polymer produced by the ring-opening metathesis polymerization with the second polymer:

(화학식 5)

(Formula 5)

), 티오우레아 링커(

), 카르바모티오에이트 링커(

또는

), 우레아 링커(

), 카르바메이트 링커(

또는

), 카르보네이트 링커(

), 티오에이트 링커(

또는

), 카르보노디티오에이트 링커(

), 안하이드라이드 링커(

), 탄소수 1 내지 10의 알킬렌 링커 또는 환원(ring-member) 원소로 O, N, S로 구성되는 헤테로 원자를 적어도 하나 이상 포함하는 탄소수 1 내지 10의 링커이다).(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

The present inventors tried to polymerize a monomer having a maleic anhydride-mimetic moiety having both cis-double bonds and anhydride groups (see FIG. 1). Ring-opening metathesis polymerization (ROMP) was selected using Grubbs' catalyst as a polymerization method without the above-described reactivity problem. Groups catalysts can polymerize electron-rich olefin monomers but do not react with electron-deficient α, β-olefins or anhydrides. In addition, possible ROMP monomers having the structure of maleic anhydride derivatives can be easily synthesized through a Diels-Alder reaction (see FIG. 2). Expected polymers can include amine-reactive maleic anhydride derivatives in all repeat units and can sufficiently gradient amine-bearing molecules to high density. Highly grafted polymers can release grafted molecules in a pH-dependent manner. The inventors were able to adjust the rate of pH-reactive release of the grafted molecule by adjusting the angle between cis-substituents. The present inventors confirmed that it is possible to synthesize a graftable ROMP polymer having a customized pH-reactive degradability as described above (see Fig. 3), and used as a polymer for pH-reactive delivery of alendronate as a model drug under in vitro conditions. The present invention was completed by demonstrating the possibility (see FIG. 4).

Hereinafter, various preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic diagram showing the synthesis, drug grafting, and pH-reactive drug release of a ring-opening metathesis polymer having an α, β-unsaturated acid anhydride group.

As shown in FIG. 1, a monomer having an α, β-unsaturated acid anhydride group according to an embodiment of the present invention can be polymerized by a ring-opening metathesis reaction, and is used for amide bonding between an acid anhydride group and an amine group of a drug. The drug can be grafted to the side chain. On the other hand, when X of the monomer shown in FIG. 1 is O, an additional ring-opening reaction may be induced during ring-opening metathesis polymerization, and in this case, in the case of the resulting polymer, pH-reactive decomposition activity may be increased.

FIG. 2 is a reaction diagram schematically showing a polymer having various ring structures and α, β-unsaturated acid anhydride groups according to an embodiment of the present invention, and a drug grafting of the polymer and a polymer prepared by ring opening metathesis thereof. The synthetic process (a), the formation of the polymer by the ring-opening metathesis reaction (b), the allyl ester decomposition process of the polymer (c), and the drug grafting process of the ring-opening metathesis polymer (d) are shown.

The most common ROMP monomers are based on ring structures such as norbornene and 4- or 5-membered rings with significant enthalpy of modification. In order to prepare a residue such as maleic anhydride having an α, β-cis-double bond after ring opening metathesis, it is necessary to prepare a cyclic monomer having two double bonds. One of them is for ring-opening metathesis polymerization and the other is for α, β-cis-double bond. The inventors have a nord with 2,3-dicarboxylic acid anhydride (monomer 1) because it can be readily synthesized from the corresponding cyclodiene and alkyne diacid by the Diels-Alder reaction (Figure 2A). The Bordiene based structure was chosen. It is rather difficult to synthesize possible cyclobutadiene or cyclopentadiene based structures due to instability or asymmetry. After ring opening of monomer 1, a ROMP polymer 3 with cyclopentene 1,2-dicarboxylic acid anhydride residues can be produced (FIG. 2B).

The pH-dependent degradability of maleic acid amide derivatives is highly dependent on the accessibility between the acid and amide groups and the substituents on the α, β-cis double bond that determine the effective concentration of each functional group in the intramolecular cyclization reaction. Cyclopentene 1,2-diacid amide has been reported to not readily degrade under mild acidic conditions (Kirby and Lancaster, J. Chem. Soc. Perk. T. 2 , 1206-1214, 1972; Aldersley et al ., J. Chem. Soc. Perk. T. 2 , 1487-1495, 1974; Kirby et al ., J. Chem. Soc. Perk. T. 2 , 1495-1504, 1974). On the other hand, since the adjacent dialkyl group can promote the cyclization reaction between acid and amide without limiting the degree of freedom of the cyclopentene ring structure (Jung and Piizzi, Chem. Rev. , 105: 1735-1766, 2005) ( Thorpe-Ingold effect), ring-free maleic acid amide exhibited rapid degradation under mildly acidic conditions. If a polymer without a five-membered ring can be obtained by ring opening, the resulting acid amide is expected to be much more unstable at slightly acidic pH conditions.

In addition, instead of cyclopentadiene, it is possible to produce monomer 2 having an oxoborodidine structure in the same Dies-Alder reaction process starting from furan (FIG. 2A). The dihydrofuran ring of the resulting ROMP polymer 4 can be decomposed by Lewis acid such as ferric chloride (FIG. 2C) because the cyclic ether is actually allyl ether. Ring-opening polymer 5 has a repeating unit similar to dialkyl maleic anhydride. The inventors have predicted that amine-grafted polymer 8 can release side chains at weaker pH conditions compared to polymers 6 and 7 with limited 5-membered ring structures.

Accordingly, the present inventors synthesized a monomer having a 5-membered ring structure having the cis-α, β-double bond acid anhydride and a polymer using them based on the above-described prediction by the method described in the following examples. Although this bicyclic mono-ene compound is frequently used in ROMP, diene compound monomer 1 was rarely tried as a ROMP monomer. Monomer 1 was polymerized using a second generation Grubbs catalyst at a monomer / initiator ratio of 25 and 50 ([M] / [I]) (FIG. 2B). The ¹ H-NMR spectrum and ¹³ C-NMR spectrum of Polymer 3 are as shown in Figs. 6 and 7, respectively. For the analysis of peak characteristics, the present inventors also synthesized a ring-opened monomer (One-mer) by treating an excess catalyst (C ₄₆ H ₆₅ Cl ₂ N ₂ PRu) (FIG. 5). The present inventors confirmed that polymer 3 was successfully synthesized based on the NMR spectrum of monomer 12 shown in FIG. 5 (FIG. 8). The degree of polymerization (DP) of the two short polymers (DP = 16 and 24) was similar to the target value (DP = 15 and 25), while the degree of polymerization of the long polymer (DP = 42) was the target value (DP = 50). Slightly lower (see Table 1). This may be one reason for the relatively poor solubility of long polymers in the reaction solvent resulting in low degree of polymerization. The E / Z ratio of polymer 3 was about 0.5, which is similar to that of ROMP polymers from monoene compounds shown in prior reports (Keitz et al ., J. Am. Chem. Soc., 134: 2040-2043, 2012. ). Ru-based metathesis catalysts generally exhibit this Z-isomer selectivity in various cross-metathesis reactions, although their mechanisms are not fully understood (Dornan et al ., Angew. Chem. Int. Ed ., 54: 7134-7138 , 2015). As a result of gel permeation chromatography (GPC) analysis, the polymer 3 sample exhibited a unimodal molecular weight distribution (FIG. 9), and the polydispersity (PD) values of the polymer 3 samples having polymerization degrees 16, 24, and 42 were respectively. It was confirmed as 1.34, 1.39 and 1.27 (see Table 1).

In addition, the present inventors reacted furan with acetylene dicarboxylic acid (see FIG. 2A) for the synthesis of oxonorbornene monomer (2). Following the flash vacuum pyrolysis following the Diels-Alder reaction, monomer 2 was produced (see Figure 10). Successful polymerization was confirmed by comparing the NMR spectrum of polymer 4 (see FIGS. 11 and 12) and the NMR spectrum of the corresponding monomers (see FIG. 13). As a result of GPC analysis, Polymer 4, which has a slightly lower PD value than Polymer 3, also showed a monomodal molecular weight distribution (see FIG. 14 and Table 1).

Subsequently, the present inventors tried to precisely control the pH reactivity by cutting the cyclic ether bond of polymer 4. Monomer 13 was treated with FeCl ₃ in acetic anhydride, one of the mildest reagents for cleavage of the cyclic ether bonds (see FIG. 5). As a result, as shown in Figure 15, the ether linkage of polymer 13 was successfully cleaved to yield a ring-opening product 14 with two acetyl esters. Polymer 4 was then reacted with the same reagent (Figure 2c). As a result, 65% of allyl ether bonds were cleaved after 3 hours reaction at room temperature. No further degradation was observed after further reactions (see FIGS. 16 and 17). GPC analysis results supported that the ROMP polymer backbone did not degrade under allyl ether cleavage conditions (see Figure 18 and Table 1).

Polymers 3, 4 and 5 contain α, β-unsaturated anhydride residues that can react with primary or secondary amine molecules in basic conditions. Each polymer was stirred at room temperature in the presence of n-propyl amine as a model compound to prepare a polymer grafted with n-propyl amine (FIG. 2D). The formation of β-carboxylic acid amides could be confirmed by ¹ H-NMR spectra of polymers 6, 7 and 8 showing characteristic amide and acid peaks at 8.6 and 9.8 ppm, respectively (see Figure 19). The anhydride portions of 79% (6), 78% (7) and 88% (8) were reacted to form acid amide bonds by simply mixing under basic conditions. The polymer backbone was maintained during the grafting reaction as shown in the GPC chromatogram (see Figure 20).

The pH-reactive degradation of the β-carboxylic acid amide bond was quantified using the amine marker fluorescamine. The release kinetics of n-propyl amine from polymers 6, 7 and 8 at different pH conditions is shown in FIG. 3. All three polymers showed negligible levels of degradation at pH 7.4, indicating stability of acid amide bonds. On the other hand, the reaction at pH 3.0 clearly promoted the hydrolysis of the bond in all polymers. At pH 3.0, polymers 6 and 7 showed about 60% degradation after 8 hours, while polymer 8 achieved 60% degradation within 4 hours. This difference was more dramatic at pH 5.5 and pH 6.5. Both polymers 6 and 7 degraded gently and hardly reached the point of decomposition of 10-20% even after 72 hours under weakly acidic conditions. However, Polymer 8 showed a decomposition degree of about 40% within 8 hours, which decomposed much faster than Polymers 6 and 7. However, the decomposition rate of the polymer did not change much in the pH range of 5.5-6.5.

As previously predicted by the inventors, 5-membered ring cleavage could clearly promote degradation of cis-α, β-unsaturated acid amide bonds between the grafted side chain and the backbone of the ROMP polymer. The bonding angle of C = CC (ONH) and (HOO) CC = C is about 132-134 ° and 131-134 ° for polymer 5 and 7 5-membered monomer units by density function theory (DFT) calculation, polymer 8 For the ring-opening monomer units of 119 ° and 123 ° were predicted (see FIG. 21). A significant decrease in the two angles between the amide and carboxylic acids can lead to an increase in the effective concentration of two reactive groups for intramolecular cyclization for pH-reactive degradation of acid amide bonds. Decomposition based on the intramolecular cyclization of acid amide bonds was further confirmed by the regenerated structures of polymers 3, 4 and 5 after release of n-propyl amine from polymers 6, 7 and 8 (see Figure 22). Decomposition by cyclization of each monomer unit is probably one reason for the result that the rate of decomposition does not depend largely on the degree of polymerization of the polymer, as in FIG. 24.

To demonstrate the possible use of graftable ROMP polymers with pH-reactive degradability, we analyzed the cytotoxicity of alendronate released from the polymer in a pH dependent manner. Alendronate is a bisphosphonate drug with a primary amine group (Figure 2D). Alendronate is known to have an inhibitory effect on osteoclasts and to show cytotoxicity against cancer cells (Fisher et al ., P roc. Natl. Acad. Sci. USA , 96: 133-138, 1999; Milner et al ., J. Vet.Intern . Med ., 18: 597-604, 2004). Because osteoporosis and cancerous tissues are characterized by an acidic environment compared to other tissues, the pH-responsive release of alendronate from target tissues reduces the possible serious side effects of alendronate in other tissues (e.g. abdominal pain, joint pain, bone necrosis of the jaw). It is believed to help (Kennel and Drake, Mayo. Clin. Proc ., 84: 632-638, 2009). Alendronate graft polymers (9, 10 and 11) were prepared by simply mixing alendronate and ROMP polymers (6, 7, 8) under basic conditions according to the same procedure as n-propyl amine grafting. The degree of grafting of alendronate was calculated to be 81% (9), 83% (10) and 83% (11), respectively (see Fig. 23). The polymer backbone was also maintained during alendronate grafting as indicated in the GPC chromatogram (FIG. 24 and Table 2).

In addition, the present inventors compared the pH-dependent bioactivity of the alendronate-grafted polymer by measuring the cytotoxicity of the alendronate-grafted ring-opening metathesis polymer in MDA-MB-231 cells (human breast cancer cells). After culturing the MDA-MB-231 cells for 24 hours, liberated alendronate exhibited a maximum maximum inhibitory concentration (IC ₅₀ ) of about 15 μM regardless of pH conditions. However, the cytotoxicity of the alendronate-grafted polymer was highly dependent on the culture conditions. At pH 7.4, all three polymers showed little cytotoxicity up to 50 μM in cancer cells (FIG. 4C). However, at pH 6.5, polymer 11 showed a survival rate of 40% at 25 μM, but polymers 9 and 10 still showed little toxicity in the experimental concentration range (FIG. 4B). On the other hand, at pH 5.8, all three polymers showed obvious toxicity. Of these, Polymer 11 showed the highest cytotoxicity similar to free alendronate (FIG. 4A). Comparison of IC ₅₀ values clearly showed the induction of toxicity at acidic pH in cells treated with alendronate-grafted polymers (Table 3). Polymer 11 has a faster decomposition rate at light acidic pH conditions than polymers 9 and 10 (FIG. 25), but non-grafted polymers 3, 4 and 5 show no toxicity to cells in the treatment concentration range (FIG. 26). . In addition, polymer 11 showed a slightly toxic effect on cells than free alendronate at pH 5.8 (FIG. 27). Thus, we could infer that the cytotoxicity of the alendronate-grafted polymer was derived from the alendronate released from the polymer in a pH-dependent manner. Although more research is needed for practical biomedical applications, it has been found that the pH-responsive release and efficacy of amine-containing drugs can be precisely controlled depending on the structure of the cis-α, β-unsaturated acid amide in the ROMP polymer. .

In summary, ROMP polymers with α, β-unsaturated acid anhydrides could be prepared to effectively incorporate amine containing side chain molecules and release the side chains in response to pH. Cis-α, β-unsaturated acid anhydride residues can be grafted with amine-containing molecules to form acid-stable β-carboxylic acid amide bonds at high density. The ring structure of the ROMP polymer can be cleaved to control the pH-reactive degradation of acid amide bonds. Transplanted ROMP polymers with different accessibility between the acid and amide groups showed a clear kinetics of pH reaction degradation, particularly under slightly acidic conditions of pH 5-6. As demonstrated in proof-of-concept experiments using alendronate-grafted ROMP polymers, ROMP polymers with α, β-unsaturated acid anhydrides can be used as useful platforms for smart functional materials in biomedical applications that require biologically resistant pH reactivity. have.

Hereinafter, the present invention will be described in more detail through the accompanying examples and experimental examples. However, the examples and experimental examples of the present invention are provided to more fully describe the present invention to those skilled in the art, and the following examples may be modified in various other forms. The scope of the invention is not limited to the following examples. Rather, these embodiments are provided to make the present disclosure more faithful and complete, and to fully convey the spirit of the present invention to those skilled in the art. In addition, the thickness or size of each layer in the drawings is exaggerated for convenience and clarity of explanation.

Example 1: Preparation of cis-α, β-unsaturated acid anhydride monomer

1-1: Preparation of ingredients

Dicyclopentadiene, furan, propyl amine, ferric chloride, acetic anhydride, triethylamine (TEA), second generation Grubbs catalyst (C ₄₆ H ₆₅ Cl ₂ N ₂ PRu), methanol, chloroform, tetrahydrofuran anhydride (THF), Fluorescamin and Sodium alendronate were purchased from Sigma-Aldrich (USA) and acetylene dicarboxylic acid from Tokyo Chemistry Industrial (Japan). Magnesium sulfate (MgSO ₄ ), sodium hydroxide (NaOH), acetone and acetic acid were purchased from Daejung Gold Co., Ltd. (Korea). Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), and fetal calf serum (FBS) were purchased from WelGENE (USA). Cell counting kits (Kit-8, CCK-8) were purchased from Dojindo Molecular Technologies (USA). The solvent was treated in a glove box under argon gas conditions. All reagents were used without further purification.

Thin film chromatography (TLC) was performed on MERCK TLC silica gel plates. The dialysis membrane (MWCO = 1000) was purchased from Spectrum Laboratories, Inc. (USA). ¹ H-NMR was measured by a Bruker (300 MHz) spectrometer. The amount of amine released from the polymer was quantified using a spectrofluorimeter (Jasco, FP-8300, Japan).

1-2: Synthesis of bicyclo [2.1.1] hepta-2,5-diene-2,3-dicarboxylic acid anhydride

1,3-cyclopentadiene was obtained by distillation of dicyclopentadiene at 180 ° C. 1,3-cyclopentadiene (1.50 mL, 17.8 mmol) was reacted with acetylene dicarboxylic acid (1.0 g, 8.8 mmol) in 20 mL of THF with vigorous stirring at room temperature. The solvent was evaporated off to produce an oily residue. The oily residue was dissolved in acetic anhydride (8.3 mL, 88 mmol) and the solution was heated at 60 ° C. for 2 hours. After the volatiles evaporated, the remaining mixture was dissolved in chloroform and washed 5 times with distilled water. The organic layer was dried over MgSO ₄ and concentrated to give the product 1, bicyclo [2.1.1] hepta-2,5-diene-2,3-dicarboxylic acid anhydride as a white powder (see FIG. 2A, yield 87%). ¹ H-NMR, ¹³ C-NMR and LC-MS data of the product 1 are as follows.

¹ H-NMR (CDCl ₃ , 300 MHz) δ (ppm): 6.95 (m, 2H), 4.04 (m, 2H), 2.35 (m, 1H), 2.19 (m, 1H),

¹³ C-NMR (CDCl ₃ , 75 MHz) δ (ppm): 168.74, 142.31, 77.99, 56.51, 162.03,

For LC MS calculated 162.03 the measured value was 162.40.

1-3: Synthesis of 7-oxabicyclo [2.2.1] hepta-2,5-diene-2,3-dicarboxylic acid anhydride

Furan (2.00 mL, 27.2 mmol) was reacted with acetylene dicarboxylic acid (1.0 g, 8.8 mmol) with 20 mL of diethyl ether for 5 days at room temperature under vigorous stirring. The crystallized white solid was filtered and washed with diethyl ether. The solid was dissolved in THF and excess acetic anhydride was added to the solution. After stirring at 60 ° C overnight, volatiles were removed by evaporation. After crystallization in ethyl acetate, the crystals were heated at 150 ° C. for 3 minutes. Product 2, 7-oxabicyclo [2.2.1] hepta-2,5-diene-2,3-dicarboxylic acid anhydride was obtained as a pale yellow oil (Figure 2b, yield 62%). ¹ H-NMR, ¹³ C-NMR and LC-MS data of the product 2 are as follows.

¹ H-NMR (THF-d, 300 MHz) δ (ppm): 6.70 (s, 2H), 5.15 (s, 2H),

¹³ C-NMR (THF-d, 75 MHz) δ (ppm): 168.13, 142.85, 138.20, 87.94,

The measured value was 164.32 for the LC MS calculated 164.01.

Example 2: Preparation of polymer

2-1: Preparation of general polymer

The ring-opening metathesis polymerization reaction (ROMP) was performed using a second-generation Grubbs catalyst. Specifically, the initial composition of the monomer and catalyst for each polymer is summarized in Table 1 below. The monomer was dissolved in dry THF under an argon atmosphere. Then, the catalyst was added to the stirred solution. After confirming the complete consumption of the monomer by TLC, the reaction was stopped by adding an excess of ethylvinyl ether. After evaporating the reaction mixture, the concentrated solution was added to methanol or diethyl ether to precipitate each polymer. The precipitated polymer (3 or 4) was dried under vacuum and then obtained as a gray solid.

The ¹ H-NMR data of the polymers 3 and 4 thus produced are as follows.

¹ H-NMR (THF- d , 300 MHz) δ (ppm)

Polymer 3 (DP = 42): 7.19-7.35 (m, 5H), 6.91 (m, 1H), 6.37-6.43 (m, 28H), 5.30-6.17 (br, 56H), 5.14-5.25 (m, 2H) , 4.99-5.06 (m, 2H), 3.90-4.23 (br, 33H), 2.94-3.58 (br, 51H), 1.86-2.07 (m, 41H), 1.53-1.84 (m, 42H)

Polymer 4 (DP = 48): 7.31-7.41 (m, 5H), 6.95 (m, 1H), 6.39-6.52 (m, 40H), 5.34-6.23 (br, 90H), 4.79-5.24 (m, 62H) , 4.63-4.77 (m, 1H), 4.32-4.58 (m, 2H)

2-2: Preparation of ring-opened polymer

Polymer 4 (30 mg) was reacted with ferric chloride (5 mg) in acetic anhydride (2 mL) at room temperature for 3 hours. The reaction mixture was dialyzed 3 times with MeOH and 2 times with distilled water using a dialysis membrane. After lyophilization, Polymer 5 shown in FIG. 2 was obtained as a white solid. The ¹ H-NMR data of the polymer 5 is as follows.

¹ H-NMR (THF-d, 300 MHz) δ (ppm): 7.17-7.41 (m, 5H), 6.94 (m, 1H), 6.18-6.72 (br, 64H), 5.61-6.14 (br, 72H) 5.25-5.59 (br, 26H), 5.15-5.23 (m, 2H), 4.97-5.12 (m, 2H), 1.88-2.19 (m, 188H)

Example 3: Preparation of grafting-polymer

3-1: Preparation of n-propylamine grafting polymer

Each polymer prepared in Example 2 was dissolved in a THF / methanol solvent and an excess of amine compound n-propylamine was added. After stirring overnight at room temperature under argon atmosphere, the reaction mixture was evaporated. The amine compound-grafted polymer was purified by dialysis twice with methanol, twice with sodium bicarbonate solution and three times with distilled water. After lyophilization, an n-propylamine-grafted polymer was obtained as a white solid. ¹ H-NMR data of grafting-polymers 6 to 8 prepared by the above method are as follows.

¹ H-NMR (THF-d, 300 MHz) δ (ppm)

Polymer 6: 8.52-8.59 (m, 20H), 7.24-7.38 (m, 5H), 6.97 (m, 1H), 6.23-6.77 (m, 28H), 5.21-6.19 (br, 56H), 5.16-5.23 ( m, 2H), 4.93-5.17 (m, 2H), 3.91-4.25 (br, 33H), 3.36-3.57 (br, 57H), 3.14-3.38 (br, 66H), 1.81-2.19 1.60-1.82 (m, 42H), 1.33-1.42 (m, 66H), 0.58-0.76 (m, 99H)

Polymer 7: 8.55-8.59 (m, 20H), 7.21-7.58 (m, 5H), 6.98 (m, 1H), 6.38-6.80 (m, 40H), 5.39-6.21 (br, 90H), 4.80-5.25 ( m, 62H), 4.69-4.80 (m, 1H), 4.42-4.65 (m, 2H), 3.22-3.49 (m, 76H), 1.19-1.42 (m, 74H), 0.00-0.43 (m, 112H)

Polymer 8: 7.19-7.49 (m, 5H), 6.92 (m, 1H), 6.15-6.75 (br, 65H), 5.51-6.17 (br, 72H), 5.22-5.52 (br, 26H), 5.13-5.203 ( m, 2H), 4.91-5.09 (m, 2H), 3.18-3.52 (m, 86H), 1.83-2.21 (m, 187H), 1.16-1.49 (m, 85H), 0.00-0.39 (m, 127H)

3-2: Preparation of alendronate-grafted polymer

Alendronate-grafted polymers (Polymers 9, 11 and 12) were prepared by reacting each polymer prepared in Example 2 with alendronate as described in Example 3-1 above.

As a result of performing NMR analysis on the polymer, as shown in FIG. 23, it was confirmed that the synthesis was normally performed.

Experimental Example 3: Molecular weight analysis by gel permeation chromatography

The polymer synthesized in Example 2-1 was analyzed for molecular weight and degree of polymerization using gel permeation chromatography (GPC) using a Shodex GPC column (KF-803, Japan). Specifically, THF was used as the mobile phase at a flow rate of 0.7 mL / min. Each sample was detected by a refraction detector (YL9170, Young Lin Instrument Co., Korea), and the molecular weight of the polymer was calculated using a polystyrene (PS) standard specimen (Agilent Technologies, USA).

As a result, as shown in Table 1, the degree of polymerization (DP) of the two short polymers (DP = 16 and 24) was similar to the target values (DP = 15 and 25), but the polymerization degree of the long polymer (DP = 42). ) Was slightly lower than the target value (DP = 50). It is believed that this is due to the relatively poor solubility of the long polymer in the reaction solvent. In addition, as a result of the gel permeation chromatography analysis, it was confirmed that the polymer 3 sample exhibited a unimodal molecular weight distribution and a very uniform distribution (FIG. 9). The polydispersity (PD) value, which is the ratio of the mass average molecular weight (Mw) and the number average molecular weight (Mn), was found to be 1.34, 1.39, and 1.27, respectively, in the polymer samples of the polymerization degrees 16, 24, and 42 (Tables). One). Likewise, Polymer 4, which has a slightly lower PD value than Polymer 3, also showed a unimodal molecular weight distribution (FIG. 14). In addition, this monomodal molecular weight distribution was confirmed by the properties of all polymers synthesized in the present invention, as can be seen in FIGS. 18, 20 and 24.

Molecular weight distribution of the ring-opening metathesis polymer with an acid anhydride having an α, β-double bond according to an embodiment of the present invention polymer goal Synthesis result M _n DP _n M _n (NMR / GPC) ^[a] M _w (GPC) ^[a] PD DP _n ^[b] 3 2.43 × 10 ³ 15 2.59 × 10 ³ /2.44×10 ³ 3.27 × 10 ³ 1.34 16 4.05 × 10 ³ 25 3.89 × 10 ³ /3.71×10 ³ 5.16 × 10 ³ 1.39 24 8.10 × 10 ³ 50 6.81 × 10 ³ /6.55×10 ³ 8.32 × 10 ³ 1.27 42 4 2.46 × 10 ³ 15 2.62 × 10 ³ /2.54×10 ³ 3.45 × 10 ³ 1.36 16 4.10 × 10 ³ 25 4.27 × 10 ³ /4.23×10 ³ 5.80 × 10 ³ 1.37 26 8.20 × 10 ³ 50 7.88 × 10 ³ /7.78×10 ³ 9.49 × 10 ³ 1.22 48 5 15 4.05 × 10 ³ /2.63×10 ³ 3.58 × 10 ³ 1.36 16 25 6.64 × 10 ³ /4.35×10 ³ 5.92 × 10 ³ 1.36 26 50 1.22 × 10 ⁴ /7.91×10 ³ 9.73 × 10 ³ 1.23 48

a: M _n and M _w (g / mol) in GPC were calculated using a polystyrene standard specimen.

b: DP was calculated from NMR data, and DPs of Examples 3, 4 and 5 were terminal phenyl peaks with -CH ₂ -of the cyclopentene ring (3) or dihydrofuran ring and-of the olefins (4 and 5). It was calculated from the comparison to the CH-peak.

Experimental Example 2: DFT (Density Function Theory) calculation

Unlimited B3LYP / 6-31G (d, p) level density function theory (DFT) calculations were performed using the quantum chemistry package Gaussian 09W. The geometry of the monomer units was fully optimized in the gas phase, and empirical dispersion correction was used in the optimization process.

As a result, as shown in FIG. 21, the bonding angle of C = CC (ONH) and (HOO) CC = C is about 132-134 ° and 131-134 ° for the 5-membered monomer units of polymers 6 and 7, For the ring-opening monomer units of Polymer 8, it was predicted to be 119 ° and 123 °. A significant decrease in the two angles between the amide and carboxylic acids can lead to an increase in the effective concentration of two reactive groups for intramolecular cyclization for pH-reactive degradation of acid amide bonds.

Experimental Example 3: Measurement of pH-dependent decomposition

The inventors quantified the pH-dependent degradation of each polymer by measuring the fluorescence of fluorescamine.

Specifically, each polymer was dissolved in phosphate buffer (100 mM, pH 3.0, 5.5, 6.5 and 7.4) at a concentration corresponding to 25 μM, the concentration of the conjugated amine. The lysate was placed in a dialysis bag and immersed in a beaker containing buffer (150 mL, pH 3.0, 5.5, 6.5 and 7.4). The dialysis was performed at room temperature. Buffers outside the dialysis membrane (20 μL) were collected for fluorescence measurements at different time points. An aliquot was mixed with a fluorescamine solution in 20 μL acetone (200 μM). After 5 minutes, the fluorescence of the mixture was measured with a spectrofluorimeter at an excitation wavelength of 390 nm and an emission wavelength of 475 nm.

As a result, as shown in FIG. 3, all three polymers showed almost negligible levels of degradation at pH 7.4, demonstrating the stability of the acid amide bond. On the other hand, the reaction at pH 3.0 clearly promoted the hydrolysis of the bond in all polymers. At pH 3.0, polymers 6 and 7 showed about 60% degradation after 8 hours, while polymer 8 achieved 60% degradation within 4 hours. This difference was more dramatic at pH 5.5 and pH 6.5. Both polymers 6 and 7 degraded gently and hardly reached the point of decomposition of 10-20% even after 72 hours under weakly acidic conditions. However, Polymer 8 showed a decomposition degree of about 40% within 8 hours, which decomposed much faster than Polymers 6 and 7. However, the decomposition rate of the polymer did not change much in the pH range of 5.5-6.5. This is exactly the same phenomenon as the inventors expected that cleavage of the zygotic molecule under weakly acidic conditions would be accelerated if the cyclic ether bond contained in the monomer was cleaved.

Experimental Example 4: In vitro bioactivity analysis of alendronate-grafted polymer

Alendronate was grafted to each polymer through a general grafting process as described in Example 3-2. The bioactivity of the alendronate-grafted polymer was evaluated by measuring the relative viability of the human breast cancer cell line MDA-MB-231 cells. Cells were plated in 96-well plates with 1 × 10 ⁴ cells per well in 90 μL of DMEM culture medium containing 10% FBS and incubated at 37 ° C. for 24 hours. The culture medium was removed and cells were washed using DPBS. The culture medium was replaced with 100 μl of DMEM (10% FBS) containing each polymer sample, and HCl was added to adjust the pH. Cells were then incubated at 37 ° C. for 24 hours. The viability at various time points of the cells incubated with the polymer sample for 24 hours was then measured. For viability analysis, cells were washed using DPBS and a 10% CCK solution in DMEM was added to each well. After reacting at 37 ° C. for 1 hour, the medium was transferred to a new plate, and absorbance at a wavelength of 450 nm was measured with a microplate reader (Molecular Devices Co., Menlo Park, CA). Relative cell viability (%) was calculated as a percentage of absorbance compared to each control cell in which only pH was adjusted without a polymer sample.

As a result, after culturing the MDA-MB-231 cells for 24 hours, liberated alendronate exhibited a maximum maximum inhibitory concentration (IC ₅₀ ) of about 15 μM regardless of pH conditions. However, as shown in Fig. 4, the cytotoxicity of the alendronate-grafted polymer showed a great dependence on the culture conditions. At pH 7.4, all three polymers showed little cytotoxicity up to 50 μM in cancer cells (FIG. 4C). However, at pH 6.5, polymer 11 showed a survival rate of 40% at 25 μM, but polymers 9 and 10 still showed little toxicity in the experimental concentration range (FIG. 4B). On the other hand, at pH 5.8, all three polymers showed obvious toxicity. Of these, Polymer 11 showed the highest cytotoxicity similar to free alendronate (FIG. 4A). Comparison of IC ₅₀ values was clearly confirmed to induce cytotoxicity at acidic pH in cells treated with alendronate-grafted polymers (Table 3). Polymer 11 has a faster decomposition rate at light acidic pH conditions than polymers 9 and 10 (FIG. 25), but non-grafted polymers 3, 4 and 5 show no toxicity to cells in the treatment concentration range (FIG. 26). . In addition, polymer 11 showed a slightly toxic effect on cells than free alendronate at pH 5.8 (FIG. 27). Thus, we could infer that the cytotoxicity of the alendronate-grafted polymer was derived from the alendronate released from the polymer in a pH-dependent manner. Although more research is needed for practical biomedical applications, it has been found that the pH-responsive release and efficacy of amine-containing drugs can be precisely controlled depending on the structure of the cis-α, β-unsaturated acid amide in the ROMP polymer. .

Minimum effect concentration according to pH conditions of the drug-grafted polymer according to an embodiment of the present invention (IC ₅₀ ) polymer pH 5.8 pH 6.5 pH7.4 9 21 - - 10 21 - - 11 17 23 - Alendronate 16 15 15

Unit: μM

As described above, the ring-opening metathesis polymer according to an embodiment of the present invention has a property capable of releasing grafted drugs in a microenvironment such as a tumor by exhibiting a pH-dependent side chain decomposition activity. It can be very useful as a smart biomaterial.

The present invention has been described with reference to the above-described Examples and Experimental Examples, but these are merely exemplary, and various modifications and equivalent other Examples and Experimental Examples are possible from those skilled in the art. Will understand. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

Claims (31)

Ring-opening metathesis polymer having a structure of any one of the following formulas 1 and 2 or a structure in which two are mixed:

(Formula 1)

(Formula 2)
(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when the formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, and aryl groups having 6 to 10 carbons. , Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthio ether having 7 to 10 carbon atoms, containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur, Aryl ester, alkyl ester, alkyl aryl ester, alkylamine, arylamine, alkylamide or arylamide). According to claim 1,
The unit of the ring-opening metathesis polymer of Formula 1 is prepared by the reaction of Scheme 2, the ring-opening metathesis polymer:

(Scheme 1). A pH-dependent compound-grafting polymer comprising the ring-opening metathesis polymer of claim 1. The pH-dependent compound-grafting polymer of claim 3, wherein the compound is a drug, a drug derivative, a bioactive substance, or a crosslinking agent. A compound-linked polymer conjugate having a structure of any one of the following Chemical Formulas 3 and 4 or a structure in which two are mixed:

(Formula 3)

(Formula 4)
(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are each independently alkyl having 1 to 10 carbons, alkyl ester, or 6 to 10 carbons. Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthio having 7 to 10 carbon atoms, containing a hetero atom selected from the group consisting of an aryl group, oxygen, nitrogen and sulfur Ether, an aryl ester having 1 to 10 carbons, an alkyl ester having 1 to 10 carbons, an alkyl aryl ester having 7 to 11 carbons, an alkylamine having 1 to 10 carbons, an arylamine having 6 to 10 carbons, an alkyl having 1 to 10 carbons Imide, and aryl amides, or guanidine group having 6 to 10 carbon atoms, wherein R is a compound having a primary or secondary amine). The method of claim 5,
The compound is a drug, a drug derivative, a bioactive substance, or a crosslinking agent, a compound-linked polymer conjugate. The method of claim 6,
The drug is an anti-cancer agent, an antibiotic or a therapeutic agent for osteoporosis, a compound-linked polymer conjugate. The method of claim 7,
The anticancer agent is doxorubicin, daunorubicin, epirubicin, idarubicin, cyclophosphamide, ifosfamide, allendronate, docetaxel, vinblastine, capecitabine, melphalan, 5-fluorouracil, temozolomide , Dacarbazine, Nitrogen Mustard, Polynin Acid, Dacarbazine, Procarbazine, Bleomycin, Trophycetron, Oxaliplatin, Bicalutamide, Flutamide, Nilutamide, Octreotide, Actinomycin D, My Tomycin C, carmustine, lomustine, methotrexate, thioguanine, hydroxyurea, azathioprine, cytarabine, pentostatin, mercaptopurine, lenalidomide, thalidomide, temozolomide, vinorelbine, amsacrine , Mitoxantrone, cladribine, vincristine, imatinib, azacitidine, bortezomib, gemcitabine, fludarabine, pemetrexed, octreotide, leu Laurel lean or anti-cancer antibody, compound-polymer conjugates connection. The method of claim 8,
The anti-cancer antibody is a cetuximab, bevacizumab, trastuzumab, rituximab, alemtuzumab, daclizumab, gemtuzumab, iritumomab, or edrepcomab, a compound-linked polymer conjugate. The method of claim 7,
The therapeutic agent for osteoporosis is cyclosporine, Odanakatip, thiazolidinedione, teriparatide, allendronate, denosumab, or calcitonin, a compound-linked polymer conjugate. The method of claim 7,
The antibiotics include ceftolozane, ceftaroline fosamil, eravacycline, ceftazidime, imipenem, brilacidin, and amibactam ), Plazomicin, penicillin, rifamycins, ciprofloxacin, trovafloxacin, garenoxacin, gatifloxacin, Gemifloxacin, hydrochlorothiazide, furosemide, sulfafurazole, sulfamethoxazole and sulfadimethoxine, sulfadimidine , Sulfamoxole, lincomycin, clindamycin, tetracycline, streptomycin, kanamycin, amikacin, tobramycin, D Becasin (dibekacin), gentamicin, sisomicin, netilmicin, neomycin, hygromycin, sephamycin, bacillomycin, and bacillomycin Echininocandin, mycosubtilin, daptomycin, iturin, surfactin, azidamfenicol, doxycycline, eperezolid , Linezolid, radezolid, ranbezolid, sutezolid, thiamphenicol, florfenicol, valnemulin, ses Ceromomycin, solithromomycin, pristinamycin, tigecycline, methicillin, vancomycin, ampicillin, cefdinir, Cefcapene, cefepime, ceftolozane, ceftobie Roll (ceftobiprole), oxacephem, cefdaloxime, ceftizoxime, cefmenoxime, cefpiramide, cefpiroxime, ceftodoxime ceftibuten, ceftitoren, cefaclor, cefotetan, cefadroxil, cefapirin, tiarcillin, piperacillin, oxalate Oxacillin, nafcillin, dicloxacillin, flucloxacillin, teicoplanin, ramoplanin, bacitracin, cyclo Serine (cycloserine), benzylpenicillin (bezylpenicillin), ticarcillin, piperacillin, ertapenem, meropenem, doripenem, cefrozil , Azteronam, imipenem, cefadroxil, cefapirin, cefachlor aclor, clavam, vaborbactam, aztrenam, amoxicillin, teixobactin, tyrothricin, or polymixin, Compound-linked polymer conjugate. A method for preparing the ring-opening metathesis polymer of claim 1, comprising the step of ring-opening and metathesizing polymerization of a maleic anhydride derivative having a cis-α, β-double bond having the structure of Formula 5 in the presence of a Groups catalyst:

(Formula 5)
(In the above formula, X is absent, O, NH, S, guanidine (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element). Ring-opening metathesis polymerization of an acid anhydride having a cis-α, β-double bond having the structure of Chemical Formula 6 in the presence of a group's catalyst; And
A method for preparing the ring-opening metathesis polymer comprising adding a ferric chloride to the ring-opening metathesis-polymerized polymer to cut the ether bond in the cycle of the polymer:

(Formula 6). Compound-grafting comprising the step of reacting a ring-opening metathesis polymer comprising a structure of any one of the following formulas 1 and 2 or a mixture of the two with a compound having a primary or secondary amine group in an aqueous solution under basic conditions Manufacturing method of ring-opening metathesis polymer:

(Formula 1)

(Formula 2)
(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms including at least one hetero atom composed of O, N, and S as a ring-member element, and n and m are each independently As an integer of 1 to 300, only when formulas 3 and 4 are mixed, n and m may all be 1, and A and A 'are alkyl having 1 to 10 carbons, alkyl esters, aryl groups having 6 to 10 carbons, Heteroalkyl having 2 to 10 carbon atoms, heteroaryl having 4 to 10 carbon atoms, arylthioester having 7 to 12 carbon atoms, arylthioether having 7 to 10 carbon atoms, aryl containing a hetero atom selected from the group consisting of oxygen, nitrogen and sulfur Esters, alkyl esters, alkyl aryl esters, alkylamines, arylamines, alkylamides or arylamides). The method of claim 14,
The compound is a drug, a bioactive substance, a crosslinking agent or an amination derivative thereof, a production method. The method of claim 15,
The drug is an anti-cancer agent, an antibiotic, or a therapeutic agent for osteoporosis. The method of claim 16,
The anticancer agent is doxorubicin, daunorubicin, epirubicin, idarubicin, cyclophosphamide, ifosfamide, allendronate, docetaxel, vinblastine, capecitabine, melphalan, 5-fluorouracil, temozolomide , Dacarbazine, Nitrogen Mustard, Polynin Acid, Dacarbazine, Procarbazine, Bleomycin, Trophycetron, Oxaliplatin, Bicalutamide, Flutamide, Nilutamide, Octreotide, Actinomycin D, My Tomycin C, carmustine, lomustine, methotrexate, thioguanine, hydroxyurea, azathioprine, cytarabine, pentostatin, mercaptopurine, lenalidomide, thalidomide, temozolomide, vinorelbine, amsacrine , Mitoxantrone, cladribine, vincristine, imatinib, azacitidine, bortezomib, gemcitabine, fludarabine, pemetrexed, octreotide, leu Laurel the lean or anti-cancer antibody, production method. The method of claim 17,
The anti-cancer antibody is cetuximab, bevacizumab, trastuzumab, rituximab, alemtuzumab, daclizumab, gemtuzumab, iritumomab, or edrepcomab. The method of claim 16,
The treatment for osteoporosis is cyclosporine, Odanakatip, thiazolidinedione, teriparatide, allendronate, denosumab, or calcitonin. The method of claim 16,
The antibiotics include ceftolozane, ceftaroline fosamil, eravacycline, ceftazidime, imipenem, brilacidin, and amibactam ), Plazomicin, penicillin, rifamycins, ciprofloxacin, trovafloxacin, garenoxacin, gatifloxacin, Gemifloxacin, hydrochlorothiazide, furosemide, sulfafurazole, sulfamethoxazole and sulfadimethoxine, sulfadimidine , Sulfamoxole, lincomycin, clindamycin, tetracycline, streptomycin, kanamycin, amikacin, tobramycin, D Becasin (dibekacin), gentamicin, sisomicin, netilmicin, neomycin, hygromycin, sephamycin, bacillomycin, and bacillomycin Echininocandin, mycosubtilin, daptomycin, iturin, surfactin, azidamfenicol, doxycycline, eperezolid , Linezolid, radezolid, ranbezolid, sutezolid, thiamphenicol, florfenicol, valnemulin, ses Ceromomycin, solithromomycin, pristinamycin, tigecycline, methicillin, vancomycin, ampicillin, cefdinir, Cefcapene, cefepime, ceftolozane, ceftobie Roll (ceftobiprole), oxacephem, cefdaloxime, ceftizoxime, cefmenoxime, cefpiramide, cefpiroxime, ceftodoxime ceftibuten, ceftitoren, cefaclor, cefotetan, cefadroxil, cefapirin, tiarcillin, piperacillin, oxalate Oxacillin, nafcillin, dicloxacillin, flucloxacillin, teicoplanin, ramoplanin, bacitracin, cyclo Serine (cycloserine), benzylpenicillin (bezylpenicillin), ticarcillin, piperacillin, ertapenem, meropenem, doripenem, cefrozil , Azteronam, imipenem, cefadroxil, cefapirin, cefachlor aclor, clavam, vaborbactam, aztrenam, amoxicillin, teixobactin, tyrothricin, or polymixin, Manufacturing method. A copolymer in which the ring-opening metathesis polymer of claim 1 and the second polymer are arranged in blocks, randomly or alternately. The method of claim 21,
The second polymer is a ring-opening metathesis polymer polymerized by ring-opening metathesis polymerization. The method of claim 21,
The second polymer is a hydrophilic polymer. The method of claim 23,
The hydrophilic polymer is polyalkyloxazoline, polyethylene glycol (PEG), polylactic glycolic acid (PLGA), polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), polyglycolic acid (PGA), or bipolar polymer , Copolymer. The method of claim 24,
The bipolar polymer is a polymethacrylphosphoryl choline (PMPC), or a polysulfonine methacrylate (PSB), a polycarboxybeta methacrylate (PCB), a copolymer. The method of claim 22,
The ring-opening metathesis polymer is a ring-opening metathesis polymer having a hydrophilic side chain, a copolymer. The method of claim 21,
The second polymer is a hydrophilic polymer comprising a bipolar polymer, a copolymer. The method of claim 21,
The second polymer is a hydrophilic polymer comprising polyalkyloxazoline, a copolymer. A compound-grafted copolymer in which a compound having a primary or secondary amine in the acid anhydride group of the copolymer of any one of claims 21 to 28 is linked by an amide bond. Adding a group catalyst to the end of the second polymer;
Ring-opening metathesis polymerization of the second polymer to which the group's catalyst is added together with a maleic anhydride derivative having a cis-α, β-double bond having the structure of Formula 5; And
Method for producing a block copolymer comprising the step of removing the group catalyst:

(Formula 5)
(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element). Polymerizing the second polymer;
Ring opening metathesis polymerization of cis-α, β-maleic anhydride derivatives having a double bond having the structure of Chemical Formula 5; And
The second polymer and the ring-opening metathesis polymerization method comprising the step of connecting to the ring-opening metathesis polymer produced by the step of producing a block copolymer:

(Formula 5)
(In the above formula, X is absent, O, NH, S, guanidine linker (

), Thiourea linker (

), Carbamothioate linker (

or

), Urea linker (

), Carbamate linker (

or

), Carbonate linker (

), Thioate linker (

or

), Carbonodithioate linker (

), Anhydride linker (

), An alkylene linker having 1 to 10 carbon atoms or a linker having 1 to 10 carbon atoms containing at least one hetero atom consisting of O, N, and S as a ring-member element).

Images