Self-assembled diblock copolymers composed of PEGMEMA and drug-loaded polymer segments

Abstract

To provide a polymer-drug conjugate for the delivery of anticancer agents, and their use for the treatment of diseases such as cancer.SOLUTION: The present invention provides a polymer-drug conjugate represented by the formula I. (R1-3 each represent CH3; x and y each represent a natural number of 1-100; n represents a natural number of 1-50; L represents a cleavable linker; D represents an anticancer drug; A represents a terminal group containing cRGDfK; and B represents a fragment of polymerization initiator).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to polymer-drug conjugates according to Formula I, aggregates comprising the polymer-drug conjugates of Formula I, methods for preparing said polymer-drug conjugates and aggregates, and their use for treating diseases such as cancer.

[0002] Most chemotherapeutic agents used in the treatment of cancer are cytotoxic. These agents can accumulate in tissues of the body in addition to the targeted area, which in turn causes a reduction in therapeutic benefit and undesirable distribution of the drug throughout healthy body tissues. Uncontrolled distribution of these agents throughout the body causes serious side effects in patients.

[0003] To address the above-mentioned problems, drug delivery systems have been developed that can deliver drugs to targeted regions of the body.For example, in some approaches to cancer treatment, these systems utilize the enhanced permeability and retention effect (EPR), which implies that drug carriers with high molecular weight and large hydrodynamic volume accumulate in solid tumors, which then leads to the passive targeting of drug molecules to tumor tissue, and minimizes the damage of chemotherapeutic agents to healthy tissue.

[0004] Since the discovery of the enhanced permeability and retention (EPR) effect, macromolecules with long blood circulation have become the primary agents for drug delivery through fenestrated blood vessels and improved accumulation in tumors.

[0005] There has been great interest in polymeric assemblies in drug delivery systems for cancer diseases, mainly due to their body distribution properties. The present invention also relates to assemblies suitable for use in drug delivery, in particular anticancer drugs for cancer treatment.

[0006] There are various kinds of techniques for preparing polymer assemblies known in the current state of the art. For example, self-assembly of polymer compounds is an attractive method for polymer assembly formation. Eisenberg and co-workers demonstrated the first example of asymmetric copolymer self-assembly in solution in the 1990s. Then, amphiphilic block copolymers with polymer blocks of different physical and chemical properties have been attracting attention to generate macromolecular structures by self-assembly.

[0007] Drug loading into such polymer assemblies can be achieved by physical encapsulation through covalent bonds or hydrophobic interactions. Considering that the total volume of these assemblies in colloidal suspensions is very small, high drug loading capacity is crucial to achieve effective drug formulations. The low drug loading capacity of polymer assemblies, mainly in micellar formulations, remains a major drawback in this field.

[0008] Some researchers have addressed this issue by modifying the drug molecules or taking advantage of π-π interactions by using additive compounds rather than providing covalent bonds. However, non-encapsulated drug aggregates in micellar polymer nanoparticle formulations are another drawback for providing safe formulations for their clinical use.

[0009] In another approach, Stenzel and co-workers have utilized drug-binding amphiphilic block copolymers to form hydrophobic segments of block copolymers to form polymer assemblies. This strategy has a lack of complete conjugation of drugs to the polymer backbone due to steric hindrance resulting in poorly defined final structures. Moreover, residual reactive groups on the polymer backbone due to incomplete conjugation may be the most important drawback of this type of post-polymerization conjugation strategy due to the possibility of undesirable biological interactions in vivo.

[0010] In light of the current state of the art summarized above, there is a need for block copolymers and assemblies formed therefrom that provide efficient conjugation of drugs that are well-defined in terms of drug content, are devoid of any remaining reactive sites, and have high drug loading capacities.

[0011] The inventors have found that the amphiphilic block copolymers according to the present invention provide for the formation of micellar type assemblies having high drug loading and well-defined drug binding.

[0012] The present invention relates to polymer-drug conjugates in the form of block copolymers for the delivery of therapeutic agents as shown in Formula I. [ka]

[0013] In other words, the present invention relates to a polymer-drug conjugate of formula I in the form of a block copolymer, and to an aggregate made from the polymer-drug conjugate of formula I, ·R 1 and R 2 are independently H or -CH 3 Selected from ·R 3 is -H or -CH 3 Selected from x is a natural number between 1 and 100 y is a natural number between 1 and 100 n is a natural number between 1 and 50 L is a cleavable linker or L can be null. D is a therapeutic agent selected from the group including combretastatins, 5-fluorouracil, gemcitabine, chloroquine, and doxorubicin. A is a terminal group or A may be null B is a terminal group or B may be null

[0014] The inventors have discovered that in addition to overcoming the shortcomings of the prior art polymer assemblies; the polymer assemblies of the present invention, consisting of polymer-drug conjugates of formula I, also reduce the amount of polymer given to the patient due to high drug loading. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing the synthesis of an NHS-modified polymer-drug conjugate (a polymer-drug conjugate of formula I having a reactive functional group) and a cRGDfK-modified polymer-drug conjugate (a polymer-drug conjugate of formula I having a targeting group). [Diagram 2] FIG. 1 is a schematic diagram showing the preparation of a non-targeted polymeric assembly of the present invention from a polymer-drug conjugate of Formula I. [Diagram 3] FIG. 1 is a schematic diagram showing the preparation of targeted polymer assemblies of the present invention from polymer-drug conjugates of formula I. [Figure 4] Fluorescence emission spectrum of Nile Red in a non-targeted polymer assembly of the present invention. [Diagram 5] Fluorescence emission spectrum of Nile Red in a targeted polymer ensemble of the present invention. [Figure 6] A plot of emission intensity at 612 nm against the logarithm of POEGMEMA-b-PCombMA concentration. [Figure 7] A plot of emission intensity at 612 nm against the logarithm of total POEGMEMA-b-PCombMA and cRGDfK-POEGMEMA-b-PCombMA concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The term "polymer-drug conjugate" refers to a polymeric structure having a therapeutic agent covalently attached to the polymer.

[0017] The term "polymer assemblies" refers to structures with diameters between 1 and 100 nm. Said assemblies consist of self-assembled polymer chains, which may or may not have hollow cavities of various sizes.

[0018] The terms "polymer backbone" and "polymer backbone" may be used interchangeably and refer to a polymer chain having side chains or pendant groups. For example, the side chains may have oligoethylene glycol units and the pendant groups may carry a therapeutic agent or any other group that may be utilized to attach therapeutic and / or diagnostic or targeting groups.

[0019] Throughout this document, the term "polymer-drug conjugate of the invention" shall be taken to mean "polymer-drug conjugate according to Formula I" or "polymer-drug conjugate of Formula I", or "Formula I", and these terms may be used interchangeably.

[0020] Throughout this document, the term "polymer assemblies" should be taken to mean "nanoparticles composed of a polymer-drug conjugate of formula I," or "nanoparticles composed of a copolymer shown in formula I," or "nanoparticles formed as a result of self-assembly of a polymer-drug conjugate of formula I," or "micellar assemblies composed of a copolymer shown in formula I," and these terms can be used interchangeably.

[0021] The term "PEG" means H-(O-CH 2 -CH 2 )n-OR 3 where n is a natural number from 1 to 200, and R 3 is H or -CH 3 PEG is defined as an oligomer or polymer of ethylene oxide. The terms "PEG", "polyethylene glycol", "polyethylene oxide", "PEO", "polyoxyethylene", and "POE" refer to the same structure and may be used interchangeably within this document.

[0022] In an embodiment of the present invention, R 1 , R 2 , and R 3 are independently H or -CH 3 is selected from.

[0023] In one embodiment, R 1 =H, R 2 =H, R 3 =H or R 1 =H, R 2 =H, R 3 =-CH 3 , or R 1 =H, R 2 =-CH 3 , R 3 =H or R 1 =H, R 2 =-CH 3 , R 3 =-CH 3 , or R 1 =-CH 3 , R 2 =H, R 3 =H or R 1 =-CH 3 , R 2 =H, R 3 =-CH 3 , or R 1 =-CH 3 , R2=-CH 3 , R 3 =H or R 1 =-CH 3 , R 2 =-CH 3 , R 3 =-CH 3 It is.

[0024] The term "block copolymer" refers to a copolymer in which all of one type of monomer is grouped together and all of the other type of monomer is grouped together. The polymer-drug conjugate of the present invention is in the form of a block copolymer. The fact that the polymer-drug conjugate of the present invention is in the form of a block copolymer allows the formation of the polymer aggregate of the present invention.

[0025] The therapeutic agent is attached to the polymer via a cleavable linker such that the therapeutic agent can be released under, for example, reducing conditions, oxidative conditions, or by hydrolysis of an ester, amide, or hydrazide.

[0026] The cleavable linker may be any hydrocarbon or substituted hydrocarbon-based compound that can be dissociated under physiological conditions. In a preferred embodiment, the linker is a compound (any C 10 ) containing an acetal or ester functional group that is cleaved under the acidic conditions of the tumor or with the help of overexpressed enzymes present in the intercellular or intracellular matrix of tumor cells. 1 ~C 10 substituted or unsubstituted and / or straight chain and / or cyclic hydrocarbons, etc.

[0027] The linker can be any type of substance that can be attached to both the polymer backbone and the drug, such as poly(ethylene glycol), an amino acid, a poly(amino acid) (e.g., a peptide or oligopeptide), or a polypeptide (e.g., a protein), such that one end of the linker can form a covalent bond with the polymer backbone and the other end can form a covalent bond with the therapeutic agent. The linker may also include a short peptide having a specific peptide sequence that is labile to cathepsin B, such as Gly-Phe-Leu-Gly (SEQ ID NO: 1), also designated GFLG or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu (SEQ ID NO: 2).

[0028] The linker may also be a C aryl group such that it contains a functional group that dissociates under physiological conditions, e.g., acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone. 1 ~C 10 Hydrocarbon or C 1 ~C 10 It may be a substituted or heterosubstituted hydrocarbon.

[0029] In an embodiment of the invention, the linker (L) is GFLG or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu.

[0030] In an embodiment of the invention, the linker is a C 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one acetal functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one ester functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one imine functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one amide functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one carbonate functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one carbamate functional group 1 ~C 10 Hetero-substituted hydrocarbons or C containing at least one hydrazone functional group 1 ~C 10 It is a hetero-substituted hydrocarbon.

[0031] In another embodiment of the invention, the linker is a C 11 containing two or more functional groups selected from the group including acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone. 1 ~C 10 It may include substituted or heterosubstituted hydrocarbons.

[0032] In another embodiment of the invention, the linker can be null, meaning that the therapeutic agent D is directly attached to the polymer chain. Direct attachment of the therapeutic agent to the polymer chain can be via an ester, imine, amide, disulfide, carbonate, carbamate, or hydrazine bond.

[0033] In a preferred embodiment of the invention, the drug molecule is attached to the polymer via a direct ester bond that dissociates under physiological conditions of the tumor. In another embodiment of the invention, the linker is a C linker containing at least one functional group selected from the group including acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone. 1 ~C 10 The substituted hydrocarbon may be in combination with a peptide chain selected from the group including GFLG, Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu. The therapeutic agent selected from combretastatin, 5-fluorouracil, gemcitabine, chloroquine, and doxorubicin may be present in an amount of 5% to 50% by weight of the polymer-drug conjugate, preferably in an amount of 6% to 48% by weight of the drug-polymer conjugate, and most preferably in an amount of 10% to 45% by weight of the drug-polymer conjugate. The therapeutic agent may be present, for example, in an amount ranging from 10% to 48% by weight, or 15% to 46% by weight, or 20% to 45% by weight, or 25% to 44% by weight of the drug-polymer conjugate.

[0034] The term "end group" refers to a functional group or unit at the end of a polymer. The end groups designated as A and B may be the same or different from each other.

[0035] In embodiments, A is an end group that is optionally a fragment of a chain transfer agent or initiator, or a fragment of a chain transfer agent or initiator conjugated to a targeting moiety, or a fragment of a chain transfer agent or initiator bearing a reactive functional group.

[0036] In embodiments, B is an end group that is optionally a chain transfer agent or a fragment of an initiator.

[0037] The term "chain transfer agent" refers to a chemical compound capable of regulating the molecular weight of a polymer by preventing uncontrolled growth of polymer chains. Chain transfer agents are well established in the literature and their structures are well known to those skilled in the art. The terms "chain transfer agent" and its abbreviation "CTA" refer to the same group of compounds and can be used interchangeably throughout this document.

[0038] A and B can optionally be fragments of chain transfer agents. In particular, when a CTA is used in a polymerization reaction, the CTA fragment can remain as an end group to the polymer. The CTA used herein can be any material known in the art suitable for initiating a polymerization reaction. In one embodiment, A and / or B can be 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, 2-cyanobutan-2-yl 4-chloro-3,5-dimethyl-1H-pyrazole-1-carbodithioate, 2-cyanobutanyl-2-yl ... 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, cyanomethyl(3,5-dimethyl-1H-pyrazole)-carbodithioate, cyanomethyldodecyltrithiocarbonate, cyanomethyl[3-(trimethoxysilyl)propyl]trithiocarbonate, 2-cyano-2-propyldodecyltrithiocarbonate, S,S-dibenzyltrithiocarbonate, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate], phthalimidomethylbutyltrithiocarbonate, 1,1,1-Tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzyl 1H-pyrrole-1-carbodithioate, cyanomethyldiphenylcarbamodithioate, cyanomethylmethyl(phenyl)carbamodithioate, cyanomethylmethyl(4-pyridyl)carbamodithioate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4- [N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, benzyl benzodithioate, cyanomethyl benzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl benzodithioate, 2-cyano-2-propyl-4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthioca 2-(4-methoxyphenylcarbonothioylthio)ethanoic acid, 2-nitro-5-(2-propynyloxy)benzyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propyl benzodithioate, cyanomethyl ... The initiator is selected from the group consisting of methyl(4-pyridyl)carbamodithioate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, or any fragment of the initiators listed herein, either by itself or conjugated to a targeting moiety or modified with a reactive group before or after the polymerization reaction.

[0039] The term "fragment" as used herein refers to a compound formed by cleavage of one or more of the covalent bonds that form the initiator molecule.

[0040] The fragmentation of the CTAs listed herein and the structures of the fragments formed are well established in the art, and therefore the structures of A and B can be determined without the use of inventive step by utilizing the state of the art.

[0041] A and / or B are optionally null (null: not present).

[0042] In an embodiment of the invention, A is null and B is a fragment of CTA.

[0043] In another embodiment, B is null and A is a fragment of CTA.

[0044] In another embodiment, A and B are both fragments of a CTA, but they are structurally different from each other, in other words, A and B are different fragments of the same CTA.

[0045] In another embodiment, A and B are both fragments of CTA, and they have the same chemical structure.

[0046] The term "targeting moiety" refers to a molecule that has a tendency to bind to a particular target site in the body. In other words, a targeting moiety is a molecule that specifically binds to a cell that has a complementary receptor.

[0047] The targeting moiety may be selected from the group comprising an antibody; an antibody fragment, or a peptide such as Cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO:3) (cRGDfK). In a preferred embodiment, cRGDfK is used as the targeting moiety.

[0048] The term "reactive group" refers to an atom or related grouping of atoms in a chemical substance that is intended or can reasonably be expected to undergo a facile chemical reaction.

[0049] The reactive group may be an acetal, hemiacetal, carboxylic acid, alcohol, amide, imide, anhydride, aryl halide, azo compound, diazo compound, hydrazine, azide, carbonate, chlorosilane, cyanide, ester, sulfate ester, phosphate ester, thiophosphate ester, isocyanate, isothiocyanate, thiocarbamate ester, dithiocarbamate ester. Preferably, the reactive group is an ester, imide, or carbonate. The reactive group may be, for example, N-hydroxysuccinimide.

[0050] Depending on the polymerization technique used to prepare the polymer conjugates of the present invention, A and / or B may be fragments of an initiator.

[0051] An initiator, as used herein, refers to a chemical compound that reacts with a monomer to form an intermediate compound that can be sequentially linked with multiple other monomers into a polymeric compound. The terms "initiator" and "polymerization initiator" can be used interchangeably within the context of this application.

[0052] Depending on the polymerization technique used to prepare the polymer conjugates of the present invention, different initiators can be used.

[0053] In embodiments, A and / or B are 4,4'-azobis(4-cyanovaleric acid), 4,4'-azobis(4-cyanovaleric acid), 1,1'-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionitrile) (also known as AIBN), ammonium persulfate, hydroxymethanesulfinic acid monosodium salt dihydrate, potassium persulfate, sodium persulfate, tert-butyl hydroperoxide, oxide, tert-butyl peracetate, cumene hydroperoxide, 2,5-di(tert-butylperoxy)-2,5-dimethyl-3-hexyne, dicumyl peroxide, 2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, 1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane, 1,1-bis(tert-amylperoxy)cyclohexane, benzoyl peroxide, 2-butanone peroxide, tert-butyl Peroxide, di-tert-amyl peroxide, lauroyl peroxide, tert-butyl peroxybenzoate, tert-butylperoxy-2-ethylhexyl carbonate, tert-butyl hydroperoxide, 2-azidoethyl-2-bromoisobutyrate, bis[2-(2-bromoisobutyryloxy)undecyl]disulfide, bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide, 2-bromoisobutanoic acid N-hydroxysuccinimide ester ester, 2-bromoisobutyric anhydride, α-bromoisobutyryl bromide, 2-(2-bromoisobutyryloxy)ethyl methacrylate, tert-butyl α-bromoisobutyrate, 3-butynyl-2-bromoisobutyrate, dipentaerythritol hexakis(2-bromoisobutyrate), dodecyl 2-bromoisobutyrate, ethyl α-bromoisobutyrate, ethylene bis(2-bromoisobutyrate), 2-hydroxyethyl 2-bromoisobutyrate, 1-(DL-1,2-isopropylideneglyceryl) 2-bromoisobutyrate, methyl α-bromoisobutyrate, octadecyl 2-bromoisobutyrate, pentaerythritol tetrakis(2-bromoisobutyrate), 1-(phthalimidomethyl) 2-bromoisobutyrate, poly(ethylene glycol) bis(2-bromoisobutyrate), propargyl 2-bromoisobutyrate, 1,1,1-tris(2-bromoisobutyryloxymethyl)ethane, 10-undecenyl 2-bromoisobutyrate, N-tert-butyl-O-[1-[4-(chloroisopropyl)-1,2-dihydro-2,3-dimethylphenyl]-1,2,3-triazabicyclo[4-(methylphenyl)-1,2,3-triazabicyclo[4-(chloroisopropyl ... methyl)phenyl]ethyl]-N-(2-methyl-1-phenylpropyl)hydroxylamine, N-tert-butyl-N-(2-methyl-1-phenylpropyl)-O-(1-phenylethyl)hydroxylamine, TEMPO, TEMPO methacrylate, 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide, 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, 4-cyano-4-[ (Dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, cyanomethyldodecyl, cyanomethyl[3-(trimethoxysilyl)propyl]trithiocarbonate, 2-cyano-2-propyldodecyltrithiocarbonate, S,S-dibenzyltrithiocarbonate, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanoic acid ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate], phthalimidomethyl butyl trithiocarbonate, 1,1,1-Tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzyl benzodithioate, cyanomethyl benzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl benzodithioate, 2-cyano-2-propyl 4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthiocarbonylthio)propionate, ethyl 2-(phenylcarbonothioylthio)-2-phenylacetate, ethyl 2-(phenylcarbonothioylthio)propionate, 1-(methoxycarbonyl)ethyl benzodithioate, 2-(4-methoxyphenylcarbonothioylthio) The initiator fragment may be selected from the group including 4-nitro-5-(2-propynyloxy)benzyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate, 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propylbenzodithioate, cyanomethylmethyl(4-pyridyl)carbamodithioate, cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, or combinations thereof, either by themselves or conjugated to a targeting moiety or modified with a reactive group before or after the polymerization reaction.

[0054] Definitions and examples of targeting moieties are given above.

[0055] The definition and examples of reactive groups are as described above.

[0056] The chain transfer agents and initiators listed within this application are given as illustrative examples of the present invention, and any chemical compounds published before or after the filing of this document that can act as CTAs and / or initiators are included within the scope of the present invention.

[0057] The amount of drug in the polymer conjugate of the present invention can be determined by using conventional techniques well known in the art, e.g., by measuring the amount of the drug in the polymer-drug conjugate. 1 This is done by calculation of drug ratios from H-NMR or by determination of the amount by forced release of drug.

[0058] In another embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 5 kDa to 60 kDa. In a preferred embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 6 Da to 50 kDa, and in a most preferred embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 7 kDa to 40 kDa.

[0059] The molecular weight of the polymer-drug conjugates of the present invention is determined by using conventional techniques known in the art, for example, by using gel permeation chromatography (GPC).

[0060] Another embodiment of the present invention is a polymer assemblies formed using a polymer-drug conjugate according to formula I, wherein: [ka] ·R 1 and R 2 are independently H or -CH 3 Selected from ·R 3 is -H or -CH 3 Selected from x is a natural number between 1 and 100 y is a natural number between 1 and 100 n is a natural number between 1 and 50 L is a cleavable linker or L can be null. D is a therapeutic agent that is a combretastatin, or 5-fluorouracil, or gemcitabine, or chloroquine, or doxorubicin A is a terminal group or A may be null B is a terminal group or B may be null

[0061] Another embodiment of the present invention is a polymeric micelle formed using a polymer-drug conjugate according to formula I, wherein: [ka] ·R 1 and R 2 are independently H or -CH 3 Selected from ·R 3 is -H or -CH 3 Selected from x is a natural number between 1 and 100 y is a natural number between 1 and 100 n is a natural number between 1 and 50 L is a cleavable linker or L can be null. D is a therapeutic agent that is a combretastatin, or 5-fluorouracil, or gemcitabine, or chloroquine, or doxorubicin A is a terminal group or A may be null B is a terminal group or B may be null

[0062] In another embodiment, the aggregates according to the present invention are used to encapsulate therapeutic molecules, in that sense, polymer aggregates consisting of polymer-drug conjugates of formula I that encapsulate therapeutic agents other than those attached to the polymer chains are another embodiment of the present invention.

[0063] The term "encapsulate" refers to the entrapment of a therapeutic agent inside the cavity of a guest molecule, e.g., a polymeric assemblies comprised of a polymer-drug conjugate of Formula I. Encapsulation preferably occurs through non-covalent interactions between the therapeutic molecule and the polymeric assemblies of the invention.

[0064] The term "therapeutic agent" refers to any compound suitable for use in the treatment of a disease. The terms "therapeutic agent," "chemotherapeutic agent," "anti-cancer agent," and "anti-neoplastic agent" all refer to compounds suitable for use in the treatment of a disease, and these terms can be used interchangeably. In one embodiment, the disease is cancer.

[0065] Furthermore, "therapeutic agent" also refers to any agent suitable for use in the treatment of disease, such as cancer. Any therapeutic agent that can be directly or indirectly bound to the polymer-drug conjugate of the present invention can be used. U.S. Patent No. 66,342,221 also describes agents related to anti-cancer drugs, and this document is incorporated herein by reference. Anti-cancer drugs can be classified as, but are not limited to, chemotherapeutic agents, cytotoxins, antimetabolites, alkylating agents, protein kinase inhibitors, anthracyclines, antibiotics, antimitotic agents (e.g., antitubulin agents), corticosteroids, radiopharmaceuticals, and proteins (e.g., cytokines, enzymes, or interferons). Specific examples of anti-cancer drugs include, for example, docetaxel, gemcitabine, imatinib, 5-fluorouracil, 9-aminocamptothecin, amine-modified geldanamycin, doxorubicin, paclitaxel, procarbazine, hydroxyurea, meso e-chlorin, cisplatin, and radionuclides (e.g., 1-131, Y-90, In-111, and Tc-99m). There are many other anti-cancer drugs known in the art, and many continue to be developed, which are also included within the scope of the present invention.

[0066] The therapeutic agent may also be selected from a subgroup including, but not limited to, nucleoside analogs, antifolates, other metabolites, topoisomerase I inhibitors, anthracyclines, podophyllotoxins, taxanes, vinca alkaloids, alkylating agents, platinates, antihormones, radiopharmaceuticals, monoclonal antibodies, tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, retinoids, immunomodulators, histone deacetylase inhibitors, and other agents.

[0067] The nucleoside analogue may be selected from the group including, but not limited to, azacitidine, cladribine, cloharabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, mercaptopurine, nelarabine, pentostatin, thioguanine, trifluridine, tipiracil.

[0068] The antifolate may be selected from the group including, but not limited to, methotrexate, pemetrexed, pralatrexed, raltitrexed.

[0069] Other metabolites can be selected from the group including, but not limited to, hydroxycarbamide.

[0070] The topoisomerase I inhibitor may be selected from the group including, but not limited to, irinotecan and topotecan.

[0071] The anthracycline may be selected from the group including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin.

[0072] The podophyllotoxin may be selected from the group including, but not limited to, etoposide and teniposide.

[0073] The taxane may be selected from the group including, but not limited to, cabazitaxel, docetaxel, paclitaxel.

[0074] The vinca alkaloid may be selected from the group including, but not limited to, vinblastine, vincristine, vindesine, vinflunine, vinorelbine.

[0075] The alkylating agent may be selected from the group including, but not limited to, bendamustine, chlorambucil, dacarbazine, melphalan, streptozotocin, trabectin.

[0076] The anti-hormonal compound may be selected from the group including, but not limited to, abiraterone, bicalutamide, cyproterone, degarelix, exemestane, fulvestrant, goserelin, hisrelin, leuprolide, mifepristone, triptorelin.

[0077] The tyrosine kinase inhibitor may be selected from the group including, but not limited to, afatinib, axitinib, bosutinib, cobimetinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, osimetinib, pazopanib, ruxolitinib, sunitinib, vandetanib.

[0078] The mammalian target of rapamycin (mTOR) inhibitor may be selected from the group including, but not limited to, everolimus, temsirolimus.

[0079] The retinoid may be selected from the group including, but not limited to, alitretinoin, bexarotene, isotretinoin, tamibarotene, tretinoin.

[0080] The immunomodulatory agent may be selected from the group including, but not limited to, lenalidomide, pomalidomide, thalidomide.

[0081] The histone deacetylase inhibitor may be selected from the group including, but not limited to, belinostat, panobinostat, valproate, vorinostat.

[0082] The platinate may be selected from the group including, but not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin.

[0083] The other agent may be selected from the group including, but not limited to, anagrelide, ceritinib, dabrafenib, idelalisib, ibrutinib, palbociclib, vemurafenib, bleomycin, bortezomib, dactinomycin, eribulin, estramustine, ixabepilone, mitomycin, procarbazine, alectinib, fluximesterone, iobegguan, imiquimod, interferon, ixazomib, lanreotide, lentinan, octreotide, omacetaxine, tegafur, gimeradil, oteracil, uracil, combretastatin, chloroquine.

[0084] In a preferred embodiment of the invention, the therapeutic agent is selected from taxanes, antifolates, tyrosine kinase inhibitors, anthracyclines, nucleoside analogs, or other agents. Most preferably, the therapeutic agent is selected from the group including docetaxel, pemetrexed, chloroquine, combretastatins, gemcitabine, doxorubicin, fluorouracil (5-FU), 5'-deoxy-5-fluorocytidine (5'-DFCR), lapatinib.

[0085] In an embodiment of the invention, the therapeutic agent is docetaxel.

[0086] In an embodiment of the invention, the therapeutic agent is carboplatin.

[0087] In an embodiment of the invention, the therapeutic agent is doxorubicin.

[0088] Another embodiment of the present invention is a method (Method I) for preparing a polymer-drug conjugate of the present invention (Formula I) comprising: (i) polymerization of a PEG (meth)acrylate monomer (Formula II), [ka] During the ceremony, R1 is H or -CH 3 Selected from R3 is selected from -H or -CH3 n is a natural number between 1 and 50 to obtain a polymer of formula IIa, [ka] During the ceremony, A is a terminal group or A may be null B is a terminal group or B may be null

[0089] (ii) further reacting Formula IIa with a (meth)acrylate LD monomer (Formula IIIa), [ka] ·In the formula, R 2 is H or CH 3 Selected from L is a cleavable linker or L may be null. · D is a therapeutic agent selected from the group consisting of combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin, resulting in a polymer-drug conjugate of formula I.

[0090] In another embodiment, a method (Method II) for preparing a polymer-drug conjugate of the present invention (Formula I) comprises: (i) polymerization of a PEG (meth)acrylate monomer (Formula II): [ka] During the ceremony, R1 is H or -CH 3 Selected from R3 is selected from -H or -CH3 n is a natural number between 1 and 50 to obtain a polymer of formula IIa, [ka] During the ceremony, A is a terminal group or A may be null B is a terminal group or B may be null

[0091] (ii) then further reacting Formula IIa with a (meth)acrylate L monomer (Formula IIIb); [ka] ·In the formula, R 2 is H or -CH 3 Selected from L is a cleavable linker or L can be null. To obtain a copolymer as shown in formula IIb [ka] In the formula, x is a natural number from 1 to 100. y is a natural number between 1 and 100

[0092] (iii) Formula IIb is then reacted with a therapeutic agent (D) selected from the group including combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin to yield the polymer conjugate shown in Formula I.

[0093] Another embodiment of the present invention is a method (Method III) for preparing a polymer-drug conjugate of the present invention (Formula I) comprising: (i) polymerization of a PEG (meth)acrylate monomer (Formula II); [ka] During the ceremony, R1 is H or -CH 3 Selected from R3 is selected from -H or -CH3 n is a natural number between 1 and 50 to obtain a polymer of formula IIa, [ka] During the ceremony, A is a terminal group or A may be null B is a terminal group or B may be null

[0094] (ii) further reacting Formula IIa with a (meth)acrylate LD monomer (Formula IIIa), [ka] ·In the formula, R 2 is H or -CH 3 Selected from L is a cleavable linker or L may be null. D is a therapeutic agent selected from the group including combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin to obtain a block copolymer, and then (iii) reacting the formed block copolymer with a targeting moiety to obtain a polymer-drug conjugate as shown in formula I.

[0095] The term "acrylate" refers to derivatives of acrylic acid. These derivatives are the same as the parent acid (CH 2 CHCO 2 H) and esters, and thus the term "acrylate-based" defines a functionality having any of the acrylate derivatives listed above.

[0096] The term "methacrylate" refers to derivatives of methacrylic acid. These derivatives are the same as the parent acid (CH 2 C(CH 3 )CO 2 H) and esters. Thus, the term "methacrylate-based" defines a functional group having any of the above methacrylate derivatives.

[0097] The term "(meth)acrylate" refers to the terms "acrylate" and "methacrylate". Thus, the term "(meth)acrylate" can be used interchangeably with "acrylate" and "methacrylate" and includes all the characteristics of these terms as described above. The term "(meth)acrylate" should be interpreted as meaning "methacrylate and / or acrylate".

[0098] Step (i) of Methods I, II, and III may further include the use of a chain transfer agent and / or an initiator.

[0099] In another aspect, the invention relates to a polymer-drug conjugate of formula I prepared by any one of methods I, II, or III.

[0100] In another embodiment, the present invention relates to a polymer-drug conjugate of formula I prepared by method I, as this method provides a polymer-drug conjugate with a very well-defined polymer structure and drug content. Also, the polymer-drug conjugate prepared by this method does not have free reactive groups on the side chains that remain unconjugated to the drug molecule.

[0101] In one embodiment, the PEG (meth)acrylate (Formula II) is preferably selected from the group including polyethylene glycol methyl ether methacrylate (CAS No.: 26915-72-0), polyethylene glycol methacrylate (CAS No.: 25736-86-1), polyethylene glycol methyl ether acrylate (CAS No.: 32171-39-4), and polyethylene glycol acrylate (CAS No.: 9051-31-4), see Table 1 for the structures of the compounds. 1 and R 3 Both are -CH 3 In the present invention, polyethylene glycol methyl ether methacrylate is used.

[0102] In an embodiment of the present invention, a PEG (meth)acrylate (formula II) having an average molecular weight of 50 to 2000 g / mol is used. In a preferred embodiment, the PEG (meth)acrylate (formula II) has an average molecular weight of 100 to 1500 g / mol, and in a most preferred embodiment, the PEG (meth)acrylate (formula II) has an average molecular weight of 150 to 1000 g / mol. The PEG (meth)acrylate (formula II) of the present invention can have an average molecular weight of, for example, 60, 70, 80, 80, 100, 150, 200 to 250, 300, 400, 500, 600, 700, 800, 900, or 1000 g / mol.

[0103] Cleavable linkers according to the present invention can be obtained from commercial sources or can be prepared by known methods provided in the literature.

[0104] In another aspect, the present invention relates to a block copolymer of formula IIb for use in the synthesis of a polymer-drug conjugate of formula I: [ka] During the ceremony; -x is a natural number between 1 and 100. -y is a natural number from 1 to 100. -R 1 and R 2 are independently H or -CH 3 Selected from: -R 3 is -H or -CH 3 Selected from -n is a natural number from 1 to 50. - L is a cleavable linker or L can be null -A is a terminal group or A can be null -B is a terminal group or B can be null.

[0105] As disclosed above, the methods for preparing the polymer-drug conjugate of formula I (methods I, II, and III) include at least two polymerization steps, in which monomers of formula II and monomers of formula IIIa or IIIb are polymerized. In embodiments of the present invention, these polymerization steps may further include an initiator and / or a polymerization agent and / or a solvent.

[0106] The polymer-drug conjugate of the present invention can be prepared by any of the known polymerization methods. Any suitable initiator and / or catalyst known in the art can be used to prepare the polymer-drug conjugate of the present invention. When a polymerization initiator is used, the initiator or a fragment thereof can be present in the resulting polymer-drug conjugate.

[0107] The polymer backbone of the polymer-drug conjugates of the present invention can be obtained, for example, by bulk, solution, and / or suspension polymerization techniques known in the art.

[0108] The polymerization technique used to prepare the polymer-drug conjugates of the present invention may be propagated by free radical polymerization or controlled / living free radical polymerization. As used herein, the term "controlled / living free radical polymerization" refers to atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, iodine transfer polymerization (ITP), selenium-centered radical-mediated polymerization, telluride-mediated polymerization (TERP), nitroxide-mediated polymerization (NMP). In a preferred embodiment of the present invention, RAFT polymerization is used to prepare the polymer-drug conjugates of the present invention.

[0109] Suitable polymerization initiators may be selected from the group described in this document.

[0110] When using RAFT polymerization, a CTA and an initiator can be used together to prepare the polymer-drug conjugates of the invention according to Methods I, II, or III provided herein.

[0111] In an embodiment of the present invention, the polymer-drug conjugates of the present invention and polymer assemblies made therefrom may further comprise a targeting group. As used herein, the term "targeting group" refers to a tumor-specific ligand that specifically binds to cells, preferably tumor cells, having a complementary receptor.

[0112] The term "targeting group" refers to a molecule that serves to deliver the polymer-drug conjugate of the present invention to a specific site for the desired activity, i.e., results in localization of the compound. Localization is mediated by specific recognition of molecular determinants, molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions, and the like. Other mechanisms for targeting drugs to specific tissues or regions are known to those skilled in the art. Targeting ligands include, for example, molecules that bind to molecules on the surface of targeted cells. Exemplary targeting ligands include antibodies, antibody fragments, small organic molecules, peptides, peptoids, proteins, polypeptides, oligosaccharides, transferrin, HS-glycoproteins, clotting factors, serum proteins, β-glycoproteins, G-CSF, GM-CSF, M-CSF, EPO, and the like. In an exemplary embodiment of the present invention, the targeting system includes covalently binding a targeting ligand, such as RGDfK, EPPT1 peptide, bisphosphonic acid, or folate, to a carrier molecule or linker.

[0113] In certain embodiments, the invention features polymer-drug conjugates with or without a targeting ligand. In some embodiments, the targeting ligand can be RGDfK, EPPT1, bisphosphonic acid, or a folate.

[0114] Another embodiment of the present invention provides a method for delivering a therapeutic agent, comprising administering an effective amount of a polymer-drug conjugate of formula I to a subject.

[0115] Another embodiment of the present invention provides a method for delivering a therapeutic agent comprising administering to a subject an effective amount of a polymeric assembly comprising a polymer-drug conjugate of formula I.

[0116] Another embodiment of the present invention is a polymer-drug conjugate of the present invention for use in the treatment of various disorders requiring the delivery of anti-cancer or similar drugs.

[0117] Another embodiment of the present invention is a polymer assemblies comprised of the polymer-drug conjugates of the present invention for use in the treatment of various disorders requiring the delivery of anti-cancer or similar drugs.

[0118] In a preferred embodiment, the present invention relates to a polymer-drug conjugate according to formula I for use as a medicament for the treatment of cancer.

[0119] In yet another preferred embodiment, the present invention relates to a polymer ensemble consisting of a polymer-drug conjugate of formula I for use as a medicament for the treatment of cancer.

[0120] As used herein, "treat" or "treating" means to inhibit, reduce, modulate, ameliorate, or prevent at least one symptom that characterizes a pathological condition in a subject threatened by or afflicted by that condition. A non-limiting list of various types of cancer is as follows: carcinoma, carcinoma of solid tissue, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, metastatic cancer, or cancer in general.

[0121] Specific examples of cancers that the disclosed compositions can be used to treat include B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, oral squamous cell carcinoma, laryngeal and larynx cancer, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, kidney cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer; testicular cancer; colon and rectal cancer, prostate cancer, or pancreatic cancer.

[0122] The polymer-drug conjugates of the invention and / or polymer assemblies comprised of the polymer-drug conjugates of the invention may also be used in the treatment of precancerous conditions such as cervical and anal dysplasia, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.

[0123] The terms "cancer" and "cancerous" as used herein refer to a malignant tumor or to the physiological condition characterized by unregulated cell growth.

[0124] As discussed herein, the polymer-drug conjugates of the present invention and / or polymer assemblies comprising the polymer-drug conjugates of the present invention find use in the treatment and / or prevention of cancer. For such uses, the polymer-drug conjugates of the present invention and / or polymer assemblies comprising the polymer-drug conjugates of the present invention are generally administered in the form of a pharmaceutical composition.

[0125] Thus, in accordance with the present invention, there is provided a pharmaceutical composition comprising a polymer-drug conjugate according to Formula I and at least one pharma- ceutically acceptable diluent, excipient, and / or carrier. The term "treatment" includes either therapeutic or prophylactic therapy.

[0126] Further, the present invention provides a pharmaceutical composition comprising a polymeric assembly consisting of a polymer-drug conjugate according to Formula I and at least one pharma- ceutically acceptable diluent, excipient, and / or carrier.

[0127] The compositions comprising the polymer-drug conjugates of the present invention and / or polymer aggregates consisting of the polymer-drug conjugates of the present invention may be in any suitable form depending on the desired method of administering it to a patient. The compositions comprising the polymer-drug conjugates of the present invention and / or polymer aggregates consisting of the polymer-drug conjugates of the present invention may be formulated for oral administration, for example in the form of a liquid dispersion or an aqueous or oily suspension, or they may be formulated for parenteral administration, for example subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, intradermal, transdermal, or other injection techniques.

[0128] The composition comprising the polymer-drug conjugate of the present invention and / or the polymer aggregates consisting of the polymer-drug conjugate of the present invention can also be formulated in the form of an aerosol or solution for administration with an inhaler or nebulizer for administration by inhalation. The polymer-drug conjugate of the present invention and / or the polymer aggregates consisting of the polymer-drug conjugate of the present invention are preferably administered to a subject transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, or via inhalation. The most suitable route for administration in any given case depends on the particular therapeutic agent present in the polymer-drug conjugate of the present invention, the subject, and the nature and severity of the subject's disease and health condition.

[0129] The polymer-drug conjugates of the invention and / or polymer assemblies consisting of the polymer-drug conjugates of the invention may be administered in combination, e.g., simultaneously, sequentially or separately, with one or more other therapeutically active compounds, which may be an anti-cancer agent, or it may be an immunomodulatory agent, an antiviral agent, an anti-infective agent, an antibacterial agent, an anti-infective agent or an anesthetic agent, or a combination thereof.

[0130] The polymer assemblies of the present invention may encapsulate a second therapeutic agent selected from the list provided above and may further be administered in combination, e.g., simultaneously, sequentially or separately, with one or more other therapeutically active compounds, which may be an anti-cancer agent, or which is an immunomodulatory agent, an anti-viral agent, an anti-infective agent, an antibacterial agent, an anti-infective agent or an anesthetic agent, or a combination thereof.

[0131] The second therapeutic agent may be selected from those listed above, provided that it is different from the therapeutic agent present in the polymer-drug conjugate of the invention.

[0132] In the context of this specification, including is intended to mean including.

[0133] Where technically appropriate, embodiments of the invention may be combined.

[0134] Embodiments are described herein as comprising certain features / elements. The disclosure also extends to separate embodiments that consist of or consist essentially of said features / elements.

[0135] Technical references, such as patents and applications, are incorporated herein by reference.

[0136] Any embodiment specifically and explicitly recited herein may alone or in combination with one or more additional embodiments form the basis of a disclaimer.

[0137] The present invention will now be described with reference to the following examples, which are merely illustrative and should not be construed as limiting the scope of the invention in any way.

[0138] example The following examples provide a step-by-step preparation of polymer-drug conjugates of Formula I and their polymeric assemblies.

[0139] Example 1A: Synthesis of polymerizable Combretastatin-A4 monomer (CombMA) Combretastatin-A4 (300 mg, 0.95 mmol), triethylamine (TEA, 191 mg, 1.89 mmol), and methacryloyl chloride (198 mg, 1.89 mmol) were dissolved in dry dichloromethane (DCM, 10 mL) in a 25 mL round-bottom flask under N2. The reaction was stirred at room temperature for 16 h. The crude product was extracted with saturated NaHCO3 (20 mL x 2) and distilled water (20 mL x 2). The organic layer was dried over Na2SO4 and the solvent was evaporated. The CombMA monomer was purified using silica column chromatography with hexane.

[0140] Example 1B: Preparation of a Chain Transfer Agent (CTA) with Reactive Functional Groups 4-Cyanopentanoic acid dithiopentadecanoate (CPDB) is a CTA that was modified with the reactive functional group N-hydroxysuccinimide (NHS) according to the following procedure.

[0141] Briefly, CPADB (200 mg, 0.72 mmol) and N-hydroxysuccinimide (125 mg, 1.07 mmol) were dissolved in anhydrous DCM (4 mL). Dicyclohexylcarbodiimide (DCC) (177 mg, 0.86 mmol) was dissolved in anhydrous DCM (1 mL). The two solutions were then mixed and the reaction mixture was stirred at room temperature in the dark for 16 h. The insoluble white by-product dicyclohexylurea (DCU) was removed by filtration. The resulting solution was dried in vacuum and the crude product was purified by silica column chromatography using hexane and EtOAc.

[0142] Example 1C: Synthesis of POEGMEMA and NHS-POEGMEMA homopolymers Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize POEGMEMA and NHS-activated POEGMEMA (NHS-POEGMEMA) homopolymers. To synthesize POEGMEMA polymer, AIBN (1.31 mg, 0.008 mmol) was added to a solution of OEGMA (600 mg, 2.0 mmol) and CPADB (20.12 mg, 0.072 mmol) in DMF (3 mL). The mixture was then cooled to 50° C. for 1 h and then cooled to 100° C. for 2 h. 2 Purge with O 2 The solvent was removed and the polymerization was stirred at 70°C. The polymerization was terminated by cooling and exposure to air. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried under vacuum to give about 35 OEGMEMA repeat units (460 mg, 77% yield). For the synthesis of NHS-POEGMEMA homopolymer, the same procedure was applied using SCPDB (27.10 mg, 0.072 mmol) as chain transfer agent to give about 35 OEGMEMA repeat units (480 mg, 80% yield).

[0143] Example 1D: Synthesis of POEGMEMA-co-CombMA and NHS-POEGMEMA-co-CombMA block copolymers RAFT polymerization was used to synthesize POEGMEMA-b-PCombMA and NHS-POEGMEMA-b-PCombMA block copolymers. To a solution of CombMA (100 mg, 0.26 mmol) and POEGMEMA polymer (50 mg, 0.005 mmol) as a macro chain transfer agent in DMF (0.75 mL) was added AIBN (0.15 mg, 0.00092 mmol). The mixture was heated at 37° C. for 1 h until 100° C. for 2 h. 2 Purge with O 2The solvent was removed and the polymerization was stirred at 65°C. The polymerization was terminated by cooling and exposure to air. The crude product was precipitated in diethyl ether. The polymer precipitate was dried in vacuum to give about 32 CombMA repeat units (98 mg, 65% yield). The same procedure was applied to synthesize NHS-POEGMEMA-b-PCombMA block copolymer using NHS-POEGMEMA (50 mg, 0.005 mmol) as macro chain transfer agent to give about 33 CombMA repeat units (105 mg, 70% yield).

[0144] Example 1E: Synthesis of targeted block copolymers NHS-POEGMEMA-b-PCombMA (50 mg, 0.002 mmol) and cRGDfK (6.4 mg, 0.01 mmol) were co-dissolved in DMF (0.25 mL), and N,N-diisopropylethylamine (6.5 mg, 0.05 mmol) was added to the reaction mixture. The reaction mixture was stirred at 30° C. for 24 h. The crude product was precipitated in diethyl ether. The polymer precipitate was dried under reduced pressure to obtain cRGDfK-POEGMEMA-b-CombMA (41 mg, 82% yield). An overall diagram showing the preparation of cRGDfK-POEGMEMA-b-CombMA block copolymer is provided as FIG. 1.

[0145] Example 1F: Preparation of Targeted and Non-Targeted Polymer Assemblies As used herein, "targeted" refers to a polymer assembly comprised of a cRGDfK-POEGMEMA-b-CombMA block copolymer, and "non-targeted" refers to a polymer assembly comprised of a POEGMEMA-b-PCombMA block copolymer.

[0146] Two solutions were prepared to obtain targeted and non-targeted assemblies. For the formation of non-targeted assemblies, 2.3 mg of POEGMEMA-b-PCombMA block copolymer was dissolved in 500 μL of THF in a glass vial, and 3 mL of water was added to the solution. The THF in the vial was then allowed to evaporate in an open atmosphere at room temperature for 24 hours to obtain micelles. Targeted assemblies were prepared in the same manner using cRGDfK-POEGMEMA-b-PCombMA and POEGMEMA-b-PCombMA mixtures (1 / 5, w / w). Schematic diagrams of the preparation of non-targeted and targeted assemblies are provided in Figures 2 and 3.

[0147] Example 1G: Critical micelle concentration (CMC) measurement A fluorescent probe method was utilized to determine the CMC values ​​of targeted and non-targeted assemblies. Block copolymer solutions (450 μL) were prepared in THF using serial dilutions in vials as described above. A 50 μL solution of Nile Red (0.03 mg / mL) in THF was added to each vial, followed by 3 mL of water. The THF was allowed to evaporate completely in the open atmosphere, and 1 × 10 -9 ~1×10 -5 Final concentration values ​​were obtained for 16 samples ranging in M. Fluorescence measurements were recorded by a fluorescence spectrophotometer at an excitation wavelength of 550 nm and emission was monitored from 580 to 660 nm.

[0148] To determine the minimum required concentration of amphiphilic polymers to form micellar polymer structures by self-assembly, the CMC values ​​of block copolymers were calculated by tracking the Nile Red fluorescence intensity as a function of polymer concentration.

[0149] Fluorescence emission spectra of Nile Red in the non-targeted and targeted assemblies were obtained at an excitation wavelength of 550 nm and emission was monitored from 580 to 660 nm (Figures 4 and 5, respectively).

[0150] To determine the CMC values, plots of emission intensity at 612 nm versus the logarithm of copolymer concentration were obtained. The CMC values ​​for non-targeted and targeted assembly formation were calculated by the intersection of the trend lines belonging to the intensity ratio with relatively constant values ​​and rapidly increasing intensity ratios (FIGS. 6 and 7, respectively). The CMC values ​​for non-targeted and targeted assembly formation were 1.796×10 -6 M and 1.566×10 -6 It turned out to be M.

[0151] Example 2A: Synthesis of polymerizable 5FU monomer (5FU-MA) 5-Fluorouracil (250 mg, 1.92 mmol) and 37% formalin (244 μL) were reacted at 60° C. for 2 hours. The mixture was then cooled and completely lyophilized to obtain a viscous compound. In the second step, this product (315 mg, 1.97 mmol) was dissolved in anhydrous acetonitrile (5.8 mL) and then triethylamine (422 μL, 3.03 mmol) was added to the solution. After the reaction mixture was cooled to 0° C., methacryloyl chloride (232 μL, 2.38 mmol) was added dropwise. The reaction was allowed to proceed overnight at room temperature and then the white precipitate was filtered off. All volatiles were evaporated under vacuum and then the crude product was dissolved in dichloromethane (20 mL) followed by 1 M HCl (2×10 mL), 1 M NaHCO 3 (10 mL), and brine (10 mL). After concentration of the organic portion, the product was isolated in pure form by silica column chromatography eluting with ethyl acetate:hexane mixture (30:70 v / v). The product was obtained as a viscous colorless oil (305 mg, 68% yield).

[0152] Example 2B: Synthesis of POEGMEMA homopolymer Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize the POEGMEMA homopolymer. To synthesize the POEGMEMA polymer, AIBN (0.78 mg, 4.76 μmol) was added to a solution of OEGMA (500 mg, 1.66 mmol) and CPADB (11.97 mg, 42.85 μmol) in DMF (2.5 mL). The mixture was then cooled to 100° C. for 2 h. 2Purge with O 2 The solvent was removed and the polymerization was stirred at 70° C. for 16 h. The polymerization was terminated by cooling and exposure to air. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried in vacuum to give 95 mg of polymer, 19% yield.

[0153] Example 2C: Synthesis of POEGMEMA-co-5FU-MA block copolymer RAFT polymerization was used to synthesize POEGMEMA-b-P5FU-MA block copolymer. To a solution of 5FU-MA (17.9 mg, 78.6 μmol) and POEGMEMA polymer (40 mg, 3.93 μmol) as a macro chain transfer agent in DMF (0.30 mL) was added AIBN (0.13 mg, 0.786 μmol). The mixture was stirred at 200° C. for 1 h at 37° C. for 2 h at 37° C. for 1 h at 37° C. 2 Purge with O 2 The polymerization was stirred at 70° C. for 16 h after removal of the solvent. The polymerization was terminated by cooling and exposure to air. The crude product was precipitated in diethyl ether. The polymer precipitate was dried in vacuum to give 16 mg of polymer in 28% yield.

[0154] Example 3A: Polymerizable Combretastatin-A4 Monomer (Comb- SS Synthesis of -MA) Add 2-hydroxyethyl methacrylate (500 mg, 3.8 mmol), 4,4'-dithiobutyric acid (1.83 g, 7.6 mmol), N,N'-dicyclohexylcarbodiimide (1.59 mg, 7.8 mmol), and 4-(dimethylamino)pyridine (281 mg, 2.3 mmol) to a 100 mL round-bottom flask in N 2 The crude product was precipitated in cold diethyl ether and left at -20°C for 20 min. The product was purified by using silica column chromatography with ethyl acetate and hexane (20:80 v / v) (426 mg, 32% yield).

[0155] The resulting product (300 mg, 0.85 mmol) and Combretastatin-A4 (270 mg, 0.85 mmol) were then added to a 25 mL round-bottom flask with N 2 The mixture was dissolved in anhydrous dichloromethane (DCM) (6 mL) together with DIPC (129 mg, 1.02 mmol) and DMAP (104 mg, 0.85 mmol) under reduced pressure. The reaction was stirred at room temperature for 16 hours. SS The -MA monomer was purified using silica column chromatography with ethyl acetate and hexane (30:70 v / v) (195 mg, 35% yield).

[0156] Example 3B: POEGMEMA and Comb- SS Synthesis of -MA-POEGMEMA homopolymer Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize POEGMEMA. To synthesize the POEGMEMA polymer, AIBN (2.17 mg, 0.0013 mmol) was added to a solution of OEGMA (1.0 g, 3.3 mmol) and CPADB (36.88 mg, 0.133 mmol) in DMF (5 mL). The mixture was then cooled to 5° C. for 24 hours. 2 Purge with O 2 The polymerization was stirred at 70° C. for 16 hours after removal of the solvent. The polymerization was terminated by cooling and exposure to air. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried in vacuum to give 715 mg of polymer, 69% yield.

[0157] Example 3C: POEGMEMA-co-Comb- SS Synthesis of -MA block copolymer POEGMEMA-b-PComb- SS RAFT polymerization was used to synthesize the -MA block copolymer. SS AIBN (0.68 mg, 0.004 mmol) was added to -MA (80 mg, 0.20 mmol) and POEGMEMA polymer (134 mg, 0.020 mmol) as a macro chain transfer agent. The mixture was heated in N 2 Purge with O 2The solvent was removed and the polymerization was stirred at 65° C. The polymerization was terminated by cooling and exposure to air. The crude product was precipitated in diethyl ether. The polymer precipitate was dried in vacuum to give 98 mg of polymer in 65% yield.

[0158] The above examples demonstrate that the invention is applicable to a range of different linkers, such as disulfides and acetals, and to a variety of drug molecules, such as combretastatins and 5-FU. [Sequence List Free Text]

[0159] Sequence Listing 1 <223> SEQ ID NO:1 Sequence Listing 2 <223> SEQ ID NO:2 Sequence Listing 3 <223> period

Claims

1. A polymer-drug conjugate of formula I in the form of a block copolymer for delivering a therapeutic agent; 【Chemistry 1】 During the ceremony, ・R １ , R ２ , and R ３ is -CH 3 ・x is a natural number between 1 and 100 ・y is a natural number between 1 and 100 ・n is a natural number from 1 to 50 L is a cleavable linker and contains GFLG D is an anticancer drug, and is a combretastatin, or 5-fluorouracil (5-FU), or gemcitabine A is a terminal group and contains cRGDfK B is a fragment of the polymerization initiator and is a part of S,S-dibenzyltrithiocarbonate.

2. A polymeric micelle comprising the polymer-drug conjugate of formula I according to claim 1. 【Chemistry 2】 During the ceremony, ・R １ , R ２ , and R ３ is -CH 3 ・x is a natural number between 1 and 100 ・y is a natural number between 1 and 100 ・n is a natural number from 1 to 50 L is a cleavable linker and contains GFLG D is an anticancer drug, and is combretastatin, or 5-fluorouracil, or gemcitabine. A is a terminal group and is a targeting moiety that includes cRGDfK B is a fragment of the polymerization initiator and is a part of S,S-dibenzyltrithiocarbonate.

3. The polymeric micelle of claim 2, further comprising an encapsulated anticancer drug as a second anticancer drug, the anticancer drug being bound to the polymer-drug conjugate of formula I.

Images