A polymer containing 8-hydroxyquinoline and amine phosphate structure, its metal complex and use

Abstract

The present application relates to a kind of polymer containing 8-hydroxyquinoline and amido phosphate structure, its metal complex and use.The complex of the present application is good when used as anticancer drug Safety, long in vivo metabolic time, good safety to healthy cell, has excellent cancer cell killing effect to multiple strains of cancer cells.The polymer of the present application is good when used as anticancer drug Safety, long in vivo metabolic time, significantly reduce copper effect, good safety to healthy cell, has excellent cancer cell proliferation inhibitory effect to multiple strains of cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of polymers and their medical applications, specifically relating to a polymer containing 8-hydroxyquinoline and aminophosphate ester structures, its metal complexes, and its uses. Background Technology

[0002] Cancer is a serious disease that severely threatens human life and health, and its effective control remains a crucial issue that the medical community urgently needs to address. Compared to healthy individuals, the abnormal copper metabolism in cancer patients has attracted widespread attention from researchers. Numerous studies have confirmed a close relationship between higher copper levels in cancer patients and the occurrence, development, metastasis, and recurrence of various cancers. For example, during cancer development, the level of ceruloplasmin in the serum of patients is 4-8 times the normal value; during cancer metastasis and recurrence, the copper content in the patient's body also increases significantly. Based on the high copper levels in cancer patients, researchers have found that copper chelators can effectively bind copper in the body, thereby inhibiting the occurrence and metastasis of cancer and playing an anti-cancer role.

[0003] Currently, research on copper chelators mainly focuses on small molecule compounds, such as tetrathiomolybdate, trientine, and penicillamine. In a recent clinical study on breast cancer metastasis, researchers demonstrated that tetrathiomolybdate can significantly inhibit cancer metastasis by binding with copper.

[0004] Furthermore, copper complexes are also considered promising anticancer drugs. For example, CN110128333 A discloses a Cu metal complex, using 2-quinoline carboxaldehyde and 2-amino-5-chlorophenol as ligands, and Cu ions as the central ion to synthesize a six-coordinate Cu metal complex with the two ligands. This literature discloses that its Cu metal complex has inhibitory effects on the activity of breast cancer cell lines and cervical cancer cell lines, exhibiting strong anticancer activity, and its anti-breast cancer and anti-cervical cancer effects are better than traditional drugs with less toxicity.

[0005] CN113943313 A discloses a dipyridylamine copper complex with anticancer activity, which exhibits good cytotoxicity against cervical cancer HeLa cells, prostate cancer PC-3 cells, and lung cancer A549 cells.

[0006] CN114106020 A discloses a highly active anticancer complex based on a pyrimidine Schiff base and o-phenanthroline-copper, which can selectively treat cancer cells such as A549 / DDP, and its IC50... 50The concentration can reach 0.97μM±0.13μM, and the in vivo antitumor effect can reach 50.6%. Its in vivo antitumor activity is significantly higher than that of the clinical drug cisplatin (33.05%), and it has very low toxicity to normal cells, which is expected to be used in the preparation of antitumor drugs. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] Existing copper chelators and copper complexes used for anticancer treatment are all small molecule drugs, which have drawbacks such as short blood half-life, low drug utilization, and high toxicity, which greatly limit their effectiveness in clinical use.

[0009] The technical problem to be solved by the present invention is to provide an anticancer drug with good safety, long in vivo metabolism time and excellent cancer cell killing effect.

[0010] Another technical problem to be solved by the present invention is to provide an anticancer drug with good safety, long in vivo metabolism time, significant copper-lowering effect and excellent cancer cell proliferation inhibition effect.

[0011] Solution for solving the problem

[0012] To address these issues, the inventors developed a macromolecule containing 8-hydroxyquinoline and aminophosphate structures, and discovered that the macromolecule has excellent copper complexing ability, and both the macromolecule and its copper complex have excellent inhibitory effects on cancer cell activity.

[0013] Specifically, the present invention solves the technical problems of the present invention through the following methods.

[0014] [1] A polymer containing 8-hydroxyquinoline and aminophosphate ester structures, characterized in that it contains structural unit I of formula I and structural unit II of formula II:

[0015]

[0016] In this context, R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted with an alkyl group, R3 and R4 each independently represent hydrogen or a methyl group, and Z represents a single bond, -O-, -S-, or -NR. 0 -, where R 0 It is a hydrogen or an alkyl group having 1 to 5 carbon atoms; B represents a single bond or an alkylene group having 1 to 10 carbon atoms; X represents a single bond, -O-, -S-, -CO-, or -NR. 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or alkylene groups with 1 to 10 carbon atoms, where G represents a hydrophilic group or hydrophilic segment.

[0017] [2] According to the polymer described in [1], characterized in that, in formula I, Z represents -O- or -NR. 0 - B represents an alkylene group with 1 to 10 carbon atoms; in Formula II, G represents a hydroxyl group, carboxyl group, sulfonic acid group, phosphate group, sulfate group, amide group, amino group, quaternary ammonium salt group, morpholino group or polyethylene glycol segment, and X represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-.

[0018] [3] According to the polymer described in [2], in Formula I, R1 and R2 each independently represent methyl, ethyl, n-butyl, isobutyl, phenyl or benzyl, Z represents -NH-, and B represents an alkylene group having 1 to 5 carbon atoms; in Formula II, X represents -CO-, -CONH- or -COO-; and G is a polyethylene glycol segment, amino group or morpholino group.

[0019] [4] A complex, characterized in that the central ion of the complex is a copper ion and the ligand is a polymer according to any one of [1] to [3].

[0020] [5] According to the complex described in [4], the molar ratio of the copper ions to structural unit I in the polymer is (0.01 to 1):1.

[0021] [6] The method for preparing the complex according to [4] or [5] is characterized by comprising the following steps:

[0022] Step 1: React compound 1 or its hydrochloride salt, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1;

[0023]

[0024] Step 2: Polymerize monomer 1 and monomer 2 in the presence of a free radical polymerization initiator;

[0025]

[0026] Step 3: Contact the polymer obtained in Step 2 with copper ions to achieve coordination;

[0027] Each of the groups has the definition in any one of claims 1 to 3.

[0028] [7] According to the preparation method described in [6], the polymer obtained in step 2 is brought into contact with copper ions in solution to coordinate.

[0029] [8] Use of the polymer according to any one of [1] to [3] or the complex according to [4] for the preparation of an anticancer drug; preferably, the anticancer drug is a drug for treating and / or preventing cancer, wherein the cancer is selected from lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer.

[0030] [9] A medicament characterized in that it comprises a polymer according to any one of [1] to [3] and / or a complex according to [4], and a pharmaceutically acceptable carrier.

[0031]

[10] The drug according to [9] is characterized in that the carrier is a solvent, preferably water.

[0032] The effects of the invention

[0033] The polymer of the present invention containing 8-hydroxyquinoline and aminophosphate structures has high copper ion complexing ability, good safety for healthy cells, and excellent cancer cell proliferation inhibition effect for multiple strains of cancer cells.

[0034] The complexes of this invention have good safety when used as anticancer drugs, long in vivo metabolism time, good safety for healthy cells, and excellent cancer cell killing effect against multiple strains of cancer cells.

[0035] The polymer of this invention has good safety when used as an anticancer drug, long in vivo metabolism time, significant copper-lowering effect, good safety for healthy cells, and excellent cancer cell proliferation inhibition effect for multiple strains of cancer cells. Attached Figure Description

[0036] Figure 1 This is the synthesis route diagram for Example 1.

[0037] Figure 2 a) is the NMR spectrum of monomer M1 in Example 1; b) is the 1H NMR spectrum of polymer P1 in Example 1.

[0038] Figure 3 In Example 4, fluorescence emission spectroscopy was used to analyze the polymer and Cu. 2+ The characterization results of the binding ability.

[0039] Figure 4 a) is the calculation diagram of the coordination ratio between small molecule model S1 and copper ions in Example 4; b) is the calculation diagram of the coordination ratio between small molecule model K1 and copper ions in Example 4.

[0040] Figure 5a) to g) are the results of the evaluation of the effect of polymer P1 on the proliferation of mouse fibroblasts, human lung cancer cells, human malignant melanoma cells, human cervical cancer cells, human liver cancer cells, human breast cancer cells, and human esophageal cancer cells by the CCK-8 assay method in Example 5.

[0041] Figure 6 a) to g) are the results of the evaluation of the effect of polymer P1 on the proliferation of mouse fibroblasts, human lung cancer cells, human malignant melanoma cells, human cervical cancer cells, human liver cancer cells, human breast cancer cells, and human esophageal cancer cells by the FDA-PI method in Example 5.

[0042] Figure 7 This is the result of evaluating the effect of polymer P1-copper complex on the proliferation of mouse fibroblasts and human breast cancer cells using the CCK-8 assay method in Example 6.

[0043] Figure 8 This is a diagram showing the distribution of tumor tissue in a mouse, captured using a small animal in vivo optical three-dimensional imaging system as described in Example 7.

[0044] Figure 9 This is the GPC effluent curve of polymer P1 in Example 1. Detailed Implementation

[0045] The present invention will now be described in detail. The description of the technical features described below is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples.

[0046] <Terminology and Definitions>

[0047] In this specification, the term "alkyl" includes straight-chain, branched, or cyclic alkyl groups, unless otherwise expressly stated.

[0048] In this specification, the term "hydrophilic group" refers to a group that imparts hydrophilicity to a compound or polymer and has a molecular weight of less than 300, and the term "hydrophilic segment" refers to a molecular chain with a molecular weight greater than 300 composed of repeating units that is hydrophilic.

[0049] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0050] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0051] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0052] In this specification, the terms "optionally" or "optionally" are used to indicate the use or non-use of certain substances, components, procedures, application conditions, etc.

[0053] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0054] In this specification, references to "preferred embodiments," "implementation methods," etc., mean that a specific element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0055] <Polymers and their preparation methods>

[0056] One object of the present invention is to provide a polymer comprising structural unit I of formula I and structural unit II of formula II:

[0057]

[0058] In this context, R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted with an alkyl group, R3 and R4 each independently represent hydrogen or a methyl group, and Z represents a single bond, -O-, -S-, or -NR. 0 -, where R 0 It is a hydrogen or an alkyl group having 1 to 5 carbon atoms; B represents a single bond or an alkylene group having 1 to 10 carbon atoms; X represents a single bond, -O-, -S-, -CO-, or -NR. 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or alkylene groups with 1 to 10 carbon atoms, where G represents a hydrophilic group or hydrophilic segment.

[0059] In the polymers of the present invention, structural unit I, containing an aminophosphate structure in its side chain as shown in Formula I, endows the polymer with the ability to coordinate with heavy metals, and structural unit II, containing a hydrophilic group in its side chain as shown in Formula II, endows the polymer with water solubility. The polymers of the present invention, by simultaneously containing structural unit I and structural unit II, possess both good water solubility and heavy metal coordination ability.

[0060] The polymers of the present invention have a weight-average molecular weight of 1,000 to 500,000, preferably 5,000 to 200,000, more preferably 20,000 to 100,000. The number-average molecular weight is 500 to 300,000, preferably 2,000 to 80,000, more preferably 20,000 to 70,000. If the molecular weight is too low, the polymer's metabolism time is too fast, resulting in decreased safety; if the molecular weight is too high, the polymer's solubility in water decreases, viscosity increases, the rate of entry into cells slows down, and the efficacy decreases.

[0061] The polymer of the present invention has a molecular weight distribution of 1.1 to 3, for example 1.2 to 2.0.

[0062] In the polymer of the present invention, the molar ratio of structural unit I to structural unit II is (0.5-1.5):(0.5-10), preferably (0.8-1.2):(1-8). By keeping the molar ratio of these two structural units within the above range, both the water solubility and heavy metal complexing ability of the polymer can be taken into account.

[0063] The ratio of structural unit I to structural unit II can be specifically selected, for example, based on the molecular weight of the hydrophilic group or hydrophilic segment in structural unit II. When structural unit II is a hydrophilic segment with a relatively large molecular weight, such as a polyethylene glycol segment, a suitable ratio of structural unit I to structural unit II is 1:(0.8–1.2), preferably 1:(0.9–1.1). When structural unit II is a hydrophilic group with a relatively small molecular weight, such as N,N-diethylacrylamide or 4-acryloylmorpholine, a suitable ratio of structural unit I to structural unit II is 1:(3–10), preferably 1:(4–8).

[0064] In one specific embodiment, the polymer is composed of structural unit I and structural unit II.

[0065] The structural units of the polymer of the present invention are described below.

[0066] Structural Unit I

[0067] In the polymers of this invention, structural unit I is represented by formula I, wherein the parameters appearing have one of the meanings given in the context. Unit I is derived from monomer 1 described below.

[0068] In one implementation, Z represents a single bond, -O-, -S-, or -NR. 0 -, preferably -O- or -NR 0 -, where R 0 It is a hydrogen or an alkyl group having 1 to 5 carbon atoms, preferably hydrogen, methyl or ethyl.

[0069] In one embodiment, B represents a single bond or an alkylene group having 1 to 10 carbon atoms, preferably an alkylene group having 1 to 5 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, cyclopentylene, hexylene, cyclohexylene, etc.

[0070] In one embodiment, R1 and R2 each independently represent an alkyl, phenyl, or alkyl-substituted phenyl group having 1 to 5 carbon atoms, preferably R1 and R2 each independently represent methyl, ethyl, n-butyl, isobutyl, phenyl, or benzyl.

[0071] In one embodiment, the number of repeating units of structural unit I in the molecular chain of the polymer of the present invention is 1 to 300, preferably 5 to 150, and more preferably 20 to 70.

[0072] In one embodiment, in the polymer of the present invention, the content of structural unit I is 5 to 70 mol% relative to the total number of moles of structural units, preferably 10 to 60 mol%.

[0073] In a preferred embodiment, structural unit I has the structure shown in equation (1):

[0074]

[0075] Structural Unit II

[0076] In the polymer of the present invention, structural unit II is represented by formula II, wherein the parameters appearing have one of the meanings given in the context. Unit II is derived from monomer 2 described below.

[0077] In one embodiment, G is a hydroxyl, carboxyl, sulfonic acid, phosphate, sulfate, amide, amino, quaternary ammonium salt, morpholino, or polyethylene glycol segment, wherein hydroxyl, carboxyl, amide, amino, morpholino, or polyethylene glycol segment is preferred, and polyethylene glycol segment, amino, or morpholino is more preferred from the perspective of imparting better water solubility and safety to the polymer.

[0078] In one embodiment, the polyethylene glycol segment is an alkyl-terminated polyethylene glycol segment, wherein the alkyl-terminated segment is an alkyl group having 1 to 5 carbon atoms, preferably methyl, ethyl, or propyl, more preferably methyl; the number of repeating oxyethyl units in the segment is 10 to 30, preferably 15 to 25.

[0079] In one embodiment, the amino group is a group represented by -NR'R”, wherein R' and R” independently represent hydrogen or an alkyl group having 1 to 10 carbon atoms, preferably hydrogen or an alkyl group having 1 to 5 carbon atoms, more preferably hydrogen, methyl, ethyl or propyl.

[0080] In one embodiment, the quaternary ammonium salt group is a trialkylammonium halide group, wherein the alkyl group has 1 to 5 carbon atoms, preferably methyl, ethyl or propyl, and the halogen balance ion is chloride ion or bromide ion.

[0081] In one implementation, X represents a single bond, -O-, -S-, -CO-, or -NR. 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or an alkyl group having 1 to 10 carbon atoms, preferably X represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-.

[0082] In the embodiment where G in Formula II is a hydrophilic segment, the number of repeating units of structural unit II in the molecular chain of the polymer of the present invention is 1 to 300, preferably 5 to 150, and more preferably 20 to 70.

[0083] In embodiments where G is a hydrophilic group in Formula II, the number of repeating units of structural unit II in the molecular chain of the polymer of the present invention is 1 to 2000, preferably 30 to 1000, and more preferably 200 to 500.

[0084] In one embodiment, in the polymer of the present invention, the content of structural unit II is 30 to 95 mol% relative to the total number of moles of structural units, preferably 40 to 90 mol%.

[0085] In one embodiment, structural unit II is a structural unit derived from (meth)acrylic acid, (meth)acrylamide, or polyethylene glycol monomethyl ether (meth)acrylate, N,N-diethyl (meth)acrylamide, (meth)acryloylmorpholine, or a vinyl alcohol structural unit.

[0086] In a preferred embodiment, structural unit II has one or more of the structures shown in equation (2), equation (3) or equation (4):

[0087]

[0088] Other structural units

[0089] The polymers of the present invention may also optionally contain other structural units, such as structural units of monomers selected from (meth)acrylates, vinyl acetates, and styrene.

[0090] In one embodiment, in the polymer of the present invention, the content of other structural units is 20 mol% or less, preferably 10 mol% or less, and more preferably 5 mol% or less, relative to the total number of moles of structural units.

[0091] In a preferred embodiment, the polymer of the present invention has the structure shown in formula (5):

[0092]

[0093] In formula (5), x and y independently represent integers from 1 to 300, preferably integers from 5 to 150, more preferably integers from 20 to 70, and n is from 10 to 30, preferably from 15 to 25.

[0094] In a preferred embodiment, the polymer of the present invention has the structure shown in formula (6):

[0095]

[0096] In formula (6), x represents an integer from 1 to 300, preferably an integer from 5 to 150, and more preferably an integer from 20 to 70; y represents an integer from 1 to 2000, preferably an integer from 30 to 1000, and more preferably an integer from 200 to 500.

[0097] In a preferred embodiment, the polymer of the present invention has the structure shown in formula (7):

[0098]

[0099] In formula (7), x represents an integer from 1 to 300, preferably an integer from 5 to 150, and more preferably an integer from 20 to 70; y represents an integer from 1 to 2000, preferably an integer from 30 to 1000, and more preferably an integer from 200 to 500.

[0100] One objective of this invention is to provide a method for preparing the polymer of this invention, comprising the following steps:

[0101] Step 1: React compound 1 or its hydrochloride salt, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1;

[0102]

[0103] Step 2: Polymerize monomer 1 with monomer 2 and optionally other monomers in the presence of a free radical polymerization initiator.

[0104]

[0105] Each group has one of the meanings given in the context.

[0106] The following describes in detail each step of the preparation method of the present invention.

[0107] Step 1

[0108] In step 1, monomer 1 is obtained by subjecting compound 1 or its hydrochloride, phosphite compound 2, and aromatic aldehyde compound 3 to a Kabachnik-Fields reaction in the presence of an acid-binding agent.

[0109] The reaction in step 1 can be carried out in an organic solvent. Examples of organic solvents include a mixture of ethanol and acetonitrile (preferably in a volume ratio of 1:1), dichloromethane, ethyl acetate, tetrahydrofuran, etc.

[0110] In one embodiment, the reaction temperature in step 1 is 35–80°C, preferably 50–70°C; the reaction time is 2–12 hours, preferably 3–8 hours. In another embodiment, the reaction temperature can be controlled by placing the reaction system in an oil bath.

[0111] In one embodiment, in step 1, the molar ratio of compound 1 or its hydrochloride salt, acid-binding agent, compound 3 and compound 2 is (0.8-1.2):(0.8-1.2):(0.9-1.5):(1.0-2.0), preferably 1:1:1.1:1.2.

[0112] The present invention does not particularly limit the acid-binding agent used, which can be any acid-binding agent known in the art for the Kabachnik-Fields reaction, including but not limited to triethylamine, pyridine, 4-dimethylaminopyridine, etc.

[0113] Step 1 may also optionally include the separation and purification of the product of the Kabachnik-Fields reaction.

[0114] In one embodiment, step 1 further includes purifying the crude product by column chromatography after the Kabachnik-Fields reaction, followed by rotary evaporation and freeze-drying. Specifically, in the column chromatography purification, the eluent can be a dichloromethane and methanol system with a volume ratio of 20:1; the rotary evaporation temperature is 30–60°C, and the rotation speed is 0.5–10 r / s; the freeze-drying temperature is -20–-50°C, the vacuum degree is 0.1–30 Pa, and the time is 12–72 h, preferably 20–30 h.

[0115] Step 2

[0116] In step 2, the polymer of the present invention is obtained by subjecting monomer 1 obtained in step 1 to a free radical polymerization reaction with water-soluble monomer 2 in the presence of a free radical polymerization initiator.

[0117] The free radical polymerization reaction in step 2 is preferably carried out under anaerobic conditions, more preferably under anhydrous and anaerobic conditions. The method for establishing anaerobic conditions can be any method known in the art, such as repeatedly evacuating the reaction vessel and introducing inert gas, bubbling the reaction system with inert gas, or performing multiple cycles of liquid nitrogen freezing-evacuation-thawing. For the method of bubbling the reaction system with inert gas, the flow rate of the inert gas can be 10–100 mL / min, and the bubbling time can be 5–60 min.

[0118] In one embodiment, the free radical polymerization reaction in step 2 is preferably carried out in an anhydrous organic solvent, such as N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0119] In one embodiment, the reaction temperature of the free radical polymerization reaction in step 2 is 50–100°C, preferably 55–75°C; the reaction time is 4–24 h, preferably 8–16 h. The reaction temperature can be controlled by placing the reaction system in an oil bath.

[0120] In one embodiment, in step 2, the molar ratio of the free radical initiator to monomer 1 is (0.01-0.1):1, and the molar ratio of monomer 1 to monomer 2 is (0.5-1.5):(0.5-10), preferably (0.8-1.2):(1-8).

[0121] When G represents a hydrophilic segment, in step 2, the molar ratio of monomer 1 to monomer 2 is 1:(0.8 to 1.2), preferably 1:(0.9 to 1.1).

[0122] When G represents a hydrophilic group, in step 2, the molar ratio of monomer 1 to monomer 2 is 1:(3-10), preferably 1:(4-8).

[0123] The present invention does not particularly limit the free radical polymerization initiator used, and it can be any suitable free radical polymerization initiator known in the art, including but not limited to azo initiators and peroxide initiators. Examples of azo initiators include azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN); examples of peroxide initiators include benzoyl peroxide (BPO) and tert-butyl hydroperoxide. These initiators can be used alone or in combination.

[0124] In one embodiment, step 2 involves polymerizing monomer 1, monomer 2, and other monomers in the presence of a free radical polymerization initiator. The invention does not particularly limit the other monomers; they can be monomers known in the art that can copolymerize with monomer 1 and monomer 2, including but not limited to (meth)acrylate monomers, vinyl acetate, styrene monomers, etc.

[0125] Step 2 may also optionally include separating and purifying the reaction system after the free radical polymerization reaction.

[0126] In one embodiment, after the free radical polymerization reaction, the reaction solution is cooled, for example, by cooling the reaction vessel in ice water. The reaction solution is then purified by dialysis in an organic solvent, followed by rotary evaporation and freeze-drying. Specifically, in the dialysis purification, the dialysis membrane used has a molecular weight cutoff of 1000–10000, and the dialysis time is 24–72 h; the organic solvent used in the dialysis purification is one or more selected from ethanol, methanol, acetonitrile, and dichloromethane. Specifically, the rotary evaporation temperature is 30–60 °C, and the rotation speed is 0.5–10 r / s. Specifically, the freeze-drying temperature is -20–-50 °C, the vacuum degree is 0.1–30 Pa, and the time is 12–72 h, preferably 20–30 h.

[0127] <Coordination Compounds and Their Preparation Methods>

[0128] Another object of the present invention is to provide a complex in which the central ion is a copper ion and the ligand is the polymer of the present invention.

[0129] The various embodiments described above for the polymers of the present invention also apply to the coordination compounds of the present invention.

[0130] In one embodiment, in the complex of the present invention, the molar ratio of copper ions as the central ion to structural unit I in the polymer as the ligand is (0.01-1):1, preferably (0.1-1):1, more preferably (0.5-1):1, further preferably (0.8-1):1, and most preferably 1:1.

[0131] Another object of the present invention is to provide a method for preparing the complex of the present invention, comprising the steps of preparing the polymer of the present invention according to the method described above, and contacting the polymer of the present invention with copper ions to perform coordination.

[0132] In one embodiment, the polymer of the present invention is contacted with copper ions in solution for coordination. For example, a solution comprising the polymer of the present invention can be mixed with a solution containing copper ions to prepare the complex of the present invention. Alternatively, a copper salt or a solution containing copper ions can be added to a solution comprising the polymer of the present invention, or the polymer of the present invention or a solution comprising the polymer of the present invention can be added to a solution containing copper ions. Preferably, the solution is an aqueous solution.

[0133] In one embodiment, the method for preparing the complex of the present invention optionally includes post-processing and purification steps, such as one or more steps selected from solvent removal, column chromatography purification, recrystallization, filtration, drying, etc.

[0134] <Drugs>

[0135] Another object of the present invention is to provide a medicament comprising a polymer according to the invention and / or a complex according to the invention, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used in this invention, also referred to as "excipient," "pharmaceuticalally acceptable excipient," "excipient," or "additional agent," refers to all additional materials used in formulating prescriptions and manufacturing pharmaceuticals, excluding the active ingredient. These are generally pharmaceutically acceptable inert components whose safety has been reasonably assessed. Pharmaceutically acceptable carriers can enhance the operational characteristics of pharmaceutical formulations, for example, by increasing flowability and / or adhesion to ensure the formulation meets process requirements. Furthermore, the "pharmaceuticalally acceptable carrier" should possess good compatibility with the active ingredient, meaning that the carrier itself or any impurities it contains will not chemically react with the structural groups in the active ingredient or cause degradation of the active ingredient, resulting in a decrease in the content of the active ingredient.

[0136] Examples of pharmaceutically acceptable carriers include, without limitation, solvents, binders, disintegrants, lubricating excipients (lubricants, flow aids, anti-adhesion agents), stabilizers, fillers (or diluents), as well as flavoring agents, thickeners, dispersants, colorants, antibacterial agents, antioxidants, pH adjusters, surfactants, fragrances, and coating materials (plasticizers, opacifiers, pigments).

[0137] In a preferred embodiment, the pharmaceutically acceptable carrier is a solvent, preferably water.

[0138] In one embodiment, the medicament of the present invention is an anticancer drug. As used herein, "anticancer drug" means a drug used to treat and / or prevent cancer.

[0139] The drugs of this invention can treat and / or prevent cancers including but not limited to lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer cells.

[0140] On the other hand, the present invention also provides a polymer, complex, or drug for treating and / or preventing cancer, wherein the cancer is selected from lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer, and the polymer, complex, or drug is as defined above.

[0141] The drug of the present invention can be administered by a suitable method, such as by injection.

[0142] <Application>

[0143] The present invention also relates to the use of polymers or complexes according to the invention in the preparation of anticancer drugs.

[0144] On the other hand, the present invention also provides a method for treating and / or preventing a patient's disease, comprising administering a therapeutically effective amount of the aforementioned polymer, complex, or drug to a patient in need. The disease includes cancer, including but not limited to lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer.

[0145] Example

[0146] Example 1: Preparation of polymer P1

[0147] according to Figure 1 The synthetic route was used to prepare polymer P1.

[0148] N-(3-aminopropyl)methacrylamide hydrochloride (3.56 g, 20 mmol), triethylamine (2.02 g, 20 mmol), 8-hydroxyquinoline-2-carboxaldehyde (3.81 g, 22 mmol), and diethyl phosphite (3.31 g, 24 mmol) were dissolved in 10 mL of a 1:1 mixture of ethanol and acetonitrile. The mixture was reacted in an oil bath at 65 °C for 5 hours. After the reaction, the product was purified by column chromatography using dichloromethane and methanol as eluents (20:1 v / v). The monomer M1 was then collected by rotary evaporation and freeze-drying, resulting in a yellow viscous liquid with a yield of 74%.

[0149] Monomer M1 1 H-NMR spectrum as shown Figure 2 As shown in figure a), the target monomer M1 was successfully prepared.

[0150] Polyethylene glycol monomethyl ether methacrylate (9.5 g, 10 mmol), monomer M1 (4.35 g, 10 mmol), and azobisisobutyronitrile (50 mg, 0.2 mmol) were dissolved in 20 mL of N,N-dimethylformamide. After deoxygenation under nitrogen for 30 minutes, the mixture was reacted in an oil bath at 65 °C for 12 hours. After the reaction was completed, the reaction solution was cooled in ice water and purified by dialyzing in methanol solution using a dialysis bag with a molecular weight cutoff of 3500 for 48 hours. Polymer P1 was collected by rotary evaporation and freeze-drying as a light yellow solid powder with a yield of 82%. Polymer P1 was characterized by NMR.

[0151] Polymer P1 1 H-NMR spectrum as shown Figure 2 As shown in b), the target polymer P1 was successfully obtained.

[0152] The molecular weight of polymer P1 was determined by gel permeation chromatography (GPC), with a number-average molecular weight of 48,000, a weight-average molecular weight of 78,000, and a molecular weight distribution of 1.63. The GPC elution profile is shown below. Figure 9 As shown. The GPC system consists of a pump (Shimadzu LC-20AD), autosampler, guard column, analytical column, and differential refractive index detector (Shimadzu RID-10A). The mobile phase is DMF (1 mL / min), and the column temperature is 50 °C. The system uses molecules with molecular weights from 200 to 10... 6 The calibration was performed using g / mol polystyrene standards.

[0153] Example 2: Preparation of polymer P2

[0154] N,N-diethylacrylamide (1.02 g, 8 mmol), monomer M1 (0.50 g, 2 mmol), and azobisisobutyronitrile (25 mg, 0.1 mmol) were dissolved in 5 mL of N,N-dimethylformamide. After deoxygenation under nitrogen for 30 minutes, the mixture was reacted in an oil bath at 65 °C for 12 hours. After the reaction was completed, the reaction solution was cooled in ice water and purified by dialyzing in methanol solution using a dialysis bag with a molecular weight cutoff of 3500 for 48 hours. Polymer P2 was collected by rotary evaporation and freeze-drying as a light yellow solid powder with a yield of 78%. Polymer P2 was characterized by NMR.

[0155] polymer P2 1 H-NMR spectrum as shown Figure 3 As shown, from Figure 3 As can be seen, the target polymer P2 was successfully obtained.

[0156] The molecular weight of polymer P2 was determined by gel permeation chromatography (GPC), with a number average molecular weight of 32,000, a weight average molecular weight of 57,000, and a molecular weight distribution of 1.45.

[0157] Example 3: Preparation of polymer P3

[0158] 4-Acryloylmorpholine (1.12 g, 8 mmol), monomer M1 (0.50 g, 2 mmol), and azobisisobutyronitrile (25 mg, 0.1 mmol) were dissolved in 5 mL of N,N-dimethylformamide. After deoxygenation under nitrogen for 30 minutes, the mixture was reacted in an oil bath at 65 °C for 12 hours. After the reaction was completed, the reaction solution was cooled in ice water and purified by dialyzing in methanol solution using a dialysis bag with a molecular weight cutoff of 3500 for 48 hours. Polymer P3 was collected by rotary evaporation and freeze-drying as a light yellow solid powder with a yield of 81%. Polymer P3 was characterized by NMR.

[0159] polymer P3 1 H-NMR spectrum as shown Figure 3 As shown, from Figure 3 As can be seen, the target polymer P3 was successfully obtained.

[0160] The molecular weight of polymer P3 was determined by gel permeation chromatography (GPC), with a number average molecular weight of 37,000, a weight average molecular weight of 49,000, and a molecular weight distribution of 1.31.

[0161] Example 4: Test of the ability of polymer P1 to bind copper ions

[0162] 1. Fluorescence emission spectroscopy of polymer P1 bound to copper ions

[0163] The polymer P1 synthesized in Example 1 was dissolved in deionized water to prepare a 2 mg / mL P1 polymer solution. This polymer solution was then reacted with 1 mg / mL copper (Cu). 2+ Equal volumes of ion-soluble aqueous solutions were mixed, and the polymer and Cu were analyzed by fluorescence emission spectroscopy. 2+ The binding ability was characterized, and the results are as follows: Figure 3 As shown.

[0164] Depend on Figure 3 It can be seen that the polymer solution exhibits a significant fluorescence emission peak at approximately 480 nm when excited by a 365 nm laser; when combined with Cu... 2+ After binding, the fluorescence emission peak at around 480 nm completely disappeared, and the quenching of fluorescence indicates that polymer P1 and Cu... 2+ It has good binding ability.

[0165] 2. Coordination ratio test of 8-hydroxyquinoline and aminophosphate ester structures with copper ions.

[0166] The coordination ratio of 8-hydroxyquinoline aminophosphate to copper was analyzed using small molecule model S1 (8-hydroxyquinoline, commercially available) and small molecule model K1 (8-hydroxyquinoline aminophosphate, synthesized by conventional methods).

[0167] Small molecule model S1 and small molecule model K1 were dissolved in methanol solutions, and then mixed with methanol solutions of copper ions (prepared by dissolving copper chloride in methanol) to prepare a series of solutions with different proportions (the total concentration of small molecule model and copper ions in the solution is (i.e., S1 + Cu)). 2+ The sum of the concentrations of K1+Cu 2+ The sum of their concentrations is 10 -4 M), using Job's plot method (fixed Cu) 2+ With the total concentration of ligands (S1 or K1) remaining constant, the intensity of the characteristic absorption peak at 250-270 nm in the UV-Vis absorption spectrum was used to evaluate the Cu content. 2+ Plotting the ratio of Cu to ligand ratio, the highest value corresponds to Cu. 2+ (The coordination ratios of two small molecule models with copper ions were calculated, with the ligands being perfectly coordinated.)

[0168] Depend on Figure 4 a) It can be seen that the maximum coordination ratio between the small molecule model S1 containing the 8-hydroxyquinoline structure and copper ions is 2:1; Figure 4 (b) It is known that the maximum coordination ratio between the small molecule model K1, containing the 8-hydroxyquinoline and aminophosphate structures, and copper ions is 1:1. These results indicate that not only does the 8-hydroxyquinoline structure itself possess good coordination ability with copper ions, but the introduction of the aminophosphate structure further enhances its coordination effect. This further proves that the polymer P1, containing the 8-hydroxyquinoline and aminophosphate structures, and Cu... 2+ It has good binding ability.

[0169] Example 5: Anti-cancer test of polymer P1

[0170] 1. CCK-8 test

[0171] The polymer P1 synthesized in Example 1 was dissolved at different concentrations (5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL) in 1640 cell culture medium. Cell culture medium without polymer P1 was used as a control. The cells were co-cultured with mouse fibroblasts (L929), human lung cancer cells (A549), human malignant melanoma cells (A375), human cervical cancer cells (HeLa), human liver cancer cells (Hepg2), human breast cancer cells (MCF-7), and human esophageal cancer cells (KYSE) for 24 hours. The culture medium was then aspirated, and the cells were washed twice with PBS solution. CCK-8 reagent diluted 10-fold with 1640 cell culture medium was then added. The absorbance at 450 nm was measured using a microplate reader to evaluate the effect of polymer P1 on the proliferation of different cells. Mouse fibroblasts (L929) were used as healthy cells to assess the safety of the polymer, while the other six different cancer cell lines were used as tumor cell models to evaluate the anti-cancer effect of the polymer. The results are as follows: Figure 5 As shown.

[0172] Depend on Figure 5 a) It can be seen that polymer P1 has good safety for healthy cells. In the presence of 15 mg / mL polymer P1, the cell proliferation rate of mouse fibroblasts (L929) reached 93%, and in the presence of 25 mg / mL polymer P1, the cell proliferation rate of mouse fibroblasts (L929) reached 80%.

[0173] Depend on Figure 5 b)~ Figure 5 g) It can be seen that after co-culturing 15 mg / mL polymer P1 with human lung cancer cells (A549), human malignant melanoma cells (A375), human cervical cancer cells (HeLa), human liver cancer cells (Hepg2), human breast cancer cells (MCF-7), and human esophageal cancer cells (KYSE), the cell proliferation rates of various cancer cells were 83%, 46%, 65%, 77%, 34%, and 53%, respectively; after co-culturing 25 mg / mL polymer P1 with human lung cancer cells (A549), human malignant melanoma cells (A375), human cervical cancer cells (HeLa), human liver cancer cells (Hepg2), human breast cancer cells (MCF-7), and human esophageal cancer cells (KYSE), the cell proliferation rates of various cancer cells were 69%, 33%, 35%, 47%, 15%, and 44%, respectively. The above results indicate that polymer P1 has varying degrees of inhibitory effects on the proliferation of various cancer cells, with particularly significant inhibitory effects on the proliferation of human breast cancer cells (MCF-7), human malignant melanoma cells (A375), and human esophageal cancer cells (KYSE).

[0174] 2. FDA-PI method test

[0175] Mouse fibroblasts (L929) were cultured in three separate culture dishes in 1640 cell culture medium for 24 hours. The cells in the first dish were subjected to an FDA-PI test (0 h). Polymer P1 dissolved in 1640 cell culture medium was added to the second culture dish to achieve a polymer concentration of 15 mg / mL. Cells in the second and third culture dishes were cultured for another 24 h, and the FDA-PI test was performed again. The second culture dish was designated as the 24-h polymer group, and the third culture dish as the 24-h blank group.

[0176] Human lung cancer cells (A549), human malignant melanoma cells (A375), human cervical cancer cells (HeLa), human liver cancer cells (Hepg2), human breast cancer cells (MCF-7), and human esophageal cancer cells (KYSE) were subjected to FDA-PI testing using the same method as the aforementioned mouse fibroblast cells (L929).

[0177] FDA-PI test method: Aspirate the culture medium, wash twice with PBS solution, then add FDA and PI reagents diluted with 1640 cell culture medium, and observe the cell viability under a fluorescence microscope.

[0178] The effect of polymer P1 on the proliferation of different cells was evaluated using the FDA-PI test. Mouse fibroblasts (L929) were used as healthy cells to assess the safety of the polymer, while six different cancer cell lines were used as tumor cell models to assess the anti-cancer effect of the polymer. The results are as follows: Figure 6 As shown.

[0179] Depend on Figure 6 a) It can be seen that polymer P1 is safe for healthy cells. Compared with the "0h group", both the "24h-blank group" and the "24h-polymer group" showed the same degree of cell proliferation and no obvious dead cells were observed, indicating that the polymer is very safe.

[0180] Depend on Figure 6 b)~ Figure 6 g) It was found that, compared with the "24h-control group," the "24h-polymer group" showed varying degrees of inhibitory effects on the proliferation of human malignant melanoma cells (A375), human cervical cancer cells (HeLa), human liver cancer cells (Hepg2), human breast cancer cells (MCF-7), and human esophageal cancer cells (KYSE), and no obvious cell death was observed in any of them. This indicates that the anticancer effect of the polymer mainly stems from inhibiting the proliferation of cancer cells. Polymer P1 showed the most significant inhibitory effect on the proliferation of human breast cancer cells (MCF-7), human malignant melanoma cells (A375), and human esophageal cancer cells (KYSE).

[0181] Example 6: Cancer cell toxicity test of polymer-copper complex

[0182] The polymer P1 synthesized in Example 1 was dissolved in 1640 cell culture medium at a concentration of 15 mg / mL. Copper chloride was added to make copper ion concentrations of 0, 25, 50, and 100 μg / mL. After co-culturing with mouse fibroblasts and human breast cancer cells for 24 hours, the safety of the polymer P1-copper complex to healthy cells and its toxicity to cancer cells were evaluated using the CCK-8 assay. The results are as follows: Figure 7 As shown.

[0183] Depend on Figure 7 a) It can be seen that for mouse fibroblasts, the polymer P1-copper complex maintains high cell safety with increasing copper ion concentration; at a concentration of 15 mg / mL polymer combined with 100 μg / mL copper ions, the survival rate of mouse fibroblasts can still reach 95%. Figure 7 (b) It was found that for human breast cancer cells, the cell survival rate decreased significantly with increasing copper ion concentration. With a polymer concentration of 15 mg / mL combined with 100 μg / mL copper ions, the cell survival rate decreased to 11%. These results indicate that the polymer P1-copper complex also exhibits good cell safety and excellent tumor cell killing effects.

[0184] Example 7: Polymer Inhibition of Breast Cancer Metastasis in Mice

[0185] Mouse breast cancer cells (bioluminescently labeled) were suspended in PBS at 2×10⁻⁶ ppm. 5 Cells / mouse concentrations were injected into Balb / C mice via tail vein to establish a mouse model of breast cancer metastasis. 24 hours after cell implantation, 0.5 mL of physiological saline, 0.5 mL of polymer P1 solution (250 mg / mL), and 0.2 mL of tetrathiomolybdate (10 mg / mL) were injected via tail vein as the control group, polymer group, and tetrathiomolybdate group, respectively. After 14 days of normal feeding, the distribution of tumor tissue in the mice was observed using a small animal in vivo optical three-dimensional imaging system. The results are as follows: Figure 8 As shown.

[0186] Depend on Figure 8 It can be seen that the control group mice showed extensive tumor tissue spread in the lungs and tails, indicating that the mouse breast cancer metastasis model was successfully established; the tetrathiomolybdate group mice had significantly less tumor tissue than the control group, but cancer cell metastasis was still observed in the lungs; the polymer group mice showed almost no breast cancer tissue, indicating that polymer P1 significantly inhibited the proliferation and metastasis of cancer cells in the mouse breast cancer metastasis model, and played a good anti-cancer role.

[0187] Industrial availability

[0188] The polymers and their metal complexes of the present invention can be widely used in the treatment and prevention of cancers such as lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer cells.

Claims

1. A polymer containing an 8-hydroxyquinoline and an aminophosphate ester structure, characterized in that, Contains structural unit I as shown in Equation I and structural unit II as shown in Equation II: In this context, R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted with an alkyl group, R3 and R4 each independently represent hydrogen or a methyl group, and Z represents a single bond, -O-, -S-, or -NR. 0 -, where R 0 It is a hydrogen or an alkyl group having 1 to 5 carbon atoms; B represents a single bond or an alkylene group having 1 to 10 carbon atoms; X represents a single bond, -O-, -S-, -CO-, or -NR. 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or alkylene groups with 1 to 10 carbon atoms, G represents a hydrophilic group or hydrophilic segment. The polymer has a molecular weight distribution of 1.1 to 3.

2. The polymer according to claim 1, characterized in that, In Equation I, Z represents -O- or -NR. 0 - B represents an alkylene group with 1 to 10 carbon atoms; in Formula II, G represents a hydroxyl group, carboxyl group, sulfonic acid group, phosphate group, sulfate group, amide group, amino group, quaternary ammonium salt group, morpholino group or polyethylene glycol segment, and X represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-.

3. The polymer according to claim 2, characterized in that, In Formula I, R1 and R2 each independently represent methyl, ethyl, n-butyl, isobutyl, phenyl, or benzyl, Z represents -NH-, and B represents an alkylene group with 1 to 5 carbon atoms; in Formula II, X represents -CO-, -CONH-, or -COO-; G is a polyethylene glycol segment, amino group, or morpholino group.

4. A complex, characterized in that, The central ion of the complex is a copper ion, and the ligand is a polymer according to any one of claims 1 to 3.

5. The complex according to claim 4, characterized in that, The molar ratio of copper ions to structural unit I in the polymer is (0.01~1):

1.

6. The method for preparing the complex according to claim 4 or 5, characterized in that, Includes the following steps: Step 1: React compound 1 or its hydrochloride salt, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1; Step 2: Polymerize monomer 1 and monomer 2 in the presence of a free radical polymerization initiator; Monomer 2 Step 3: Contact the polymer obtained in Step 2 with copper ions to achieve coordination; Each of the groups has the definition in any one of claims 1 to 3.

7. The preparation method according to claim 6, characterized in that, In step 3, the polymer obtained in step 2 is brought into contact with copper ions in solution to coordinate.

8. Use of the polymer according to any one of claims 1 to 3 or the complex according to claim 4 in the preparation of an anticancer drug.

9. The use according to claim 8, characterized in that, The anticancer drug is a drug for treating and / or preventing cancer, wherein the cancer is selected from lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer.

10. A drug, characterized in that, It includes the polymers according to any one of claims 1 to 3 and / or the complexes according to claim 4, as well as pharmaceutically acceptable carriers.

11. The medicament according to claim 10, characterized in that, The carrier is a solvent.

12. The medicament according to claim 11, characterized in that, The carrier is water.

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