A macromolecular anticancer drug and its controllable molecular weight preparation method

Abstract

This invention relates to a macromolecular anticancer drug and a method for preparing it with controllable molecular weight. The macromolecular copper chelating agent of this invention possesses excellent copper ion binding capacity, significant copper-reducing effect, good safety profile, and good therapeutic and preventative effects against cancer. The method for preparing the controllable molecular weight of this invention is simple, has high yield, uses inexpensive and readily available raw materials, and operates under mild reaction conditions. It enables the large-scale, simple, and efficient preparation of the polymer of this invention, and allows for precise control of its molecular weight, thereby obtaining a macromolecular anticancer drug with a long metabolic time in vivo and good therapeutic effect on cancer metastasis.

Description

Technical Field

[0001] This invention relates to a macromolecular anticancer drug and a method for preparing the drug with controllable molecular weight, belonging to the field of polymers and their medical applications. Background Technology

[0002] Cancer is a serious disease that threatens human life and health, and how to effectively control cancer has always been a crucial problem that the medical community urgently needs to solve. Compared with healthy individuals, the abnormal copper metabolism in cancer patients has attracted widespread attention from researchers. During the metastasis and recurrence of cancer, the copper content in patients' bodies also increases significantly. Therefore, it has been found that copper chelation therapy can effectively bind copper in the human body, thereby inhibiting the occurrence and metastasis of cancer and playing an anti-cancer role.

[0003] Currently, research on copper chelating agents mainly focuses on small molecule compounds, such as tetrathiomolybdate, trientine, and penicillamine.

[0004] For example, Patent Document 1 discloses a tetrapropylammonium tetrathiomolybdate compound and a method for using it to prevent and treat vascular diseases such as cancer. Patent Document 2 discloses a metal chelating agent combination therapy for treating cancer. Patent Document 3 discloses a composition comprising triethylenetetramine disuccinate and a method for using them to prevent and treat copper-related diseases, conditions, and symptoms.

[0005] However, these copper chelators are all small molecule drugs, with short blood half-lives and low drug utilization, which greatly limits their clinical efficacy.

[0006] References:

[0007] Patent Document 1: CN1688303A

[0008] Patent Document 2: CN113631193A

[0009] Patent Document 3: US20220280451A1 Summary of the Invention

[0010] The problem the invention aims to solve

[0011] Existing copper chelators are all small molecule drugs, which have disadvantages such as short blood half-life and low drug utilization, which greatly limit their clinical efficacy.

[0012] The purpose of this invention is to provide a macromolecular copper chelating agent that has good safety, significant copper-lowering effect, and can effectively inhibit the proliferation and metastasis of cancer cells.

[0013] The present invention also provides a method for preparing such a macromolecular copper chelating agent with controllable molecular weight. By precisely controlling the molecular weight of the polymer, the metabolic time of the drug in vivo can be further improved, thereby enhancing the therapeutic effect of the drug on cancer metastasis.

[0014] Solution for solving the problem

[0015] To address the aforementioned problems, the inventors have developed a polymer containing 8-hydroxyquinoline and aminophosphate ester structures, and a method for preparing the polymer with controllable molecular weight.

[0016] Specifically, the present invention solves the technical problems of the present invention through the following methods.

[0017] [1] A polymer containing 8-hydroxyquinoline and aminophosphate ester structures, comprising structural unit I of formula I and structural unit II of formula II:

[0018]

[0019] 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 having 1 to 10 carbon atoms, where G represents a hydrophilic group or hydrophilic segment;

[0020] The polymer has a weight-average molecular weight of 15,000 or higher.

[0021] [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 having 1 to 10 carbon atoms; in Formula II, G represents a hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid 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-;

[0022] The polymer has a weight-average molecular weight of 30,000 or more, preferably 40,000 or more, and more preferably 50,000 or more.

[0023] [3] The polymer according to [1] or [2] is characterized in that one end of the molecular chain of the polymer has the following end-capping groups:

[0024]

[0025] The dashed lines represent connecting bonds, and R5 represents alkyl groups with 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and neopentyl.

[0026] [4] The method for preparing the polymer according to any one of [1] to [3] is characterized by comprising the following steps:

[0027] 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;

[0028]

[0029] Step 2: Monomer 1 and monomer 2 undergo reversible addition-fragmentation chain transfer polymerization in the presence of a free radical polymerization initiator and a chain transfer agent;

[0030]

[0031] Each of the groups has the definition in any one of claims 1 to 3.

[0032] [5] According to the preparation method described in [4], the free radical initiator is characterized in that, in step 2, the free radical initiator is one or more selected from azo initiators and peroxide initiators; the azo initiator is preferably selected from azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN); the peroxide initiator is preferably selected from benzoyl peroxide (BPO) and tert-butyl hydroperoxide; the molar ratio of the free radical initiator to the monomer 1 is 0.001 to 0.05:1.

[0033] [6] The preparation method according to [4] or [5] is characterized in that the chain transfer agent is selected from one or more thiocarbonyl thio compounds, preferably the chain transfer agent has the structure shown in formula (9):

[0034]

[0035] Wherein, R5 represents an alkyl group having 1 to 5 carbon atoms; R6 represents an alkyl carboxylic acid group optionally substituted with -CN;

[0036] More preferably, the chain transfer agent is 4-cyano-4-(((ethylthio)thiocarbonyl)thio)valerate;

[0037] The molar ratio of the chain transfer agent to the monomer M1 is 0.002 to 0.1:1, preferably 0.01 to 0.05:1.

[0038] [7] The preparation method according to [4] or [5] is characterized in that the reversible addition-fragmentation chain transfer polymerization is carried out under oxygen-free and preferably anhydrous conditions; the reaction temperature is 55 to 100°C and the reaction time is 4 to 24 h.

[0039] [8] Use of the polymer according to any one of [1] to [3] in 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.

[0040] [9] A drug characterized in that it comprises a polymer according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

[0041]

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

[0042] The effects of the invention

[0043] The macromolecular copper chelating agent of the present invention has excellent copper ion binding ability, significant copper reduction effect, good safety, and good therapeutic and preventive effects on cancer.

[0044] The controllable molecular weight preparation method of the present invention is simple, has a high yield, uses inexpensive and readily available raw materials, and operates under mild reaction conditions. It can prepare the polymer of the present invention in large quantities, simply and efficiently, and can precisely control its molecular weight, thereby obtaining a large molecular anticancer drug with a long metabolic time in vivo and good therapeutic effect on cancer metastasis. Attached Figure Description

[0045] Figure 1 This is the synthesis route diagram for Example 1.

[0046] Figure 2 These are the hydrogen nuclear magnetic resonance spectra of polymers P1 and P2 in Example 1.

[0047] Figure 3 These are the gel permeation chromatograms of polymers P1 and P2 in Example 1.

[0048] Figure 4 In Example 2, the UV-Vis absorption spectroscopy was used to analyze the interaction between polymers P1 and P2 and Cu. 2+ The characterization results of the binding ability.

[0049] Figure 5 a) and b) are the results of the evaluation of the effects of polymers P1 and P2 on the proliferation of mouse fibroblasts and mouse breast cancer cells by the CCK-8 assay method in Example 3.

[0050] Figure 6a) and b) are the results of the evaluation of the effects of polymers P1 and P2 on the proliferation of mouse fibroblasts and mouse breast cancer cells by the FDA-PI method in Example 3.

[0051] Figure 7 a) and b) are images of the distribution of tumor tissue in mice taken using a small animal in vivo optical three-dimensional imaging system in Example 4.

[0052] Figure 8 This is the survival curve of the experimental mice in Example 4.

[0053] Figure 9 These are the results of the metabolic experiments of polymers P1 and P2 in mice in Example 5. Detailed Implementation

[0054] 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.

[0055] <Terminology and Definitions>

[0056] In this specification, the term "alkyl" includes straight-chain, branched, or cyclic alkyl groups, unless otherwise expressly stated.

[0057] 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.

[0058] 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.

[0059] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0060] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] <Polymers containing 8-hydroxyquinoline and aminophosphate structures>

[0065] One objective of this invention is to provide a polymer containing an 8-hydroxyquinoline and an aminophosphate ester structure, which contains structural unit I (monomer unit I) of Formula I and structural unit II (monomer unit II) of Formula II:

[0066]

[0067] 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 having 1 to 10 carbon atoms, where G represents a hydrophilic group or hydrophilic segment;

[0068] The polymer has a weight-average molecular weight of 15,000 or higher.

[0069] 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 copper, 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 good water solubility and copper chelating ability.

[0070] The polymer of the present invention has a weight-average molecular weight of 15,000 or more, preferably 20,000 or more, even more preferably 21,000 or more, more preferably 30,000 or more, further preferably 40,000 or more, and even more preferably 50,000 or more. The polymer of the present invention has a weight-average molecular weight of 200,000 or less, preferably 100,000 or less, more preferably 80,000 or less, further preferably 70,000 or less, and even more preferably 68,000 or less. 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, its viscosity increases, its entry into cells slows down, and its efficacy decreases.

[0071] The polymer of the present invention has a molecular weight distribution of 1.1 to 3, for example 1.2 to 2.0.

[0072] 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 copper chelating ability of the polymer can be taken into account.

[0073] 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:(0.5–2), preferably 1:(0.8–1.2), and more 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).

[0074] In one specific embodiment, the polymer consists of structural unit I, structural unit II, and end-capping groups.

[0075] The structural units and end-capping groups of the polymer of the present invention are described below.

[0076] Structural Unit I

[0077] In the polymers of this invention, structural unit I is as shown in Formula I, wherein the parameters appearing have one of the meanings given in the context. Structural unit I is derived from monomer 1 described below.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 10 to 70.

[0082] 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%.

[0083] In a preferred embodiment, structural unit I has the structure shown in equation (1):

[0084]

[0085] Structural Unit II

[0086] In the polymers of this invention, structural unit II is as shown in Formula II, wherein the parameters appearing therein have one of the meanings given in the context. Structural unit II is derived from monomer 2 described below.

[0087] 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.

[0088] 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 units of oxyethyl in the segment is 10 to 40, preferably 15 to 30, more preferably 15 to 25.

[0089] 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.

[0090] 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.

[0091] 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-.

[0092] In the embodiment where G is a hydrophilic segment 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 300, preferably 5 to 150, and more preferably 10 to 70.

[0093] 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.

[0094] 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%.

[0095] 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.

[0096] In a preferred embodiment, structural unit II has one or more of the structures shown in equation (2), equation (3) or equation (4):

[0097]

[0098] Other structural units

[0099] 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.

[0100] 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.

[0101] End capping groups

[0102] In one embodiment, one end of the molecular chain of the polymer of the present invention has the following end-capping groups:

[0103]

[0104] The dashed lines represent connecting bonds, and R5 represents alkyl groups with 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and neopentyl.

[0105] In one embodiment, the polymer of the present invention has the following end-capping groups at both ends of its molecular chain:

[0106]

[0107] The dashed lines represent connectors.

[0108] In one embodiment, the end-capping groups of the polymer of the present invention are derived from the chain transfer agent described below.

[0109] In a preferred embodiment, the polymer of the present invention has a repeating unit structure (end groups not shown) as shown in formula (5):

[0110]

[0111] 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.

[0112] In a preferred embodiment, the polymer of the present invention has a repeating unit structure (end groups not shown) as shown in formula (6):

[0113]

[0114] 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.

[0115] In a preferred embodiment, the polymer of the present invention has a repeating unit structure (end groups not shown) as shown in formula (7):

[0116]

[0117] 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.

[0118] In the preferred embodiments of the polymer of the present invention described above, the polymer molecular chain has end capping groups derived from the chain transfer described below at both ends.

[0119] In a preferred embodiment, the polymer of the present invention has the structure shown in formula (8):

[0120]

[0121] In formula (8), x and y 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.

[0122] <Preparation Method of Controllable Molecular Weight>

[0123] One object of the present invention is to provide a method for preparing the polymer of the present invention, which includes the following steps:

[0124] 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;

[0125]

[0126] Step 2: Monomer 1 and monomer 2 undergo reversible addition-fragmentation chain transfer polymerization in the presence of a free radical polymerization initiator and a chain transfer agent;

[0127]

[0128] Each group has one of the meanings given in the context.

[0129] The preparation method of the present invention employs reversible addition-fragmentation chain transfer polymerization (RAFT), thereby enabling precise control of the polymer molecular weight.

[0130] The following describes in detail each step of the preparation method of the present invention.

[0131] Step 1

[0132] 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.

[0133] 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.

[0134] In one embodiment, the Kabachnik-Fields reaction in step 1 is carried out at a temperature of 35–80°C, preferably 50–70°C, for a time of 2–12 h, preferably 3–8 h. In another embodiment, the reaction temperature can be controlled by placing the reaction system in an oil bath.

[0135] 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.

[0136] 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.

[0137] Step 1 may also optionally include the separation and purification of the product of the Kabachnik-Fields reaction.

[0138] 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 (10-30):1, preferably 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 to -50°C, the vacuum degree is 0.1-30 Pa, and the time is 12-72 h, preferably 20-30 h.

[0139] Step 2

[0140] In step 2, the polymer of the present invention is obtained by subjecting monomer 1 obtained in step 1 to a RAFT polymerization reaction with water-soluble monomer 2 in the presence of a free radical polymerization initiator and a chain transfer agent.

[0141] The RAFT polymerization in step 2 is 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 an inert gas, bubbling the reaction system with an inert gas, or performing multiple cycles of liquid nitrogen freezing-evacuation-thawing. For the method of bubbling the reaction system with an inert gas, the flow rate of the inert gas can be 10–100 mL / min, and the bubbling time can be 5–60 min. The inert gas can be nitrogen or argon.

[0142] In one embodiment, the RAFT polymerization reaction in step 2 is preferably carried out in an anhydrous organic solvent, such as N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0143] In one embodiment, the reaction temperature of the RAFT 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.

[0144] In one embodiment, in step 2, the molar ratio of monomer 1 to monomer 2 is (0.5-1.5):(0.5-10), preferably (0.8-1.2):(1-8).

[0145] When G represents a hydrophilic segment, in step 2, the molar ratio of monomer 1 to monomer 2 is 1:(0.5-2), preferably 1:(0.8-1.2), and more preferably 1:(0.9-1.1).

[0146] 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).

[0147] 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.

[0148] In one embodiment, the molar ratio of the free radical initiator to monomer 1 is 0.001 to 0.05:1.

[0149] The present invention does not particularly limit the chain transfer agent used, which can be any chain transfer agent known in the art suitable for RAFT polymerization, such as thiocarbonyl thio compounds, including but not limited to dithiobenzoates, trithiosulfates and dithiocarbamates, with trithiocarbonates being preferred.

[0150] In one embodiment, the chain transfer agent used in this invention has the structure shown in formula (9):

[0151]

[0152] R5 represents an alkyl group with 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl; R6 represents an alkyl carboxylic acid group optionally substituted with -CN, such as 4-cyanopentanoic acid group.

[0153] Specific chain transfer agents include 4-cyano-4-(((ethylthio)thiocarbonyl)thio)valeric acid, 4-cyano-4-[(dodecylthioalkylthiocarbonyl)thioalkyl]valeric acid, 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanovaleric acid, 4-cyano-4-(phenylcarbonylthio)valeric acid, etc.

[0154] In one embodiment, in step 2, the molar ratio of the chain transfer agent to monomer M1 is (0.002-0.2):1, preferably 0.01-0.1:1, more preferably (0.01-0.05):1.

[0155] In one embodiment, step 2 involves polymerizing monomer 1, monomer 2, and other monomers in the presence of a free radical polymerization initiator and a chain transfer agent. 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.

[0156] Step 2 may also optionally include separating and purifying the reaction system after the free radical polymerization reaction.

[0157] 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.

[0158] <Drugs>

[0159] Another object of the present invention is to provide a medicament comprising the polymer 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 and manufacturing pharmaceuticals, excluding the active ingredient. These are generally pharmaceutically acceptable inert components whose safety has been reasonably assessed. A pharmaceutically acceptable carrier can enhance the operational characteristics of a pharmaceutical formulation, 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.

[0160] Non-limiting examples of pharmaceutically acceptable carriers include 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).

[0161] In a preferred embodiment, the pharmaceutically acceptable carrier is a solvent, preferably water.

[0162] 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.

[0163] 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.

[0164] On the other hand, the present invention also provides a polymer 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 or drug is as defined above.

[0165] The drug of the present invention can be administered by a suitable method, such as by injection.

[0166] <Applications>

[0167] The present invention also relates to the use of the polymers according to the invention in the preparation of anticancer drugs.

[0168] 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 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.

[0169] Example

[0170] The following specific embodiments further illustrate the present invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0171] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0172] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0173] Example 1: Preparation of polymers P1 and P2

[0174] according to Figure 1 The synthetic route shown is used to prepare polymers P1 and P2, as detailed below.

[0175] 1. Preparation of polymeric monomer M1

[0176] 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 20 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%.

[0177] 2. Preparation of polymer P1

[0178] Polyethylene glycol monomethyl ether methacrylate (9.5 g, 10 mmol), monomer M1 (4.35 g, 10 mmol), 4-cyano-4-(((ethylthio)thiocarbonyl)thio)valeric acid (53 mg, 0.2 mmol), and azobisisobutyronitrile (13 mg, 0.08 mmol) were dissolved in 20 mL of N,N-dimethylformamide (chain transfer agent: M1 = 0.02:1). After deoxygenation under nitrogen for 30 minutes, the mixture was reacted in an oil bath at 75 °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, obtained by rotary evaporation and freeze-drying, was a light yellow solid powder with a yield of 83%.

[0179] 3. Preparation of polymer P2

[0180] Polyethylene glycol monomethyl ether methacrylate (9.5 g, 10 mmol), monomer M1 (4.35 g, 10 mmol), 4-cyano-4-(((ethylthio)thiocarbonyl)thio)valeric acid (176 mg, 0.66 mmol), and azobisisobutyronitrile (43 mg, 0.26 mmol) were dissolved in 20 mL of N,N-dimethylformamide (chain transfer agent: M1 = 0.066:1). After deoxygenation under nitrogen for 30 minutes, the mixture was reacted in an oil bath at 75 °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. The polymer P2 obtained by rotary evaporation and freeze-drying was a light yellow solid powder with a yield of 89%.

[0181] Polymers P1 and P2 were characterized by NMR and gel permeation chromatography.

[0182] Depend on Figure 2 shown 1 The H-NMR spectrum showed that the target polymers P1 and P2 were successfully prepared.

[0183] Depend on Figure 3 The gel permeation chromatography chromatograms shown indicate that the weight-average molecular weight of polymer P1 is 67,200, and the weight-average molecular weight of polymer P2 is 22,100.

[0184] Example 2: Test of the ability of polymers P1 and P2 to bind copper ions

[0185] The polymer P1 synthesized in Example 1 was dissolved in deionized water to prepare a 0.1 mg / mL P1 polymer solution; copper sulfate was dissolved in this solution to prepare a solution containing 0.5 mM Cu. 2+ A solution of P1 polymer (0.1 mg / mL).

[0186] The polymer P2 synthesized in Example 1 was dissolved in deionized water to prepare a 0.1 mg / mL P2 polymer solution; copper sulfate was dissolved in this solution to prepare a solution containing 0.5 mM Cu. 2+ A solution of P2 polymer (0.1 mg / mL).

[0187] The two solutions were characterized by ultraviolet-visible absorption spectroscopy, and the results are as follows: Figure 4 As shown.

[0188] Depend on Figure 4 It can be seen that, in relation to Cu 2+ After bonding, polymers P1 and P2 exhibit significant UV absorption peaks at 261 nm and 262 nm, respectively, indicating that polymers P1 and P2 react with Cu. 2+ It has good binding ability.

[0189] Example 3: Inhibition of cancer cells by polymers P1 and P2

[0190] 1. CCK-8 test

[0191] Polymer P1 synthesized in Example 1 was dissolved in 1640 cell culture medium at different concentrations (1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL), with cell culture medium without polymer P1 used as a control. After co-culturing with mouse fibroblasts (L929) and mouse breast cancer cells (4T1) for 24 hours, the effect of polymer P1 on the proliferation of different cells was evaluated using the CCK-8 assay. The effect of polymer P2 on the proliferation of different cells was evaluated using the same method. The results are as follows. Figure 5 As shown in a) and b).

[0192] Depend on Figure 5a) It can be seen that polymer P1 has good safety for healthy cells, with a cell survival rate of 94% at 20 mg / mL; polymer P1 has a significant inhibitory effect on the proliferation of mouse breast cancer cells, with a cell survival rate of 46% at 20 mg / mL. Figure 5 b) It can be seen that polymer P2 has good safety for healthy cells, with a cell survival rate of 92% at 20 mg / mL polymer P2; polymer P2 has a significant inhibitory effect on the proliferation of mouse breast cancer cells, with a cell survival rate of 42% at 20 mg / mL polymer P2.

[0193] The results above show that the cell survival rates of polymers P1 and P2 are close to their concentration curves, indicating that the difference in molecular weight between polymers P1 and P2 does not significantly affect the inhibition of cancer cells.

[0194] 2. FDA-PI method test

[0195] The polymers P1 and P2 synthesized in Example 1 were dissolved in 1640 cell culture medium at a concentration of 20 mg / mL and co-cultured with mouse fibroblasts (L929) and mouse breast cancer cells (4T1) for 24 hours.

[0196] Four groups of samples were tested using the FDA-PI method: 0h (cells before polymer addition), 24h (control group, blank cultured cells), 24h (polymer P1 group, cells co-cultured with polymer P1), and 24h (polymer P2 group, cells co-cultured with polymer P2). The effects of polymer P1 and polymer P2 on the proliferation of different cells were evaluated. The results are as follows: Figure 6 As shown in a) and b).

[0197] 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.

[0198] Mouse fibroblasts (L929) were used as healthy cells to assess the safety of the polymer. Figure 6 a) It can be seen that polymer P1 is safe for healthy cells. The "24h-control group", "24h-polymer P1 group" and "24h-polymer P2 group" all showed similar levels of cell proliferation compared with the "0h group", and no obvious dead cells were observed, indicating that polymer P1 and polymer P2 are very safe.

[0199] Mouse breast cancer cells (4T1) were used as cancer cells to evaluate the anti-cancer effects of polymers P1 and P2. Figure 6b) It can be seen that the "24h-polymer P1 group" and "24h-polymer P2 group" showed significantly less cancer cell proliferation compared with the "24h-control group", which indicates that polymers P1 and P2 have a significant inhibitory effect on the proliferation of mouse breast cancer cells.

[0200] No obvious dead cells were observed in either group of experiments, indicating that the anti-cancer mechanism of polymers P1 and P2 mainly stems from inhibiting the proliferation of cancer cells.

[0201] Example 4: Polymer P1 and P2 inhibition of mouse breast cancer metastasis test

[0202] 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. Twenty-four hours before cell implantation, prophylactic protection was provided by tail vein injection of 0.5 mL of physiological saline (control group), 0.5 mL of polymer P1 solution (100 mg / mL), 0.5 mL of polymer P2 solution (100 mg / mL), and 0.2 mL of tetrathiomolybdate (5 mg / mL). Ten and fifteen days after cancer cell injection and 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 7 As shown in a) and b).

[0203] Depend on Figure 7 a) It was found that in the control group mice, extensive tumor tissue spread in the lungs 10 days after cancer cell injection, indicating the successful establishment of the mouse breast cancer metastasis model. The tumor tissue in the tetrathiomolybdate group mice was not significantly different from that in the control group, indicating that the small molecule drug tetrathiomolybdate was not very effective. The mice in the polymer P2 group had significantly less lung cancer tissue than the control group, but a small amount of cancer cell metastasis was still observed in the lungs. Almost no breast cancer tissue was observed in the lungs of the mice in the polymer P1 group. These results indicate that in the mouse breast cancer metastasis model, compared with the small molecule drug tetrathiomolybdate, polymers P1 and P2 can significantly inhibit the proliferation and metastasis of cancer cells, demonstrating a good anti-cancer effect.

[0204] Depend on Figure 7 (b) It was found that 15 days after injection of cancer cells, significant lung metastasis was observed in the lungs of mice in the polymer P2 group, while no breast cancer tissue was observed in the lungs of mice in the polymer P1 group. This indicates that the molecular weight of the polymer can significantly affect the inhibitory effect on breast cancer metastasis; the larger the molecular weight, the better the anti-cancer effect.

[0205] The survival status of mice in each experimental group was recorded over 60 days, and the results are as follows: Figure 8 As shown.

[0206] Depend on Figure 8Survival curves showed that the median survival times of the control group and the tetrathiomolybdate group were 12.5 days and 12 days, respectively, with no significant difference between the two curves (p = 0.2932), indicating that the small molecule drug tetrathiomolybdate was not effective. The median survival times of the polymer P1 group and the polymer P2 group were 18 days and 25.5 days, respectively, showing a significant increase in survival time compared to the control group (P1, p ~ 0.0001, P2, p = 0.2932), indicating that polymers P1 and P2 can effectively inhibit breast cancer metastasis. The significantly longer survival time of the polymer P1 group compared to the polymer P2 group further demonstrates that the molecular weight of the polymer significantly affects the inhibitory effect on breast cancer metastasis; the larger the molecular weight, the better the anti-cancer effect.

[0207] Example 5: Metabolic behavior test of polymer P1 and P2 mice

[0208] The trithioester at the P1 end group of the polymer synthesized in Example 1 was converted to a thiol functional group via ammonolysis, thereby further modifying the fluorescent dye Cy7 to prepare the fluorescent polymer P1-Cy7. The fluorescent polymer P2-Cy7 was prepared using the same method.

[0209] 0.5 mL of fluorescent polymer P1-Cy7 (100 mg / mL), 0.5 mL of fluorescent polymer P2-Cy7, and 0.5 mL of small molecule Cy7 (1 mg / mL) were injected into mice via the tail vein. Fluorescence signals in mice were recorded using a small animal in vivo optical three-dimensional imaging system 1 hour (day 0) and 1–10 days after tail vein injection to observe the metabolic behavior of P1-Cy7, P2-Cy7, and Cy7. The results are as follows: Figure 9 As shown.

[0210] Depend on Figure 9 The fluorescence distribution in mice showed that the fluorescence signal of the small molecule Cy7 rapidly decreased and disappeared after one day, a phenomenon similar to that of the small molecule drug ammonium tetrathiomolybdate, indicating that the metabolism time of small molecule drugs is short. Mice injected with P1-Cy7 and P2-Cy7 showed strong fluorescence signals throughout their bodies on day 1, and the fluorescence signals could still be detected after a long period of time.

[0211] Quantitative data analysis showed that P1-Cy7 exhibited a longer in vivo circulation time, retaining 80.5% of the polymer on day 1 and 53.8% on day 3, while P2-Cy7 had a relatively shorter circulation time, retaining 46.2% of the polymer on day 1 and 28.2% on day 3. This indicates that polymer P1 has a longer in vivo metabolic behavior than polymer P2, a phenomenon consistent with the conclusions of the cancer metastasis model in Example 4, further demonstrating that increasing the molecular weight can effectively increase the metabolic time of the polymer and enhance its effect in inhibiting cancer cell metastasis.

[0212] Industrial availability

[0213] The polymer of this 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 method for preparing a polymer containing 8-hydroxyquinoline and an aminophosphate ester structure, 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; ， wherein R1and R2each independently represent an alkyl group having 1 to 10 carbon atoms; R3and R4each independently represent hydrogen or a methyl group; Z represents -O- or -NR 0 -, wherein R 0 is hydrogen or an alkyl group having 1 to 5 carbon atoms; B represents an alkylene group having 1 to 10 carbon atoms; Step 2: Monomer 1 and monomer 2 undergo reversible addition-fragmentation chain transfer polymerization in the presence of a free radical polymerization initiator and a chain transfer agent; ， Where G represents an amino group, a quaternary ammonium salt group, a morpholino group, or a polyethylene glycol segment; X represents -CO- or -C(=O)O-. The chain transfer agent is 4-cyano-4-(((ethylthio)thiocarbonyl)thio)valerate; the molar ratio of the chain transfer agent to monomer 1 is 0.01~0.05:1; The free radical polymerization initiator is selected from one or more of azo initiators and peroxide initiators; the molar ratio of the free radical polymerization initiator to the monomer 1 is 0.001~0.05:1; The reversible addition-fragmentation chain transfer polymerization is carried out under anaerobic conditions; the reaction temperature is 55–100℃, and the reaction time is 4–24 h. The polymer has a weight-average molecular weight of 15,000 or more and 70,000 or less.

2. The preparation method according to claim 1, characterized in that, The azo initiator is selected from azobisisobutyronitrile (AIBN) and azobisisoheptanenitrile (ABVN); the peroxide initiator is selected from benzoyl peroxide (BPO) and tert-butyl hydroperoxide.

3. The preparation method according to claim 1, characterized in that, The reversible addition-fragmentation chain transfer polymerization is carried out under anaerobic and anhydrous conditions.

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