Macromolecular anticancer drug and controllable molecular weight preparation method thereof

Abstract

The invention relates to a macromolecular anticancer drug and a controllable molecular weight preparation method thereof. The macromolecular copper chelating agent disclosed by the invention has excellent copper ion binding capacity, remarkable copper reducing effect and good safety, and has good cancer treatment and prevention effects. The preparation method of the controllable molecular weight is simple in process, high in yield, cheap and easily available in raw materials and mild in reaction condition, the polymer can be massively, simply and efficiently prepared, and the molecular weight of the polymer can be accurately controlled, so that the macromolecular anticancer drug which is long in metabolism time in vivo and good in cancer metastasis treatment effect is obtained.

Description

Technical Field

[0001] The invention relates to a macromolecular anticancer drug and a method for preparing a controllable molecular weight thereof, belonging to the field of polymers and their medical uses. Background Art

[0002] Cancer is a disease that seriously endangers human life and health. How to effectively control cancer has always been an important issue that needs to be solved in the medical community. Compared with healthy individuals, the abnormal copper metabolism in cancer patients has attracted extensive attention from researchers. During the metastasis and recurrence of cancer, the copper content in the patient's body will also increase significantly. Therefore, it has been found that copper chelation therapy can effectively bind to the copper element in the human body, thereby inhibiting the occurrence and metastasis of cancer and playing an anti-cancer role.

[0003] At present, the copper chelators studied are mainly concentrated on small molecule compounds, such as tetrathiomolybdate, trientine, penicillamine, etc.

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

[0005] However, currently these copper chelators are small molecule drugs with short blood half-life and low drug utilization, which greatly limits their effectiveness in clinical use.

[0006] References:

[0007] Patent document 1: CN1688303A

[0008] Patent document 2: CN113631193A

[0009] Patent document 3: US20220280451A1 Summary of the invention

[0010] Problem that the invention aims to solve

[0011] The copper chelators in the prior art are all small molecule drugs, which have the disadvantages of short blood half-life and low drug utilization, which greatly limits their clinical use effect.

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

[0013] The present invention also provides a controllable molecular weight preparation method for the macromolecular copper chelating agent. By precisely controlling the molecular weight of the polymer, the metabolism time of the drug in the body can be further increased, thereby improving the effect of the drug in treating cancer metastasis.

[0014] Solutions for solving problems

[0015] In order to solve the above problems, the present inventors have developed a polymer containing 8-hydroxyquinoline and aminophosphonate structures and a method for preparing the polymer with controllable molecular weight.

[0016] Specifically, the present invention solves the technical problem of the present invention through the following method.

[0017] [1] A polymer containing 8-hydroxyquinoline and aminophosphonate structures, which contains a structural unit I represented by formula I and a structural unit II represented by formula II:

[0018]

[0019] wherein R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted by 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 is 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, and X represents a single bond, -O-, -S-, -CO-, -NR 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or an alkylene group having 1 to 10 carbon atoms, G represents a hydrophilic group or a hydrophilic segment;

[0020] The weight average molecular weight of the polymer is 15,000 or more.

[0021] [2] The polymer according to [1], wherein in the formula I, Z represents -O- or -NR 0 -, B represents an alkylene group having 1 to 10 carbon atoms; in the formula II, G represents a hydroxyl group, a carboxyl group, a sulfonic acid group, a phosphoric acid group, a sulfate group, an amide group, an amine group, a quaternary ammonium salt group, a morpholine group or a polyethylene glycol segment, and X represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-;

[0022] The weight average molecular weight of the polymer is 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], characterized in that one end of the molecular chain of the polymer has the following end-capping group:

[0024]

[0025] The dotted line represents a connecting bond, and R5 represents an alkyl group having 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 a polymer according to any one of [1] to [3], characterized in that it comprises the following steps:

[0027] Step 1: reacting compound 1 or its hydrochloride, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1;

[0028]

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

[0030]

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

[0032] [5] The preparation method according to [4] 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 dibenzoyl 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], characterized in that the chain transfer agent is one or more selected from thiocarbonylthio compounds, and preferably the chain transfer agent has a structure represented by the following 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)pentanoic acid;

[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 anaerobic and preferably anhydrous conditions; the reaction temperature is 55 to 100°C and the reaction time is 4 to 24 hours.

[0039] [8] Use of the polymer described in any one of [1] to [3] for preparing an anticancer drug; preferably, the anticancer drug is a drug for treating and / or preventing cancer, and the cancer is selected from lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, and esophageal cancer.

[0040] [9] A drug comprising the polymer according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

[0041]

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

[0042] 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 cancer treatment and prevention effects.

[0044] The controllable molecular weight preparation method of the present invention has the advantages of simple process, high yield, cheap and readily available raw materials, and warm reaction conditions. The polymer of the present invention can be prepared in large quantities, simply and efficiently, and its molecular weight can be precisely controlled, thereby obtaining a macromolecular anticancer drug with a long metabolism time in the body and a good effect in treating cancer metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the synthesis route diagram of Example 1.

[0046] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of polymers P1 and P2 in Example 1.

[0047] Figure 3 is the gel permeation chromatography spectrum of polymers P1 and P2 in Example 1.

[0048] Figure 4 The UV-visible absorption spectra of polymers P1 and P2 and Cu 2+ The characterization results of the binding ability.

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

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

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

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

[0053] Fig. 9 These are the results of the metabolism experiments of polymers P1 and P2 in Example 5 in mice. DETAILED DESCRIPTION

[0054] The following is a detailed description of the present invention. The technical features described below are described 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] <Terms and Definitions>

[0056] In the present specification, the term "alkyl group" includes a linear, branched or cyclic alkyl group, unless otherwise specifically 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 300 or less, and the term "hydrophilic segment" refers to a molecular chain with a molecular weight greater than 300 and composed of repeating units having hydrophilicity.

[0058] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0059] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0060] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0061] In this specification, the use of "optionally" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0062] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0063] In this specification, the references to "preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0064] <Polymer containing 8-hydroxyquinoline and aminophosphonate structure>

[0065] One of the purposes of the present invention is to provide a polymer containing 8-hydroxyquinoline and aminophosphonate structure, which contains structural unit I (monomer unit I) shown in formula I and structural unit II (monomer unit II) shown in formula II:

[0066]

[0067] wherein R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted by 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 is 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, and X represents a single bond, -O-, -S-, -CO-, -NR 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or an alkylene group having 1 to 10 carbon atoms, G represents a hydrophilic group or a hydrophilic segment;

[0068] The weight average molecular weight of the polymer is 15,000 or more.

[0069] In the polymer of the present invention, the structural unit I having an aminophosphoric acid ester structure in the side chain shown in formula I can give the polymer the ability to coordinate with copper, and the structural unit II having a hydrophilic group in the side chain shown in formula II can give the polymer water solubility. The polymer of the present invention has good water solubility and copper chelating ability by containing both structural units I and II.

[0070] The weight average molecular weight of the polymer of the present invention is 15,000 or more, preferably 20,000 or 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 weight average molecular weight of the polymer of the present invention is 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 metabolism time of the polymer is too fast, and the safety is reduced; if the molecular weight is too high, the solubility of the polymer in water is reduced, the viscosity is increased, the speed of entering the cell is slowed down, and the efficacy is reduced.

[0071] The molecular weight distribution of the polymer of the present invention is 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 making the molar ratio of these two structural units within the above range, 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, according to the molecular weight of the hydrophilic group or hydrophilic segment in structural unit II. For the case where structural unit II is a hydrophilic segment with a relatively large molecular weight, such as a polyethylene glycol segment, the suitable ratio of structural unit I to structural unit II is 1: (0.5-2), preferably 1: (0.8-1.2), preferably 1: (0.9-1.1). For the case where structural unit II is a hydrophilic group with a relatively small molecular weight, such as N, N-diethylacrylamide and 4-acryloylmorpholine, the suitable ratio of structural unit I to structural unit II is 1: (3-10), preferably 1: (4-8).

[0074] In one embodiment, the polymer is composed 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 respectively.

[0076] Structural unit I

[0077] In the polymer of the present invention, the structural unit I is represented by formula I, wherein the parameters appearing therein have one of the meanings given above and below. The structural unit I is derived from the monomer 1 described below.

[0078] In one embodiment, Z represents a single bond, -O-, -S- or -NR 0 -, preferably represents -O- or -NR 0-, where R 0 It is 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, for example, 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 group having 1 to 5 carbon atoms, a phenyl group or an alkyl-substituted phenyl group. Preferably, R1 and R2 each independently represent a methyl group, an ethyl group, an n-butyl group, an isobutyl group, a phenyl group or a benzyl group.

[0081] In one embodiment, in the molecular chain of the polymer of the present invention, the number of repeating units of the structural unit I is 1-300, preferably 5-150, and more preferably 10-70.

[0082] In one embodiment, in the polymer of the present invention, the content ratio of the structural unit I is 5 to 70 mol %, preferably 10 to 60 mol %, based on the molar number of the total structural units.

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

[0084]

[0085] Structural Unit II

[0086] In the polymer of the present invention, the structural unit II is represented by formula II, wherein the parameters appearing therein have one of the meanings given above and below. The structural unit II is derived from the following monomer 2.

[0087] In one embodiment, G is a hydroxyl group, a carboxyl group, a sulfonic acid group, a phosphoric acid group, a sulfate group, an amide group, an amine group, a quaternary ammonium salt group, a morpholine group or a polyethylene glycol segment, wherein a hydroxyl group, a carboxyl group, an amide group, an amine group, a morpholine group or a polyethylene glycol segment is preferred, and from the perspective of imparting better water solubility and safety to the polymer, a polyethylene glycol segment, an amine group or a morpholine group is more preferred.

[0088] In one embodiment, the polyethylene glycol segment is an alkyl-terminated polyethylene glycol segment, wherein the terminal alkyl group is an alkyl group having 1 to 5 carbon atoms, preferably a methyl group, an ethyl group or a propyl group, more preferably a methyl group; the number of repeating units of the oxyethyl group in the segment is 10 to 40, preferably 15 to 30, and more preferably 15 to 25.

[0089] In one embodiment, the amine 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, and 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 a methyl group, an ethyl group or a propyl group, and the halogen counter ion is a chloride ion or a bromide ion.

[0091] In one embodiment, X represents a single bond, -O-, -S-, -CO-, -NR 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or an alkyl group having 1 to 10 carbon atoms, and X preferably represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-.

[0092] 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 1-300, preferably 5-150, and more preferably 10-70.

[0093] In the embodiment where G in formula II is a hydrophilic group, 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 ratio of the structural unit II is 30 to 95 mol %, preferably 40 to 90 mol %, based on the molar number of the total structural units.

[0095] In one embodiment, the 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 the following formula (2), formula (3) or formula (4):

[0097]

[0098] Other structural units

[0099] The polymer of the present invention may also optionally contain other structural units, for example structural units from monomers selected from (meth)acrylates, vinyl acetate, styrenes.

[0100] In one embodiment, in the polymer of the present invention, the content ratio of other structural units is 20 mol% or less, preferably 10 mol% or less, and more preferably 5 mol% or less, based on the molar number of the total structural units.

[0101] Capping Group

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

[0103]

[0104] The dotted line represents a connecting bond, and R5 represents an alkyl group having 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 molecular chains of the polymer of the present invention each have the following end-capping groups:

[0106]

[0107] The dotted lines represent connecting bonds.

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

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

[0110]

[0111] In formula (5), x and y each independently represent an integer of 1 to 300, preferably an integer of 5 to 150, more preferably an integer of 20 to 70, and n is 10 to 30, preferably 15 to 25.

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

[0113]

[0114] In formula (6), x represents an integer of 1 to 300, preferably an integer of 5 to 150, and more preferably an integer of 20 to 70; y represents an integer of 1 to 2000, preferably an integer of 30 to 1000, and more preferably an integer of 200 to 500.

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

[0116]

[0117] In formula (7), x represents an integer of 1 to 300, preferably an integer of 5 to 150, and more preferably an integer of 20 to 70; y represents an integer of 1 to 2000, preferably an integer of 30 to 1000, and more preferably an integer of 200 to 500.

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

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

[0120]

[0121] In formula (8), x and y each independently represent an integer of 1 to 300, preferably an integer of 5 to 150, and more preferably an integer of 20 to 70; and n is 10 to 30, and preferably 15 to 25.

[0122] <Controllable Molecular Weight Preparation Method>

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

[0124] Step 1: reacting compound 1 or its hydrochloride, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1;

[0125]

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

[0127]

[0128] Therein the individual radicals have one of the meanings given above and below.

[0129] The preparation method of the invention adopts reversible addition fragmentation chain transfer polymerization (RAFT), so that the molecular weight of the polymer can be accurately controlled.

[0130] The various steps of the preparation method of the present invention are described in detail below.

[0131] Step 1

[0132] In step 1, the compound 1 or its hydrochloride, the phosphite compound 2 and the aromatic aldehyde compound 3 are subjected to a Kabachnik-Fields reaction in the presence of an acid-binding agent to obtain a monomer 1.

[0133] The reaction in step 1 can be carried out in an organic solvent. Examples of the organic solvent include a mixed solvent of ethanol and acetonitrile (preferably in a volume ratio of 1:1), dichloromethane, ethyl acetate, and tetrahydrofuran.

[0134] In one embodiment, the reaction temperature of the Kabachnik-Fields reaction in step 1 is 35 to 80° C., preferably 50 to 70° C., and the reaction time is 2 to 12 hours, preferably 3 to 8 hours. In one 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, 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 has no particular limitation on the acid-binding agent used, and the acid-binding agent may be an acid-binding agent known in the art for Kabachnik-Fields reaction, including but not limited to triethylamine, pyridine, 4-dimethylaminopyridine, and the like.

[0137] Step 1 may also optionally include an operation of separating and purifying the product of the Kabachnik-Fields reaction.

[0138] In one embodiment, step 1 also includes, after the Kabachnik-Fields reaction is completed, purifying the crude product by column chromatography, and then performing rotary evaporation and freeze drying. Specifically, in the column chromatography purification, the eluent may be a dichloromethane and methanol system, and the volume ratio may be (10-30):1, preferably 20:1; the temperature of the rotary evaporation is 30-60°C, and the rotation speed is 0.5-10r / s; the temperature of the freeze drying is -20--50°C, and the vacuum degree is 0.1-30Pa; the time is 12-72h, preferably 20-30h.

[0139] Step 2

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

[0141] The RAFT polymerization of step 2 is carried out under anaerobic conditions, more preferably under anhydrous and anaerobic conditions. For the method of forming anaerobic conditions, it can be any method known in the art, such as by repeatedly evacuating the reaction vessel and introducing an inert gas, bubbling the reaction system with an inert gas, or performing multiple liquid nitrogen freeze-vacuum-thaw cycles. For the method of bubbling the reaction system with an inert gas, the flow rate of the inert gas can be 10 to 100 mL / min, and the bubbling time can be 5 to 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, and examples of the organic solvent include N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[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 4-24h, preferably 8-16h, and 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 the monomer 1 to the monomer 2 is 1:(3-10), preferably 1:(4-8).

[0147] The present invention has no particular limitation on 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. For azo initiators, azobisisobutyronitrile (AIBN for short), azobisisoheptanenitrile (ABVN for short) and the like can be listed; for peroxide initiators, dibenzoyl peroxide (BPO for short) and tert-butyl hydroperoxide and the like can be listed. These initiators can be used alone or in combination.

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

[0149] The present invention has no particular limitation on the chain transfer agent used, and the chain transfer agent may be any chain transfer agent known in the art that is suitable for RAFT polymerization reaction, such as thiocarbonylthio compounds, etc., specifically including but not limited to dithiobenzoates, trithiosulfates and dithiocarbamates, etc., preferably trithiocarbonates.

[0150] In one embodiment, the chain transfer agent used in the present invention has a structure represented by the following formula (9):

[0151]

[0152] Among them, 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, and neopentyl; R6 represents an alkyl carboxylic acid group optionally substituted with -CN, such as 4-cyanopentanoyl, etc.

[0153] Specific examples of the chain transfer agent include 4-cyano-4-(((ethylthio)thiocarbonyl)thio)pentanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-((((2-carboxyethyl)thio)carbonylthio)thio)-4-cyanopentanoic acid, and 4-cyano-4-(phenylcarbonylthio)pentanoic acid.

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

[0155] In one embodiment, step 2 is to polymerize monomer 1, monomer 2 and other monomers in the presence of a free radical polymerization initiator and a chain transfer agent. The present invention has no particular limitation on other monomers, which may be monomers known in the art that can be copolymerized with monomer 1 and monomer 2, including but not limited to (meth)acrylate monomers, vinyl acetate, styrene monomers, etc.

[0156] Step 2 optionally further comprises the operation of 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, the reaction vessel is cooled in ice water, and then the reaction solution is dialyzed and purified in an organic solvent, and then rotary evaporated and freeze-dried. Specifically, in the dialysis purification, the molecular weight cutoff of the dialysis membrane used is 1000 to 10000, and the dialysis time is 24-72h; the organic solvent used in the dialysis purification is one or more selected from ethanol, methanol, acetonitrile, and dichloromethane. Specifically, the temperature of the rotary evaporation is 30 to 60°C, and the rotation speed is 0.5 to 10r / s. Specifically, the temperature of the freeze drying is -20 to -50°C, the vacuum degree is 0.1 to 30Pa, and the time is 12-72h, preferably 20 to 30h.

[0158] <Medication>

[0159] Another object of the present invention is to provide a drug, which includes a polymer according to the present invention, and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" described in the present invention may also be referred to as an "excipient", "pharmaceutically acceptable adjuvant", "adjuvant" or "additive", etc., which refers to the general term for all additional materials used in the preparation of prescriptions and the production of drugs, except for the active ingredient. It is generally a pharmaceutically inert ingredient, and a reasonable evaluation has been conducted in terms of safety. Pharmaceutically acceptable carriers can enhance the handling characteristics of pharmaceutical preparations, for example, by increasing fluidity and / or adhesion so that the preparation meets the process requirements. Furthermore, the "pharmaceutically acceptable carrier" should have good compatibility with the active ingredient, that is, the carrier itself or the impurities contained therein will not react chemically with the structural groups in the active ingredient or cause the active ingredient to degrade, 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, glidants, anti-adhesive agents), stabilizers, fillers (or diluents), as well as flavoring agents, thickeners, dispersants, colorants, antibacterial agents, antioxidants, pH adjusters, surfactants, flavors and coating materials (plasticizers, opacifiers, pigments) and other substances.

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

[0162] In one embodiment, the drug of the present invention is an anticancer drug. "Anticancer drug" herein refers to a drug used to treat and / or prevent cancer.

[0163] Cancers that can be treated and / or prevented by the drug of the present invention include, but are not limited to, lung cancer, malignant melanoma, cervical cancer, liver cancer, breast cancer, esophageal cancer cells, etc.

[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, for example, by injection.

[0166] <Purpose>

[0167] The present invention also relates to the use of the polymer according to the present invention for preparing 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 above polymer or drug to a patient in need thereof. 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 present invention is further described by enumerating specific implementation cases below. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the present invention equally.

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

[0172] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

[0174] according to Figure 1 The synthetic routes shown were used to prepare polymers P1 and P2 as follows.

[0175] 1. Preparation of polymer 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), diethyl phosphite (3.31 g, 24 mmol) were dissolved in 20 mL of a mixed solvent system of ethanol and acetonitrile in a volume ratio of 1:1, and placed in a 65°C oil bath for reaction for 5 hours. After the reaction, column chromatography was used for purification, and the eluent was dichloromethane and methanol in a volume ratio of 20:1. The monomer M1 was collected by rotary evaporation and freeze drying, which was 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)pentanoic acid (53 mg, 0.2 mmol), azobisisobutyronitrile (13 mg, 0.08 mmol) were dissolved in 20 mL N,N-dimethylformamide (chain transfer agent: M1 = 0.02:1), deoxygenated with nitrogen for 30 minutes, and then placed in a 75°C oil bath for 12 hours. After the reaction was completed, the reaction solution was cooled in ice water, and the reaction solution was dialyzed and purified in a methanol solution with a dialysis bag with a molecular weight cutoff of 3500 for 48 hours. The 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)pentanoic acid (176 mg, 0.66 mmol), azobisisobutyronitrile (43 mg, 0.26 mmol) were dissolved in 20 mL N,N-dimethylformamide (chain transfer agent: M1 = 0.066: 1), deoxygenated with nitrogen for 30 minutes, and then placed in a 75°C oil bath for 12 hours. After the reaction was completed, the reaction solution was cooled in ice water, and the reaction solution was dialyzed and purified in a methanol solution with 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] The polymers P1 and P2 were characterized by NMR and GPC.

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

[0183] Depend on Figure 3 The gel permeation chromatography chart shown shows 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 the solution to prepare a 0.5 mM Cu 2+ 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 the solution to prepare a 0.5 mM Cu 2+ of P2 polymer (0.1 mg / mL).

[0187] The above two solutions were characterized by UV-visible absorption spectroscopy. Figure 4 shown.

[0188] Depend on Figure 4 It can be seen that in the 2+ After the combination, polymers P1 and P2 showed a significant UV absorption peak at 261nm and 262nm, respectively, indicating that polymers P1 and P2 bind to Cu 2+ Have good bonding ability.

[0189] Example 3: Cancer cell inhibition test of polymers P1 and P2

[0190] 1. CCK-8 test

[0191] The 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). The cell culture medium without polymer P1 was used as a reference. After co-culture 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 by CCK-8 test method. The effect of polymer P2 on the proliferation of different cells was evaluated by the same method. The results are shown in Figure 2. Figure 5 As shown in a) and b).

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

[0193] From the above results, it can be seen that the cell survival rate and concentration curve of polymers P1 and P2 are close, indicating that the difference in molecular weight between polymers P1 and P2 has no significant effect on the inhibition of cancer cells.

[0194] 2. FDA-PI method test

[0195] The polymer P1 and polymer 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, including 0h (cells before adding polymer), 24h-control group (blank cultured cells), 24h-polymer P1 group (polymer P1 co-cultured cells), and 24h-polymer P2 group (polymer P2 co-cultured cells), were tested for cell viability using the FDA-PI method to evaluate the effects of polymer P1 and polymer P2 on the proliferation of different cells. The results are shown in the figure. 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 life and death of cells under a fluorescence microscope.

[0198] Mouse fibroblasts (L929) were used as healthy cells to evaluate the safety of the polymers. 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 degrees of cell proliferation compared to the "0h group", and no obvious dead cells were observed, indicating that polymers P1 and 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 to the "24h-control group", which indicates that polymers P1 and P2 have a significant proliferation inhibitory effect on mouse breast cancer cells.

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

[0201] Example 4: Test on the inhibition of breast cancer metastasis in mice by polymers P1 and P2

[0202] Mouse breast cancer cells (bioluminescent marker) were suspended in PBS at 2×10 5 The concentration of cells / mouse was injected into Balb / C mice through the tail vein to construct a mouse breast cancer metastasis model. 24 hours before implanting the cells, 0.5mL of normal saline (control group), 0.5mL of polymer P1 solution (100mg / mL), 0.5mL of polymer P2 solution (100mg / mL) and 0.2mL of tetrathiomolybdate (5mg / mL) were injected through the tail vein for preventive protection. After 10 and 15 days of normal feeding after cancer cell injection, the distribution of tumor tissue in the mice was observed using a small animal in vivo optical three-dimensional imaging system. The results are shown in the figure. Figure 7 As shown in a) and b).

[0203] Depend on Figure 7 a) It can be seen that 10 days after the injection of cancer cells, mice in the control group had large-scale tumor tissue spread in the lungs, indicating that the mouse breast cancer metastasis model was successfully established; the tumor tissue of mice in the tetrathiomolybdate group was not significantly different from that in the control group, indicating that the small molecule drug tetrathiomolybdate had a poor therapeutic effect. The mice in the polymer P2 group had significantly less lung cancer tissue than those in the control group, but a small amount of cancer cell metastasis was still observed in the lungs; breast cancer tissue was almost not observed in the lungs of mice in the polymer P1 group. The above results show 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 and have a good anti-cancer effect.

[0204] Depend on Figure 7 b) It can be seen that 15 days after the cancer cell injection, obvious lung metastasis of cancer can be observed in the lungs of mice in the polymer P2 group, while no breast cancer tissue can be observed in the lungs of mice in the polymer P1 group. This shows that the molecular weight of the polymer can significantly affect the inhibitory effect of breast cancer metastasis. The larger the molecular weight, the better the anti-cancer effect.

[0205] The survival of mice in each experimental group within 60 days was recorded. Figure 8 shown.

[0206] Depend on Figure 8From the survival curve, it can be seen that the median survival time of mice in the control group and the tetrathiomolybdate group was 12.5 days and 12 days, respectively, and there was no significant difference between the two curves (p = 0.2932), which indicates that the small molecule drug tetrathiomolybdate has a poor therapeutic effect. The median survival time of mice in the polymer P1 group and the polymer P2 group was 18 days and 25.5 days, respectively, showing a significant extension of the survival time relative to the control group mice (P1, p ~ 0.0001, P2, p = 0.2932), which indicates that polymers P1 and P2 can effectively inhibit the metastasis of breast cancer. The polymer P1 group showed a significant extension of the survival time relative to the polymer P2 group, which further indicates that the molecular weight of the polymer can significantly affect the inhibitory effect of breast cancer metastasis, and the larger the molecular weight, the better the anti-cancer effect.

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

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

[0209] 0.5mL fluorescent polymer P1-Cy7 (100mg / mL), 0.5mL fluorescent polymer P2-Cy7 and 0.5mL small molecule Cy7 (1mg / mL) were injected into mice through the tail vein. The fluorescence signal in the mouse body was recorded by the small animal in vivo optical three-dimensional imaging system 1 hour (0 day) and 1 to 10 days after the tail vein injection, and the metabolic behavior of P1-Cy7, P2-Cy7 and Cy7 was observed. The results are shown in Fig. 9 shown.

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

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

[0212] Industrial Applicability

[0213] The polymer 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, esophageal cancer cells, etc.

Claims

1. A polymer containing 8-hydroxyquinoline and aminophosphoric acid ester structure, characterized in that: Containing structural unit I shown in formula I and structural unit II shown in formula II: wherein R1 and R2 each independently represent an alkyl group having 1 to 10 carbon atoms or a phenyl group optionally substituted by 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 is 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, and X represents a single bond, -O-, -S-, -CO-, -NR 0 -, -C(=O)NH-, -C(=O)O-, -OC(=O)- or an alkylene group having 1 to 10 carbon atoms, G represents a hydrophilic group or a hydrophilic segment; The weight average molecular weight of the polymer is 15,000 or more.

2. The polymer according to claim 1, characterized in that In the formula I, Z represents -O- or -NR 0 -, B represents an alkylene group having 1 to 10 carbon atoms; in the formula II, G represents a hydroxyl group, a carboxyl group, a sulfonic acid group, a phosphoric acid group, a sulfate group, an amide group, an amine group, a quaternary ammonium salt group, a morpholine group or a polyethylene glycol segment, and X represents a single bond, -CO-, -C(=O)NH- or -C(=O)O-; The weight average molecular weight of the polymer is 30,000 or more, preferably 40,000 or more, and more preferably 50,000 or more.

3. The polymer according to claim 1 or 2, characterized in that One end of the molecular chain of the polymer has the following end-capping group: The dotted line represents a connecting bond, and R5 represents an alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and neopentyl.

4. The method for preparing a polymer according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: reacting compound 1 or its hydrochloride, compound 2 and compound 3 in the presence of an acid-binding agent to obtain monomer 1; Step 2: subjecting monomer 1 and monomer 2 to reversible addition-fragmentation chain transfer polymerization in the presence of a free radical polymerization initiator and a chain transfer agent; Each group has the definition in any one of claims 1 to 3.

5. The preparation method according to claim 4, 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 dibenzoyl 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.

6. The preparation method according to claim 4 or 5, characterized in that: The chain transfer agent is one or more selected from thiocarbonylthio compounds, and preferably the chain transfer agent has a structure shown in the following formula (9): Wherein, R5 represents an alkyl group having 1 to 5 carbon atoms; R6 represents an alkyl carboxylic acid group optionally substituted with -CN; More preferably, the chain transfer agent is 4-cyano-4-(((ethylthio)thiocarbonyl)thio)pentanoic acid; 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.

7. The preparation method according to claim 4 or 5, characterized in that: The reversible addition-fragmentation chain transfer polymerization is carried out under anaerobic and preferably anhydrous conditions; the reaction temperature is 55 to 100° C., and the reaction time is 4 to 24 hours.

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

9. A drug, characterized in that The invention comprises the polymer according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

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

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