PH-responsive polypeptide simulant as well as preparation method and application thereof

Abstract

The invention relates to a pH responsive polypeptide simulant as well as a preparation method and application thereof. Specifically, the invention provides a pH responsive polymer, and the skeleton structure of the polymer is an alpha amino acid polymer, polyoxazoline, a beta amino acid polymer, an alpha / beta amino acid polymer, a gamma amino acid polymer or polyoxazine. In the polymer, side chains of at least part of polymerization units contain amino groups amidated by pH responsive molecules; the polymer has low toxicity to mammalian cells and prolonged blood circulation time in a physiological environment; pH responsive charge reversal occurs in a microbial infection part or an acidic microenvironment of a tumor, so that efficient antibacterial activity to gram-negative bacteria and / or gram-positive bacteria and / or fungi is recovered, or the antitumor activity has a good application prospect in the aspects of improving drug specific delivery and treating infectious diseases and cancers.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a preparation method of a pH-responsive polypeptide mimetic and its antibacterial / antitumor application. Background Art

[0002] Drug-resistant bacterial infections and cancers have become a huge threat to human life and health. With the rapid development of microbial resistance, infections that could have been effectively treated with antibiotics have become difficult to control. At the same time, cancer, as a complex and stubborn disease, has become a major challenge in the field of global public health due to its high morbidity and mortality. In addition, due to the existence of tumor heterogeneity and multidrug resistance, existing treatments face significant limitations, leading to increased risks of treatment failure and recurrence. Therefore, there is an urgent need to develop effective new antibacterial / antitumor drugs to protect human life safety and health.

[0003] Host defense peptides are an important component of the innate immune system in organisms. They have broad-spectrum antibacterial, antifungal, and tumor cell inhibition biological activities and are not easy to induce drug resistance in microorganisms / tumor cells. They have been widely studied as promising new antibacterial / antitumor drugs. However, the inherent disadvantages of natural host defense peptides, such as easy degradation by proteases, cumbersome preparation, and high cost, limit their practical application. In addition, in complex clinical environments, cationic host defense peptides may interact nonspecifically with host cells with polyanionic surface structures such as red blood cells or polyanionic macromolecules such as serum proteins, which may lead to reduced antibacterial / antitumor activity in vivo, and even undesirable characteristics such as hemolytic toxicity or short half-life.

[0004] Therefore, there is a need in the art to provide drugs with antibacterial, antitumor activity and safety. Summary of the invention

[0005] The present invention provides a class of polypeptide mimetics with novel structure, pH responsiveness and excellent antibacterial / antitumor activity, as well as a preparation method and application thereof.

[0006] In a first aspect of the present invention, a pH-responsive polymer, wherein the backbone structure of the polymer is polyoxazoline, α-amino acid polymer, β-amino acid polymer, α / β-amino acid polymer, γ-amino acid polymer or polyoxazine;

[0007] And in the polymer, at least part of the side chains of the polymerized units contain the A1 structure;

[0008]

[0009] L is a substituted or unsubstituted group selected from the group consisting of: none, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 alkyl-O-C0-C4 alkyl, C1-C4 alkyl-S-C0-C4 alkyl, C1-C4 alkyl-NH-C0-C4 alkyl, C1-C4 alkyl-CO-C0-C4 alkyl, C1-C4 alkyl-COO-C0-C4 alkyl, C3-C10 cycloalkyl, 3-10 membered heterocyclic group including 1-3 heteroatoms selected from O, N, S, C6-C10 aryl, 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S, C1-C6 alkyl-Rc, C1-C6 alkyl-COO-Rc, C1-C6 alkyl-O-Rc; each Rc is independently selected from the group consisting of substituted or unsubstituted benzyl, C3-C10 cycloalkyl, 3-10 membered heterocyclic group including 1-3 heteroatoms selected from O, N, S, C6-C10 aryl, 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S; the substitution means that one or more H on the group are independently and optionally substituted by a group selected from the group consisting of OH or C1-C4 alkyl;

[0010] X is NH, NR, guanidyl biguanidyl wherein R is C1-C6 alkyl;

[0011] Y is a pH-responsive molecular fragment, and Y is selected from the group consisting of: preferably

[0012] In another preferred embodiment, L is a substituted or unsubstituted group selected from the group consisting of: none, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 alkyl-O-C0-C4 alkyl, C1-C4 alkyl-S-C0-C4 alkyl, C1-C4 alkyl-NH-C0-C4 alkyl, C1-C4 alkyl-CO-C0-C4 alkyl, C1-C4 alkyl-COO-C0-C4 alkyl; wherein the substitution means that one or more H on the group are independently and optionally substituted by a group selected from the group consisting of OH or C1-C4 alkyl.

[0013] In another preferred embodiment, L is C1-C8 alkyl, preferably C1-C4 alkyl.

[0014] In another preferred embodiment, the degree of polymerization of the polymer is a positive integer from 2 to 500, preferably from 3 to 300, such as 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 150, 200 or 250.

[0015] In another preferred example, in the polymer, the proportion x% of the polymerization units containing side chains with the A1 structure among all the polymerization units is at least 1%, at least 5% or at least 10%, such as 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0016] In another preferred example, when the polymerization unit has a side chain containing the A1 structure, the polymerization unit has only a single side chain, or also has one or two side chains selected from C1-C8 alkyl groups.

[0017] In another preferred example, in addition to the polymerization units containing side chains with the A1 structure, the polymer further contains one or two types of polymerization units with other side chains, and the polymerization units independently have the following characteristics:

[0018] The side chain of the polymerization unit contains the structure: -L-X-H(A2); where L and X are defined as above, the polymerization unit has only a single side chain, or also has one or two side chains selected from C1-C8 alkyl groups;

[0019] The polymerization unit has 1, 2 or 3 side chains, and the side chains independently contain the structure: L-R2; where L is defined as above, and R2 is selected from the following group: H, OH, COOH, C1-C8 alkyl group, substituted or unsubstituted C3-C10 cycloalkyl group, substituted or unsubstituted 3-10 membered heterocyclic group containing 1-3 heteroatoms selected from O, N, S, substituted or unsubstituted benzyl group, substituted or unsubstituted biphenyl group, substituted or unsubstituted C6-C10 aryl group, substituted or unsubstituted 5-10 membered heteroaryl group containing 1-3 heteroatoms selected from O, N, S; wherein, the substitution means that one or more H on the group are optionally substituted by a group selected from the following group: OH, COOH or C1-C4 alkyl group; or two side chain groups on adjacent backbone carbon atoms together with the connected carbon atom form a substituted or unsubstituted C3-C10 cycloalkyl group or a substituted or unsubstituted 3-10 membered heterocyclic group containing 1-3 heteroatoms selected from O, N, S, wherein, the substitution means that one or more H on the group are optionally substituted by a group selected from the following group: OH, COOH or C1-C4 alkyl group.

[0020] In another preferred example, in the amino acid polymer, the first connection site of the side chain on each polymerization unit is shown as Z1 in any of the following structures:

[0021] wherein, m is independently 0, 1 or 2; when the polymerization unit has 2 or 3 side chains, the second and third side chains can be located at other substitutable positions of the backbone carbon atoms, and when present, A1 or A2 is located at the Z1 site.

[0022] In another preferred example, in polyoxazoline or polyoxazine, the first linking site of the side chain on each polymerization unit is as shown by Z1:

[0023] wherein, m is independently 1 or 2; when there are 2 or 3 side chains on the polymerization unit, the second and third side chains may be located at other substitutable positions of the backbone carbon atoms, and when present, A1 or A2 is located at the Z1 site.

[0024] In another preferred example, the polymer has a segment shown by Formula I, Formula II or Formula III:

[0025]

[0026]

[0027] In Formula I, m is independently 1 or 2;

[0028] In Formula II and Formula III, m is independently 0, 1 or 2;

[0029] In each formula, n is independently a positive integer from 2 to 500;

[0030] x is independently from 1 to 100, y is independently from 0 to 100, z is independently from 0 to 99, and x + y + z = 100;

[0031] L, X, Y, R2 are independently defined as above.

[0032] In another preferred example, one end of the polymer is H, a monomer or an initiator residue (such as p-tert-butylbenzyl), and the other end is H, a monomer residue or other reactive end groups, such as -R a , C1-C8 alkylene R a (such as -tBu tert-butyl R a ), C1-C8 haloalkyl R a (preferably C3-C8 fluoroalkyl R a ), C6-C10 aryl-R a , C1-C8 alkylene C6-C10 aryl-R a , -NHC1-C8 alkylene R a , -OC1-C8 alkylene R a , -COC1-C8 alkylene R a ; wherein each R a is independently selected from the group consisting of: -SH, -NH 2 , -COOH, CHO, -Br, -Cl, -OH, 3-6 membered epoxy group, alkenyl, alkynyl, -COCl, azide group, maleimide group, o-dithiopyridyl (OPSS), cyclodextrin, adamantane.

[0033] In another preferred embodiment, the initiator is selected from the group consisting of: LiHMDS, NaHMDS, KHMDS, trityl mercaptoethylamine, p-tert-butylbenzylamine, and triphenylmercaptopropyl 3-bromide.

[0034] In another preferred embodiment, x is independently 1 - 100, preferably 5 - 90, such as 10, 15, 20, 25, 30, 40, 50, 60, 70, or 80.

[0035] In another preferred embodiment, y is independently 0 - 99, preferably 5 - 60, such as 10, 15, 20, 25, 30, 40, or 50.

[0036] In another preferred embodiment, z is independently 0 - 99, preferably 5 - 60, such as 10, 15, 20, 25, 30, 40, or 50.

[0037] In another preferred embodiment, n (degree of polymerization) is independently 3 - 300, such as 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 150, 200, or 250.

[0038] In another preferred embodiment, each chiral center in the polymer can be of the S configuration or the R configuration.

[0039] In another preferred embodiment, the polymer includes homopolymers, random copolymers, and block copolymers.

[0040] In another preferred embodiment, the polymer is in the form of a linear polymer, a dendritic polymer, or a polymer brush. In another preferred embodiment, the pH-responsive polymer structure includes:

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] In a second aspect of the present invention, there is provided a method for preparing the polymer as described in the first aspect of the present invention, comprising the following steps:

[0048] Providing an aqueous solution of the backbone polymer and adjusting the pH value to 8.5 - 9;

[0049] Add pH-responsive molecules to the polymer solution, and adjust the pH to always remain between 8.5 and 9 with a base (such as NaOH) during the addition.

[0050] React and stir for 6 - 24 h (preferably 8 - 16 h).

[0051] Dialyze the reaction solution in deionized water with a pH of 8.5 - 9, and then dry the sample to obtain the pH-responsive polypeptide mimetic polymer.

[0052] In another preferred example, the method includes the steps of:

[0053] Dissolve the polymer in deionized water, and adjust the pH value to 8.5 - 9 with a sodium hydroxide solution.

[0054] After stirring for 10 minutes under an ice bath, add different molar amounts of pH-responsive molecules to the polymer solution in batches, and adjust the pH to always remain between 8.5 and 9 with a sodium hydroxide solution during the addition.

[0055] React and stir overnight.

[0056] Dialyze the reaction solution in deionized water with a pH of 8.5 - 9, and then lyophilize the sample to obtain a powdery pH-responsive polypeptide mimetic product.

[0057] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the polymer according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0058] In a fourth aspect of the present invention, there is provided the use of the polymer according to the first aspect of the present invention in the preparation of a pharmaceutical composition for antibacterial and / or anti-tumor purposes.

[0059] The application of the polymer according to the first aspect of the present invention in the preparation of a pharmaceutical composition or material having one or more functions selected from the following group:

[0060] The pH responsiveness improves the specific delivery of polypeptide mimetics to tissues with an acidic microenvironment such as the site of microbial infection and tumor tissues.

[0061] The pH responsiveness causes charge inversion to penetrate the mucus barrier and improves pulmonary drug delivery.

[0062] The pH responsiveness has antibacterial effects.

[0063] The pH responsiveness has anti-tumor effects.

[0064] In another preferred example, antibacterial includes Gram-positive bacteria, Gram-negative bacteria, fungi, spores, and dormant cells.

[0065] In another preferred example, Gram-positive bacteria include Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus, Bacillus, Listeria, Bacillus anthracis; Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, Vibrio cholerae; fungi include Candida albicans, Cryptococcus neoformans, Candida tropicalis, Candida glabrata, Cryptococcus gattii, Aspergillus, dermatophyte, Histoplasma capsulatum.

[0066] In another preferred example, the above-mentioned tumor is a solid cancer or a blood cancer, such as gastric cancer, cervical cancer, melanoma, endometrial cancer, esophageal cancer, liver cancer, prostate cancer, breast cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, colon cancer, uterine tumors and soft tissue sarcoma, lymphoma, leukemia, multiple myeloma, mesothelioma, malignant rhabdoid tumor, cholangiocarcinoma and gallbladder cancer, bladder cancer; brain tumors, neuroblastoma, schwannoma, glioma, glioblastoma and astrocytoma.

[0067] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 In vivo hemolysis results of polyoxazoline before and after modification with 2,3-dimethylmaleic anhydride.

[0069] Figure 2 In vivo acute toxicity test results of pH-responsive polyoxazoline. Where a is the maximum acceptable dose of a single intravenous injection of polyoxazoline before and after modification with 2,3-dimethylmaleic anhydride; b is the body weight change of mice after intravenous injection of pH-responsive polyoxazoline.

[0070] Figure 3 Serum biochemical indexes of mice after intravenous injection of pH-responsive polyoxazoline.

[0071] Figure 4 Histological analysis of the liver and kidneys of mice after intravenous injection of pH-responsive polyoxazoline.

[0072] Figure 5 Results of the interaction between polyoxazoline before and after modification with 2,3-dimethylmaleic anhydride and plasma proteins.

[0073] Figure 6 Blood circulation time of polyoxazoline before and after modification with 2,3-dimethylmaleic anhydride.

[0074] Figure 7The experimental results of pH-responsive polyoxazoline. Among them, a is the characterization of 1H NMR spectrum; b is the characterization of Zeta potential.

[0075] Figure 8 The time-killing kinetics of pH-responsive polyoxazoline at different pH values.

[0076] Figure 9 The in vivo transformation study of pH-responsive polyoxazoline. Among them, a is the schematic diagram of acid-responsive deprotection of dual-fluorescent dye polyoxazoline; b is the fluorescence confocal imaging of infected tissues; c is the curve of relative fluorescence intensity changing with time; d is the removal rate of pH-responsive small molecules.

[0077] Figure 10 The pH-responsive accumulation of fluorescently labeled polyoxazoline in infected tissues.

[0078] Figure 11 The treatment results of pH-responsive polyoxazoline on subcutaneous abscess bacterial infection in mice.

[0079] Figure 12 The treatment results of pH-responsive peptidomimetic polymers on fungal infection of skin abrasions in mice.

[0080] Figure 13 The treatment results of pH-responsive polyoxazoline on bacterial infection of thigh muscles in mice.

[0081] Figure 14 The treatment results of pH-responsive polyoxazoline on bacterial infection of lungs in mice.

[0082] Figure 15 The treatment results of pH-responsive polyoxazoline on bacterial infection of kidneys in mice.

[0083] Figure 16 The treatment results of pH-responsive polyoxazoline on systemic bacterial infection in mice. Among them, a is the survival rate of infected mice after treatment; b is the bacterial load in various organs, blood and peritoneal lavage fluid of infected mice after treatment; c is the histological analysis of various organs of infected mice after treatment. Detailed implementation methods

[0084] Through extensive and in-depth research, the present inventor provides the use of a class of pH-responsive polypeptide mimics in the treatment of microbial infections or anti-tumor applications. The inventor obtained a class of pH-responsive polymers by amidating the side-chain amino groups of pH-responsive small-molecule polypeptide polymers. Surprisingly, the polymers have high biocompatibility and extended blood circulation time in the physiological environment, and can restore high antibacterial / anti-tumor activity by removing the pH-responsive small molecules in the acidic microenvironment of the infection site or tumor tissue, showing good application prospects in improving drug-specific delivery and treating infectious diseases and cancers. Based on the above findings, the inventor completed the present invention.

[0085] Term

[0086] In the present invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0087] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left. For example, -CH 2 O- is equivalent to -OCH 2 -.

[0088] As used herein, the term "plurality" means two or more.

[0089] As used herein, the compounds in the present invention can be expanded in scope by taking any number of substituents or functional groups. Generally, the term "substituted", whether it appears before or after the term "optional", includes the general formula of substituents in the present invention, which means replacing a hydrogen radical with a specified structural substituent. When multiple positions in a specific structure are substituted by multiple specific substituents, each position of the substituent can be the same or different. The term "substituted" used herein includes all allowable substitutions of organic compounds. Broadly speaking, allowable substituents include acyclic, cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, a heteroatom such as nitrogen can have a hydrogen substituent or any allowable organic compound described above to supplement its valence state. In addition, the present invention is not intended to limit the allowable substituted organic compounds in any way.

[0090] Unless otherwise stated, it is assumed that any heteroatom with an unsatisfied valence state has sufficient hydrogen atoms to supplement its valence state.

[0091] As used in the text, the term "statistic coplymer" or "random copolymer" refers to a polymer formed by the simultaneous polymerization of two or more monomers and connected irregularly.

[0092] Prefix "C u -C v” means that the following group has u to v carbon atoms, such as “C 1 -C 6 ” can be C 1 、C 2 、C 3 、C 4 、C 5 or C 6 。 For example, “C 1 -C 6 alkyl” means that the alkyl has 1 to 6 carbon atoms. C0-C4 can be C 0 、C 1 、C 2 、C 3 、C 4 。

[0093] The term “plurality” refers to 2 or more, such as 2, 3, 4, 5 or 6.

[0094] The term “halogen” or “halo” refers to fluorine, chlorine, bromine, iodine.

[0095] The term “alkyl” refers to a straight-chain or branched-chain unsubstituted hydrocarbon group having 1-8 carbon atoms (i.e., C 1 -C 8 alkyl), preferably a hydrocarbon group having 1-6 carbon atoms (i.e., C 1 -C 6 alkyl), more preferably a hydrocarbon group having 1-4 carbon atoms (i.e., C 1 -C 4 alkyl). Examples of “alkyl” include but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl, etc.

[0096] The term “alkenyl” refers to a straight-chain or branched-chain hydrocarbon group having 2-8 carbon atoms (i.e., C 2 -C 8 alkenyl), preferably 2-6 carbon atoms (i.e., C 2 -C 6 alkenyl) or 2-4 carbon atoms (i.e., C 2 -C 4 alkenyl), and having 1-2 carbon-carbon double bonds. Examples of alkenyl include but are not limited to vinyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0097] The term “alkynyl” refers to a straight-chain or branched-chain hydrocarbon group having 2-8 carbon atoms (i.e., C 2 -C 8 alkynyl), preferably 2-6 carbon atoms (i.e., C 2 -C 6alkynyl) or 2 to 4 carbon atoms (i.e., C 2 -C 4 alkynyl), and is a straight-chain or branched-chain hydrocarbon group having 1 to 2 carbon-carbon triple bonds.

[0098] The term "alkylene" refers to a saturated divalent hydrocarbon group derived by removing two hydrogen atoms from an alkyl group as defined above, such as C 1 -C 8 alkylene, preferably a hydrocarbon group having 1 to 6 carbon atoms (i.e., C 1 -C 6 alkylene), more preferably 1 to 3 carbon atoms (i.e., C 1 -C 3 alkylene). Examples of "alkylene" include but are not limited to methylene, ethylene, isopropylidene, etc. Alkenylene and alkynylene are divalent groups derived by removing two hydrogen atoms from an alkenyl group and an alkynyl group as defined above, respectively.

[0099] The term "cycloalkyl" refers to a non-aromatic, saturated or partially unsaturated cyclo-hydrocarbon group, which may optionally be substituted by one or more substituents described in the present application, and has 3 to 6 carbon atoms to form a monocyclic ring, or 7 to 12 carbon atoms to form a bicyclic ring. As used herein, cycloalkyl has 3 to 8 ring carbon atoms (i.e., C 3 -C 8 cycloalkyl) or 3 to 6 ring carbon atoms (i.e., C 3 -C 6 cycloalkyl). Examples of monocyclic cycloalkyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, cycloheptyl, cyclooctyl. Exemplary bridged bicyclic cycloalkyls include but are not limited to bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane.

[0100] The terms "aromatic ring" and "aryl" refer to aromatic carbocyclic groups having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused systems. As used herein, aryl has 6 to 10 ring carbon atoms (i.e., C 6 -C 10 aryl). Aryl includes bicyclic groups that include aromatic rings fused to saturated or partially unsaturated carbocyclic or heterocyclic rings (such as benzo C 3 -C 6 cycloalkyl or benzo 4-6 membered heterocyclic group). Generally, aryl groups include but are not limited to the following groups: benzene, naphthalene, anthracene, biphenyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthalenyl, 2,3-dihydrobenzofuranyl, and the like. The "aryl" category includes structures of aryl rings fused to cycloalkyl and heterocycloalkyl.

[0101] The terms "heterocyclic", "heterocyclic group" and "heterocyclic moiety" refer to optionally substituted, fully saturated or partially unsaturated non-aromatic ring groups, for example, it can be a 3-7 membered monocyclic, 7-11 membered bicyclic or 10-15 membered tricyclic system, which has at least one heteroatom in at least one carbon-containing ring. Each ring of the heterocyclic moiety containing a heteroatom can have 1, 2 or 3 heteroatoms selected from oxygen, nitrogen and sulfur. As used herein, the heterocyclic moiety has 3 to 10 ring atoms (i.e., 3-10 membered heterocyclic moiety), 3 to 8 ring atoms (i.e., 3-8 membered heterocyclic moiety), 3-8 membered heterocyclic moiety or 3 to 6 ring atoms (i.e., 3-6 membered heterocyclic moiety) or 5 to 6 ring atoms (i.e., 5-6 membered heterocyclic moiety). The "heterocyclic moiety" can be optionally substituted by one or more substituents described in this application. Examples of the "heterocyclic moiety" include but are not limited to pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholino, thiomorpholino, piperazinyl, homopiperazinyl, glycidyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, N-pyridylurea, pyrimidinone and 1,1-dioxo-thiomorpholino.

[0102] The term "heteroaryl" or "heteroaromatic ring" refers to a heteroaromatic system containing one or more heteroatoms selected from oxygen, nitrogen and sulfur, including monocyclic, bicyclic or polycyclic fused systems. The heteroaryl can be optionally substituted by one or more substituents described in this application. As used herein, the heteroaryl can have 5 to 10 ring atoms (i.e., 5-10 membered heteroaryl), 5 to 8 ring atoms (i.e., 3-8 membered heteroaryl), or 5 to 6 ring atoms (i.e., 5-6 membered heteroaryl). The heteroaryl can have 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms or 1 ring heteroatom, wherein the ring heteroatoms are independently selected from oxygen, nitrogen and sulfur. Examples of the "heteroaryl" include but are not limited to pyrrolyl, pyridyl, pyrazolyl, imidazolyl, pyrazinyl, imidazopyridyl, benzofuranyl, pyrimidinyl, triazolyl, tetrazolyl, furanyl, thiophenyl, thiazolyl, quinolinyl, isoquinolinyl, indolyl and the like.

[0103] The term "substituted" means that one or more hydrogen atoms in a specific group are substituted by any substituent mentioned in the specification of the present invention. Unless otherwise specified, the "substitution" means that one or more (such as 2, 3 or 4) hydrogens on the group are independently substituted by a group selected from the following group: H, OH, COOH, C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocyclic group containing 1-3 heteroatoms selected from O, N, S, substituted or unsubstituted benzyl, substituted or unsubstituted biphenyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl containing 1-3 heteroatoms selected from O, N, S; wherein, the substitution means that one or more H on the group are optionally substituted by a group selected from the following group: OH, COOH or C1-C4 alkyl.

[0104] pH-responsive polymer

[0105] The present invention provides an active polymer with pH general responsiveness. By simulating the key structural features of positive charge / amphiphilicity of host defense peptides, the side-chain amino groups of pH-responsive small molecule amide polypeptides are polymerized to obtain a special class of pH-responsive antibacterial / antitumor polypeptide mimics. Due to reasons such as the metabolism of microorganisms or tumor cells, the infected site and tumor tissue both have a unique acidic microenvironment. The pH-responsive polypeptide mimics of the present invention can be invisible in the physiological environment, have high in vivo safety and extended blood circulation time; they are activated in the acidic microenvironment of the infected site / tumor tissue, remove the pH-responsive group, restore high antibacterial / antitumor activity and achieve cumulative enhancement at the same time, providing a new strategy for the treatment of drug-resistant bacterial infections / tumors.

[0106] Specifically, the backbone structure of the polymer of the present invention is based on polyoxazoline, polyoxazine, α-amino acid polymer, β-amino acid polymer, α / β-amino acid polymer, γ-amino acid polymer, preferably α-amino acid polymer, polyoxazoline.

[0107] Furthermore, in the polymer of the present invention, at least part of the side chains of the polymerization units contain the A1 structure;

[0108]

[0109] Wherein, L, X and Y are defined as above.

[0110] L is a linker group;

[0111] X is a group that can carry a positive charge under physiological conditions, such as NH, NR, guanidine group Diguanidine group Y is a pH-responsive molecular fragment.

[0112] Common pH-responsive molecules include 2,3-dimethylmaleic anhydride, citraconic anhydride, cis-aconitic anhydride, 2-propionic acid-3-methylmaleic anhydride, etc., which react with the precursor groups NH 2 、NHR, guanidyl, biguanidylamide to form the corresponding groups:

[0113] (which may contain corresponding counter cations to form acids or salts, such as H + 、Na+, K+, etc., but the carboxyl group will ionize to form anions in solution or in vivo).

[0114] In an acidic environment, the amide bond between X and Y is broken, and the pH-responsive group is removed, restoring the X group to its precursor state, which will carry a positive charge in solution or in vivo, thus restoring its high antibacterial / antitumor activity.

[0115] The polymers of the present invention can have various side chain groups. In addition to the groups having pH-responsive molecular fragments described above, which are necessary, the side chains can also contain side chains with open positively charged groups (-L-X-H), and / or side chains with lipophilicity (-L-R 2 ) groups (which can adjust the hydrophilic-lipophilic balance).

[0116] Usually, in one polymerization unit, only one L-X-Y side chain or one -L-X-H side chain is contained, but one or more (such as 2 or 3) -L-R 2 side chains can coexist with the aforementioned groups on one polymerization unit, or can be located on other polymerization units.

[0117] Usually, in the polymer, the proportion x% of the polymerization units containing side chains with A1 structure accounts for at least 1%, at least 5% or at least 10% of all polymerization units, such as 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0118] In another preferred example, the proportion y% of the polymerization units containing side chains with -L-X-H structure independently ranges from 0-99%, preferably 5-60%, such as 10%, 15%, 20%, 25%, 30%, 40% or 50%.

[0119] In the present invention, each side chain group can be located on any substitutable atom of the polymer backbone, such as on the backbone C or N atom. Preferably, in the amino acid polymer, the first connection site of the side chain on each polymerization unit is shown as Z1 in any of the following structures:

[0120] wherein, m is independently 0, 1 or 2 (representing α - amino acid, β - amino acid, γ - amino acid polymerization units respectively); when there are multiple side chains on the polymerization unit, other side chains can be located at other substitutable positions of the backbone carbon atoms. Preferably, when present, -L-X-H or -L-X-H is located at the Z1 position. In addition, Z 1 can also be located at the ortho - position of CO.

[0121] In another preferred example, in polyoxazoline or polyoxazine, the first connection site of the side chain on each polymerization unit is as shown in Z1:

[0122] wherein, m is independently 1 or 2; when there are multiple side chains on the polymerization unit, other side chains can be located at other substitutable positions of the backbone carbon atoms. Preferably, when present, -L-X-H or -L-X-H is located at the Z1 position.

[0123] Preferably, the polymer has a segment shown in Formula I, Formula II or Formula III:

[0124]

[0125]

[0126] In Formula I, m is independently 1 or 2; (when m are all 1, it corresponds to polyoxazoline, and when m are all 2, it represents polyoxazine)

[0127] In Formula II and Formula III, m is independently 0, 1 or 2;

[0128] In each formula, n is independently a positive integer from 2 to 500;

[0129] x is independently 1 - 100, y is independently 0 - 100, z is independently 0 - 99, and x + y + z = 100;

[0130] L, X, Y, R2 are independently as defined herein.

[0131] In Formula I, Formula II or Formula III, m can be the same or different, preferably the same.

[0132] In the present invention, the L - X - Y structures on different polymerization units in the same polymer can be the same or different, preferably the same. When L - X - Y and L - X - H are present on different polymerization units in the same polymer respectively, the L - X - parts in each L - X - Y and L - X - H can be the same or different, preferably the same.

[0133] Generally, both ends of the polymer have end groups, usually H, monomer or initiator residues. In particular, one end of the polymer of the present invention can have a reactive end group, such as -R a , C1 - C8 alkylene Ra (such as -tBu tert-butyl R a ), C1-C8 haloalkyl R a (preferably C3-C8 fluoroalkyl R a ), C6-C10 aryl-R a , C1-C8 alkylene C6-C10 aryl-R a , -NHC1-C8 alkylene R a , -OC1-C8 alkylene R a , -COC1-C8 alkylene R a ; wherein each R a is independently selected from the group consisting of: -SH, -NH 2 , -COOH, CHO, -Br, -Cl, -OH, 3-6 membered epoxy group, alkenyl, alkynyl, -COCl, azide group, maleimide group, o-dithiopyridyl (OPSS), cyclodextrin, adamantane. Those skilled in the art understand that the monomer residues, initiator residues, functional reactive end groups, etc. on the end groups are related to the monomer types and initiator types, but will not significantly affect the activity of the polymer segments of the present invention.

[0134] Each chiral center in the polymer can be of the S configuration or the R configuration. The polymer includes homopolymers, random copolymers and block copolymers. The polymer can be in the form of a linear polymer, a dendritic polymer or a polymer brush.

[0135] In addition, the polymer of the present invention has basic groups such as amino groups and acidic groups such as COOH, and they can exist in various salt forms, such as hydrochloride, bromate, trifluoroacetate, phosphate, lithium salt, sodium salt, potassium salt, etc.

[0136] Preparation method

[0137] The polymer of the present invention has no particular requirement for the preparation method and can be prepared by the methods commonly used in the present invention.

[0138] Preferably, the preparation method of the polymer comprises the following steps:

[0139] Provide an aqueous solution of the backbone polymer and adjust the pH value to 8.5-9;

[0140] Add the pH-responsive molecule to the polymer solution and adjust the pH to always remain at 8.5-9 with a base (such as NaOH) during the addition;

[0141] React and stir for 6-24 h (preferably 8-16 h);

[0142] Dialyze the reaction solution in deionized water with a pH of 8.5-9, and then dry the sample to obtain the pH-responsive polypeptide mimetic polymer.

[0143] It is understandable that the backbone polymer has one or more L-X-H side chains before the reaction, and after reacting with the pH-responsive molecule, the corresponding L-X-Y side chains are formed. In addition, the reaction degree of the L-X-H side chains on the backbone polymer can be controlled by controlling the feeding ratio of the pH-responsive molecule to the backbone polymer, so that it is partially or completely converted into L-X-Y.

[0144] In another preferred embodiment, the method comprises the steps of:

[0145] Dissolve the polymer in deionized water, and add sodium hydroxide solution to adjust the pH value to 8.5-9;

[0146] After stirring for 10 minutes under an ice bath, different molar amounts of the pH-responsive molecule are added to the polymer solution in batches, and the pH is adjusted with sodium hydroxide solution to always remain at 8.5-9 during the addition;

[0147] React and stir overnight;

[0148] Dialyze the reaction solution in deionized water with a pH of 8.5-9, and then lyophilize the sample to obtain a powdery pH-responsive polypeptide mimetic product.

[0149] Applications

[0150] The polymer of the present invention has one or more functions or properties selected from the following group:

[0151] The pH responsiveness improves the specific delivery of polypeptide mimetics to tissues with an acidic microenvironment such as the site of microbial infection and tumor tissues;

[0152] The pH responsiveness causes charge inversion to penetrate the mucus barrier and improves pulmonary drug delivery;

[0153] pH-responsive antibacterial;

[0154] pH-responsive anti-tumor.

[0155] Therefore, the present invention also provides the use of the polymer as described in the first aspect of the present invention in the preparation of a pharmaceutical composition for antibacterial and / or anti-tumor.

[0156] In another preferred embodiment, antibacterial includes Gram-positive bacteria, Gram-negative bacteria, fungi, spores, and dormant cells.

[0157] In another preferred example, Gram-positive bacteria include Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus, Bacillus, Listeria, Bacillus anthracis; Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, Vibrio cholerae; fungi include Candida albicans, Cryptococcus neoformans, Candida tropicalis, Candida glabrata, Cryptococcus gattii, Aspergillus, dermatophyte, Histoplasma capsulatum.

[0158] In another preferred example, the above-mentioned tumors are solid cancers or blood cancers, such as gastric cancer, cervical cancer, melanoma, endometrial cancer, esophageal cancer, liver cancer, prostate cancer, breast cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, colon cancer, uterine tumors and soft tissue sarcoma, lymphoma, leukemia, multiple myeloma, mesothelioma, malignant rhabdoid tumor, bile duct and gallbladder cancer, bladder cancer; brain tumors, neuroblastoma, schwannoma, glioma, glioblastoma multiforme and astrocytoma.

[0159] In addition, the geopolymers of the present invention can be modified to the material surface through reactive groups on the end groups, such as forming polymer brushes, so that the material has the biological functions of the polymers of the present invention, such as antibacterial and anti-tumor properties. Generally, the material of the material surface can be selected from (but not limited to): inorganic non-metallic biomaterials (such as bioceramics, bioactive glasses, graphene, bone cements and medical carbon materials), bio-metallic materials (such as stainless steel, cobalt-based and titanium-based alloys, shape memory alloys, precious metals such as silver, platinum, tantalum, niobium, zirconium, palladium, platinum), natural polymer materials (such as hyaluronic acid, chitosan, alginic acid, cellulose, collagen, gelatin), synthetic polymer materials (polyetheretherketone, polycaprolactone, polylactic acid, polycarbonate, polyurethane, polyester, polyanhydride, polydimethylsiloxane, polymethyl methacrylate, polyphosphazene, polyamide, polyethylene, polypropylene, polytetrafluoroethylene, polyethylene terephthalate, resin), or combinations thereof (such as their composite materials, assembled materials). The material can generally be used as (but not limited to) orthopedic implants, dental implants, medical catheters, dressings, gels, and other medical materials that can be implanted into the body.

[0160] Drug composition

[0161] The drug composition of the present invention comprises the polymer of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0162] The "safe and effective amount" herein refers to: an amount of the polymer sufficient to significantly improve the condition without causing serious side effects. Generally, the drug composition contains 1-2000 mg of the polymer of the present invention per dose, more preferably, 10-500 mg of the polymer of the present invention per dose. Preferably, the "per dose" is a capsule or a tablet.

[0163] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the composition can be blended with the polymers of the present invention and with each other without significantly reducing the efficacy of the polymers. Some examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0164] There is no particular limitation on the mode of administration of the polymers or pharmaceutical compositions of the present invention. Representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0165] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active polymer is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or calcium phosphate, or is mixed with the following components: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxypropylmethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0166] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and casings, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active polymer or polymer in such compositions can be delayed and released in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and wax substances. If necessary, the active polymer can also be in the form of microcapsules with one or more of the above excipients.

[0167] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active polymer, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifying agents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0168] In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0169] In addition to the active polymer, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances, etc.

[0170] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and their suitable mixtures.

[0171] Dosage forms of the polymers of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required, if necessary.

[0172] The polymers of the present invention can be administered alone or in combination with other pharmaceutically acceptable polymers. The pharmaceutical compositions also contain one or more antibacterial agents, anticancer agents and / or immunosuppressive agents.

[0173] The general range of the therapeutically effective dose of the polymer or polymer-containing composition of the present invention will be: about 1 - 2000 mg / day, about 10 - about 1000 mg / day, about 10 - about 500 mg / day, about 10 - about 250 mg / day, about 10 - about 100 mg / day, or about 10 - about 50 mg / day. The therapeutically effective dose will be administered in one or more doses. However, it should be understood that the specific dose of the polymer of the present invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health, diet, individual response, time of administration, severity of the disease to be treated, activity of the specific polymer administered, dosage form, mode of application, and concomitant medications. The therapeutically effective amount for a given situation can be determined by conventional experimentation and is within the competence and judgment of a clinician or physician. In any case, the compound or composition will be administered in multiple doses based on the individual circumstances of the patient and in a manner that permits delivery of a therapeutically effective amount.

[0174] The main advantages of the present invention are as follows:

[0175] 1. The polypeptide mimetic of the present invention relies on existing polymerization methods, can efficiently and rapidly complete a large amount of polymerization, the synthesis raw materials are cheap and easily available, and has the potential to achieve industrial mass production.

[0176] 2. The polypeptide mimetic of the present invention itself has broad-spectrum antimicrobial / antitumor cell activity and is not easily induced to produce drug resistance in microorganisms / tumor cells.

[0177] 3. The preparation method provided by the present invention can conveniently and efficiently obtain pH-responsive polypeptide mimetics by amidating the primary amine on the side chain of pH-responsive small molecule polymers.

[0178] 4. The pH-responsive polypeptide mimetic of the present invention can significantly reduce hemolytic toxicity, improve biocompatibility, and will not cause obvious blood toxicity or organ damage when intravenously injected into mice.

[0179] 5. The pH-responsive polypeptide mimetic of the present invention can significantly reduce the interaction with plasma proteins, prolong the blood circulation time, and improve the in vivo utilization rate.

[0180] 6. The pH-responsive polypeptide mimetic of the present invention can rapidly remove the pH-responsive small molecule under acidic conditions and restore high-efficiency antibacterial activity.

[0181] 7. The pH-responsive polypeptide mimetic of the present invention can achieve in vivo transformation through pH response in infected tissues and achieve enhanced accumulation at the infection site.

[0182] 8. The pH-responsive polypeptide mimetic of the present invention exhibits high antibacterial activity and good in vivo safety in animal infections, can significantly improve the survival rate of mice with systemic infections, and reduce the bacterial / fungal burden in locally infected tissues, providing a new strategy for the development of novel antibacterial drugs.

[0183] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0184] Example 1: Preparation of α-amino acid polymer (LysNle) by polymerization of N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyanhydride and DL-norleucine-N-carboxyanhydride initiated by lithium hexamethyldisilazide

[0185]

[0186] Accurately weigh lithium hexamethyldisilazide and prepare a 0.1 M solution with tetrahydrofuran for later use. Weigh N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyanhydride and DL-norleucine-N-carboxyanhydride, using tetrahydrofuran as the solvent. Take 0.8 mL of N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyanhydride (0.2 M) and 1.2 mL of DL-norleucine-N-carboxyanhydride (0.2 M), mix them and add a magnetic stirrer for stirring. In the stirred reaction flask, add 0.8 mL of a 0.1 M lithium hexamethyldisilazide solution. Stir the mixture in a glove box at room temperature for 5 minutes. After the polymerization reaction is completed, pour 45 mL of cold petroleum ether into the above reaction mixture. The precipitated white floccules are collected by centrifugation, dried in a stream of air, and redissolved in 1.5 mL of tetrahydrofuran, and then precipitated with a large amount of cold petroleum ether. This dissolution-precipitation process is repeated three times.

[0187] Add 2 mL of trifluoroacetic acid to the dried polymer, shake it at room temperature for 2 hours, then blow off the excess trifluoroacetic acid. The obtained viscous liquid is dissolved in 0.5 mL of methanol, and then 45 mL of ice-cooled methyl tert-butyl ether is added to precipitate a white solid. The dissolution-precipitation process is repeated three times to obtain a random polymer with deprotected side-chain amino groups. The deprotected polymer is dissolved again in 5 mL of ultrapure water, filtered and freeze-dried for subsequent pH-responsive small molecule modification.

[0188] Example 2: Preparation of α - amino acid polymer (LysHoPhe) by polymerization of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride and DL - norleucine - N - carboxy anhydride initiated by lithium hexamethyldisilazide

[0189]

[0190] The experimental method was the same as that in Example 1, except that 0.8 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 1.2 mL of DL - norleucine - N - carboxy anhydride (0.2 M) were replaced with 1.2 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 0.8 mL of DL - homophenylalanine - N - carboxy anhydride (0.2 M).

[0191] Example 3: Preparation of α - amino acid polymer (LysTrp) by polymerization of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride and DL - tryptophan - N - carboxy anhydride initiated by lithium hexamethyldisilazide

[0192]

[0193] The experimental method was the same as that in Example 1, except that 0.8 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 1.2 mL of DL - norleucine - N - carboxy anhydride (0.2 M) were replaced with 1.2 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 0.8 mL of DL - tryptophan - N - carboxy anhydride (0.2 M).

[0194] Example 4: Preparation of α - amino acid polymer (LysNap) by polymerization of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride and L - alanine - 3 - (1 - naphthyl) - N - carboxy anhydride initiated by lithium hexamethyldisilazide

[0195]

[0196] The experimental method was the same as that in Example 1, except that 0.8 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 1.2 mL of DL - norleucine - N - carboxy anhydride (0.2 M) were replaced with 1.2 mL of N - ε - tert - butoxycarbonyl - DL - lysine - N - carboxy anhydride (0.2 M) and 0.8 mL of L - alanine - 3 - (1 - naphthyl) - N - carboxy anhydride (0.2 M).

[0197] Example 5: Guanidinated α - amino acid polymer

[0198]

[0199] Weigh lithium hexamethyldisilazide accurately and prepare a 0.1 M solution with tetrahydrofuran for later use. Weigh N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyanhydride and DL-norleucine-N-carboxyanhydride, using tetrahydrofuran as the solvent. Take 0.6 mL of N-ε-tert-butoxycarbonyl-DL-lysine-N-carboxyanhydride (0.2 M) and 1.4 mL of DL-norleucine-N-carboxyanhydride (0.2 M), mix them, and add a magnetic stir bar to stir. In the stirred reaction flask, add 0.8 mL of a 0.1 M lithium hexamethyldisilazide solution. Stir the mixture in the glove box at room temperature for 5 minutes. After the polymerization reaction is completed, pour 45 mL of cold petroleum ether into the above reaction mixture. The precipitated white flocculent substance is collected by centrifugation, dried in a stream of air, and redissolved in 1.5 mL of tetrahydrofuran, and then precipitated again by adding a large amount of cold petroleum ether. This dissolution-precipitation process is repeated three times.

[0200] Add 2 mL of trifluoroacetic acid to the polymer dried by suction. After shaking at room temperature for 2 hours, blow off the excess trifluoroacetic acid. The obtained viscous liquid is dissolved in 0.5 mL of methanol, and then 45 mL of ice-cold methyl tert-butyl ether is added to precipitate a white solid. The dissolution-precipitation process is repeated three times to obtain a random polymer with deprotected side-chain amino groups.

[0201] Dissolve the deprotected polymer in a methanol solution, and successively add 4 equivalents of N,N-diisopropylethylamine (DIEA) and 3 equivalents of 1H-pyrazole-1-carboxamidine hydrochloride relative to the number of amine units per polymer chain. Stir the mixture at 60 °C for 24 hours. Remove the solvent under vacuum. The concentrate is dissolved in 0.5 mL of methanol, and then 45 mL of cold acetone is added to precipitate the solid. The dissolution-precipitation process is repeated three times to obtain a guanidinylated α-amino acid polymer.

[0202] Example 6: Biguanide α-amino acid polymer

[0203]

[0204] The experimental method is the same as that in Example 5. The difference is that the deprotected polymer is dissolved in an aqueous solution, and 4 equivalents of N,N-diisopropylethylamine (DIEA) and 3 equivalents of a biguanidating reagent are successively added relative to the number of amine units per polymer chain. Stir the mixture at 80 °C for 30 minutes. After the reaction solution is cooled to room temperature, add 45 mL of cold acetone to precipitate the solid. After drying the precipitate, dissolve the solid in as little water as possible and precipitate again with cold acetone. The dissolution-precipitation process is repeated three times to obtain a biguanidated α-amino acid polymer.

[0205] Example 7: Preparation of α-peptide polymer (NaegNpfb) by polymerization of N-substituted-N-carboxy anhydride Naeg and N-substituted-N-carboxy anhydride Npfb initiated by p-tert-butylbenzylamine

[0206]

[0207] Accurately weigh N-substituted-N-carboxy anhydride Naeg and N-substituted-N-carboxy anhydride Npfb, and prepare a 1M solution with tetrahydrofuran for standby. Take 1 mL of Naeg and 1 mL of Npfb, mix them and add a magnetic stirrer for stirring. In the stirred reaction flask, quickly add 0.1 mL of a 1M p-tert-butylbenzylamine solution. Stir the mixture at 60 °C for reaction. After the reaction is completed, pour cold petroleum ether (45 mL) into the above reaction mixture. The precipitated white flocculent substance is collected by centrifugation, dried in a stream of air, and redissolved in tetrahydrofuran (1 mL), and then precipitated with a large amount of cold petroleum ether. This dissolution-precipitation process is repeated three times in total.

[0208] Add 1 mL of acetic acid solution of hydrogen bromide (33 wt.%) and 1 mL of trifluoroacetic acid to the polymer after drying by suction. Shake overnight at room temperature and then blow off the solvent. The obtained viscous liquid is dissolved in 1 mL of methanol, and then 45 mL of ice-cooled methyl tert-butyl ether is added to precipitate a white solid. The dissolution-precipitation process is repeated three times to obtain a random polymer with deprotected side-chain amino groups. The deprotected polymer is dissolved again in 5 mL of ultrapure water, filtered, freeze-dried and used for the subsequent pH-responsive small molecule modification.

[0209] Example 8: Preparation of β-amino acid polymer (DMBu) by polymerization of β-lactam monomer DM and β-lactam monomer Bu initiated by p-tert-butylbenzoyl chloride and lithium hexamethyldisilazide

[0210]

[0211] Accurately weigh β-lactam monomer DM and β-lactam monomer Bu, and prepare a 0.2M solution with tetrahydrofuran for standby. Take 1.6 mL of DM and 0.4 mL of Bu, mix them and add a magnetic stirrer for stirring. In the stirred reaction flask, quickly add 0.1 mL of a 0.2M p-tert-butylbenzoyl chloride solution and 0.1 mL of a 0.5M lithium hexamethyldisilazide solution. Stir the mixture at room temperature for 6 hours, take it out of the glove box and quench it with 1 drop of methanol. Pour cold petroleum ether (45 mL) into the above reaction mixture. The precipitated white flocculent substance is collected by centrifugation, dried in a stream of air, and redissolved in tetrahydrofuran (1 mL), and then precipitated with a large amount of cold petroleum ether. This dissolution-precipitation process is repeated three times in total.

[0212] The dried polymer was added with 2 mL of trifluoroacetic acid and shaken at room temperature for 2 hours. Then the excess trifluoroacetic acid was blown off. The resulting viscous liquid was dissolved in 0.5 mL of methanol, and then 45 mL of ice-cooled methyl tert-butyl ether was added to precipitate a white solid. The dissolution-precipitation process was repeated three times to obtain a random polymer with deprotected side-chain amino groups. The deprotected polymer was redissolved in 5 mL of ultrapure water, filtered, freeze-dried, and used for the subsequent pH-responsive small molecule modification.

[0213] Example 9: Preparation of α / β amino acid polymer LysCH by p-tert-butylbenzylamine-initiated N-ε-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride and β 2,3 -cyclohexyl-N-carboxythiocarbonyl anhydride

[0214]

[0215] N-ε-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride and β 2,3 -cyclohexyl-N-carboxythiocarbonyl anhydride were accurately weighed and dissolved in N,N-dimethylformamide to prepare a 0.2 M solution for standby. 0.8 mL of N-ε-tert-butoxycarbonyl-L-lysine-N-carboxyanhydride and 1.2 mL of β 2,3 -cyclohexyl-N-carboxythiocarbonyl anhydride were mixed and stirred with a magnetic stirrer. In the stirred reaction flask, 0.1 mL of a 0.2 M p-tert-butylbenzylamine solution was quickly added. The mixture was stirred at room temperature for 4 days. After the reaction was completed, cold petroleum ether (45 mL) was poured into the above reaction mixture. The precipitated white floccules were collected by centrifugation, dried in a stream of air, redissolved in tetrahydrofuran (1 mL), and then precipitated with a large amount of cold petroleum ether. This dissolution-precipitation process was repeated three times.

[0216] The dried polymer was added with 2 mL of trifluoroacetic acid and shaken at room temperature for 2 hours. Then the solvent was blown off. The resulting viscous liquid was dissolved in 1 mL of methanol, and then 45 mL of ice-cooled methyl tert-butyl ether was added to precipitate a white solid. The dissolution-precipitation process was repeated three times to obtain a random polymer with deprotected side-chain amino groups. The deprotected polymer was redissolved in 5 mL of ultrapure water, filtered, freeze-dried, and used for the subsequent pH-responsive small molecule modification.

[0217] Example 10: Preparation of polyoxazoline (NMePentyl) by methyl trifluoromethanesulfonate-initiated N-ε-tert-butoxycarbonyl-2-(aminomethyl)oxazoline and 2-(cyclopentyl)oxazoline

[0218]

[0219] Accurately weigh N-ε-Boc-2-(aminomethyl)oxazoline and 2-(cyclopentyl)oxazoline, and prepare a 0.2 M solution with N,N-dimethylacetamide for standby. Take 1 mL of N-ε-Boc-2-(aminomethyl)oxazoline and 1 mL of 2-(cyclopentyl)oxazoline, mix them and add a magnetic stir bar for stirring. In the stirred reaction flask, quickly add 0.1 mL of a 0.2 M methyl trifluoromethanesulfonate solution. Stir the mixture at 100 °C for 16 hours. After the reaction is completed, pour 45 mL of cold petroleum ether into the above reaction mixture. The precipitated white flocculent substance is collected by centrifugation, dried in a stream of air, redissolved in 1 mL of tetrahydrofuran, and then precipitated by adding a large amount of cold petroleum ether. This dissolution-precipitation process is repeated three times.

[0220] Add 2 mL of trifluoroacetic acid to the polymer dried by suction, shake it at room temperature for 2 hours, then blow off the excess trifluoroacetic acid. The obtained viscous liquid is dissolved in 0.5 mL of methanol, and then 45 mL of ice-cooled methyl tert-butyl ether is added to precipitate a white solid. The dissolution-precipitation process is repeated three times to obtain a random polymer with deprotected side-chain amino groups. The deprotected polymer is dissolved again in 5 mL of ultrapure water, filtered, freeze-dried, and used for the following pH-responsive small molecule modification.

[0221] Example 11: Preparation of polyoxazoline (NPrBu) by methyl trifluoromethanesulfonate-initiated N-ε-Boc-2-(aminopropyl)oxazoline and 2-(butyl)oxazoline

[0222]

[0223] The experimental method is the same as that in Example 8, except that 1 mL of N-ε-Boc-2-(aminomethyl)oxazoline (0.2 M) and 1 mL of 2-(cyclopentyl)oxazoline (0.2 M) are replaced by 0.6 mL of N-ε-Boc-2-(aminopropyl)oxazoline (0.2 M) and 1.4 mL of 2-(butyl)oxazoline (0.2 M).

[0224] Example 12: Modification of pH-responsive small molecule 2,3-dimethylmaleic anhydride on α-amino acid polymer LysNle

[0225]

[0226] Dissolve the α-amino acid polymer LysNle in deionized water and adjust the pH to 8.5 - 9 with 3M NaOH solution. After stirring in an ice bath for 10 minutes, add 1.5 molar equivalents of the pH-responsive small molecule 2,3-dimethylmaleic anhydride to the polymer solution in several portions. During this period, maintain the pH of the reaction mixture at 8.5 - 9 by adding 3M NaOH solution, and then stir overnight at room temperature. Dialyze the mixture in deionized water at pH 8.5 - 9 for one day to remove the excess 2,3-dimethylmaleic anhydride, and obtain the pH-responsive α-amino acid polymer after lyophilization.

[0227] Example 13: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the α-amino acid polymer LysHoPhe

[0228] The preparation steps of the above pH-responsive α-amino acid polymer are basically the same as those in Example 12.

[0229] Example 14: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the α-amino acid polymer LysTrp

[0230] The preparation steps of the above pH-responsive α-amino acid polymer are basically the same as those in Example 12.

[0231] Example 15: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the α-amino acid polymer LysNap

[0232] The preparation steps of the above pH-responsive α-amino acid polymer are basically the same as those in Example 12.

[0233] Example 16: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the guanidinated α-amino acid polymer

[0234] The preparation steps of the above pH-responsive α-amino acid polymer are basically the same as those in Example 12.

[0235] Example 17: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the biguanidinated α-amino acid polymer

[0236] The preparation steps of the above pH-responsive α-amino acid polymer are basically the same as those in Example 12.

[0237] Example 18: Modification of the pH-responsive small molecule 2,3-dimethylmaleic anhydride on the α-peptoid polymer NaegNpfb

[0238] The preparation steps of the above pH-responsive α-peptoid polymer are basically the same as those in Example 12.

[0239] Example 19: Modification of pH-responsive small molecule 2,3-dimethylmaleic anhydride on β-amino acid polymer DMBu

[0240] The preparation steps of the above pH-responsive β-amino acid polymer are basically the same as those in Example 12.

[0241] Example 20: Modification of pH-responsive small molecule 2,3-dimethylmaleic anhydride on LysCH of α / β-amino acid polymer

[0242] The preparation steps of the above pH-responsive α / β-amino acid polymer are basically the same as those in Example 12.

[0243] Example 21: Modification of pH-responsive small molecule 2,3-dimethylmaleic anhydride on polyoxazoline NMePentyl

[0244] The preparation steps of the above pH-responsive polyoxazoline are basically the same as those in Example 12.

[0245] Example 22: Modification of pH-responsive small molecule 2,3-dimethylmaleic anhydride on polyoxazoline NMeBu

[0246] The preparation steps of the above pH-responsive polyoxazoline are basically the same as those in Example 12.

[0247] Example 23: Modification of pH-responsive small molecule citraconic anhydride on polyoxazoline NMePentyl

[0248]

[0249] The preparation steps of the above pH-responsive polyoxazoline are basically the same as those in Example 12. The difference is that the pH-responsive small molecule 2,3-dimethylmaleic anhydride is replaced by citraconic anhydride.

[0250] Example 24: pH-responsive small molecule modification significantly reduces the in vitro hemolytic toxicity of peptide mimics

[0251] The selected peptide polymers are α-amino acid polymers of Examples 1-6, α-peptide polymers of Example 7, β-amino acid polymers of Example 8, α / β-amino acid polymers of Example 9, polyoxazolines of Examples 10-11, and the corresponding polymers modified with pH-responsive small molecule 2,3-dimethylmaleic anhydride including Examples 12-22.

[0252] Wash fresh mouse blood 3 times with Tris-buffered saline (TBS, pH = 7.4), and finally dilute to obtain a 5% (v / v) working suspension of red blood cells (RBCs). In a 96-well plate, serially dilute the polymer to be tested twofold (50 μL), and add an equal volume of the RBCs suspension to each well to achieve the desired polymer concentration. Incubate the plate at 37 °C for 1 hour, then centrifuge at 3700 rpm / min for 5 minutes, and transfer 80 μL of the supernatant from each well to a new 96-well plate. The well containing a mixture of 0.1% TX-100 and RBCs serves as a positive control; the well containing only the RBCs suspension is the blank control. Finally, measure the OD of the supernatant using an enzyme-linked immunosorbent assay (ELISA) reader 405 , and calculate the percentage of hemolysis according to the formula. HC 50 value is defined as the lowest concentration of the polymer that causes 50% lysis of RBCs.

[0253]

[0254] The experimental results are shown in Table 1-5. Amidating the primary amines on the side chains with the pH-responsive small molecule 2,3-dimethylmaleic anhydride can significantly reduce the hemolytic toxicity of the polypeptide mimics.

[0255] Table 1 Hemolytic toxicity of α-amino acid polymers before and after modification with pH-responsive small molecules

[0256] Polymer Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 <![CDATA[HC 50 (μg / mL)]]> 15.6 15.6 200 15.6 15.6 15.6 Polymer Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 <![CDATA[HC 50 (μg / mL)]]> >4000 >4000 >4000 >4000 >4000 >4000

[0257] Table 2 Hemolytic toxicity of α-peptide polymers before and after modification with pH-responsive small molecules

[0258] Polymer Example 7 Example 18 <![CDATA[HC 50 (μg / mL)]]> 50 350

[0259] Table 3 Hemolytic toxicity of β-amino acid polymers before and after modification with pH-responsive small molecules

[0260] Polymer Example 8 Example 19 <![CDATA[HC 50 (μg / mL)]]> 100 >4000

[0261] Table 4 Hemolytic toxicity of α / β-amino acid polymers before and after modification with pH-responsive small molecules

[0262] Polymer Example 9 Example 20 <![CDATA[HC 50 (μg / mL)]]> 200 1000

[0263] Table 5 Hemolytic toxicity of polyoxazolines before and after modification with pH-responsive small molecules

[0264] Polymer Example 10 Example 11 Example 21 Example 22 Example 23 <![CDATA[HC 50 (μg / mL)]]> 150 100 >4000 500 >4000

[0265] Example 25: Modification with pH-responsive small molecules significantly reduces the in vivo hemolytic toxicity of polypeptide mimics

[0266] Prepare female ICR mice aged 6 - 8 weeks and raise them for two weeks before the experiment to acclimatize them to the environment. Prepare polymer solutions of Example 10 and Example 21 with physiological saline for standby. For each group of 3 mice, inject the polymer solutions of Example 10 and Example 21 with a dose of 30 mg / kg via the tail vein. Inject physiological saline as the control group. After 3 hours, euthanize the mice, collect blood by cardiac puncture, and collect the plasma supernatant by centrifuging at 3000 rpm for 15 minutes. Dilute the plasma 30 - fold with PBS and measure the OD value of the plasma at a wavelength of 540 nm using an enzyme - linked immunosorbent assay (ELISA) reader to analyze the release of hemoglobin. The OD value of the diluted plasma is as Figure 1 shown. Different from Example 10, after injecting the polymer solution of Example 21 via the tail vein for 3 hours, the increase in the release of hemoglobin in the mouse blood is almost negligible, indicating that the modification with the pH - responsive small molecule 2,3 - dimethylmaleic anhydride can reduce the in - vivo hemolytic toxicity of the polypeptide mimetic.

[0267] Example 26: pH - responsive small - molecule modification reduces the in - vivo acute toxicity of polypeptide mimetic

[0268] Prepare female ICR mice aged 6 - 8 weeks and raise them for two weeks before the experiment to acclimatize them to the environment. Prepare polymer solutions of Example 11 and Example 22 with different doses using physiological saline for standby.

[0269] For each group of 3 mice, inject different doses of the solutions of Example 11 and Example 22 via the tail vein and observe the survival rate of the mice to determine the maximum acceptable dose of the polymer solution for single - dose intravenous injection in mice.

[0270] Randomly divide healthy mice into two groups of 7 each, and inject the polymer solution of Example 22 (25 mg / kg) and physiological saline via the tail vein respectively. Then observe the physiological state of the mice and record the change in body weight of the mice within 14 days.

[0271] The survival rate of the mice after intravenous injection of the polymer solution is as Figure 2 shown in a. After modification with 2,3 - dimethylmaleic anhydride, the maximum acceptable dose for intravenous injection of the polymer is significantly increased. The change in the body weight of the mice is as Figure 2 shown in b. There is no significant difference between the polymer group and the physiological saline group. These experimental results indicate that the pH - responsive polypeptide mimetic has no obvious in - vivo acute toxicity.

[0272] Example 27: pH - responsive small - molecule modification improves the in - vivo safety of polypeptide mimetic

[0273] Prepare female ICR mice aged 6 - 8 weeks and house them for two weeks before the experiment to acclimatize them to the environment. Randomly divide the healthy mice into three groups of 6 mice each. Two of the groups are intravenously injected with the polymer solution of Example 22 (25 mg / kg), and the other group serves as a control. On the 2nd and 14th days, euthanize the two groups of mice injected with the polymer solution of Example 22, and collect blood (0.5 - 0.8 mL) by cardiac puncture. Let the collected whole blood stand at room temperature for 2 hours, and centrifuge at 3000 rpm for 15 minutes to collect the serum. Analyze the clinically significant biomarkers in the blood, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), blood urea nitrogen (BUN), sodium ion (Na + +) and potassium ion (K + +) content. In addition, collect the livers and kidneys of the mice on the 2nd and 14th days, fix them with 4% paraformaldehyde, and prepare H&E pathological section staining.

[0274] Serum biochemical indicators are as Figure 3 shown. Compared with the normal saline group, there are no significant differences in the physiological indicators representing liver function, kidney function, and electrolyte balance after intravenous injection of the pH-responsive polymer. The H&E-stained pathological sections of the liver and kidney tissues are as Figure 4 shown, and no obvious liver and kidney tissue damage is caused in the polymer group. These experimental results indicate that the pH-responsive polypeptide mimetic has good biocompatibility in vivo.

[0275] Example 28: Reduction of the binding of polypeptide mimetic to plasma proteins by pH-responsive small molecule modification

[0276] Host defense peptide mimetics may have strong interactions with plasma proteins, leading to obvious aggregation and precipitation of proteins, resulting in reduced or lost biological activity, pulmonary embolism, organ dysfunction, and even death. Therefore, test the binding ability of the polypeptide mimetic to plasma proteins. Dissolve the freeze-dried powders of the polymer (Example 11) and the corresponding pH-responsive polymer (Example 22) in deionized water to prepare polymer solutions with a concentration of 0.2 mg / mL. Mix the polymer solution with fresh mouse plasma at a ratio of 1:1, and after incubating for 15 minutes, measure its hydrodynamic diameter using a Malvern nano particle size analyzer. The experimental results are as Figure 5 shown. There are obvious differences in the binding force of Example 11 and Example 22 to plasma proteins. Example 11 has a strong interaction with plasma proteins, resulting in the change of the hydrodynamic diameter of the protein from 7.0 nm to 23.1 nm. In contrast, Example 22 modified with 2,3-dimethylmaleic anhydride does not cause an increase in the hydrodynamic diameter of plasma proteins and reduces the binding to plasma proteins.

[0277] Example 29: Extension of the blood circulation time of polypeptide mimetic by pH-responsive small molecule modification

[0278] Six- to eight-week-old female ICR mice were prepared and raised for two weeks before the experiment to acclimatize them to the environment. The synthesis of fluorescently labeled polyoxazoline and pH-responsive fluorescently labeled polyoxazoline is as shown in Figure 6 a. Fluorescently labeled polyoxazoline and pH-responsive fluorescently labeled polyoxazoline solutions were prepared with physiological saline for standby. Three mice were in each group, and the polyoxazoline solution was injected into the tail vein at a dose of 5 mg / kg. Blood was collected by cardiac puncture at different time points after administration, and plasma was separated by centrifugation at 3000 rpm for 10 minutes. The fluorescence intensity was measured on a microplate reader (excitation wavelength 410 nm, emission wavelength 540 nm), and the concentration of fluorescently labeled polyoxazoline in plasma was calculated. The in vivo plasma clearance curve is as shown in Figure 6 b. The plasma half-lives of polyoxazoline before and after modification with 2,3-dimethylmaleic anhydride were 6.2 ± 0.8 minutes and 46.1 ± 6.4 minutes, respectively, significantly prolonging the blood circulation time of the polypeptide mimetic.

[0279] Example 30: pH-responsive polypeptide mimetic enables rapid acid response

[0280] 5 mg of the lyophilized powder of Example 22 was dissolved in 0.6 mL of heavy water, and the pH was adjusted to 5.5 with deuterated hydrochloric acid, and nuclear magnetic resonance characterization was performed at different time points.

[0281] 1 mg of the lyophilized powder of Example 22 was dissolved in 1 mL of deionized water, and the pH was adjusted to 5.5 or 7.4 with 1 mM HCl. The Zeta potential of the polymer solution was measured with a Malvern nanoparticle size analyzer at different time points.

[0282] The proton nuclear magnetic resonance spectrum is as shown in Figure 7 a. In an acidic aqueous solution with pH = 5.5, Example 22 was rapidly hydrolyzed to remove the pH-responsive small molecule, and the degree of hydrolysis reached 84% within 30 minutes. The change in Zeta potential is as shown in Figure 7 b. Different from the physiological pH environment, the Zeta potential of Example 22 rapidly increased from about -12 mV to +19 mV under acidic conditions, showing excellent pH responsiveness.

[0283] Example 31: pH-responsive polypeptide mimetic rapidly restores bactericidal activity through acid response

[0284] The selected antibacterial agent is the pH-responsive polyoxazoline of Example 22. The selected strains are Pseudomonas aeruginosa O1 and Escherichia coli ATCC25922. The pH of the MH medium is adjusted to 5.5 or 7.4 with 1 mM HCl and reserved. First, the bacteria are cultured in LB medium in a shaker at 37 °C for 10 hours, centrifuged at 4000 rpm for 5 minutes, the supernatant is discarded, and the bacteria at the bottom are dispersed with a small amount of the test medium, and the OD value is measured on an enzyme-linked immunosorbent assay (ELISA) reader. According to the OD value, the bacterial suspension is diluted with MH medium of different pH values to 2×10 5 CFU / mL for use. Subsequently, an equal volume of the solution of Example 22 (dissolved in MH medium of different pH values) is quickly added to make the final concentration 25 μg / mL and the bacterial concentration 1×10 5 CFU / mL, and incubated in a shaker at 37 °C and 200 rpm. At different time points, the bacterial suspension is taken out, diluted and spread on plates, incubated overnight in a mold incubator at 37 °C, and then the CFU is counted. The amount of bacteria at the corresponding time points is calculated according to the dilution factor. The group containing only bacteria without Example 22 is used as a control. The time-kill kinetic results are as Figure 8 shown. Under the condition of pH 5.5, Example 22 with a concentration of 25 μg / mL can completely kill Escherichia coli or Pseudomonas aeruginosa within 60 minutes and 120 minutes respectively; while at pH 7.4, there is no obvious bactericidal effect even after 180 minutes.

[0285] Example 32: Rapid in vivo transformation of pH-responsive polypeptide mimics at the infection site

[0286] To verify the in vivo transformation of the pH-responsive polypeptide mimics, as Figure 9 shown in a, we synthesized polyoxazoline labeled with dual fluorescent dyes, where rhodamine B is attached to the terminal group of the polymer and 5-aminofluorescein is labeled on the pH-responsive 2,3-dimethylmaleic anhydride group on the side chain of the polymer. The rhodamine B dye as the terminal group of the polymer is stable under acidic conditions, while 5-aminofluorescein can be removed as the pH-responsive small molecule is removed. Therefore, the in vivo transformation of the pH-responsive polymer in the acidic infection environment can be verified by detecting the fluorescence intensity changes of these two dyes.

[0287] Six- to eight-week-old female ICR mice are prepared and raised for two weeks before the experiment to adapt to the environment. First, Escherichia coli is cultured in LB medium in a shaker at 37 °C until the growth phase, washed three times with physiological saline, and the bacterial suspension is diluted to 10 8CFU / mL for use. Before surgery, mice were anesthetized by intraperitoneal injection of 75 mg / kg sodium pentobarbital. The right dorsal area of the mice was shaved, the remaining hair was removed using depilatory cream, and the area was disinfected with 75% alcohol. A syringe was used to aspirate the Escherichia coli bacterial suspension, and 50 μL of the suspension was injected subcutaneously. At 24 hours after infection, a pH-responsive polyoxazoline labeled with a dual-fluorescent dye was injected into the abscess. Tissues were collected at different time points, and the fluorescence intensities of 5-aminofluorescein derivatives (green fluorescence) and rhodamine B derivatives (red fluorescence) were detected using a fluorescence confocal microscope at wavelengths of 488 nm and 561 nm, respectively. The experimental results are as Figure 9 shown in b. The fluorescence intensity of rhodamine B in the infected tissues did not decrease significantly over time, indicating that the polymer remained at the infection site; in sharp contrast, the fluorescence intensity of 5-aminofluorescein decreased rapidly ( Figure 9 c). Based on the difference in the relative fluorescence intensity changes of the two dyes, approximately 90% of the 5-aminofluorescein-labeled 2,3-dimethylmaleic anhydride groups at the infection site were cleared within 4 hours ( Figure 9 d), indicating that in vivo transformation of the pH-responsive polypeptide mimetic occurred in the infected tissues.

[0288] Example 33: Enhancement of the accumulation of polypeptide mimetic at the infection site by pH-responsive small molecule modification

[0289] Female ICR mice aged 6 - 8 weeks were prepared and allowed to acclimatize to the environment for two weeks before the experiment. Four days and one day before infection, the mice were intraperitoneally injected with 150 and 100 mg / kg cyclophosphamide, respectively, to induce neutropenia. Escherichia coli was cultured in LB medium in a shaker at 37 °C until the growth phase, washed three times with physiological saline, and the bacterial suspension was diluted to 2×10 5 CFU / mL for use. Before surgery, mice were anesthetized by intraperitoneal injection of 75 mg / kg sodium pentobarbital. The hair on the right thigh of the mice was removed using depilatory cream, and the area was disinfected with 75% alcohol, and then 50 μL of the bacterial suspension was injected intramuscularly. At 24 hours after infection, a pH-responsive fluorescently labeled polyoxazoline solution (10 mg / kg) of Example 29 was injected intravenously. At 1, 3, and 6 hours after administration, the mice were euthanized. The infected and non-infected thigh tissues were collected separately, weighed, and homogenized in physiological saline. The supernatant was separated by centrifugation at 3000 rpm for 10 minutes, and the fluorescence intensity (excitation wavelength 410 nm, emission wavelength 540 nm) was measured on a microplate reader, and the concentration of the fluorescently labeled polyoxazoline in the thigh tissues was calculated. The experimental results are as Figure 10As shown, the fluorescently labeled polyoxazoline rapidly accumulates at the thigh infection site and maintains a high concentration for a long time, about 4 times higher than that in the uninfected thigh. This may be because the polyoxazoline prodrug undergoes pH-responsive deacetylation of 2,3-dimethylmaleic anhydride in the acidic infection microenvironment, resulting in a charge inversion to a positively charged state, and attaches to the negatively charged bacterial surface through electrostatic interactions, thereby further enhancing the accumulation of the drug at the infection site.

[0290] Example 34: pH-responsive polypeptide mimetic effectively treats bacterial infection of subcutaneous abscess in mice

[0291] Female BALB / c mice aged 6 - 8 weeks were prepared and allowed to acclimatize for two weeks before the experiment. First, Pseudomonas aeruginosa was cultured in LB medium in a shaker at 37 °C until the growth phase, then washed three times with physiological saline, and the bacterial suspension was diluted to 10 7 CFU / mL for use. Before the operation, 75 mg / kg of sodium pentobarbital was intraperitoneally injected to anesthetize the mice. The right dorsal area of the mice was shaved, the remaining hair was removed using depilatory cream, and the area was disinfected with 75% alcohol. A syringe was used to aspirate the Pseudomonas aeruginosa bacterial suspension, and 50 μL of the suspension was subcutaneously injected. At 24 hours after infection, the mice were randomly divided into three groups of 6 each: the group injected with physiological saline, the group injected with the polymer of Example 22 (20 mg / kg), and the group injected with the antibiotic meropenem (20 mg / kg). Intravenous administration was performed once every 12 hours for a total of 3 times. At 12 hours after the last dose, the mice were euthanized. Samples were taken from the subcutaneous abscess site, weighed, and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenate was serially diluted and spread on plates, and CFU counting was performed after incubation overnight in a 37 °C incubator to calculate the bacterial load at each infection site. The experimental results are as Figure 11 shown. Compared with the physiological saline group, the treatment with the polymer of Example 22 could significantly reduce the bacterial load at the abscess site by about 1.9 Log, which was comparable to the treatment effect of the antibiotic meropenem. This indicates that the pH-responsive polypeptide mimetic can effectively treat bacterial infection of subcutaneous abscess.

[0292] Example 35: pH-responsive polypeptide mimetic effectively treats fungal infection of skin abrasion in mice

[0293] Female ICR mice aged 6 - 8 weeks were prepared and allowed to acclimatize for two weeks before the experiment. First, Candida albicans was cultured in YPD medium in a shaker at 30 °C until the growth phase, then washed three times with physiological saline, and the bacterial suspension was diluted to 3×10 8 CFU / mL for use. Before the operation, 75 mg / kg of sodium pentobarbital was intraperitoneally injected to anesthetize the mice. The dorsal area of the mice was shaved, the remaining hair was removed using depilatory cream, and the area was disinfected with 75% alcohol. The skin was continuously peeled off using a sterile needle (1×1 cm 2), 10 μL of Candida albicans suspension was dropped onto the abraded site, and covered with a transparent dressing to prevent contamination. At 24 hours after infection, the mice were randomly divided into three groups of 6 each: the group administered with normal saline, the group administered with the polymer of Example 18 (5 mg / mL), and the group administered with the antifungal drug miconazole nitrate (5 mg / mL). Administration was carried out by local dropwise addition once every 12 hours for a total of 4 times. At 12 hours after the last administration, the mice were euthanized. Samples were taken from the subcutaneous abscess site, weighed and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenate was serially diluted and spread on plates, and CFU counts were performed after incubation overnight in a mold incubator at 30 °C to calculate the fungal burden at each infected site. The experimental results are as Figure 12 shown. Compared with the normal saline group, polymer treatment could significantly reduce the fungal burden at the abraded site by about 1.3 Log, which was comparable to the treatment effect of miconazole nitrate. This indicates that the pH-responsive polypeptide mimetic can effectively treat skin abrasion fungal infections.

[0294] Example 36: pH-responsive polypeptide mimetic effectively treats bacterial infection in the thigh muscle of mice

[0295] Female ICR mice aged 6 - 8 weeks were prepared and raised for two weeks before the experiment to adapt to the environment. Four days and one day before infection, the mice were intraperitoneally injected with 150 and 100 mg / kg of cyclophosphamide respectively to cause neutropenia. Escherichia coli was cultured in LB medium in a shaker at 37 °C until the growth phase, washed three times with normal saline, and the bacterial suspension was diluted to 6×10 5 CFU / mL for use. Before the operation, the mice were anesthetized by intraperitoneal injection of 75 mg / kg of sodium pentobarbital. The hair on the right thigh of the mice was removed using depilatory cream, disinfected with 75% alcohol, and then 50 μL of the bacterial suspension was intramuscularly injected. At 1 hour after infection, the mice were randomly divided into three groups of 6 each: the group injected with normal saline, the group injected with the polymer of Example 22 (20 mg / kg), and the group injected with the antibiotic meropenem (20 mg / kg). Administration was carried out by intravenous injection once every 24 hours for a total of 3 times. At 24 hours after the last administration, the mice were euthanized. Samples were taken from the infected thigh tissue, weighed and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenate was serially diluted and spread on plates, and CFU counts were performed after incubation overnight in a mold incubator at 37 °C to calculate the bacterial burden at each infected site. The experimental results are as Figure 13 shown. Compared with the normal saline group, polymer treatment could significantly reduce the bacterial burden at the infected thigh site by about 2.8 Log, which was comparable to the treatment effect of meropenem. This indicates that the pH-responsive polypeptide mimetic can effectively treat bacterial infection in the thigh muscle.

[0296] Example 37: pH-responsive polypeptide mimetic effectively treats bacterial infection in the lungs of mice

[0297] Six- to eight-week-old female ICR mice were prepared and housed for two weeks before the experiment to acclimatize to the environment. Four days and one day before infection, the mice were intraperitoneally injected with 150 and 100 mg / kg of cyclophosphamide respectively to induce neutropenia. Escherichia coli was cultured in LB medium in a shaker at 37 °C until the growth phase, washed three times with physiological saline, and the bacterial suspension was diluted to 6×10 5 CFU / mL for use. Before the operation, the mice were intraperitoneally injected with 500 mg / kg of tribromoethanol to anesthetize them. The hair on the necks of the mice was removed using depilatory cream and disinfected with 75% alcohol. The trachea was exposed surgically, and then 30 μL of the bacterial suspension was injected intratracheally. Two hours after infection, the mice were randomly divided into three groups of 6 each: the group injected with physiological saline, the group injected with the polymer of Example 22 (20 mg / kg), and the group injected with the antibiotic levofloxacin (20 mg / kg). Intravenous administration was carried out once every 12 hours for a total of 2 times. Twelve hours after the last dose, the mice were euthanized. The lung tissues were collected, weighed, and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenate was serially diluted and spread on plates, incubated overnight in a 37 °C mold incubator, and then CFU counting was performed to calculate the bacterial load at each infection site. The experimental results are as Figure 14 shown. Compared with the physiological saline group, polymer treatment could significantly reduce the bacterial load in the lungs of mice by about 1.3 Log, which was comparable to the therapeutic effect of levofloxacin. This indicates that the pH-responsive polypeptide mimetic can effectively treat pulmonary bacterial infections.

[0298] Example 38: The pH-responsive polypeptide mimetic effectively treats bacterial kidney infections in mice

[0299] Six- to eight-week-old female ICR mice were prepared and housed for two weeks before the experiment to acclimatize to the environment. Seven days before infection, 0.2 mL of 2 mg / mL λ-carrageenan was injected into the tail vein of the mice to enhance their susceptibility to bacterial kidney infections. Escherichia coli was cultured in LB medium in a shaker at 37 °C until the growth phase, washed three times with physiological saline, and the bacterial suspension was diluted to 6×10 7 CFU / mL for use. Each mouse was intravenously injected with 200 μL of the bacterial suspension to induce kidney infections. Four hours after infection, the mice were randomly divided into three groups of 6 each: the group injected with physiological saline, the group injected with the polymer of Example 22 (20 mg / kg), and the group injected with the antibiotic meropenem (20 mg / kg). Intravenous administration was carried out once every 24 hours for a total of 3 times. Twenty-four hours after the last dose, the mice were euthanized. The kidney tissues were collected, weighed, and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenate was serially diluted and spread on plates, incubated overnight in a 37 °C mold incubator, and then CFU counting was performed to calculate the bacterial load at each infection site. The experimental results are asFigure 15 As shown, compared with the normal saline group, polymer treatment can significantly reduce the bacterial load in the infected thigh area by about 1.9 Log, which is comparable to the therapeutic effect of meropenem. This indicates that the pH-responsive polypeptide mimetic can effectively treat renal bacterial infections.

[0300] Example 39: pH-responsive polypeptide mimetic effectively treats systemic infections in mice

[0301] Female ICR mice aged 6 - 8 weeks were prepared and allowed to acclimatize to the environment for two weeks before the experiment. Acinetobacter baumannii was cultured in LB medium in a shaker at 37 °C until the growth phase, then washed three times with normal saline, and then diluted with normal saline containing 5% mucin to 2×10 6 CFU / mL for use. Each mouse was intraperitoneally injected with 200 μL of the bacterial suspension to cause systemic infection. At 0.5 hour after infection, the mice were randomly divided into three groups of 6 each: the normal saline injection group, the polymer injection group of Example 22 (25 mg / kg), and the antibiotic tigecycline injection group (10 mg / kg). The mice were administered once by intravenous injection and then fed normally. Infected mice were dissected immediately once they died, and the remaining surviving mice were euthanized 24 hours later. 3 mL of normal saline was injected into the abdominal cavity and the abdomen was massaged for peritoneal lavage. Subsequently, the abdominal cavity was opened and the lavage fluid was recovered for quantitative analysis of bacterial load. Blood was collected by cardiac puncture for quantitative analysis of bacterial load. The heart, liver, spleen, lungs, and kidneys of the mice were dissected, weighed, and homogenized in PBS buffer containing 0.1% TX-100. The tissue homogenates were serially diluted and plated, and CFU counts were performed after incubation overnight in a 37 °C incubator for calculating the bacterial load in each organ. In addition, the heart, liver, spleen, lungs, and kidneys were collected from mice in the three different dosing groups, fixed with 4% paraformaldehyde, and stained with H&E for pathological sectioning. For the survival rate experiment, the mice were fed normally after dosing, their physiological status was observed, and the survival of the infected mice within 7 days was recorded.

[0302] The results of the survival rate experiment are as Figure 16 shown in a. By infecting mice with a lethal dose of Acinetobacter baumannii by intraperitoneal injection, all untreated mice died within 38 hours. After a single intravenous injection of the polymer and tigecycline, the survival rates of the infected mice were significantly increased to 83% and 100% respectively. The bacterial load analysis is as Figure 16 shown in b. Compared with the normal saline group, the polymer was as effective as tigecycline, reducing Acinetobacter baumannii in all five major organs, blood, and peritoneal fluid by about 3.2 - 5.5 Log. In addition, the histological analysis is as Figure 16As shown in c, Acinetobacter baumannii infection can cause various organ lesions, such as necrosis of some hepatocytes, disappearance of the normal white pulp structure of the spleen, thickening of the alveolar wall, and degeneration of some renal tubular epithelial cells; after treatment with the polymer or tigecycline, typical pathological changes including necrosis and inflammatory infiltration were effectively alleviated. These experimental results indicate that the pH-responsive polypeptide mimetic can effectively treat systemic bacterial infections.

[0303] Example 40: Cytotoxicity of the polypeptide mimetic against cancer cells B16

[0304] The B16 mouse melanoma cell line was selected and cultured until the logarithmic growth phase. After digestion with trypsin, these cells were diluted into a cell suspension of 30,000 cells per milliliter. They were inoculated into 96-well plates at 100 μL per well, with 3 replicates per group, and cultured overnight in a 37 °C, 5% CO 2 cell incubator. The polymers of Example 9 and Example 11 of the present invention were dissolved in ultrapure water to prepare an aqueous solution of 4 mg / mL, diluted with culture medium to 200 μg / mL and then serially diluted two-fold, and the original culture medium was replaced with the culture medium containing the polymer. The culture medium without the polymer was used as a negative control, and pure DMSO was used as a blank control. After treating the cells with different concentrations of the polymer for 24 hours, the original culture medium was discarded, and 100 μL of serum-free medium containing the cell viability detection reagent MTT solution (0.5 mg / mL) was added to each well, and incubated at 37 °C in the dark for 4 hours. Finally, the OD at a wavelength of 570 nm was detected with an enzyme-linked immunosorbent assay (ELISA) 570 , and the cell survival rate was calculated according to the formula.

[0305]

[0306] Table 6 Cytotoxicity of the polymer against cancer cells B16

[0307] Polymer Example 9 Example 11 <![CDATA[IC 50 (μg / mL)]]> 34 50

[0308] The obtained IC 50 As shown in Table 6, the polymers all have high anti-cancer activity against cancer cells B16.

[0309] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A pH-responsive polymer, wherein the backbone structure of the polymer is an α-amino acid polymer, a polyoxazoline, a β-amino acid polymer, an α / β-amino acid polymer, a γ-amino acid polymer or a polyoxazine; And in the polymer, at least part of the side chains of the polymerized units contain the A1 structure; L is a substituted or unsubstituted group selected from the following group: none, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 alkyl-O-C0-C4 alkyl, C1-C4 alkyl-S-C0-C4 alkyl, C1-C4 alkyl-NH-C0-C4 alkyl, C1-C4 alkyl-CO-C0-C4 alkyl, C1-C4 alkyl-COO-C0-C4 alkyl, C3-C10 cycloalkyl, a 3-10 membered heterocyclic group including 1-3 heteroatoms selected from O, N, and S, a C6-C10 aryl, a 3-10 membered heterocyclic group including 1-3 heteroatoms selected from O, N, S heteroatom 5-10 membered heteroaryl, C1-C6 alkyl-Rc, C1-C6 alkyl-COO-Rc, C1-C6 alkyl-O-Rc; each Rc is independently selected from the following groups: substituted or unsubstituted: benzyl, C3-C10 cycloalkyl, 3-10 membered heterocyclyl including 1-3 heteroatoms selected from O, N, S, C6-C10 aryl, 5-10 membered heteroaryl including 1-3 heteroatoms selected from O, N, S; the substitution means that one or more H on the group is optionally independently substituted by a group selected from the following group: OH or C1-C4 alkyl; X is NH, NR, guanidine Biguanidine Wherein R is a C1-C6 alkyl group; Y is a pH responsive molecular fragment, and Y is selected from the following group: Preferably 2. The polymer according to claim 1, characterized in that In the polymer, the proportion x% of the polymerization units containing side chains of the A1 structure to all the polymerization units is at least 1%, at least 5% or at least 10%, such as 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

3. The polymer according to claim 1, characterized in that When the polymerized unit has a side chain containing the A1 structure, the polymerized unit has only a single side chain, or further has one or two side chains selected from C1-C8 alkyl groups.

4. The polymer according to claim 1, characterized in that In addition to the polymerized units containing the side chains of the A1 structure, the polymer further contains one or two polymerized units having other side chains, and the polymerized units independently have the following characteristics: The side chain of the polymerized unit contains the structure: -LXH(A2); wherein L and X are as defined above, and the polymerized unit has only a single side chain, or further has one or two side chains selected from C1-C8 alkyl groups; The polymerized unit has 1, 2 or 3 side chains, and the side chains independently contain the structure: L-R2; wherein L is as defined above, and R2 is selected from the following group: H, OH, COOH, C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted 3-10 membered heterocyclic group including 1-3 heteroatoms selected from O, N, and S, substituted or unsubstituted benzyl, substituted or unsubstituted biphenyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5- 10-membered heteroaryl; wherein the substitution means that one or more H on the group is optionally substituted by a group selected from the group: OH, COOH or C1-C4 alkyl; or two side chain groups on adjacent backbone carbon atoms and the connected carbon atoms together form a substituted or unsubstituted C3-C10 cycloalkyl or a substituted or unsubstituted 3-10-membered heterocyclic group including 1-3 heteroatoms selected from O, N, and S, wherein the substitution means that one or more H on the group is optionally substituted by a group selected from the group: OH, COOH or C1-C4 alkyl.

5. The polymer according to claim 1, characterized in that The first attachment site of the side chain on each polymeric unit is shown as Z1 in any of the following structures: Wherein, m is independently 0, 1 or 2; when there are 2 or 3 side chains on the polymerized unit, the second and third side chains may be located at other substitutable positions of the backbone carbon atom, and when present, A1 or A2 is located at the Z1 position; In polyoxazoline or polyoxazine, the first attachment site of the side chain on each polymeric unit is shown as Z1: Wherein, m is independently 1 or 2; when there are 2 or 3 side chains on the polymer unit, the second and third side chains may be located at other substitutable positions of the backbone carbon atom, and when present, A1 or A2 is located at the Z1 site.

6. The polymer according to claim 1, characterized in that The polymer has a chain segment shown in Formula I, Formula II or Formula III: In Formula I, m is independently 1 or 2; In Formula II and Formula III, m is independently 0, 1 or 2; In each formula, n is independently a positive integer of 2-500; x is independently 1-100, y is independently 0-100, z is independently 0-99, x+y+z=100; L, X, Y, and R2 are independently as defined above.

7. The polymer according to claim 1, characterized in that The polymer has H, monomer or initiator residue (such as p-tert-butylbenzyl) at one end and H, monomer residue or other reactive end group (such as -R a 、C1-C8 alkylene R a (such as -tBu tert-butyl R a ), C1-C8 haloalkyl R a (Preferably C3-C8 fluorinated alkyl R a ), C6-C10 aryl-R a 、C1-C8 alkylene C6-C10 aryl-R a 、-NHC1-C8 alkylene R a 、-OC1-C8 alkylene R a 、-COC1-C8 alkylene R a ; Each R a Independently selected from the following group: -SH, -NH2, -COOH, CHO, -Br, -Cl, -OH, 3-6 membered epoxy, alkenyl, alkynyl, -COCl, azido, maleimide, o-pyridyl disulfide (OPSS), cyclodextrin, adamantane.

8. The polymer according to claim 1, characterized in that pH-responsive polymer structures include:

9. The method for preparing a polymer as claimed in claim 1, comprising the following steps: Providing an aqueous solution of the backbone polymer, adjusting the pH to 8.5-9; The pH responsive molecule is added to the polymer solution, and during the addition, the pH is adjusted with a base (such as NaOH) to always be maintained at 8.5-9; The reaction is stirred for 6-24h (preferably 8-16h); The reaction solution was dialyzed in deionized water with a pH value of 8.5-9, and then the sample was dried to obtain the pH-responsive polypeptide mimetic polymer.

10. A pharmaceutical composition comprising the polymer according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. Use of the polymer according to claim 1 in preparing an antibacterial and / or antitumor pharmaceutical composition, preferably the bacteria include Gram-positive bacteria, Gram-negative bacteria, fungi, spores, dormant cells; the tumor is solid cancer or blood cancer, such as gastric cancer, cervical cancer, melanoma, endometrial cancer, esophageal cancer, liver cancer, prostate cancer, breast cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, colon cancer, uterine tumor and soft tissue sarcoma, lymphoma, leukemia, multiple myeloma, mesothelioma, malignant rhabdoid tumor, bile duct and gallbladder cancer, bladder cancer; brain tumor, neuroblastoma, neurilemmoma, glioma, glioblastoma and astrocytoma.

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