A cationic polymer, and lipid compositions and uses comprising the same

Abstract

The present disclosure relates to the technical field of targeted delivery lipids, in particular to a cationic polymer, a lipid composition comprising the same and applications thereof. The cationic polymer provided by the present disclosure has a structure shown in formula (I), and the lipid nanoparticles prepared therefrom can be used for RNA targeted delivery, and can significantly enhance the targeting effect of mRNA drugs on liver or spleen.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field, and specifically relates to a cationic polymer and lipid compositions comprising the same and their applications. Background Technology

[0002] Lipid nanoparticles (LNPs) serve as a nucleic acid drug delivery platform, and their technological value lies in: (1) the combination of cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids to form spherical vesicles with a particle size of about 100 nm, thereby achieving efficient encapsulation of single-chain or circular mRNA and significantly inhibiting its degradation by ribonuclease; (2) the adsorption of apolipoprotein E (ApoE) on the surface of LNPs, followed by recognition and endocytosis by low-density lipoprotein receptor (LDLR), and the protonation of ionizable lipids triggered by pH changes in the acidic environment of the endosome, inducing membrane instability, thereby achieving cytoplasmic release and transient expression of mRNA, thus making them suitable for the delivery of mRNA vaccines or for delivering mRNA capable of expressing therapeutic proteins to cells via LNPs.

[0003] However, existing LNPs still face significant technical bottlenecks. For example, their biodistribution is biased towards hepatocytes, with a targeting efficiency of less than 5% in non-hepatic tissues, thus limiting the precise delivery of LNPs for indications such as tumors, lungs, and central nervous systems. Their endosomal escape efficiency is only 2–3%, leading to an exponential increase in the amount of active substance that can exert an effective dose in vivo, thereby increasing the risk of dose-dependent inflammatory responses. Empty LNPs or high-dose repeated administration pose dose-limiting toxicity risks.

[0004] Therefore, there is a need for LNPs that can deliver active substances to non-hepatic tissues, preferably those that can improve endosomal escape rate and reduce immunogenicity, thereby enhancing their safety and specificity. Summary of the Invention

[0005] This disclosure provides a cationic polymer, a lipid composition comprising the same, and applications. The cationic polymer can be used to assist mRNA-LNP delivery systems, improve LNP delivery efficiency, achieve high transfection efficiency in transfecting cells, and exhibit low cytotoxicity. It can enhance delivery efficiency and safety, and has enhanced liver or spleen targeting effects.

[0006] This invention provides a polymer or a pharmaceutically acceptable salt thereof having the structure shown in formula (I):

[0007] ;

[0008] in, for , for ;

[0009] or, for , for ;

[0010] symbol" " indicates that the repeating units in the polymer backbone, which are divided by it, are connected in a disordered manner;

[0011] R is ;

[0012] R2 is -COOH, -C 1-6 Alkylene -NH2 or -CONH(CH2) p1 NH(CH2) p2 NH(CH2) p3 NH2;

[0013] p1, p2, and p3 are independent integers from 1 to 5;

[0014] R3 is -NR 3a R 3b ;

[0015] R 3a For H or C 1-6 alkyl;

[0016] R 3b C 1-6 Alkyl or with one or more R 3-1 Replacement C 1-6 alkyl;

[0017] R 3-1 It can be independently -OH, a 5-10 membered heteroaryl group, or a 4-10 membered heterocyclic alkyl group;

[0018] Or R 3a R 3b Together with the nitrogen atom it is attached to, it forms a 5-10 membered heteroaryl group, a 4-10 membered heterocyclic alkyl group, and is bonded to one or more R atoms. 3-2 Substituted 5-10 heteroaryl groups, or substituted with one or more R groups 3-3 Substituted 4-10 membered heterocyclic alkyl groups;

[0019] R 3-2 and R 3-3 Independent of halogen, C 1-6 Alkyl, C 1-6 alkoxy or hydroxy substituted C 1-6 alkyl;

[0020] X is -NHC(O)- or -C(O)NH-;

[0021] Y can be -C(O)O-, -NH-, or -NH(CH). q1 NH-, -NH(CH) q2 NH(CH) q3 NH- or not present;

[0022] q1, q2, and q3 are independent integers from 1 to 5;

[0023] Z is -NH-, -C(O)NH-, or -NHC(O)-;

[0024] m and n are each integers from 1 to 100;

[0025] a is an integer from 0 to 5;

[0026] b is an integer from 0 to 10;

[0027] c is an integer from 0 to 10;

[0028] The heteroatoms in the 5-10 membered heteroaryl and 4-10 membered heterocyclic alkyl groups are independently one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0029] In some embodiments, repeating units A1 and A2 in the polymer are connected in a disordered manner.

[0030] In some embodiments, the polymer is a random copolymer.

[0031] In some embodiments, the polymer has an end group of -NHR1 on one side and an end group of H on the other side; R1 is C. 1-10 Alkyl, benzyl or -C 1-6 Alkylene-(OCH2CH2)t-OC 1-6 Alkyl; t is an integer from 1 to 100.

[0032] In some embodiments, the polymer has the structure shown in formula (Ia):

[0033] ;

[0034] Where R1 is C 1-10 Alkyl, benzyl or -C 1-6 Alkylene-(OCH2CH2)t-OC 1-6 Alkyl group; t is an integer from 1 to 100;

[0035] The definitions of R2, R3, X, Y, Z, m, n, a, b, and c are as described above.

[0036] In some implementations, t is an integer from 40 to 50.

[0037] In some implementations, R1 is , or .

[0038] In some implementations, R2 is , or .

[0039] In some implementation schemes, R 3a It can be H, -CH3, or -CH2CH3.

[0040] In some implementation schemes, R 3-2 and R 3-3 Independently for C 1-6 alkyl or hydroxy substituted C 1-6 alkyl.

[0041] In some implementations, R3 is , , , , , , , , , , or .

[0042] In some implementations, Y is -NH(CH)2NH-, -NH(CH)2NH(CH)2NH-, -C(O)O-, -NH-, or is not present.

[0043] In some implementations, Z is -NH- or -C(O)NH-.

[0044] In some implementations, a is 0 or 3.

[0045] In some implementations, b is 0, 2, 3, 5, or 6.

[0046] In some implementations, b is 0, 2, or 5; preferably 5.

[0047] In some implementations, c is 5 or 6; preferably 6.

[0048] In some implementations, m is 5-60, such as 10, 12, 13, 14, 15, 16, 17, 18, 22, 24 or 47.

[0049] In some implementations, n is 5-60, such as 6, 8, 12, 13, 14, 15, 16, 17, 18, 20, 26, 28, 35 or 53.

[0050] In some implementations, the sum of m and n is 20-120, such as 30, 50, or 100.

[0051] In some embodiments, the polymer has the structure shown in formula (Ib):

[0052] ;

[0053] Where R1 is C 1-10 Alkyl or -C 1-6 Alkylene -(OCH2CH2)t-OCH3; t is an integer from 1 to 100;

[0054] R3 is -NR 3a R 3b ;

[0055] R 3a For H or C 1-6 alkyl;

[0056] R 3b C 1-6 Alkyl or with one or more R 3-1 Replacement C 1-6 alkyl;

[0057] R 3-1 It is independently a 5-10 membered heteroaryl or a 4-10 membered heterocyclic alkyl;

[0058] Or R 3a R 3b Together with the nitrogen atom it is attached to, it forms a 5-10 membered heteroaryl group, a 4-10 membered heterocyclic alkyl group, and is bonded to one or more R atoms. 3-2 Substituted 5-10 heteroaryl groups, or substituted with one or more R groups 3-3 Substituted 4-10 membered heterocyclic alkyl groups;

[0059] R 3-2 and R 3-3 Independently for C 1-6 alkyl;

[0060] b is an integer from 4 to 8;

[0061] c is an integer from 4 to 8;

[0062] m is 5-60;

[0063] n is 5-60;

[0064] The heteroatoms in the 5-10 membered heteroaryl and 4-10 membered heterocyclic alkyl groups are independently one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0065] In some embodiments, the polymer is any of the following polymers:

[0066] ,

[0067] ,

[0068] ,

[0069] ,

[0070] ,

[0071] ,

[0072] ,

[0073] ,

[0074] ,

[0075] ,

[0076] ,

[0077] ,

[0078] ,

[0079] ,

[0080] ,

[0081] ,

[0082] ,

[0083] ,

[0084] ,

[0085] ,

[0086] ,

[0087] ,

[0088] ,

[0089] ,

[0090] ,

[0091] or

[0092] .

[0093] The present invention provides a carrier comprising a cationic polymer, said cationic polymer being a polymer as described above or a pharmaceutically acceptable salt thereof.

[0094] In some embodiments, the carrier further includes cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0095] In some embodiments, the molar ratio of the cationic polymer to the cationic lipid is 1:(1~500), preferably 1:(1~100), for example 1:10 or 1:20.

[0096] In some embodiments, the molar ratio of the cationic polymer to the neutral lipid is 0.01:1 to 5:1, preferably 0.05:1 to 5:1, more preferably 0.075:1 or 0.125:1.

[0097] In some embodiments, the molar ratio of the cationic polymer to the structural lipid is 0.01:1 to 1.5:1, preferably 0.033:1 or 0.019:1.

[0098] In some embodiments, the molar ratio of the polymer conjugated lipid to the carrier is 0.005:1 to 0.1:1, preferably 0.0075:1, 0.0125:1 or 0.015:1.

[0099] In some embodiments, the cationic lipid is SM-102, MC3 or YK-009, preferably YK-009.

[0100] In some embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0101] In some embodiments, the neutral lipid is selected from any or a combination of at least two of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl -2-Oleoyl-sn-glycerol-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine, 1-Oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphocholine, 1-hexadecyl-sn-glycerol-3-phosphocholine, 1,2-dilinanoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphocholine), 1,2-dioleoyl- sn-glycerol-3-phosphate ethanolamine, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine Sodium rac-(1-glycerol), dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0102] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphocholine.

[0103] In some embodiments, the structural lipid is selected from any or at least a combination of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids; preferably cholesterol.

[0104] In some embodiments, the polymeric conjugated lipid is selected from any or at least a combination of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, preferably any or at least a combination of distearylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol bistetradecylacetamide; more preferably 1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol 2000.

[0105] In some embodiments, the molar ratio of the cationic polymer, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid in the carrier is (0.2~25): (25~75): (5~25): (15~65): (0.5~10), preferably (0.05~5): (35~49): (7.5~15): (35~55): (0.5~5).

[0106] In some embodiments, the molar ratio of the cationic polymer, cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid in the carrier is 1.25: 49.5: 10: 38: 1.25 or 0.75: 50: 10: 38.5: 0.75.

[0107] The present invention provides a composition comprising an active ingredient and a carrier as described above.

[0108] In some embodiments, the composition is a nanoparticle formulation with an average particle size of 10 nm to 300 nm, preferably 40 nm to 240 nm.

[0109] In some embodiments, the polydispersity index of the nanoparticle formulation is ≤0.5, preferably ≤0.4.

[0110] In some embodiments, the active ingredient comprises a therapeutic or preventative agent.

[0111] In some embodiments, the mass ratio of the carrier to the therapeutic or preventative agent is 5:1 to 15:1, preferably 10:1.

[0112] In some implementations, the therapeutic or preventative agent is a vaccine or compound capable of evoking an immune response.

[0113] In some embodiments, the therapeutic or preventative agent is selected from any one or a combination of at least two of the groups consisting of nucleic acids, small molecule compounds, or polypeptides.

[0114] In some embodiments, the therapeutic agent or the preventive agent is a nucleic acid, preferably ribonucleic acid.

[0115] In some embodiments, the ribonucleic acid is selected from any or a combination of at least two of the following: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA, preferably messenger RNA.

[0116] In some embodiments, the composition further includes pharmaceutically available excipients and / or diluents.

[0117] The present invention provides the use of the polymer as described above or a pharmaceutically acceptable salt thereof, the carrier as described above, or the composition as described above in the preparation of a medicine.

[0118] The present invention provides the use of the polymer or carrier described above in improving cell transfection efficiency and / or reducing cytotoxicity.

[0119] The present invention provides the use of the polymer or carrier described above in improving the targeting of nucleic acids to a target, and / or in increasing the expression level of nucleic acids in a target, wherein the target is selected from any one of the group consisting of target organs, target tissues and target cells or a combination of at least two of them.

[0120] In some implementations, the target organ or target tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph nodes and muscle.

[0121] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this disclosure, the use and terminology of this disclosure shall prevail.

[0122] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0123] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0124] Unless otherwise indicated in the working embodiments or elsewhere, all numerical values ​​for quantitative properties such as dosages set forth in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range enumerated in this disclosure is intended to include all subranges within that range and any combination of the endpoints of that range or subranges. When a numerical range is disclosed herein, the range is considered continuous and includes both the minimum and maximum values ​​of the range, and every value between such minimum and maximum. Further, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0125] As used herein, the words “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms “containing,” “including,” or “comprising” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the above terms also include “consistently composed of” or “composed of.”

[0126] The terms “subject” or “patient” in this disclosure include humans and mammals.

[0127] As used herein, the term "treatment" means the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of said disease, in order to cure, alleviate, reduce, improve, or affect said disease or its symptoms. In the context of this disclosure, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0128] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0129] The term "alkyl" in this disclosure refers to a branched and straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. n-m This refers to groups that include carbon atoms from n to m. For example, C 2-5 Alkyl groups include C2 alkyl, C3 alkyl, C4 alkyl, and C5 alkyl.

[0130] The term "alkylene" refers to the further substitution of one hydrogen atom of an alkyl group.

[0131] The term "alkoxy" refers to -O-alkyl, and the definition of alkyl is as described above.

[0132] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group containing 3-10 ring carbon atoms, for example 3-8 ring carbon atoms, or even 3-6 ring carbon atoms, which may have one or more rings, for example, one or two rings. For example, "C 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3-6 ring carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and similar groups.

[0133] The term "heterocyclic alkyl" refers to a group in which one or more carbon atoms of a cycloalkyl group are substituted with heteroatoms. "3-8 membered heterocyclic alkyl" means that in a cycloalkyl group having 3-8 carbon atoms, any one or more carbon atoms are substituted with heteroatoms or heterogroups. In some embodiments, the heteroatoms and heterogroups are selected from one or more of O, N, S, and -SO2-. Examples of heterocyclic alkyl groups include, but are not limited to, azirrobutyl, piperidinyl, piperazine, tetrahydropyrrole, etc. wait.

[0134] The term "heteroaryl" refers to a heteroaryl system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from one or more of O, S, and N. Examples of heteroaryl groups include, but are not limited to, imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, pyrrolel, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, etc.

[0135] "Therapeutic effective amount" is the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" is the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can determine the therapeutic and prophylactic effective amounts conventionally based on their knowledge and this disclosure.

[0136] In this disclosure, when the name of a compound differs from its structural formula, the structural formula shall prevail.

[0137] As used in this article, the term "cationic polymer" refers to a polymer that carries a positive charge within a specific pH range (e.g., pH 4.5–6.8).

[0138] Cationic polymers readily bind to negatively charged nucleic acids, that is, they interact with the negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs).

[0139] This disclosure provides a composition comprising a carrier, said carrier including cationic polymers, cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0140] In one embodiment, the composition is a nanoparticle formulation, wherein the average size of the nanoparticle formulation is 10 nm to 300 nm, preferably 40 nm to 240 nm; and the polydispersity index of the nanoparticle formulation is ≤0.5, preferably ≤0.4.

[0141] cationic polymers

[0142] In one embodiment of the composition / carrier disclosed herein, the cationic polymer is any one of YK-POL-001 to YK-POL-027.

[0143] In one embodiment of this disclosure, the cationic polymer accounts for about 0.2% to 25% of the carrier molar ratio, for example about 1%, 5%, 10%, or 20%.

[0144] This carrier can be used for the delivery of active ingredients such as therapeutic and / or preventative agents. The active ingredient can be encapsulated within the carrier or bound to the carrier in any form.

[0145] For example, examples of the therapeutic agent or the preventive agent may be one or more of nucleic acid molecules, small molecule compounds, or peptides. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0146] cationic lipids

[0147] The carrier also comprises cationic lipids. In one embodiment of the composition / carrier disclosed herein, the cationic lipid is: (a) one or more selected from compounds in CN 114044741 B or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers; (b) the cationic lipid compound may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, (b) the cationic lipid compound may be SM-102 in CN102625696B, or MC3 in CN102625696B.

[0148] In one embodiment, the molar ratio of the cationic polymer to the cationic lipid is about 1:(1~500), for example about 1:1.1, 1:10, 1:100, 1:300, 1:400, or 1:499.

[0149] neutral lipids

[0150] The carrier may contain neutral lipids. In this disclosure, neutral lipids refer to lipids that exist in a charge-free form or as neutral ions within a specific pH value or range. These neutral lipids may modulate the flowability of nanoparticles to form a lipid bilayer and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0151] In one embodiment, the molar ratio of the cationic polymer to the neutral lipid is about 0.01:1 to 5:1, for example, about 0.02:1, 0.1:1, 1:1, 2:1, and 4.9:1. In another preferred embodiment, the molar ratio of the cationic polymer to the neutral lipid is about 0.125:1, and in yet another preferred embodiment, the molar ratio is about 0.075:1.

[0152] For example, neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0153] The carrier component of a composition comprising cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0154] Neutral lipids may be selected from the non-restrictive group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. Fatty acids may be selected from the non-restrictive group consisting of lauric acid, myristic acid, myristenoic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzanoic acid, docosapentaenoic acid, and docosahexaenoic acid. Also encompassing are non-natural species including natural species with modifications and substitutions, such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of a composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components such as targeting or imaging components (e.g., dyes).

[0155] The neutral lipids that can be used in these compositions may be selected from the non-limiting group of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether 1,2-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatid ...phosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphat Phosphatidylethanolamine (POPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0156] In some embodiments, neutral lipids include DSPC. In some embodiments, neutral lipids include DOPE. In some embodiments, neutral lipids include both DSPC and DOPE.

[0157] structural lipids

[0158] The carrier of the composition containing the cationic polymer may also include one or more structural lipids. The structural lipids can enhance the stability of the nanoparticles by filling the gaps between the lipids.

[0159] In one embodiment, the molar ratio of the cationic polymer to the structural lipid is about 0.01:1 to 1.5:1, for example, about 0.02:1, 0.05:1, 0.1:1, 0.2:1, 1:1, 2:1, 3:1, 4:1, and 4.9:11. In another preferred embodiment, the molar ratio of the cationic polymer to the structural lipid is about 0.033:1, and in yet another preferred embodiment, the molar ratio of the cationic polymer to the neutral lipid is about 0.019:1.

[0160] Structural lipids may be selected from, but are not limited to, the group consisting of: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0161] Polymer conjugated lipids

[0162] The carrier of the composition containing the cationic polymer may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages.

[0163] In one embodiment, the polymeric conjugated lipid comprises about 0.5% to 10% of the carrier, for example, about 0.6%, 0.9%, 1.5%, 3%, 9%, and 9.9%. In another preferred embodiment, the polymeric conjugated lipid comprises about 1.25% of the carrier, and in yet another preferred embodiment, the polymeric conjugated lipid comprises about 0.75% of the carrier.

[0164] In one embodiment, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350-5000 Da.

[0165] For example, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0166] Therapeutic agents and / or preventative agents

[0167] The composition may include one or more therapeutic and / or preventive agents. In one embodiment, the mass ratio of the cationic polymer to the therapeutic or preventive agent is 5:1 to 15:1, for example, 6:1, 7:1, 10:1, or 14.9:1.

[0168] In another embodiment, the mass ratio of the cationic polymer to the therapeutic or preventative agent is 10:1.

[0169] The therapeutic or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, or polypeptides.

[0170] For example, the therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

[0171] The carriers disclosed herein can deliver therapeutic and / or preventative agents to mammalian cells or organs. Accordingly, this disclosure provides the use of the polymers of this disclosure or pharmaceutically acceptable salts thereof or compositions of this disclosure in the preparation of medicaments for treating a disease or condition of a subject in need.

[0172] This disclosure also provides the use of the polymers or compositions of this disclosure in the preparation of nucleic acid drugs, vaccines, chemical drugs or peptide drugs.

[0173] Therapeutic agents and / or preventative agents include bioactive substances and are alternatively referred to as "active agents." Therapeutic agents and / or preventative agents can be substances that, upon delivery to a cell or organ, induce a desired change in that cell or organ or other body tissue or system. Such species can be used to treat and / or prevent one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventative agents are small molecule pharmaceutical products that can be used to treat a specific disease, condition, or illness.Examples of pharmaceuticals that can be used in a composition include, but are not limited to, anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside). Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0174] In some implementations, the therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraquinone. Anthracindione, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Examples of vaccines include compounds and formulations that provide immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. These may include, for example, mRNA encoding pathogenic antigens and / or their epitopes. Vaccines may also include compounds and formulations that direct an immune response against cancer cells, such as mRNA encoding tumor cell-derived antigens, epitopes, and / or novel epitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and / or compound capable of evoking an immune response is administered intramuscularly by a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), or (III) (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122).Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, lactamazole (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), and cyclophosphamide. Phosphoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP, cisplatin), anthracyclines (e.g. daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), and antimitotic agents (e.g. vincristine, vinblastine, paclitaxel and levothyroxine).

[0175] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to this disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA) and its hybrids; RNAi inducible factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribonuclease; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribonuclease, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some implementations, the RNA is mRNA.

[0176] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any polypeptide that is naturally or non-naturally present or otherwise modified. The polypeptide encoded by the mRNA may have any size and may possess any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells.

[0177] In other embodiments, the therapeutic and / or preventative agent is siRNA. siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA may be chosen such that, upon administration of a composition comprising the siRNA to a subject in need, a gene associated with a specific disease, symptom, or condition is silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0178] In some implementations, the therapeutic and / or preventative agents are sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0179] In some implementations, the therapeutic and / or prophylactic agent is shRNA or its encoding vector or plasmid. shRNA can be generated within the target cell after delivery of an appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant field.

[0180] Disease or ailment

[0181] The compositions / carriers disclosed herein can deliver therapeutic or preventative agents to subjects or patients. These therapeutic or preventative agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, or peptides. Therefore, the compositions disclosed herein can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, or peptide drugs. Due to the wide variety of such therapeutic or preventative agents, the compositions disclosed herein can be used to treat or prevent a variety of diseases or conditions.

[0182] In one embodiment, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0183] For example, the disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0184] In one implementation, the infectious disease is selected from diseases caused by influenza virus, hepatitis virus, immunodeficiency virus, rabies virus, human papillomavirus, respiratory syncytial virus, herpesvirus, etc., as well as pneumonia, Rift Valley fever, yellow fever, etc.

[0185] Other components

[0186] The composition may include one or more components other than those described in the foregoing sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0187] The composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0188] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), and mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, and eprazinone). The composition may contain mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, streptococcal DNase α (dornasealfa), neltenexine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0189] The composition may also contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0190] In addition to these components, the composition may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0191] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0192] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0193] The compositions disclosed herein can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. For example, a sterile injectable solution can be prepared by incorporating the desired amount of the therapeutic or prophylactic agent with the various other components described above into a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Surfactants may also be added to promote the formation of a homogeneous solution or suspension.

[0194] For example, the compositions disclosed herein can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the compositions are administered intravenously.

[0195] The compositions disclosed herein are administered in therapeutically effective amounts, which can vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, the therapeutic or prophylactic agent can be administered to a subject (preferably a mammal, such as a human) at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0196] It should be understood that the uses provided in this disclosure may include both therapeutic and diagnostic uses, as well as non-therapeutic and non-diagnostic uses. For example, therapeutic uses may include using the polymers or lipid compositions provided in this disclosure to encapsulate and deliver the (drug) active ingredient to target organs / tissues / cells, or using the compositions of this disclosure to deliver the contained active ingredient to target organs / tissues / cells, thereby achieving the effects of treating diseases, improving symptoms, and regulating physiological activities in the body; diagnostic uses may include encapsulating the active ingredient for disease diagnosis in the cationic polymers provided in this disclosure, so that the active ingredient is delivered to target organs / tissues / cells, thereby achieving the purpose of disease diagnosis; non-therapeutic / non-diagnostic uses may include using the cationic polymers or lipid compositions provided in this disclosure to encapsulate the active ingredient, thereby delivering it to target organs / tissues / cells, for non-therapeutic and non-diagnostic purposes such as scientific research and detection (e.g., conducting disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0197] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0198] The reagents and raw materials used in this invention are all commercially available.

[0199] The positive and progressive effects of this invention are that the cationic polymer and lipid composition disclosed herein can be used for the encapsulation and delivery of active pharmaceutical ingredients such as nucleic acids (e.g., mRNA).

[0200] Compared with the prior art, the mRNA-LNP composition prepared by the cationic polymer disclosed herein has one or more of the following advantages: it can significantly increase the expression level of protein in vivo and in vitro, has significant spleen targeting, and has improved biosafety or significantly improved cell survival rate. Detailed Implementation

[0201] The present disclosure is further described below with reference to embodiments, but the present disclosure is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. In the specific embodiments of the present disclosure, the raw materials used are all commercially available. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0202] In the following embodiments, the abbreviations have the following meanings:

[0203] Boc2O: ditert-butyl dicarbonate; DMAP: 4-dimethylaminopyridine; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HBTU: O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; Cbz: benzyloxycarbonyl; TEA: triethylamine; HEX: n-hexane; EA: ethyl acetate; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; MeOH: methanol; DMSO: dimethyl sulfoxide; NMP: N-methylpyrrolidone; TFA: trifluoroacetic acid.

[0204] Example 1: Synthesis of cationic polymers

[0205] 1. Synthesis of intermediate INT-1

[0206]

[0207] The symbol " in the INT-1 structure "" indicates that the repeating units divided by it in the main chain are connected in an unordered manner.

[0208] Step 1: Synthesis of INT-1-PM1

[0209] L-Aspartic acid-4-benzyl ester (10.00 g, 44.80 mmol) was dissolved in tetrahydrofuran (150 mL), propylene oxide (13.00 g, 223.83 mmol) was added, followed by the slow addition of triphosgene (6.60 g, 22.24 mmol). The reaction was carried out at room temperature for 4 h until the solution became clear. The reaction was monitored by TLC until the starting material had completely reacted. The reaction was stopped, and the solution was concentrated under reduced pressure. A small amount of the solution was added dropwise to n-hexane at 0 °C to precipitate the product. After filtration, the crude product was dissolved in tetrahydrofuran and crystallized again with n-hexane. The product was filtered, and the white crystalline product was collected and concentrated to give INT-1-PM1 (10.11 g, 40.57 mmol, 90.6%). 12 H 11 NO5, MS(ES): m / z (M+H+ 250.1.

[0210] Step 2: Synthesis of INT-1-PM2

[0211] INT-1-PM1 (2.00 g, 8.03 mmol) was dissolved in N,N-dimethylformamide (20 mL), and nitrogen gas was introduced to replace the nitrogen atmosphere. Then, n-hexylamine (27 mg, 0.27 mmol) was added, and the reaction was carried out at room temperature for 24 h. The reaction was characterized as complete by infrared spectroscopy. The reaction was stopped, and the reaction mixture was added dropwise to a mixture of diethyl ether and n-hexane at 0 °C. After centrifugation, the residue was removed. This process was repeated three times. The residue was dried under vacuum to obtain a white solid INT-1-PM2 (1.98 g, >100%). Infrared characteristic peak signal: carbonyl signal 1840 cm⁻¹. -1 The peak disappears and is replaced by the characteristic peak of amide at 1680 cm⁻¹. -1 . 1 H NMR (400 MHz, DMSO-d6) δ 8.44-7.98 (m, 87 H), 7.42-7.22(m, 58 H), 5.18 (s, 59 H), 4.25 (m, 30 H), 2.79-2.51 (m, 61 H), 1.63-1.26 (m,8 H), 0.78 (t, 3H).

[0212] Step 3: Synthesis of INT-1

[0213] INT-1-PM2 (1.00 g, 0.16 mmol) was dissolved in DMSO, and then TEA (1.46 g, 14.40 mmol) and triethylenetetramine (2.10 g, 14.40 mmol) were added sequentially. The mixture was heated to 40 °C and stirred for 24 hours. The reaction progress was monitored by infrared spectroscopy at 1740 cm⁻¹. -1 The peak disappeared. The reaction solution was added to 3.5 kDa for dialysis purification, and then freeze-dried to obtain product INT-1 (400 mg, 0.05 mmol, 33.8%). 1 H NMR (400 MHz, DMSO-d6) δ 4.28 (m, 30 H), 3.44-3.18 (m, 56H), 2.77-2.43 (m, 320 H), 1.52-1.29 (m, 8 H), 0.85 (t, 3H).

[0214] 2. Synthesis of intermediate INT-2

[0215]

[0216] Step 1: Synthesis of INT-2-PM1

[0217] Using N6-Cbz-L-lysine (20.00 g, 71.35 mmol) as a starting material, the product was collected and concentrated according to the synthesis method of INT-1-PM1 to obtain INT-2-PM1 (10.00 g, 32.65 mmol, 45.8%). 15 H 18 N₂O₅, MS(ES): m / z (M+H) + )307.1.

[0218] Step 2: Synthesis of INT-2-PM2

[0219] Using INT-2-PM1 (1.00 g, 3.26 mmol) as the starting material, a white solid INT-2-PM2 (0.80 g, 92.3%) was obtained according to the synthesis method of INT-1-PM2. Infrared characteristic peak signal: carbonyl signal 1840 cm⁻¹. -1 The peak disappears and is replaced by the characteristic peak of amide at 1680 cm⁻¹. -1 .

[0220] Step 3: Synthesis of INT-2

[0221] Dissolve INT-2-PM2 (790 mg, 0.10 mmol) in trifluoroacetic acid (9 mL), and slowly add HBr / EA solution (3 mL). Stir overnight at room temperature. The reaction is complete when the solution becomes clear. Slowly add saturated sodium bicarbonate solution to neutralize the large amount of acid. Transfer the solution to a 30 kDa dialysis bag and dialysis for purification. Repeat three times. Freeze-dry to obtain the white product INT-2 (300 mg, 0.08 mmol, 76.7%). 1 H NMR (400 MHz, D2O) δ 4.58- 4.05 (m, 29 H), 3.09-2.88 (m, 58 H), 1.89-1.52 (m, 102 H), 1.44-1.20 (m, 77 H), 0.77 (t, 3 H).

[0222] 3. Synthesis of intermediate INT-3

[0223]

[0224] Step 1: Synthesis of INT-3-PM1

[0225] Using INT-2-PM1 (1.00 g, 3.26 mmol) as the starting material, INT-3-PM1 (720 mg, 0.05 mmol, 83.4%) was obtained by following the synthesis method of INT-1-PM2.

[0226] Step 2: Synthesis of INT-3

[0227] Using INT-3-PM1 (720 mg, 0.05 mmol) as a starting material, INT-3 (310 mg, 0.05 mmol, 87.4%) was obtained by following the synthesis method of INT-2. 1 H NMR (400 MHz, D2O) δ 4.58- 3.99 (m, 47 H), 3.13-2.88 (m, 98H), 1.91-1.50 (m, 176 H), 1.46-1.20 (m, 132 H), 0.77 (t, 3 H).

[0228] 4. Synthesis of intermediate INT-4

[0229]

[0230] Step 1: Synthesis of INT-4-PM1

[0231] Using INT-2-PM1 (1.00 g, 3.26 mmol) as a starting material, INT-4-PM1 (680 mg, 0.03 mmol, 79.1%) was obtained by following the synthesis method of INT-1-PM2.

[0232] Step 2: Synthesis of INT-4

[0233] Using INT-4-PM1 (680 mg, 0.03 mmol) as a starting material, INT-4 (300 mg, 0.02 mmol, 89.9%) was obtained by following the synthesis method of INT-2. 1 H NMR (400 MHz, D2O) δ 4.58- 3.99 (m, 96 H), 3.13-2.88 (m, 193 H), 1.91-1.50 (m, 340 H), 1.46-1.20 (m, 260 H), 0.77 (t, 3 H).

[0234] 5. Synthesis of intermediate INT-5

[0235]

[0236] Step 1: Synthesis of INT-5-PM1

[0237] Using INT-2-PM1 (1.00 g, 3.26 mmol) as a starting material, INT-5-PM1 (750 mg, 0.09 mmol, 86.4%) was obtained by following the synthesis method of INT-1-PM2.

[0238] Step 2: Synthesis of INT-5

[0239] Using INT-5-PM1 (750 mg, 0.09 mmol) as a starting material, INT-5 (290 mg, 0.07 mmol, 78.0%) was obtained by following the synthesis method of INT-2. 1 H NMR (400 MHz, D2O) δ 8.47 (m, 1 H), 8.26 (m, 2 H), 7.27 (m, 2H), 5.34 (s, 2 H), 4.25 (s, 30 H), 3.09-2.68 (m, 63 H), 1.78-1.48 (m, 98 H),1.44-1.20 (m, 76 H).

[0240] 6. Synthesis of intermediate INT-6

[0241]

[0242] Step 1: Synthesis of INT-6-PM1

[0243] Using INT-2-PM1 (1.00 g, 3.26 mmol) as a starting material, INT-6-PM1 (800 mg, 0.06 mmol, 92.7%) was obtained by following the synthesis method of INT-1-PM2.

[0244] Step 2: Synthesis of INT-6

[0245] Using INT-6-PM1 (800 mg, 0.06 mmol) as a starting material, INT-6 (210 mg, 0.03 mmol, 53.3%) was obtained by following the synthesis method of INT-2. 1 H NMR (400 MHz, D2O) δ 8.47 (m, 1 H), 8.26 (m, 2 H), 7.28 (m,2H), 5.33 (s, 2 H), 4.22 (s, 46 H), 3.09-2.68 (m, 98 H), 1.78-1.48 (m, 160H), 1.44-1.20 (m, 126H).

[0246] 7. Synthesis of intermediate INT-7

[0247]

[0248] Step 1: Synthesis of INT-7-PM1

[0249] 2(S)-1-amino-3-chloroprop-2-ol (60.00 g, 410.93 mmol) was dissolved in ultra-dry DCM (600 mL), cooled to 0 °C, and triethylamine (124.75 g, 1232.79 mmol) was slowly added dropwise, followed by Boc2O (358.74 g, 1643.72 mmol). After the addition was complete, the temperature was raised to 40 °C and the reaction was carried out for 16 h. The reaction was monitored by TLC until the starting material was completely reacted. The reaction was stopped by cooling, and the reaction solution was quenched by adding dropwise to a saturated sodium bicarbonate aqueous solution. Dichloromethane was added for extraction, and the solution was dried over anhydrous sodium sulfate. The organic phase was then concentrated after filtration. The residual liquid was wet-processed and purified by silica gel column chromatography with an elution gradient of HEX / EA = 0-60%. The product was collected and concentrated to obtain INT-7-PM1 (62.00 g, 295.70 mmol, 72.0%). 16 ClNO3, MS(ES): m / z (M+H) + -56) 154.1.

[0250] Step 2: Synthesis of INT-7-PM2

[0251] INT-7-PM1 (4.00 g, 19.08 mmol) was dissolved in acetonitrile (20 mL), followed by the weighing of potassium carbonate (7.91 g, 57.23 mmol) and N-methylethanolamine (1.58 g, 21.06 mmol). The mixture was heated to 70 °C and reacted for 12 h. The reaction was monitored for completeness by TLC. After the starting materials had completely reacted, the mixture was slowly cooled to room temperature, filtered to remove salts, and the organic phase was concentrated by rotary evaporation under reduced pressure. The residual liquid was loaded onto a silica gel column for purification using a DCM / MeOH elution gradient of 0-15%. The product was collected and concentrated to obtain INT-7-PM2 (4.20 g, 16.91 mmol, 88.7%). 11 H 24 N₂O₄, MS(ES): m / z (M+H) + 249.2.

[0252] Step 3: Synthesis of INT-7-PM3

[0253] INT-7-PM2 (4.20 g, 16.91 mmol) was dissolved in ultra-dry dichloromethane (10 mL), and HCl / EA (15 mL) was slowly added dropwise. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by TLC until the reactants had completely reacted. The reaction was stopped, the product was collected by rotary evaporation and concentrated. The pH was adjusted to neutral with saturated sodium bicarbonate, and the product was extracted three times with EA. After drying with anhydrous sodium sulfate, the product was filtered and concentrated to obtain INT-7-PM3 (2.50 g, 16.87 mmol, 99.7%). C6H 16 N2O2, MS(ES): m / z (M+H)+ 149.1.

[0254] Step 4: Synthesis of INT-7

[0255] Hexabromohexanoic acid (1.37 g, 7.04 mmol) was dissolved in acetonitrile (10 mL), and then HBTU (2.67 g, 7.04 mmol) and N,N-diisopropylethylamine (1.82 g, 14.08 mmol) were added sequentially. INT-7-PM3 (870 mg, 5.87 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 2 h. The reaction was characterized as complete by TLC. The reaction was stopped, and the mixture was extracted three times with dichloromethane after adding saturated sodium bicarbonate aqueous solution. The organic phase was separated and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated by rotary evaporation under reduced pressure. The residual liquid was wet-processed and purified by silica gel column chromatography with an elution gradient of DCM / MeOH = 0-15%. The product INT-7 (950 mg, 2.92 mmol, 49.8%) was obtained by rotary evaporation. 12 H 25 BrN2O3, MS (ES): m / z (M+H) + 325.2.

[0256] 8. Synthesis of intermediate INT-8

[0257]

[0258] Step 1: Synthesis of INT-8-PM1

[0259] Using INT-7-PM1 (2.00 g, 9.54 mmol) as the starting material, the product INT-8-PM1 (1.51 g, 6.18 mmol, 64.8%) was obtained by synthesizing INT-7-PM2 using the same method. 12 H 24 N₂O₃, MS (ES): m / z (M+H) + 245.1.

[0260] Step 2: Synthesis of INT-8-PM2

[0261] Using INT-8-PM1 (1.51 g, 6.18 mmol) as a starting material, INT-8-PM2 (850 mg, 5.89 mmol, 95.4%) was synthesized following the method used for INT-7-PM3. C7H 16 N₂O, MS(ES): m / z (M+H) + 145.1.

[0262] Step 3: Synthesis of INT-8

[0263] Using INT-8-PM2 (420 mg, 2.91 mmol) as a starting material, INT-8 (450 mg, 1.40 mmol, 48.1%) was synthesized according to the method for INT-7. 13 H 25 BrN2O2, MS(ES): m / z (M+H) + 321.2.

[0264] 9. Synthesis of intermediate INT-9

[0265]

[0266] Step 1: Synthesis of INT-9-PM1

[0267] Using INT-7-PM1 (6.50 g, 31.00 mmol) as the starting material, INT-9-PM1 (6.4 g, 26.0 mmol, 83.8%) was synthesized according to the same method as INT-7-PM2. 12 H 26 N₂O₃, MS (ES): m / z (M+H) + 247.2.

[0268] Step 2: Synthesis of INT-9-PM2

[0269] Using INT-9-PM1 (6.40 g, 26.0 mmol) as a starting material, INT-9-PM2 (3.78 g, 25.85 mmol, 99.5%) was synthesized following the same method as INT-7-PM3. C7H 18 N₂O, MS(ES): m / z (M+H) + 147.1.

[0270] Step 3: Synthesis of INT-9

[0271] Using INT-9-PM2 (590 mg, 4.04 mmol) as a starting material, INT-9 (950 mg, 2.94 mmol, 72.8%) was synthesized according to the method for INT-7. 13 H 27 BrN2O2, MS(ES): m / z (M+H) + 323.2.

[0272] 10. Synthesis of intermediate INT-10

[0273]

[0274] Step 1: Synthesis of INT-10-PM1

[0275] Using INT-7-PM1 (5.00 g, 23.85 mmol) as the starting material, the product INT-10-PM1 (6.00 g, 21.95 mmol, 92.0%) was obtained by synthesizing INT-7-PM2 using the same method. 13 H 27 N3O3, MS (ES): m / z (M+H) + 274.2.

[0276] Step 2: Synthesis of INT-10-PM2

[0277] Using INT-10-PM1 (6.00 g, 21.95 mmol) as a starting material, INT-10-PM2 (3.45 g, 19.91 mmol, 90.7%) was synthesized according to the method used for INT-7-PM3. C8H 19 N3O, MS(ES): m / z (M+H) + 174.1.

[0278] Step 3: Synthesis of INT-10

[0279] Using INT-10-PM2 (2.00 g, 11.54 mmol) as a starting material, INT-10-PM3 (1.65 g, 4.71 mmol, 40.8%) was synthesized according to the method for INT-7. C 14 H 28 BrN3O2, MS(ES): m / z (M+H) + 350.1.

[0280] 11. Synthesis of intermediate INT-11

[0281]

[0282] Step 1: Synthesis of INT-11-PM1

[0283] Using INT-7-PM1 (3.00 g, 14.31 mmol) as the starting material, the product INT-11-PM1 (1.2 g, 4.70 mmol, 32.8%) was obtained by synthesizing INT-7-PM2 using the same method. 12 H 21 N3O3, MS (ES): m / z (M+H) + 256.1.

[0284] Step 2: Synthesis of INT-11-PM2

[0285] Using INT-11-PM1 (1.20 g, 4.70 mmol) as a starting material, INT-11-PM2 (720 mg, 4.64 mmol, 98.7%) was synthesized according to the method for INT-7-PM3. C7H 13 N3O, MS(ES): m / z (M+H) + 156.2.

[0286] Step 3: Synthesis of INT-11

[0287] Using INT-11-PM2 (420 mg, 2.71 mmol) as a starting material, INT-11 (400 mg, 1.20 mmol, 44.5%) was synthesized according to the method for INT-7. 13 H 22 BrN3O2, MS(ES): m / z (M+H) + 332.1.

[0288] 12. Synthesis of intermediate INT-12

[0289]

[0290] Step 1: Synthesis of INT-12-PM1

[0291] Using INT-7-PM1 (2.00 g, 9.54 mmol) as the starting material, the product INT-12-PM1 (1.20 g, 4.41 mmol, 46.2%) was obtained by synthesizing INT-7-PM2 using the same method. 14 H 28 N₂O₃, MS (ES): m / z (M+H) + 273.2.

[0292] Step 2: Synthesis of INT-12-PM2

[0293] Using INT-12-PM1 (1.2 g, 4.41 mmol) as a starting material, INT-11-PM2 (740 mg, 4.30 mmol, 97.5%) was synthesized according to the method for INT-7-PM3. 20 N₂O, MS(ES): m / z (M+H) + 173.2.

[0294] Step 3: Synthesis of INT-12

[0295] Using INT-12-PM2 (400 mg, 2.32 mmol) as a starting material, INT-12 (600 mg, 1.72 mmol, 74.0%) was synthesized according to the method for INT-7. 15 H 29 BrN2O2, MS(ES): m / z (M+H) + 349.1.

[0296] 13. Synthesis of intermediate INT-13

[0297]

[0298] 6-Bromohexanol (10.10 g, 55.78 mmol) was dissolved in dichloromethane (100 mL). Tert-butoxycarbonyl-6-aminohexanoic acid (12.90 g, 55.78 mmol), EDCI (12.83 g, 66.94 mmol), and DMAP (1.36 g, 11.16 mmol) were added sequentially. After addition, the mixture was stirred overnight at room temperature, and the reaction was monitored by TLC until complete. The reaction solution was slowly quenched with saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane, and the organic phase was separated and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated by rotary evaporation under reduced pressure. The residual liquid was loaded onto a silica gel column for purification using a HEX / EA elution gradient of 0-40%. The product was collected and concentrated to obtain INT-13 (11.50 g, 29.16 mmol, 52.3%). 17 H 32 BrNO4, MS(ES): m / z (M+H) + 395.2.

[0299] 14. Synthesis of intermediate INT-14

[0300]

[0301] Step 1: Synthesis of INT-14-PM1

[0302] INT-7-PM3 (380 mg, 2.56 mmol) was dissolved in acetonitrile (3 mL), followed by the weighing of potassium carbonate (1.06 g, 7.68 mmol) and INT-13 (1.20 g, 3.04 mmol). The mixture was heated to 70 °C and reacted for 12 h. The reaction was characterized as complete by TLC. The reaction solution was slowly cooled to room temperature, filtered to remove salt, concentrated by rotary evaporation under reduced pressure, and the residue was wet-processed and purified by silica gel column chromatography with an elution gradient of DCM / MeOH = 0-15%. The product was collected and concentrated to obtain INT-14-PM1 (286 mg, 0.62 mmol, 24.2%). 23 H 47N3O6, MS(ES): m / z (M+H) + 447.1.

[0303] Step 2: Synthesis of INT-14

[0304] INT-14-PM1 (286 mg, 0.62 mmol) was dissolved in DCM (3 mL), and HCl / EA (3 mL) was added. The mixture was stirred at room temperature for 3 h, and the reaction was completed by TLC. The pH was adjusted to neutral with saturated sodium bicarbonate, and the mixture was extracted three times with DCM. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated to obtain INT-14 (205 mg, 0.57 mmol, 91.5%). 18 H 39 N3O, MS(ES): m / z (M+H) + 347.2.

[0305] 15. Synthesis of intermediate INT-15

[0306]

[0307] Step 1: Synthesis of INT-15-PM1

[0308] Using INT-7-PM1 (4.19 g, 19.98 mmol) as the starting material, the product INT-15-PM1 (3.92 g, 14.94 mmol, 74.8%) was synthesized according to the same method as INT-7-PM2. 12 H 26 N₂O₄, MS (ES): m / z (M+H) + ) 263.1.

[0309] Step 2: Synthesis of INT-15-PM2

[0310] Using INT-15-PM1 (3.92 g, 14.94 mmol) as a starting material, INT-15-PM2 (2.31 g, 14.24 mmol, 95.3%) was synthesized according to the method for INT-7-PM3. C7H 18 N₂O₂, MS (ES): m / z (M+H) + 163.2.

[0311] Step 3: Synthesis of INT-15-PM3

[0312] Using INT-15-PM2 (310 mg, 1.91 mmol) as a starting material, the product INT-15-PM3 (265 mg, 0.56 mmol, 29.2%) was synthesized according to the same method as INT-14-PM1. 24H 48 N3O6, MS (ES): m / z (M+H) + 461.2.

[0313] Step 4: Synthesis of INT-15

[0314] Using INT-15-PM3 (265 mg, 0.56 mmol) as a starting material, INT-15 (195 mg, 0.52 mmol, 93.2%) was obtained by synthesizing INT-14 using the same method. 19 H 41 N3O4, MS (ES): m / z (M+H) + 361.2.

[0315] 16. Synthesis of intermediate INT-16

[0316]

[0317] Step 1: Synthesis of INT-16-PM1

[0318] Using INT-9-PM2 (800 mg, 5.47 mmol) as a starting material, the product INT-16-PM1 (900 mg, 1.96 mmol, 35.8%) was obtained by synthesizing INT-14-PM1 using the same method. 24 H 49 N3O5, MS (ES): m / z (M+H) + 445.3.

[0319] Step 2: Synthesis of INT-16

[0320] Using INT-16-PM1 (300 mg, 0.65 mmol) as a starting material, INT-16 (205 mg, 0.57 mmol, 87.4%) was synthesized according to the method for INT-14. 19 H 41 N3O3, MS (ES): m / z (M+H) + 360.5.

[0321] 17. Synthesis of intermediate INT-17

[0322]

[0323] Step 1: Synthesis of INT-17-PM1

[0324] Using INT-7-PM1 (2.51 g, 12.00 mmol) as the starting material, the product INT-17-PM1 (2.32 g, 10.63 mmol, 88.8%) was obtained by synthesizing INT-7-PM2 using the same method. 10 H 22 N₂O₃, MS (ES): m / z (M+H) + ) 219.2.

[0325] Step 2: Synthesis of INT-17-PM2

[0326] Using INT-17-PM1 (2.32 g, 10.63 mmol) as a starting material, INT-17-PM2 (1.08 g, 9.14 mmol, 86.0%) was synthesized according to the method for INT-7-PM3. C5H 14 N₂O, MS(ES): m / z (M+H) + 119.1.

[0327] Step 3: Synthesis of INT-17-PM3

[0328] Using INT-17-PM2 (333 mg, 2.82 mmol) as a starting material, the product INT-15-PM3 (300 mg, 0.70 mmol, 24.7%) was synthesized according to the same method as INT-14-PM1. 22 H 45 N3O5, MS (ES): m / z (M+H) + 417.3.

[0329] Step 4: Synthesis of INT-17

[0330] Using INT-15-PM3 (300 mg, 0.70 mmol) as a starting material, INT-17 (210 mg, 0.63 mmol, 91.1%) was obtained according to the synthesis method of INT-14. 17 H 37 N3O3, MS (ES): m / z (M+H) + 317.1.

[0331] 18. Synthesis of intermediate INT-18

[0332]

[0333] Step 1: Synthesis of INT-18-PM1

[0334] Using INT-10-PM2 (310 mg, 1.79 mmol) as a starting material, the product INT-18-PM1 (500 mg, 1.03 mmol, 57.4%) was synthesized according to the same method as INT-14-PM1. 25 H 50 N4O5, MS (ES): m / z (M+H) + 472.3.

[0335] Step 2: Synthesis of INT-18

[0336] Using INT-18-PM1 (500 mg, 1.03 mmol) as a starting material, INT-18 (350 mg, 0.91 mmol, 88.1%) was synthesized according to the method for INT-14. 20 H 42 N4O3, MS (ES): m / z (M+H) + 371.2.

[0337] 19. Synthesis of intermediate INT-19

[0338]

[0339] Step 1: Synthesis of INT-19-PM1

[0340] Using INT-11-PM2 (300 mg, 1.93 mmol) as a starting material, the product INT-19-PM1 (226 mg, 0.48 mmol, 24.9%) was obtained by synthesizing INT-14-PM1 using the same method. 24 H 44 N4O5, MS (ES): m / z (M+H) + 469.6.

[0341] Step 2: Synthesis of INT-19

[0342] Using INT-19-PM1 (226 mg, 0.48 mmol) as a starting material, INT-19 (160 mg, 0.43 mmol, 90.0%) was synthesized according to the method for INT-14. 19 H 36 N4O3, MS (ES): m / z (M+H) + 354.3.

[0343] 20. Synthesis of intermediate INT-20

[0344]

[0345] Step 1: Synthesis of INT-20-PM1

[0346] Using INT-7-PM1 (2.05 g, 9.78 mmol) as the starting material, the product INT-20-PM1 (1.65 g, 5.44 mmol, 55.6%) was synthesized according to the same method as INT-7-PM2. 14 H 29 N3O4, MS (ES): m / z (M+H) + 304.2.

[0347] Step 2: Synthesis of INT-20-PM2

[0348] Using INT-20-PM1 (1.65 g, 5.44 mmol) as a starting material, INT-20-PM2 (1.05 g, 5.17 mmol, 95.0%) was synthesized according to the method for INT-7-PM3. C9H 21 N3O2, MS(ES): m / z (M+H) + 204.2.

[0349] Step 3: Synthesis of INT-20-PM3

[0350] Using INT-20-PM2 (315 mg, 1.55 mmol) as a starting material, the product INT-20-PM3 (285 mg, 0.55 mmol, 35.6%) was synthesized according to the same method as INT-14-PM1. 26 H 52 N4O6, MS (ES): m / z (M+H) + 502.3.

[0351] Step 4: Synthesis of INT-20

[0352] Using INT-20-PM3 (285 mg, 0.55 mmol) as a starting material, INT-20 (195 mg, 0.47 mmol, 84.9%) was synthesized according to the method for INT-14. 21 H 44 N4O4, MS (ES): m / z (M+H) + 402.3.

[0353] 21. Synthesis of intermediate INT-21

[0354]

[0355] Step 1: Synthesis of INT-21-PM1

[0356] Using INT-12-PM2 (300 mg, 1.74 mmol) as a starting material, the product INT-21-PM1 (246 mg, 0.51 mmol, 29.1%) was obtained by synthesizing INT-14-PM1 using the same method. 26 H 51 N3O5, MS (ES): m / z (M+H) + 486.3.

[0357] Step 2: Synthesis of INT-21

[0358] Using INT-21-PM1 (246 mg, 0.51 mmol) as a starting material, INT-21 (175 mg, 0.45 mmol, 89.6%) was synthesized according to the method for INT-14. 21 H 43 N3O3, MS (ES): m / z (M+H) + 386.3.

[0359] 22. Synthesis of intermediate INT-22

[0360]

[0361] Step 1: Synthesis of INT-22-PM1

[0362] Using INT-7-PM1 (3.0 g, 14.31 mmol) as the starting material, the product INT-20-PM1 (1.15 g, 3.82 mmol, 26.7%) was synthesized according to the same method as INT-7-PM2. 15 H 31 N3O3, MS (ES): m / z (M+H) + 302.2.

[0363] Step 2: Synthesis of INT-22-PM2

[0364] Using INT-22-PM1 (1.15 g, 3.82 mmol) as a starting material, INT-22-PM2 (724 mg, 3.60 mmol, 94.3%) was synthesized according to the method for INT-7-PM3. 10 H 23 N3O, MS(ES): m / z (M+H) + ) 202.3.

[0365] Step 3: Synthesis of INT-22-PM3

[0366] Using INT-22-PM2 (724 mg, 3.60 mmol) as a starting material, the product INT-22-PM3 (160 mg, 0.31 mmol, 8.6%) was synthesized according to the same method as INT-14-PM1. 27 H 54 N4O5, MS (ES): m / z (M+H) + 515.2.

[0367] Step 4: Synthesis of INT-22

[0368] Using INT-22-PM3 (160 mg, 0.31 mmol) as a starting material, INT-22 (120 mg, 0.29 mmol, 93.1%) was synthesized according to the method for INT-14. 22 H 46 N4O3, MS (ES): m / z (M+H) + 415.3.

[0369] 23. Synthesis of intermediate INT-23

[0370]

[0371] Step 1: Synthesis of INT-23-PM1

[0372] Using INT-7-PM1 (4.2 g, 20.03 mmol) as the starting material, the product INT-23-PM1 (1.69 g, 6.00 mmol, 30.0%) was obtained by synthesizing INT-7-PM2 using the same method. 14 H 23 N3O3, MS (ES): m / z (M+H) + ) 282.1.

[0373] Step 2: Synthesis of INT-23-PM2

[0374] Using INT-23-PM1 (1.69 g, 6.00 mmol) as a starting material, INT-23-PM2 (960 mg, 5.30 mmol, 88.2%) was synthesized according to the method for INT-7-PM3. C9H 15 N3O, MS(ES): m / z (M+H) + 182.2.

[0375] Step 3: Synthesis of INT-23-PM3

[0376] Using INT-23-PM2 (872 mg, 4.81 mmol) as a starting material, the product INT-23-PM3 (249 mg, 0.50 mmol, 10.5%) was synthesized according to the method for INT-14-PM1. 26 H 46 N4O5, MS (ES): m / z (M+H) + 495.3.

[0377] Step 4: Synthesis of INT-23

[0378] Using INT-23-PM3 (249 mg, 0.50 mmol) as a starting material, INT-23 (116 mg, 0.29 mmol, 58.4%) was synthesized according to the method for INT-14. 21 H 38 N4O3, MS (ES): m / z (M+H) + 395.1.

[0379] 24. Synthesis of intermediate INT-24

[0380]

[0381] Step 1: Synthesis of INT-24-PM1

[0382] Using INT-7-PM1 (4.01 g, 19.12 mmol) as a starting material, INT-24-PM1 (2.02 g, 7.05 mmol, 36.9%) was synthesized according to the same method as INT-7-PM2. Molecular formula: C 15 H 30 N₂O₃, MS (ES): m / z (M+H) + 287.1.

[0383] Step 2: Synthesis of INT-24-PM2

[0384] Using INT-24-PM1 (2.02 g, 7.05 mmol) as a starting material, INT-24-PM2 (1.25 g, 6.71 mmol, 95.1%) was synthesized according to the method for INT-7-PM3. C 10 H 22 N₂O, MS(ES): m / z (M+H) + 187.1.

[0385] Step 3: Synthesis of INT-24-PM3

[0386] Using INT-24-PM2 (600 mg, 3.22 mmol) as a starting material, the product INT-24-PM3 (420 mg, 0.84 mmol, 26.1%) was obtained by synthesizing INT-14-PM1 using the same method. 27 H 53 N3O5, MS (ES): m / z (M+H) + 500.4.

[0387] Step 4: Synthesis of INT-24

[0388] Using INT-24-PM3 (210 mg, 0.42 mmol) as a starting material, INT-24 (155 mg, 0.39 mmol, 92.3%) was synthesized according to the method for INT-14. 27 H 53 N3O5, MS (ES): m / z (M+H) + 400.3.

[0389] 25. Synthesis of intermediate INT-25

[0390]

[0391] Step 1: Synthesis of INT-25-PM1

[0392] INT-12 (350 mg, 1.00 mmol) was dissolved in acetonitrile (4 mL), and N-tert-butoxycarbonyl-1,2-ethylenediamine (192 mg, 1.20 mmol) and potassium carbonate (415 mg, 3.00 mmol) were added sequentially. After the addition was complete, the mixture was heated to 70 °C and reacted for 10 h. The reaction was monitored by TLC until complete. The reaction solution was slowly cooled to room temperature, filtered to remove salts, and the residual liquid was concentrated and then loaded onto the sample using a wet method. The solution was purified by chromatography with an elution gradient of DCM / MeOH (10% NH3). . H2O) = 0-12%, collect and concentrate the product to obtain INT-25-PM1 (260 mg, 0.61 mmol, 60.5%). Molecular formula: C 22 H 44 N4O4, MS (ES): m / z (M+H) + 429.3.

[0393] Step 2: Synthesis of INT-25

[0394] Using INT-24-PM1 (260 mg, 0.61 mmol) as a starting material, INT-25 (185 mg, 0.56 mmol, 92.8%) was obtained following the synthesis method of INT-14. 17 H36 N4O2, MS (ES): m / z (M+H) + 329.3.

[0395] 26. Synthesis of intermediate INT-26

[0396]

[0397] Step 1: Synthesis of INT-26-PM1

[0398] INT-7-PM3 (1.00 g, 6.75 mmol) was dissolved in acetonitrile (4 mL), and tert-butyl 6-bromohexanoate (1.70 g, 6.75 mmol) and potassium carbonate (2.80 g, 20.25 mmol) were added sequentially. After the addition was complete, the mixture was heated to 70 °C and reacted for 10 h. The reaction was monitored by TLC until complete. The reaction solution was slowly cooled to room temperature, filtered to remove salts, and the residual liquid was concentrated and then loaded onto the sample using a wet method. The solution was purified by chromatography with an elution gradient of DCM / MeOH (10% NH3). . H2O) = 0-12%, collect and concentrate the product to obtain INT-26-PM1 (550mg, 1.73mmol, 25.6%). Molecular formula: C 16 H 34 N₂O₄, MS (ES): m / z (M+H) + 319.2.

[0399] Step 2: Synthesis of INT-26

[0400] Using INT-26-PM1 (550 mg, 1.73 mmol) as a starting material, INT-26 (320 mg, 1.22 mmol, 70.6%) was synthesized according to the method for INT-14. 12 H 26 N₂O₄, MS (ES): m / z (M+H) + ) 263.1.

[0401] 27. Synthesis of intermediate INT-27

[0402]

[0403] Step 1: Synthesis of INT-27-PM1

[0404] Using INT-10 (1.00 g, 2.85 mmol) as a starting material, INT-27-PM1 (540 mg, 1.26 mmol, 44.0%) was synthesized according to the method for INT-25-PM1. Molecular formula: C 21 H 43N5O4, MS(ES): m / z (M+H) + 430.2.

[0405] Step 2: Synthesis of INT-27-PM2

[0406] Using INT-27-PM1 (540 mg, 1.26 mmol) as a starting material, INT-27-PM2 (414 mg, 1.26 mmol, 99.9%) was synthesized according to the method for INT-14. 16 H 35 N5O2, MS(ES): m / z (M+H) + 330.5.

[0407] Step 3: Synthesis of INT-27-PM3

[0408] INT-27-PM2 (414 mg, 1.26 mmol) was dissolved in acetonitrile (4 mL), and tert-butyl 6-bromohexanoate (316 mg, 1.26 mmol) and potassium carbonate (522 mg, 3.78 mmol) were added sequentially. After the addition was complete, the mixture was heated to 70 °C and reacted for 10 h. The reaction was monitored by TLC until complete. The reaction solution was slowly cooled to room temperature, filtered to remove salts, and the residual liquid was concentrated and then loaded onto the sample using a wet method. The solution was purified by chromatography with an elution gradient of DCM / MeOH (10% NH3). . H2O) = 0-12%, the product was collected and concentrated to obtain INT-27-PM3 (150mg, 0.30mmol, 23.9%). Molecular formula: C 26 H 53 N5O4, MS(ES): m / z (M+H) + 500.3.

[0409] Step 4: Synthesis of INT-27

[0410] Using INT-27-PM3 (150 mg, 0.30 mmol) as a starting material, INT-27 (110 mg, 0.25 mmol, 82.6%) was synthesized according to the method for INT-14. 22 H 45 N5O4, MS(ES): m / z (M+H) + 445.3.

[0411] 28. Synthesis of intermediate INT-28

[0412]

[0413] Step 1: Synthesis of INT-28-PM1

[0414] Using INT-9 (800 mg, 2.47 mmol) as a starting material, INT-28-PM1 (600 mg, 1.49 mmol, 60.2%) was synthesized following the same method as INT-25-PM1. Molecular formula: C 20 H 42 N4O4, MS (ES): m / z (M+H) + 403.2.

[0415] Step 2: Synthesis of INT-28-PM2

[0416] Using INT-28-PM1 (600 mg, 1.49 mmol) as a starting material, INT-28-PM2 (435 mg, 1.44 mmol, 96.5%) was synthesized according to the method for INT-14. 15 H 34 N4O2, MS (ES): m / z (M+H) + 303.2.

[0417] Step 3: Synthesis of INT-28-PM3

[0418] INT-28-PM2 (435 mg, 1.44 mmol) was dissolved in acetonitrile (4 mL), and tert-butyl 6-bromohexanoate (362 mg, 1.44 mmol) and potassium carbonate (597 mg, 4.32 mmol) were added sequentially. After the addition was complete, the mixture was heated to 70 °C and reacted for 10 h. The reaction was monitored by TLC until complete. The reaction solution was slowly cooled to room temperature, filtered to remove salts, and the residual liquid was concentrated and then loaded onto the sample using a wet method. The solution was purified by chromatography with an elution gradient of DCM / MeOH (10% NH3). . H2O) = 0-15%, collect and concentrate the product to obtain INT-28-PM3 (235mg, 0.50mmol, 34.6%). Molecular formula: C 25 H 52 N4O4, MS (ES): m / z (M+H) + 473.4.

[0419] Step 4: Synthesis of INT-28

[0420] Using INT-28-PM3 (235 mg, 0.50 mmol) as a starting material, INT-28 (201 mg, 0.48 mmol, 97.1%) was synthesized according to the method for INT-14. 21 H 44 N4O4, MS (ES): m / z (M+H) + 417.3.

[0421] 29. Synthesis of intermediate INT-29

[0422]

[0423] Step 1: Synthesis of INT-29-PM1

[0424] Using INT-10-PM2 (1.50 g, 8.66 mmol) as a starting material, INT-29-PM1 (650 mg, 1.89 mmol, 21.9%) was synthesized following the same method as INT-26-PM1. Molecular formula: C 18 H 37 N3O3, MS (ES): m / z (M+H) + 344.2.

[0425] Step 2: Synthesis of INT-29

[0426] Using INT-29-PM1 (650 mg, 1.89 mmol) as a starting material, INT-29 (515 mg, 1.79 mmol, 94.7%) was synthesized according to the method for INT-14. 14 H 29 N3O3, MS (ES): m / z (M+H) + 288.2.

[0427] 30. Synthesis of intermediate INT-30

[0428]

[0429] Step 1: Synthesis of INT-30-PM1

[0430] Using INT-9-PM2 (1.60 g, 10.94 mmol) as a starting material, INT-30-PM1 (960 mg, 3.03 mmol, 27.7%) was synthesized following the same method as INT-26-PM1. Molecular formula: C 17 H 36 N₂O₃, MS (ES): m / z (M+H) + 317.4.

[0431] Step 2: Synthesis of INT-30

[0432] Using INT-30-PM1 (960 mg, 3.03 mmol) as a starting material, INT-30 (650 mg, 2.50 mmol, 82.3%) was synthesized according to the method for INT-14. 13 H 28N₂O₃, MS (ES): m / z (M+H) + ) 261.3.

[0433] 31. Synthesis of YK-POL-001

[0434]

[0435] Weigh INT-1 (50 mg, 0.01 mmol) and dissolve it in DMF (1 mL). Weigh and add potassium carbonate (82 mg, 0.60 mmol) and INT-7 (290 mg, 0.90 mmol). After the addition is complete, heat to 70 °C and react for 10 h. After the reaction is complete, add the reaction solution dropwise to diethyl ether to precipitate the product. Purify by dialysis with 3.5 kDa, repeating three times. Freeze-dry the solution to obtain YK-POL-001 (79 mg, 77.2%). 1 H NMR(400 MHz, DMSO-d6) δ 8.21 - 8.35 (m, 48 H), 7.21 - 7.37 (m, 42 H), 4.36 -4.63 (m, 16 H), 4.05 - 4.26 (m, 28 H), 3.87 - 4.02 (m, 32 H), 3.57 - 3.71 (m, 32 H), 3.11 - 3.38 (m, 75 H), 2.97 - 3.00 (m, 48 H), 2.62 - 2.71 (m, 58 H), 2.27 - 2.53 (m, 90 H), 2.04 - 2.15 (m, 88 H), 1.85 - 2.02 (m, 48H), 1.74 -1.82 (m, 14 H), 1.26 - 1.57 (m, 32 H), 1.09 - 1.21 (m, 14 H), 0.89 - 1.01 (m,10 H), 0.79 - 0.81 (m, 3H). 1 H NMR calculation: m=16, n=14.

[0436] 32. Synthesis of YK-POL-002

[0437]

[0438] Using INT-1 (50 mg, 0.01 mmol) as the starting material, YK-POL-002 (21 mg, 21.3%) was obtained by following the synthesis method of YK-POL-001. 1¹H NMR (400 MHz, DMSO-d⁶) δ 4.64 (m, 27 H), 3.92–2.66 (m, 92 H), 2.60–2.12 (m, 320 H), 1.86–1.29 (m, 60 H), 0.78 (t, 3 H). 1 H NMR calculation: m=17, n=13.

[0439] 33. Synthesis of YK-POL-003

[0440]

[0441] Using INT-1 (50 mg, 0.01 mmol) as a starting material, YK-POL-003 (85 mg, 83.5%) was obtained by following the synthesis method of YK-POL-001. 1 H NMR (400 MHz, DMSO-d6) δ 4.64 (m, 28 H), 4.11 - 2.65 (m, 88H), 2.50 - 2.12 (m, 360 H), 1.86 - 1.55 (m, 28 H), 1.48 (m, 78 H), 0.78 (t, 3H). 1 H NMR calculation: m=17, n=13.

[0442] 34. Synthesis of YK-POL-004

[0443]

[0444] Using INT-1 (50 mg, 0.01 mmol) as a starting material, YK-POL-004 (17 mg, 15.8%) was obtained by following the synthesis method of YK-POL-001. 1H NMR (400 MHz, DMSO-d6) δ 8.78 - 8.83 (m, 48 H), 7.57 - 7.07(m, 42 H), 4.46 - 4.42 (m, 16 H), 4.09 - 4.23 (m, 113 H), 3.61 -3.88 (m, 219H), 3.21 - 3.57 (m, 158 H), 3.01 - 3.18 (m, 64 H), 2.64 - 2.82 (m, 10 H), 2.40- 2.52 (m, 64 H), 1.89 - 2.18 (m, 168 H), 1.31 - 1.57 (m, 6 H), 0.77 -0.81 (m, 3 H). through 1 H NMR calculation: m=16, n=14.

[0445] 35. Synthesis of YK-POL-005

[0446]

[0447] Using INT-1 (40 mg, 0.01 mmol) as the starting material, YK-POL-005 (18 mg, 21.8%) was obtained by following the synthesis method of YK-POL-001. 1 H NMR (400 MHz, DMSO-d6) δ 8.03 (m, 29 H), 7.10 (d, 12 H), 6.75 (d, 12 H), 4.35 (m, 30 H), 4.05 - 2.60 (m, 420 H), 2.52 (s, 54 H), 2.45- 2.12 (m, 244 H), 1.70 - 1.29 (m, 10 H), 0.78 (t, 3 H). 1 H NMR calculation: m=18, n=12.

[0448] 36. Synthesis of YK-POL-006

[0449]

[0450] Using INT-1 (60 mg, 0.01 mmol) as a starting material, YK-POL-006 (71 mg, 55.2%) was obtained by following the synthesis method of YK-POL-001. 1H NMR (400 MHz, DMSO-d6) δ 7.96 - 8.59 (m, 24 H), 7.81 - 7.93 (m, 48 H), 7.70 - 7.75 (m, 12 H), 7.54 - 7.67 (m, 24 H), 5.58 - 5.90 (m, 24H), 4.35 - 4.87 (m, 164 H), 3.93 - 4.22 (m, 92 H), 3.58 - 3.87 (m, 98 H), 3.17 - 3.52 (m, 96 H), 3.02- 3.11 (m, 96 H), 2.28-2.96 (m, 36 H), 2.12 - 2.24(m, 96 H), 1.77 - 1.96 (m, 96 H), 1.58 - 1.67 (m, 152 H), 1.40 - 1.49 (m, 10H), 1.21 - 1.37 (m, 88 H), 0.91 - 0.97 (m, 3 H). 1 H NMR calculation: m=24, n=6.

[0451] 37. Synthesis of YK-POL-007

[0452]

[0453] Weigh INT-1-PM2 (60 mg, 0.01 mmol) into a reaction flask and add NMP (1 mL). Then, weigh in potassium carbonate (119 mg, 0.86 mmol) and INT-14 (155 mg, 0.43 mmol) sequentially. After addition, heat to 70 °C and react for 12 h. IR spectroscopy is performed at 1736 cm⁻¹. -1 The peak disappeared. The precipitate was added to ice-cold ether, dialyzed through a 3.5 kDa dialysis bag, dialyzed three times in water, and then freeze-dried. Product YK-POL-007 (30 mg, 22.6%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 4.29 (m, 30 H), 4.01 (m, 27 H), 3.52 - 2.24 (m, 320 H), 2.15 (s, 42 H), 2.29 - 2.18 (m, 38H), 1.65 - 1.01 (m, 120 H), 0.81 (t, 3 H). 1 H NMR calculation: m=14, n=16.

[0454] 38. Synthesis of YK-POL-008

[0455]

[0456] Using INT-1-PM2 (60 mg, 0.01 mmol) as the raw material, according to the synthesis method of YK-POL-007, YK-POL-008 (65 mg, 47.5 %) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.63 - 9.10 (m, 60 H), 7.42 - 7.57 (m, 17 H), 6.41 - 7.02 (m, 13 H), 7.54 - 7.67 (m, 24 H), 4.04 - 4.52 (m, 64 H), 3.72 - 3.96 (m, 39 H), 3.04 - 3.64 (m, 62 H), 2.80 - 3.00 (m, 31 H), 2.57 - 2.77 (m, 58 H), 2.25 - 2.52 (m, 53 H), 2.08 - 2.20 (m, 39 H), 1.92 - 2.05 (m, 26 H), 1.32 - 1.54 (m, 45 H), 1.05 - 1.30 (m, 27 H), 0.78 - 0.92 (m, 10 H), 0.69 - 0.75 (m, 3 H). By 1 Calculated by 1H NMR: m = 13, n = 17.

[0457] 39. Synthesis of YK-POL-009

[0458]

[0459] Using INT-1-PM2 (60 mg, 0.01 mmol) as the raw material, according to the synthesis method of YK-POL-007, YK-POL-009 (21 mg, 15.9 %) was obtained. 1H NMR (400 MHz, DMSO-d6) δ 7.49 - 8.84 (m, 28 H), 4.96 -5.08 (m, 14 H), 4.35 - 4.59 (m, 16 H), 4.11 - 4.26 (m, 16 H), 4.00 - 4.06 (m, 48 H), 3.83 - 3.98 (m, 64 H), 3.09 - 3.45 (m, 16 H), 2.97 - 3.00 (m, 62 H), 2.82 - 2.85 (m, 48 H), 2.66 - 2.78 (m, 84 H), 2.57 - 2.63 (m, 81 H), 2.34 -2.56 (m, 67 H), 2.15 - 2.28 (m, 32 H), 2.05 - 2.12 (m, 16 H), 1.74 - 1.88 (m, 28 H), 1.36 - 1.58 (m, 32 H), 1.09 - 1.32 (m, 14 H), 0.86 - 1.02 (m, 10 H),0.77 - 0.79 (m, 3H). 1 H NMR calculation: m=16, n=14.

[0460] 40. Synthesis of YK-POL-010

[0461]

[0462] Using INT-1-PM2 (60 mg, 0.01 mmol) as a raw material, YK-POL-010 (34 mg, 27.4%) was obtained by following the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 7.63 - 9.42 (m, 66 H), 7.57 -7.07 (m, 24 H), 4.08 - 4.49 (m, 22 H), 3.80 - 4.03 (m, 77 H), 3.45 -3.67 (m, 116 H), 3.15 - 3.37 (m, 131 H), 2.84 - 3.05 (m, 93 H), 2.58-2.79 (m, 10 H), 2.08- 2.44 (m, 88 H), 1.46 (s, 132 H), 1.05-1.31 (m, 67 H), 0.67-0.83 (m, 3H). 1 H NMR calculation: m=22, n=8.

[0463] 41. Synthesis of YK-POL-011

[0464]

[0465] Using INT-1-PM2 (60 mg, 0.01 mmol) as the raw material, according to the synthesis method of YK-POL-007, YK-POL-010 (21 mg, 15.0%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.70 - 8.78 (m, 40 H), 6.13 - 6.51 (m, 20 H), 4.99 - 5.18 (m, 10 H), 4.96 - 5.05 (m, 10 H), 4.57 - 4.79 (m, 10 H), 4.03 - 4.51 (m, 40 H), 3.80 - 3.96 (m, 40 H), 3.03 - 3.58 (m, 20 H), ...... 1 Calculated by 1H NMR: m = 10, n = 20.

[0466] 42. Synthesis of YK-POL-012

[0467]

[0468] Using INT-1-PM2 (40 mg, 0.01 mmol) as the raw material, according to the synthesis method of YK-POL-007, YK-POL-012 (38 mg, 42.3%) was obtained. 1 It should be noted that in the original text, the content in the middle of the "H NMR (400 MHz, DMSO-d6)......" in line 10 seems to be incomplete. I have translated it as best as possible according to the existing content. If there are any inaccuracies, please provide more complete information.1H NMR (400 MHz, DMSO-d6) δ 7.66 - 8.56 (m, 45 H), 6.96 - 6.99 (m, 15 H), 6.66 - 6.68 (m, 15 H), 4.96 - 5.05 (m, 18 H), 4.14 - 4.35 (m, 30 H), 3.71 - 3.99 (m, 60 H), 3.47 - 3.53 (m, 30 H), 3.24 - 3.32 (m, 58 H), 2.62 - 2.78 (m, 48 H), 2.35 – 2.52 (m, 87 H), 2.13 - 2.27 (m, 88 H), 1.39 - 1.54 (m, 10 H), 1.13 - 1.22 (m, 22 H), 0.97 - 1.00 (m, 30 H), 0.77 - 0.79 (m, 3H). By 1 1H NMR calculation: m = 15, n = 15.

[0469] 43. Synthesis of YK-POL-013

[0470]

[0471] Using INT-1-PM2 (60 mg, 0.01 mmol) as the raw material and following the synthesis method of YK-POL-007, YK-POL-013 (50 mg, 33.6%) was obtained. 1 1H NMR (400 MHz, DMSO-d6) δ 7.71 - 9.37 (m, 90 H), 4.43 - 4.73 (m, 16 H), 4.04 - 4.52 (m, 48 H), 3.36 - 3.53 (m, 79 H), 3.04 - 3.27 (m, 62 H), 2.83 - 3.01 (m, 31 H), 2.54 - 2.76 (m, 59 H), 2.21 - 2.46 (m, 54 H), 2.08 - 2.14 (m, 33 H), 1.92 - 2.05 (m, 24 H), 1.55 - 1.78 (m, 112 H), 1.15 - 1.32 (m, 28 H), 1.01 – 1.21 (m, 10 H), 0.82 - 0.91 (m, 3 H). By 1 1H NMR calculation: m = 14, n = 16.

[0472] 44. Synthesis of YK-POL-014

[0473]

[0474] Using INT-1-PM2 (50 mg, 0.01 mmol) as a raw material, YK-POL-014 (38 mg, 32.6%) was obtained according to the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 8.43 - 8.22 (m, 29 H), 4.08 -4.49 (m, 29 H), 4.11 - 3.82 (m, 77 H), 3.65 - 3.37 (m, 140 H), 3.36 - 3.10(m, 120 H), 3.05-2.60 (m, 112 H), 2.45- 2.08 (m, 88 H), 1.78-1.60 (m, 64 H), 1.43-1.05 (m, 67 H), 0.81 (t, 3 H). 1 H NMR calculation: m=10, n=20.

[0475] 45. Synthesis of YK-POL-015

[0476]

[0477] Using INT-1-PM2 (35 mg, 0.01 mmol) as a raw material, YK-POL-015 (43 mg, 49.8%) was obtained according to the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 7.73 - 8.91 (m, 90 H), 4.27 -4.42 (m, 12 H), 3.99 - 4. 15 (m, 55 H), 3.44 -3.71 (m, 76 H), 3.12 - 3.34 (m, 67 H), 2.68 - 3.10 (m, 99 H), 2.33 - 2.54 (m, 42 H), 2.10 - 2.24 (m, 54 H), 1.98 - 2.04 (m, 33 H), 1.79 - 1.88 (m, 24 H), 1.55 - 1.62 (m, 28 H), 1.22 -1.34 (m, 10 H), 0.82 - 0.85 (m, 3 H). (via) 1 H NMR calculation: m=12, n=18.

[0478] 46. ​​Synthesis of YK-POL-016

[0479]

[0480] Using INT-1-PM2 (50 mg, 0.01 mmol) as a raw material, YK-POL-016 (28 mg, 23.6%) was obtained according to the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 8.61 - 8.40 (m, 15 H), 7.87 -7.69 (m, 30 H), 7.61 - 7.11 (m, 30 H), 4.40 (d, J = 76.1 Hz, 33 H), 4.09 -3.89 (m, 30 0.92 -0.83 (m, 3 H). 1 H NMR calculation: m=15, n=15.

[0481] 47. Synthesis of YK-POL-017

[0482]

[0483] Using INT-1-PM2 (60 mg, 0.01 mmol) as a raw material, YK-POL-017 (28 mg, 19.5%) was obtained according to the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 4.69 - 4.11 (m, 52 H), 4.08 (m, 30H), 4.02 - 3.85 (m, 45 H), 3.75 (m, 10H), 3.65 - 3.55 (m, 10 H), 3.37 (m, 40 H),3.01 (s,35 H), 2.93 - 2.65 (m, 35 H), 2.31 (d, J = 30.6 Hz, 40 H), 1.56 -0.98 (m, 220 H), 0.85 (m, 33 H). 1 H NMR calculation: m=10, n=20.

[0484] 48. Synthesis of YK-POL-018

[0485]

[0486] Using INT-1-PM2 (50 mg, 0.01 mmol) as a raw material, YK-POL-018 (35 mg, 30.0%) was obtained according to the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 4.57 (t, J = 6.5 Hz, 30 H), 4.31- 4.22(m, 13 H), 4.15 - 3.90 (m, 52 H), 3.85 - 3.8(m, 13 H), 3.70 (s, 13 H), 3.65 - 3.31 (m, 120 H), 3.00 - 2.10 (m, 130 H), 1.58 - 1.23 (m, 286 H), 0.88 (s, 3 H). 1 H NMR calculation: m=13, n=17.

[0487] 49. Synthesis of YK-POL-019

[0488]

[0489] Using INT-3 (30 mg, 0.01 mmol) as a starting material, dissolved in DMF (3 mL), HATU (131 mg, 0.35 mmol) and DIEA (89 mg, 0.69 mmol) were added sequentially by weighing. The mixture was stirred at room temperature for 5 min, and then INT-26 (73 mg, 0.28 mmol) was added by weighing. The mixture was stirred at room temperature overnight. The reaction system was quenched with water, and the mixture was added to a 3.5 kDa dialysis bag and dialyzed in EtOH / H2O = 1 / 1. The mixture was dialyzed three times in water, and then freeze-dried to obtain product YK-POL-019 (30 mg, 34.8%). 1H NMR (400 MHz, DMSO-d6)δ 9.97 - 8.36 (m, 65 H), 4.51 - 4.31(m, 35 H), 4.26 (s, 50 H), 3.37 (s, 15H),3.86 - 3.75(m, 33 H), 3.51 (m, 125 H), 2.93 - 2.85 (m, 35 H), 2.77 (s, 45 H), 2.75 - 2.61 (m, 150 H), 1.62 - 1.45 (m, 160 H), 1.41 - 1.15 (m, 84 H), 1.12 -0.93 (m, 116 H), 0.92 - 0.80 (m, 3 H). 1 H NMR calculation: m=15, n=35.

[0490] 50. Synthesis of YK-POL-020

[0491]

[0492] Using INT-3 (30 mg, 0.01 mmol) as a starting material, YK-POL-020 (25 mg, 19.5%) was obtained by following the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.50 (d, 25 H), 8.28 (d, 24 H), 8.11(s, 25 H), 7.30 (d, 1 H), 5.21 - 5.18(s, 24 H), 4.27 (s, 50 H), 4.15 - 3.75(m, 24 H), 3.63 - 3.49 (m, 192 H), 3.37 (s, 25 H), 3.16 (m, 230 H), 2.79 (t,75 H), 3.12 - 2.55(140 H), 2.38 (m, 70 H), 2.07 (m, 84H ), 1.59 - 1.45 (m,294H), 1.42 - 1.31 (m, 294 H), 0.87 (m, 3 H). (The last part, "northwest," appears to be a typo and can be left as is.) 1 H NMR calculation: m=24, n=26.

[0493] 51. Synthesis of YK-POL-021

[0494]

[0495] Using INT-3 (15 mg, 0.01 mmol) as a starting material, YK-POL-021 (35 mg, 57.5%) was obtained by following the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1 H), 8.40 (s, 1 H), 8.26 -7.84 (m, 45 H), 7.38 (m, 3 H), 5.35 - 5.18(s, 22 H), 4.71 - 4.53(m, 22 H),4.21 (s, 50 H), 3.91 (m, 88 H), 3.64 (m, 88 H), 3.28 - 3.09 (m, 140 H), 2.97- 2.75 (m, 176 H), 2.10 (m, 36 H), 1.73 - 1.23 (m, 564 H), 1.19 (t, 132 H), 0.87 (t, 3 H). 1 H NMR calculation: m=22, n=28.

[0496] 52. Synthesis of YK-POL-022

[0497]

[0498] Using INT-2 (50 mg, 0.01 mmol) as a starting material, YK-POL-022 (105 mg, 73.5%) was obtained by following the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.38 - 7.95 (m, 16 H), 8.21(d, 1 H)7.28 (d, 1 H), 4.71 (s, 16 H), 4.35(s, 16 H), 4.28 (s, 30 H), 3.82 - 3.41 (m,64 H), 2.80 (s, 28 H), 2.65 - 2.35 (m, 102 H), 1.49 - 1.08 (m, 276 H), 0.87 (s, 3 H). 1 H NMR calculation: m=16, n=14.

[0499] 53. Synthesis of YK-POL-023

[0500]

[0501] Using INT-2 (50 mg, 0.01 mmol) as a starting material, YK-POL-023 (62 mg, 40.7%) was obtained by following the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 3.6 Hz, 1 H), 8.23 ​​(d,1 H), 8.13 (s, 30 H), 7.26 (m, 2 H), 5.35 - 5.21(s, 15 H), 4.27 (s, 15 H),3.44 (d, 15 H), 3.33 - 2.81 (m, 100 H), 2.74 (d, 45 H), 2.65 - 2.31 (m, 140H), 1.60 - 1.13 (m, 370 H), 0.86 (m, 3 H). 1 H NMR calculation: m=15, n=15.

[0502] 54. Synthesis of YK-POL-024

[0503]

[0504] Using INT-5 (50 mg, 0.01 mmol) as a starting material, YK-POL-024 (65 mg, 42.7%) was obtained according to the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.46 (d, 2 H), 8.23 ​​- 8.30 (m, 26H), 8.12 (s, 2 H), 7.26 (m, 1H), 5.18 - 5.10 (s, 13 H), 4.26 (s, 30 H), 4.16-3.85(m, 56 H), 3.75 - 3.70(m, 13 H), 3.57 - 2.94 (m, 116 H), 2.74 (s, 39 H), 2.44 - 2.12 (m, 110 H), 1.86 - 1.07 (m, 256 H), 0.87 (m,3 H). 1 H NMR calculation: m=13, n=17.

[0505] 55. Synthesis of YK-POL-025

[0506]

[0507] Using INT-6 (50 mg, 0.01 mmol) as a starting material, YK-POL-025 (60 mg, 42.0%) was obtained by following the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.16 (m, 48 H), 7.28 (m, 5 H), 5.16 (s, 2 H), 4.22 (s, 50 H), 4.16-3.85 (m, 30 H), 3.66 - 2.12 (m, 368 H), 1.88 -1.17 (m, 460 H), 1.12 (m, 144 H). 1 H NMR calculation: m=24, n=26.

[0508] 56. Synthesis of YK-POL-026

[0509]

[0510] Using INT-4 (50 mg, 0.01 mmol) as a starting material, YK-POL-025 (65 mg, 45.2%) was obtained according to the synthesis method of YK-POL-019. 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (m, 92 H), 4.26 (s, 95 H), 4.16- 3.85(m, 56 H), 3.75 - 3.70 (m, 13 H), 3.66 - 2.12 (m, 760 H), 1.86 - 1.21(m, 870 H), 1.12 (m, 240 H), 0.79 (t, 3 H). 1 H NMR calculation: m=47, n=53.

[0511] 57. Synthesis of YK-POL-027

[0512]

[0513] Step 1: Synthesis of YK-POL-027-PM1

[0514] Using INT-1-PM1 (2.00 g, 8.03 mmol) as a raw material, YK-POL-027-PM1 (2.0 g, 91.5%) was obtained by following the synthesis method of INT-1-PM2. 1H NMR (400 MHz, DMSO-d6) δ 8.19 (m, 30 H), 7.33 (m, 148H), 5.15 (s, 60 H), 4.63 (m, 30 H), 3.68 (m, 180 H), 3.40 (s, 3 H), 2.88 -2.51 (m, 61H).

[0515] Step 1: Synthesis of YK-POL-027

[0516] Using YK-POLY-027-PM1 (60 mg, 0.01 mmol) as the raw material, YK-POL-017 (35 mg, 28.8%) was obtained by following the synthesis method of YK-POL-007. 1 H NMR (400 MHz, DMSO-d6) δ 8.17 (m, 26 H), 4.58 (m, 30 H), 3.64 (m, 180 H), 3.40 (s, 3 H), 2.89 - 2.36 (m, 210 H), 2.16 (s, 42H), 1.70 - 1.18 (m, 186 H). 1 H NMR calculation: m=16, n=14.

[0517] In the cationic polymers (YK-POL-001~YK-POL-027) prepared above, the repeating units are connected in a disordered manner.

[0518] 58. Synthesis of cationic lipid YK-009

[0519] Following the synthesis method of YK-009 in CN114044741 B, 3.0g of YK-009 was synthesized.

[0520] Example 2: Optimization of mRNA-LNP composition

[0521] 2.1 Optimization of the ratio of cationic polymer to mRNA

[0522] Step 1:

[0523] Following a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5, SM-102 (Xiamen Sinobond Biotechnology Co., Ltd.) or YK-009 (synthesized in Example 1) was dissolved in ethanol along with DSPC (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 to obtain solution A. Solution A was then rapidly added to citrate buffer (pH=4~5) using the ethanol injection method, and vortexed for 30 s to obtain an ethanol lipid solution.

[0524] Step 2:

[0525] eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH=4~5) to obtain an aqueous solution of eGFP-mRNA.

[0526] Step 3:

[0527] The cationic lipid carrier obtained in step 1 (molar ratio of cationic lipid:DSPC:cholesterol:DMG-PEG2000 49:10:39.5:1.5) was mixed with the ethanol lipid solution prepared in step 1 and the mRNA aqueous solution prepared in step 2 at a flow rate of 10 mL / min using a microfluidic device to obtain the corresponding liposome solution, namely solution B.

[0528] Step 4:

[0529] The cationic polymers YK-POL-001, YK-POL-007, YK-POL-012 and YK-POL-022 synthesized in Example 1 were dissolved in DMSO to obtain different solutions C.

[0530] Step 5:

[0531] Solution C was injected into solution B according to cationic polymer:mRNA mass ratios of 5:1, 10:1, and 15:1. The solution was dialyzed in neutral PBS using a 10 kDa dialysis bag to remove ethanol and DMSO. The dialyzed lipid solution was concentrated in a 300 kDa dialysis tube, then brought to a suitable volume with PBS, and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP compositions encapsulated with eGFP-mRNA using cationic polymers (YK-POL-001, YK-POL-007, YK-POL-012, or YK-POL-022) / DSPC / cholesterol / DMG-PEG2000.

[0532] Cell transfection experiments showed that when the mass ratio of cationic polymer to mRNA was in the range of 5:1 to 15:1, the corresponding mRNA-LNP compositions all exhibited good transfection effects, with the best effect observed at a ratio of 10:1. The transfection effects were essentially consistent when the cationic lipids were SM-102 or YK-009.

[0533] 2.2 Optimization of the ratio of cationic polymers to cationic lipids

[0534] mRNA-LNP compositions encapsulated with eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the molar ratio of the cationic polymer (YK-POL-001, YK-POL-007, YK-POL-012 or YK-POL-022) to the cationic lipid SM-102 or YK-009 was adjusted to 1:100, 1:20, 1:10 and 1:1, respectively.

[0535] Cell transfection experiments showed that when the molar ratio of cationic polymer to cationic lipid was within the range of 1:100 to 1:1, the corresponding mRNA-LNP compositions could all transfect cells, with the highest transfection efficiency at a ratio of 1:20. The transfection effect was basically consistent when the cationic lipid was SM-102 or YK-009.

[0536] 2.3 Optimization of the ratio of cationic polymers to polymer-conjugated lipids

[0537] mRNA-LNP compositions encapsulated with eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the molar percentage of polymer conjugated lipid DMG-PEG2000 in the carrier was 1.5%, wherein the molar ratio of cationic polymer (YK-POL-001, YK-POL-007, YK-POL-012 or YK-POL-022) to polymer conjugated lipid DMG-PEG2000 was adjusted to 5:1, 1:1, 1:5 and 1:10, respectively.

[0538] Cell transfection experiments showed that when the molar ratio of cationic polymer to polymer-conjugated lipid was within the range of 5:1 to 1:10, the corresponding mRNA-LNP compositions could all transfect cells, with the highest transfection efficiency at a ratio of 1:1. The transfection effect was basically consistent when the cationic lipid was SM-102 or YK-009.

[0539] Example 3: Cell transfection with mRNA-LNP formulation encapsulated with eGFP-mRNA

[0540] Step 1: Cell resuscitation and passage: Resuscitate 293T cells and passage them in culture dishes to the required number of cells.

[0541] Step 2: Plating: Digest and count the cells in the culture dish, and plate 10,000 cells per well in a 96-well plate or 150,000 cells per well in a 12-well plate. Incubate overnight until the cells adhere.

[0542] Step 3: Cell transfection: Add 1.5 μg of the mRNA-LNP preparation containing eGFP-mRNA prepared in Example 2 to the cell culture medium of a 12-well plate and continue culturing for 24 hours. Observe under a fluorescence microscope and examine the transfection efficiency based on the fluorescence intensity.

[0543] Example 4: Preparation of mRNA-LNP composition

[0544] 4.1 Preparation of mRNA-LNP compositions containing cationic polymers in the carrier

[0545] 4.1.1 Preparation Method 1

[0546] Step 1:

[0547] Following a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5, SM-102 (Xiamen Sinobond Biotechnology Co., Ltd.) or YK-009 (synthesized in Example 1) was dissolved in ethanol along with DSPC (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 to obtain solution A. Solution A was then rapidly added to citrate buffer (pH=4~5) using the ethanol injection method, and vortexed for 30 s to obtain an ethanol lipid solution.

[0548] Step 2:

[0549] eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH=4~5) to obtain an aqueous solution of eGFP-mRNA.

[0550] Step 3:

[0551] The cationic lipid carrier obtained in step 1 (molar ratio of cationic lipid:DSPC:cholesterol:DMG-PEG2000 49:10:39.5:1.5) was mixed with the ethanol lipid solution prepared in step 1 and the mRNA aqueous solution prepared in step 2 at a flow rate of 10 mL / min using a microfluidic device to obtain the corresponding liposome solution, namely solution B.

[0552] Step 4:

[0553] The cationic polymers YK-POL-001, YK-POL-007, YK-POL-012 and YK-POL-022 synthesized in Example 1 were dissolved in DMSO to obtain different solutions C.

[0554] Step 5:

[0555] Solution C was injected into solution B at a cationic polymer:mRNA mass ratio of 10:1. The solution was dialyzed in neutral PBS using a 10 kDa dialysis bag to remove ethanol and DMSO. The dialyzed lipid solution was concentrated in a 300 kDa dialysis tube, then brought to a suitable volume with PBS, and filtered through a 0.2 μm sterile filter to obtain an mRNA-LNP composition encapsulating eGFP-mRNA in cationic polymer / DSPC / cholesterol / DMG-PEG2000.

[0556] 4.1.2 Preparation Method 2

[0557] Step 1: Dissolve the cationic polymers (YK-POL-001 ~ YK-POL-027 and INT-1 prepared in Example 1) in DMSO, and vortex to completely dissolve the cationic polymers to obtain a DMSO solution of polymer YK-POL with a concentration of 20 mg / mL.

[0558] Step 2: Dissolve cationic lipids SM-102 or YK-009, DSPC, cholesterol, and DMG-PEG2000 in ethanol, with each component having a concentration of 20 mg / mL.

[0559] Step 3: Mix cationic polymer: cationic lipid: DSPC: cholesterol: DMG-PEG2000 in a molar ratio of 0.75: 50: 10: 38.5: 0.75 to prepare a lipid solution.

[0560] Step 4: Quickly inject 150 μL of the above lipid solution into 450 μL of sodium citrate (10 mM, pH = 4.0) and vortex to mix.

[0561] Step 5: Dilute the Fluc-mRNA solution with enzyme-free water to obtain a vaccine solution of 0.22 μg / mL.

[0562] Step 6: Inject the nanoparticle solution obtained in Step 4 into the mRNA solution obtained in Step 5, vortex to mix, and incubate at room temperature for 15 minutes. Transfer the solution to a 10 kDa dialysis bag and dialyze in neutral PBS to remove ethanol and DMSO. Concentrate the dialyzed lipid solution in a 300 kDa dialysis tube, then bring it to a suitable volume with PBS, and filter using a 0.2 μm sterile filter to obtain an mRNA-LNP composition encapsulating Fluc-mRNA, consisting of cationic polymer / cationic lipid / DSPC / cholesterol / DMG-PEG2000 (molar ratio of 0.75: 50:10: 38.5: 0.75).

[0563] 4.2 Preparation of mRNA-LNP compositions without cationic polymers in the carrier

[0564] The molar ratio of SM-102 or YK-009:DSPC:cholesterol:DMG-PEG2000 in the carrier was 49:10:39.5:1.5, and the preparation method was the same as in Example 4 of CN115745820A (paragraph

[0489] of the specification). SM-102 was purchased from Xiamen Sinobond Biotechnology Co., Ltd., and is compound 25 in WO20170409245A2. The synthesis method of YK-009 is described in Example 1.

[0565] Example 5: Determination of mRNA-LNP particle size, polydispersity index (PDI), and encapsulation efficiency

[0566] Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering.

[0567] Take 10 μL of the mRNA-LNP solution prepared in Example 4, dilute it to 1 mL with RNase-free deionized water, add it to the sample well, and measure each sample three times. The measurement conditions are: 90° scattering angle, 25°C.

[0568] According to the manufacturer's instructions, the encapsulation efficiency of LNPs was determined using the Quant it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK). The molar ratios of cationic polymer: YK-009: DSPC: cholesterol: DMG-PEG2000 were 1.25:49.5:10:38:1.25, and the results for carriers without cationic polymers (cationic lipid YK-009) are shown in Table 1. The molar ratios of cationic polymer: YK-009:DSPC: cholesterol: DMG-PEG2000 were 0.75:50:10:38.5:0.75, and the results for carriers without cationic polymers (cationic lipid YK-009) are shown in Table 2. When the cationic lipid was SM-102 or YK-009, the particle size, polydispersity index, and encapsulation efficiency of the corresponding mRNA-LNPs were basically consistent.

[0569] Table 1. Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP.

[0570]

[0571]

[0572] As shown in Table 1, except for the cationic polymer YK-POL-013, the nanolipid particles prepared with a carrier molar ratio of cationic polymer: cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 1.25:49.5:10:38:1.25 had particle sizes between 70 and 130 nm and could all be used for mRNA delivery. The polydispersity index was less than 0.2, indicating good particle size uniformity. The encapsulation efficiency of the above mRNA-LNP compositions varies. The mRNA-LNP compositions containing vectors YK-POL-001, YK-POL-002, YK-POL-003, YK-POL-004, YK-POL-005, YK-POL-008, YK-POL-009, YK-POL-010, YK-POL-011, YK-POL-012, YK-POL-015, YK-POL-016, YK-POL-017, and YK-POL-027 have a high encapsulation efficiency, exceeding 90%.

[0573] Table 2. Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP.

[0574]

[0575] Table 2 shows that the nanolipid particles prepared with a cationic polymer:cationic lipid:DSPC:cholesterol:DMG-PEG2000 molar ratio of 0.75:50:10:38.5:0.75 in the carrier have a particle size between 120 and 180 nm and can all be used for mRNA delivery. The mRNA-LNP compositions prepared from YK-POL-008, YK-POL-009, YK-POL-010, YK-POL-012, YK-POL-015, YK-POL-016, YK-POL-017, YK-POL-018, YK-POL-022, and YK-POL-027 all have polydispersity indices less than 0.15, indicating good particle size uniformity. Furthermore, they exhibit high encapsulation efficiency, all exceeding 70%.

[0576] Example 6: In vitro delivery performance and toxicity of LNP

[0577] The methods for cell resuscitation, passage, and plating are the same as those in Step 1 and Step 2 of Example 3.

[0578] In step 2, an appropriate volume of 293T cell culture medium was added to the 96-well plate containing 293T cells. An mRNA-LNP preparation containing 0.3 μg Fluc-mRNA (prepared in Example 4) was added to the 96-well plate. After culturing for 24 h, the appropriate reagents were added according to the Gaussian Luciferase Assay Kit instructions. The relative fluorescence intensity of each well was detected using an IVIS fluorescence detection system. Finally, 10 μL of CCK-8 solution was added to each well of the plate after 24 hours of culture. The plate was incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader to detect cell viability. The results of relative fluorescence intensity and cell viability are shown in Table 3 (cationic polymer: YK-009: DSPC: cholesterol: DMG-PEG2000 molar ratio of 1.25:49.5:10:38:1.25; when there is no cationic polymer in the carrier, the cationic lipid is SM-102 or YK-009) and Table 4 (cationic polymer: YK-009: DSPC: cholesterol: DMG-PEG2000 molar ratio of 0.75:50:10:38.5:0.75; when there is no cationic polymer in the carrier, the cationic lipid is SM-102 or YK-009). When the cationic lipid is SM-102 or YK-009, the mRNA-LNP fluorescence detection results and cell viability are basically consistent.

[0579] Table 3. Fluorescence detection results and cell viability of Fluc-mRNA-LNP -1

[0580]

[0581]

[0582] Table 3 shows significant differences in the relative fluorescence intensity (corresponding to mRNA translation efficiency) of the mRNA-LNP compositions. The mRNA-LNP compositions prepared with carriers containing cationic polymers YK-POL-001, YK-POL-007, YK-POL-011, YK-POL-012, YK-POL-016, and YK-POL-018 exhibited significantly higher relative fluorescence intensity than the mRNA-LNP composition containing cationic polymer INT-1 (number 28). Specifically:

[0583] The cell transfection efficiency of mRNA-LNP compositions containing cationic polymers YK-POL-001, YK-POL-007, YK-POL-011, YK-POL-012, YK-POL-016, and YK-POL-018 was significantly improved compared to that of mRNA-LNP compositions containing cationic polymer INT-1 (composition number 28).

[0584] The cell viability of the above-mentioned mRNA-LNP compositions was significantly improved, and they exhibited superior biosafety. Specifically, the mRNA-LNP compositions containing cationic polymers YK-POL-007, YK-POL-008, YK-POL-009, YK-POL-010, YK-POL-011, YK-POL-012, YK-POL-013, YK-POL-014, YK-POL-015, YK-POL-016, YK-POL-017, YK-POL-023, YK-POL-024, YK-POL-025, YK-POL-026, and YK-POL-027 as vectors all showed higher cell viability than the mRNA-LNP composition containing the cationic polymer INT-1 (Sequence 28).

[0585] The introduction of long alkyl cationic side chains reduces the cytotoxicity of cationic polymers. For example, the cell viability of mRNA-LNP compositions prepared by YK-POL-001 and YK-POL-007 is significantly higher than that of mRNA-LNP compositions prepared by INT-1.

[0586] Table 4. Fluorescence detection results of Fluc-mRNA-LNP and cell viability -2

[0587]

[0588] Table 4 shows significant differences in the relative fluorescence intensity (corresponding to mRNA translation efficiency) of the mRNA-LNP compositions. The mRNA-LNP compositions containing cationic polymers YK-POL-009, YK-POL-010, YK-POL-012, YK-POL-015, YK-POL-016, and YK-POL-017 in the carrier exhibited significantly higher relative fluorescence intensity than those without cationic polymers in the carrier. Specifically:

[0589] The cell transfection efficiency of mRNA-LNP compositions containing cationic polymers YK-POL-009, YK-POL-010, YK-POL-012, YK-POL-015, YK-POL-016, and YK-POL-017 was significantly improved compared to the mRNA-LNP composition (No. 18) without cationic polymers. For example, the cell transfection efficiency of the mRNA-LNP composition containing YK-POL-015 was up to 1.5 times that of the mRNA-LNP composition (No. 18) without cationic polymers.

[0590] The cell viability of the above-mentioned mRNA-LNP compositions is significantly improved, and they have better biocompatibility. The mRNA-LNP compositions containing YK-POL-002, YK-POL-007, YK-POL-008, YK-POL-009, YK-POL-010, YK-POL-011, YK-POL-012, YK-POL-015, YK-POL-016, YK-POL-017 and YK-POL-027 have significantly higher cell viability than the mRNA-LNP composition (number 18) that does not contain cationic polymers.

[0591] Example 7: In vivo delivery performance of LNP

[0592] The Fluc-mRNA-LNP composition prepared in Example 4 was injected via tail vein into 4-6 week old female BALB / c albino mice weighing 17-19g (approximately 5 μg Fluc-mRNA / mouse). Six hours after administration, the fluorescent imaging substrate was injected intravenously into the mice. The mice were allowed free movement for 5 minutes, and then the total radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the proteins expressed by the mRNA carried by the mRNA-LNP composition in the mice was detected using an IVIS Spectrum small animal in vivo imaging system. After sampling, the mice were euthanized by cervical dislocation and dissected, and the liver and spleen were precisely isolated. The total radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the proteins expressed by Fluc-mRNA in the various organs of the mice was detected using an IVIS Spectrum small animal in vivo imaging system. The results of mouse in vivo imaging and protein expression detection in the liver and spleen are shown in Table 5 (cationic polymer: YK009: DSPC: cholesterol: DMG-PEG2000 molar ratio of 1.25:49.5:10:38:1.25; when there is no cationic polymer in the carrier, the cationic lipid is SM-102 or YK-009) and Table 6 (cationic polymer: YK-009: DSPC: cholesterol: DMG-PEG2000 molar ratio of 0.75:50:10:38.5:0.75; when there is no cationic polymer in the carrier, the cationic lipid is SM-102 or YK-009). The data from mouse in vivo and organ imaging experiments were basically consistent when the cationic lipid was SM-102 or YK-009.

[0593] Table 5. Mouse in vivo and organ imaging experimental data - 1

[0594]

[0595] As shown in Table 5, mRNA-LNP compositions with carriers containing cationic polymers YK-POL-001, YK-POL-007, YK-POL-012 or YK-POL-016 can efficiently deliver mRNA to the liver, showing improvements compared to mRNA-LNP compositions without carriers containing cationic polymers (number 7).

[0596] Table 6. Mouse in vivo and organ imaging experimental data - 2

[0597]

[0598] As shown in Table 6, the mRNA-LNP compositions with carriers containing cationic polymers YK-POL-009, YK-POL-010, YK-POL-016 or YK-POL-027 can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with the mRNA-LNP compositions with carriers not containing cationic polymers (serial number 8). Specifically, compared with mRNA-LNP compositions (No. 8) whose carriers do not contain cationic polymers, mRNA-LNP compositions prepared with carriers containing cationic polymers YK-POL-009, YK-POL-010, YK-POL-016 or YK-POL-027 all showed significantly enhanced total spleen radiation intensity. For example, the total spleen radiation intensity of mRNA-LNP compositions prepared with carriers containing YK-POL-010, YK-POL-016 and YK-POL-027 was 10 times, 10 times and 5 times that of mRNA-LNP compositions (No. 8) whose carriers did not contain cationic polymers, respectively.

[0599] Although this application has been described through the above embodiments, it should not be construed as being limited thereto. Rather, this application covers the general aspects previously disclosed and can be modified and varied in many ways without departing from the spirit and scope of this application.

Claims

1. A polymer or a pharmaceutically acceptable salt thereof, characterized in that, The polymer has a structure represented by Formula (Ia): ； R1is C 1-10 alkyl or -C 1-6 alkylene-(OCH2CH2) t -OC 1-6 alkyl; t is an integer from 1 to 100; R2 is -COOH; R3 is , , , , , , , or ; X is -C(O)NH-; Y is -C(O)O-; Z is -NH-; m is 5-60; n is 5-60; a is 0; b is 5; c is 6.

2. The polymer or pharmaceutically acceptable salt thereof according to claim 1, wherein R1is or .

3. The polymer or pharmaceutically acceptable salt thereof according to claim 1, characterized by, The polymer has any one of the following structures: 、 、 、 、 、 、 、 、 or 。 4. The polymer or pharmaceutically acceptable salt thereof according to claim 1, characterized by, The polymer is any one of the following structures: 、 、 、 、 、 、 、 、 or 。 5. A carrier comprising a cationic polymer, the cationic polymer being the polymer of any one of claims 1-4 or a pharmaceutically acceptable salt thereof.

6. The carrier of claim 5, wherein, The mole percentage of the cationic polymer in the carrier is 0.2% to 25%.

7. The carrier of claim 5, wherein, The carrier further comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

8. The carrier of claim 7, wherein, The carrier satisfies any one or a combination of at least two of the following (1) to (4): (1) the mole ratio of the cationic polymer to the cationic lipid is 1: (1 to 500); (2) the mole ratio of the cationic polymer to the neutral lipid is 0.01:1 to 5:1; (3) the mole ratio of the cationic polymer to the structural lipid is 0.01:1 to 1.5:1; (4) the mole percentage of the polymer-conjugated lipid to the carrier is 0.005:1 to 0.1:

1.

9. The carrier of claim 7, wherein, The carrier satisfies any one or a combination of at least two of the following (1) to (5): (1) the cationic lipid is SM-102, MC3, or YK-009; (2) the neutral lipid is any one or a combination of at least two selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol; (3) the structural lipid is any one or a combination of at least two selected from the group consisting of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and corticosteroid; (4) the polymer-conjugated lipid is any one or a combination of at least two selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; (5) the mole ratio of the cationic polymer, the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid in the carrier is (0.2 to 25) : (25 to 75) : (5 to 25) : (15 to 65) : (0.5 to 10).

10. The carrier of claim 9, wherein, The carrier satisfies any one or a combination of at least two of the following (1) to (5): (1) the cationic lipid is YK-009; (2) the neutral lipid is selected from the group consisting of any one or a combination of at least two of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-didodecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyloleoyl phosphatidyl ethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine; (3) the structural lipid is cholesterol; (4) the polymeric conjugated lipid is any one or a combination of at least two of distearoylphosphatidyl ethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol 2000, and methoxypolyethylene glycol bimyristyl acetamide; (5) the molar ratio of the cationic polymer, the cationic lipid, the neutral lipid, the structural lipid, and the polymeric conjugated lipid in the carrier is (0.05-5) : (35-49) : (7.5-15) : (35-55) : (0.5-5).

11. The carrier of claim 10, wherein, The carrier satisfies any one or a combination of at least two of the following (1)-(3): (1) the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine; (2) the polymeric conjugated lipid is 1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol 2000; (3) in the vector, the molar ratio of the cationic polymer, the cationic lipid, the neutral lipid, the structural lipid, and the polymeric conjugated lipid is 1.25: 49.5: 10: 38: 1.25 or 0.75: 50: 10: 38.5: 0.

75.

12. A composition characterized in that, The composition comprises an active ingredient and a carrier, and the carrier is the carrier according to any one of claims 5-11.

13. The composition according to the preceding claim 12, characterized in that, The composition is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10-300 nm; the polydispersity index of the nanoparticle preparation is ≤0.

5.

14. The composition of claim 12, wherein, The active ingredient comprises a therapeutic agent or a prophylactic agent.

15. The composition of claim 14, wherein, The mass ratio of the carrier to the therapeutic agent or prophylactic agent is 5:1-15:

1. Alternatively, the therapeutic agent or prophylactic agent is a vaccine or a compound capable of causing an immune response.

16. The composition of claim 14, wherein, The therapeutic agent or prophylactic agent is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, or polypeptides. Alternatively, the mass ratio of the carrier to the therapeutic agent or prophylactic agent is 10:

1.

17. The composition of claim 16, wherein, The therapeutic agent or prophylactic agent is a ribonucleic acid.

18. The composition of claim 17, wherein, The ribonucleic acid is selected from any one or a combination of at least two of the group consisting of small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA.

19. The composition according to the preceding claim 12, characterized in that, The composition further comprises a pharmaceutically acceptable excipient and / or diluent.

20. Use of the polymer or pharmaceutically acceptable salt thereof according to any one of claims 1-4, or the carrier according to any one of claims 5-11, in the preparation of a carrier for improving cell transfection efficiency and / or reducing cytotoxicity.

21. Use of the polymer according to any one of claims 1-4, or the carrier according to any one of claims 5-11, for improving cell transfection efficiency and / or reducing cytotoxicity for non-disease diagnosis and treatment purposes.

22. Use of the polymer of any one of claims 1 to 4, or the carrier of any one of claims 5 to 11, for increasing the targeting of a nucleic acid to a target, and / or, for increasing the expression amount of a nucleic acid in a target, for a non-disease diagnostic treatment purpose, wherein, The target is selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue, and a target cell.

23. Use according to claim 22, characterized in that, The target organ or target tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph, and muscle.

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