Polyamine, polyhydroxy ionizable cationic lipids, compositions comprising the same, and uses

Abstract

The application discloses a kind of polyamine, polyhydroxy ionizable cationic lipids, compositions comprising and purposes, specifically discloses a kind of cationic lipids shown in formula (I). The cationic lipids provided by the application can be used as RNA targeted delivery, can significantly enhance the spleen targeting of mRNA drug.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a polyamine- and polyhydroxy ionizable cationic lipid, compositions containing the same, and their uses. Background Technology

[0002] In the pharmaceutical field, the efficient targeted delivery of small molecule drugs, peptides, proteins, and nucleic acids remains a persistent challenge. The delivery of nucleic acids, in particular, faces significant challenges due to their low cell permeability and high sensitivity to degradation by nucleases such as RNase.

[0003] Compositions containing cationic lipids, liposomes, and lipoplexes serve as transport media, effectively delivering bioactive substances such as small molecule drugs, peptides, proteins, and nucleic acids into cells and / or intracellular compartments. These compositions typically contain one or more cationic and / or ionizable lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, readily ionizable amine-containing lipids. Although various such lipid-containing nanoparticle compositions have been demonstrated, their safety, efficacy, and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, a major concern that could limit their clinical application. For example, nucleic acid drugs, exemplified by patisiran (trade name onpattro®), require prior administration of steroids and antihistamines to patients to eliminate unwanted immune responses.

[0004] Recent studies have designed ionizable lipids for selective mRNA delivery in the spleen, thereby reducing off-target accumulation in organs such as the liver. Nevertheless, further research is needed to optimize LNP formulations, improve their efficacy and safety, and ultimately meet the stringent standards required for medical applications. Summary of the Invention

[0005] The technical problem this disclosure aims to solve is to overcome the poor safety or delivery efficiency of ionizable lipids in the prior art, and provides a polyamine- or polyhydroxy ionizable cationic lipid, compositions comprising the same, and their uses. The ionizable cationic lipid provided by this disclosure can be used to deliver therapeutic or preventative agents such as nucleic acid molecules, small molecule compounds, peptides, or proteins. It has a simple preparation method, strong targeting to the spleen, can carry active pharmaceutical ingredients to transfect cells with high transfection efficiency, and exhibits low cytotoxicity, thus improving delivery efficiency and safety.

[0006] This disclosure provides a compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer;

[0007] ,

[0008] in:

[0009] R1 is -N(R 1a )-C 1-8 Alkylene-N(R) 1b -, -(5-10-membered heterocyclic alkylene)-(5-10-membered heterocyclic alkylene)- or -(5-10-membered heterocyclic alkylene)-;

[0010] R 1a and R 1b Independently for C 1-6 alkyl;

[0011] R2 and R3 are independently C 10-20 Alkyl or C 6-16 Alkenyl groups, where R2 and R3 may be the same or different;

[0012] L1 and L2 are independently C 2-8 Straight-chain alkylene;

[0013] M1 is -(CH2) n1 -or-(CH2) n2 NHC(O)O(CH2) m - where n1, n2 and m are each independently 0, 1, 2, 3 or 4;

[0014] M2 and M3 are independently -CH=CH- or -C(O)O-, and M2 and M3 may be the same or different;

[0015] The heteroatoms in the 5-10 member heterocyclic alkyl group are independently one, two, or three of N, O, and S, and the number of heteroatoms is independently one, two, or three.

[0016] In some implementations, R1 is -N(R 1a )-C 3-6 Alkylene-N(R) 1b -, -(5-6-membered heterocyclic alkyl)-(5-6-membered heterocyclic alkyl)- or -(5-6-membered heterocyclic alkyl)-.

[0017] In some embodiments, the heteroatom in the 5-6 membered heterocyclic alkylene group is N, and the number of heteroatoms is one or two, for example... or .

[0018] In some implementation schemes, R 1a and R 1b Independently for C 1-3 Alkyl groups, such as ethyl groups.

[0019] In some implementations, R2 and R3 are independently C 10-20 straight-chain alkyl, C 10-20 Branched alkyl, C 6-16 Straight-chain alkenyl or C 6-16 Branched alkenyl groups.

[0020] In some implementations, R2 and R3 are independently C 16-20 Branched alkyl, C 10 Straight-chain alkyl or C8 straight-chain alkenyl.

[0021] In some implementations, L1 and L2 are independently C 3-8 Straight-chain alkylene groups.

[0022] In some implementations, n1, n2, and m are each independently 1 or 2.

[0023] In some implementations, R1 is -N(R 1a )-C 3-6 Alkylene-N(R) 1b -, -(5-6-membered heterocyclic alkylene)-(5-6-membered heterocyclic alkylene)- or -(5-6-membered heterocyclic alkylene)-;

[0024] R 1a and R 1b Independently for C 1-3 alkyl;

[0025] R2 and R3 are independently C 16-20 Branched alkyl, C 10 Straight-chain alkyl or C8 straight-chain alkenyl;

[0026] L1 and L2 are independently C 3-8 Straight-chain alkylene;

[0027] M1 is -(CH2) n1 -or-(CH2) n2 NHC(O)O(CH2) m - where n1, n2 and m are each independently 1 or 2;

[0028] M2 and M3 are independently -CH=CH- or -C(O)O-, and M2 and M3 may be the same or different;

[0029] The heteroatom in the 5-6 membered heterocyclic alkyl group is N, and the number of heteroatoms is 1 or 2.

[0030] In some implementations, R1 is , , or .

[0031] In some implementations, R2 is , , , or .

[0032] In some implementations, R3 is , ... , or .

[0033] In some implementations, L1 is -(CH2)3-, -(CH2)5-, -(CH2)7-, or -(CH2)8-.

[0034] In some implementations, L2 is -(CH2)3-, -(CH2)5-, -(CH2)7-, or -(CH2)8-.

[0035] In some implementations, M1 is -CH2- or -CH2NHC(O)O(CH2)2- a The a terminal is connected to the N terminal.

[0036] In some implementations, M2 is -CH=CH- or -C(O)O- b b end and R 2 connect.

[0037] In some implementations, M3 is -CH=CH- or -C(O)O- c c end and R 3 connect.

[0038] In some embodiments, the compound represented by formula (I) is any of the following compounds:

[0039] ,

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] ,

[0046] ,

[0047] ,

[0048] ,

[0049] ,

[0050] ,

[0051] ,

[0052] ,

[0053] ,

[0054] ,

[0055] ,

[0056] , or .

[0057] This disclosure also provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, said cationic lipid being a compound of formula (I) as described above or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0058] In some embodiments, the composition is a lipid composition or a pharmaceutical composition.

[0059] In some embodiments, the molar ratio of the cationic lipid to the carrier is 0.25:1 to 0.75:1; preferably 0.35:1 to 0.65:1; for example, 0.4:1, 0.45:1 or 0.49:1.

[0060] In some embodiments, the carrier also comprises neutral lipids.

[0061] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1; preferably 3:1 to 10:1; for example, 3.5:1, 4:1 or 4.9:1.

[0062] 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 and sterol.

[0063] 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-phosphate choline, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate choline, n-Glyceryl-3-phosphate ethanolamine, 1,2-Diphylanoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-Distearatel-sn-glyceryl-3-phosphate ethanolamine, 1,2-Dilinoleoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-Dilinoleoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-Diarachidonicoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-bis(docohexanoyl)-sn-glyceryl-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phosphate ethanolamine Sodium rac-(1-glycerol) salt, 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.

[0064] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and / or 1,2-distearate-sn-glycerol-3-phosphate choline.

[0065] In some embodiments, the carrier also comprises structural lipids.

[0066] In some embodiments, the molar ratio of the structural lipid to the carrier is 0.15:1 to 0.65:1; preferably 0.2:1 to 0.535:1; for example, 0.25:1, 0.395:1, 0.435:1 or 0.485:1.

[0067] 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.

[0068] In some implementations, the structural lipid is cholesterol.

[0069] In some embodiments, the carrier further comprises polymeric conjugated lipids.

[0070] In some embodiments, the molar ratio of the polymer conjugated lipid to the carrier is 0.005:1 to 0.1:1; preferably 0.015:1 to 0.05:1; for example, 0.025:1 or 0.035:1.

[0071] In some embodiments, the polymeric conjugated lipid is selected from any or a combination of at least two of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0072] In some embodiments, the polymeric conjugated lipid is selected from any or a combination of at least two of the following: distearate phosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol bistetradecylacetamide.

[0073] In some embodiments, the polymeric conjugated lipid is 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000.

[0074] In some embodiments, the composition comprises the cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid; the molar ratio of the cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid is (25~75):(5~25):(15~65):(0.5~10); preferably (35-49):(7.5-15):(35-55):(1-5); more preferably 49:10:39.5:1.5.

[0075] In some embodiments, the composition comprises the cationic lipid, DSPC, cholesterol, and DMG-PEG2000; the molar ratio of the cationic lipid, DSPC, cholesterol, and DMG-PEG2000 may be (25~75):(5~25):(15~65):(0.5~10); preferably (35-49):(7.5-15):(35-55):(1-5); more preferably 49:10:39.5:1.5.

[0076] In some embodiments, the composition further includes one or more other ionizable lipid compounds.

[0077] In some embodiments, the composition further comprises a therapeutic or preventative agent.

[0078] In some embodiments, the mass ratio of the carrier to the therapeutic or preventative agent is 10:1 to 30:1; preferably 15:1 to 20:1.

[0079] In some embodiments, the therapeutic agent or the preventive agent is a vaccine or compound capable of evoking an immune response.

[0080] In some embodiments, the therapeutic agent or the preventive agent is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides, or proteins.

[0081] In some embodiments, the therapeutic agent or the preventive agent is a nucleic acid; preferably ribonucleic acid.

[0082] In some embodiments, the nucleic 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, and messenger RNA; preferably messenger RNA.

[0083] In some embodiments, the composition is a nanoparticle formulation with an average particle size of 10-300 nm, preferably 40 nm-240 nm, and more preferably 90 nm-150 nm.

[0084] In some embodiments, the polydispersity index of the nanoparticle formulation is ≤0.5, preferably ≤0.4, and more preferably less than 0.10.

[0085] In some embodiments, the composition further includes pharmaceutically available excipients and / or diluents.

[0086] This disclosure also provides the use of a compound of formula (I) as described above, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or pharmaceutical composition as described above, in the preparation of a medicament for treating a disease or condition.

[0087] In some implementations, the disease or condition is characterized by dysfunction or abnormality of a protein or polypeptide.

[0088] In some implementations, the disease or condition is selected from any one or a combination of at least two of the following: infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, renal and vascular diseases, and metabolic diseases.

[0089] In some embodiments, the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation; subcutaneous administration is preferred.

[0090] In some embodiments, the therapeutic or preventative agent in the composition is administered at a dose of about 0.001 mg / kg to 10 mg / kg.

[0091] This disclosure also provides the use of the compound of formula (I) as described above, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or pharmaceutical composition as described above, in improving cell transfection efficiency and / or reducing cytotoxicity, preferably in improving cell transfection efficiency and / or reducing cytotoxicity in vitro.

[0092] This disclosure also provides the use of the compound of formula (I) as described above, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the pharmaceutical composition as described above, in increasing the targeting of a nucleic acid drug to any or at least a combination of the group consisting of target organs, target tissues and target cells, and / or in increasing the expression level of a nucleic acid in any or at least a combination of the group consisting of target organs, target tissues and target cells.

[0093] 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 compounds provided in this disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers, or using the lipid compositions provided in this disclosure to package and deliver the (pharmaceutical) active ingredient to target organs / tissues / cells, or using the compositions disclosed in this disclosure to deliver the contained active ingredient to target organs / tissues / cells, thereby achieving therapeutic effects, improving symptoms, regulating physiological activities in the body, etc.; diagnostic uses may include packaging active ingredients for disease diagnosis into the compounds provided in this disclosure, or their N-oxides, solvates, or pharmaceutically acceptable salts, or stereoisomers. The active ingredient is delivered to target organs / tissues / cells in a pharmaceutically acceptable salt, stereoisomer, or lipid composition to achieve the purpose of disease diagnosis. Non-therapeutic / non-diagnostic purposes may include encapsulating the active ingredient with the compound provided in this disclosure, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, or with the lipid composition provided in this disclosure, thereby delivering it to target organs / tissues / cells for scientific research, detection, and other non-therapeutic and non-diagnostic purposes (such as conducting disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0094] Terminology Definition

[0095] 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.

[0096] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0097] 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.

[0098] 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.

[0099] Furthermore, in this invention, when a numerical range is used in the general formula and / or structural formula of a compound, it means that the number of the corresponding groups within that numerical range can be any natural number within that range, such as "C". A-B "" refers to any integer number of carbon atoms within the range from the starting point to the ending point, where A and B are both integers; for example, C 1-5 The number of carbon atoms indicates 1, 2, 3, 4, or 5; that is, when combined with other groups in the general formula and / or structural formula of a compound to form various possible compounds, C A-B It can be used in conjunction with any group containing carbon atoms to specify the number of carbon atoms, such as C. 1-5 Alkyl / alkylene compounds represent various possibilities of alkyl / alkylene compounds having 1 carbon, 2 carbon, 3 carbon, 4 carbon, and / or 5 carbon.

[0100] 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.”

[0101] The term “pharmaceutically acceptable” in this disclosure means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0102] The terms “subject” or “patient” in this disclosure include both humans and mammals. In this document, “subject” or “patient” also appears in some instances as “object”.

[0103] As used herein, the term "treatment" refers to 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. As used herein, the term includes prevention of the worsening of said disease, condition, or related symptoms. It should be understood that treatment, as used herein, may not be effective for all subjects to be treated. However, preferably, the term should require that a statistically significant proportion of subjects suffering from the disease or condition described herein can be successfully treated. Statistical significance can be determined by a variety of well-known statistical assessment tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In some embodiments, treatment includes inhibiting the proliferation of cancer cells, preferably including killing cancer cells. Preferably, treating cancer is reducing the tumor and / or cancer cell burden in the subject. As those skilled in the art will understand, the effectiveness of cancer treatment depends on a variety of factors, including, for example, cancer stage and cancer type. It is also preferred that cancer treatment further includes at least one of chemotherapy, immunotherapy, surgery, and radiation therapy.

[0104] The term "prevention" refers to maintaining health associated with the disease or condition described herein in a subject for a period of time. It should be understood that this period of time may depend on the amount of medication administered and individual factors of the subject. It should be understood that prevention may not be effective in all subjects treated. However, preferably, the term requires the effective prevention of a statistically significant proportion of a group or population of subjects suffering from the disease or condition described herein or its accompanying symptoms. Statistical significance can be determined by various well-known statistical assessment tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In the case of cancer treatment, prevention specifically relates to preventing cancer development, preventing metastasis formation, and / or preventing recurrence, preferably involving the prevention of metastasis formation and / or preventing recurrence.

[0105] The term "infectious disease" refers to a disease state or condition caused by a foreign organism (e.g., a microorganism) that enters a subject, multiplies, and elicits a response (e.g., an inflammatory response) in the subject. In some embodiments, infectious diseases are caused by viruses (or viral particles), bacteria, fungi, or parasites. Examples of "infectious diseases" include, but are not limited to, diseases caused by coronaviruses (e.g., SARS-CoV-2), influenza viruses, hepatitis viruses (e.g., hepatitis B virus), immunodeficiency viruses (e.g., human immunodeficiency virus), rabies virus, papillomavirus (e.g., human papillomavirus), respiratory syncytial virus (RSV), herpesviruses (e.g., herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, etc.), pneumonia (e.g., viral pneumonia, bacterial pneumonia, etc.), Rift Valley fever (RVF), yellow fever, etc.

[0106] "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.

[0107] The term "solvent" in this disclosure refers to a complex formed by the combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield the compound of formula (I) once absorbed into the subject, such that the use of the compound of formula (I) respectively encompasses the use of any solvate of the compound of formula (I).

[0108] The term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water.

[0109] It should be further understood that compounds of formula (I) or pharmaceutically acceptable salts thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this disclosure. For example, compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in an unsolvated form or in a solvated form formed by combining with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0110] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds disclosed herein. Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, etc. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with the compounds shown in formula (I).

[0111] The nitrogen-containing compounds of formula (I) of this disclosure can be converted into N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Therefore, provided that the valence state and structure allow, the compounds claimed in this disclosure include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0112] Some of the compounds disclosed herein can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in this disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, the compounds of this disclosure can be separated by adding chiral acid resolving reagents such as chiral tartaric acid or chiral malic acid to form salts, utilizing the physicochemical properties of the products, such as differences in solubility.

[0113] This disclosure also includes all suitable isotopic variants of the compounds disclosed herein. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but whose atomic mass differs from that of atoms commonly or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively, for example…2 H (deuterium) 3 H (tritium) 11 C 13 C 14 C 15 N、 17 O and 18 O.

[0114] The term "alkyl" in this disclosure refers to a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in this disclosure refers to a branched or straight-chain saturated aliphatic divalent 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 Alkylenes include C2 alkylenes, C3 alkylenes, C4 alkylenes, and C5 alkylenes.

[0115] In this disclosure, when the name of a compound differs from its structural formula, the structural formula shall prevail.

[0116] It should be understood that the term "compound of the present disclosure" as used herein may, depending on the context, include: compounds of formula (I), their N-oxides, their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0117] The term “cationic lipid” as used in this article refers to lipids that are positively charged at a selected pH value or range.

[0118] Cationic lipids 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).

[0119] The inventors found that screening a large number of compounds was extremely difficult, particularly for suitable cationic lipid compounds that met the following criteria: structurally different from existing cationic lipids, exhibiting high transfection efficiency and low cytotoxicity, and demonstrating high and sustained expression in mice. The inventors discovered several compounds, such as YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820, which, compared to existing cationic lipids, significantly improved intracellular transfection efficiency, significantly increased expression levels in animals, and spleen targeting.

[0120] This disclosure is based on at least the following findings:

[0121] The cationic lipid compounds disclosed herein can be used to deliver nucleic acid molecules, small molecule compounds, peptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds disclosed herein exhibit higher transfection efficiency and lower cytotoxicity, and significantly increased expression levels in animal spleens, thereby improving delivery efficiency.

[0122] cationic lipids

[0123] In one embodiment of the composition / carrier disclosed herein, the cationic lipid is one or more selected from the compounds of formula (I) above, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above. For example, the cationic lipid is a compound. In a preferred embodiment, the cationic lipid is compound YK-1801 to YK-1820, and in another preferred embodiment, the cationic lipid is compound YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820.

[0124] In another embodiment of the composition / carrier disclosed herein, the cationic lipid comprises: (a) one or more selected from the compounds of formula (I) above, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers; and (b) one or more other ionizable lipid compounds different from (a). (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 of CN102625696B, or MC3 of CN102625696B.

[0125] In one embodiment, the cationic lipid accounts for 25% to 75% of the molar ratio of the carrier, for example, 30%, 40%, 50%, 55%, 60%, 65%, or 70%.

[0126] 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.

[0127] For example, examples of the therapeutic agent or the preventive agent may be one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. 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.

[0128] neutral lipids

[0129] The carrier may contain neutral lipids. In this disclosure, neutral lipids refer to lipids present in a charge-free or neutral ionic form within a selected pH 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.

[0130] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. In another preferred embodiment, for example, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0131] For example, neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0132] 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.

[0133] 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).

[0134] 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.

[0135] In some embodiments, neutral lipids include DSPC. In some embodiments, neutral lipids include DOPE. In some embodiments, neutral lipids include both DSPC and DOPE.

[0136] structural lipids

[0137] The carrier of the composition comprising cationic lipids may also include one or more structural lipids. In this disclosure, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0138] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1.

[0139] 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.

[0140] Polymer conjugated lipids

[0141] The carrier of the composition containing cationic lipids 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.

[0142] 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.

[0143] For example, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0144] In one embodiment of the composition / carrier disclosed herein, the polymeric conjugated lipid is DMG-PEG2000.

[0145] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is (25~75):(5~25):(15~65):(0.5~10), for example (30~49):(7.5~15):(35~55):(1~5), more preferably (40~49):(8~12):(39~45):(1~3). The total molar percentage of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is 100.

[0146] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is 40:10:48.5:1.5 or 49:10:39.5:1.5.

[0147] Therapeutic agents and / or preventative agents

[0148] The composition may include one or more therapeutic and / or preventive agents. In one embodiment, the mass ratio of the carrier to the therapeutic or preventive agent is 10:1 to 30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0149] In one embodiment, the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1 to 20:1, preferably 13 to 17:1, and more preferably 15:1.

[0150] The therapeutic or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0151] For example, the therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

[0152] The carriers disclosed herein can deliver therapeutic and / or preventative agents to mammalian cells or organs; therefore, this disclosure also provides methods for treating diseases or conditions in mammals in need, including administering a composition comprising therapeutic and / or preventative agents to the mammal and / or contacting mammalian cells with the composition. Accordingly, this disclosure provides the use of the compounds of this disclosure or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or compositions of this disclosure in the preparation of medicaments for treating diseases or conditions in subjects in need.

[0153] This disclosure also provides the use of the compounds of this disclosure or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or compositions thereof, in the preparation of nucleic acid drugs, vaccines, chemical drugs, peptide drugs, or protein drugs.

[0154] Therapeutic agents and / or preventive agents include bioactive substances and are alternatively referred to as "active agents". Therapeutic agents and / or preventive 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 one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventive 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.

[0155] 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.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).

[0156] In other embodiments, the therapeutic and / or preventative agents are proteins. Therapeutic proteins that may be used in the nanoparticles of this disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] Disease or ailment

[0163] 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, peptides, or proteins. Therefore, the compositions disclosed herein can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein 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.

[0164] In one embodiment, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0165] 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.

[0166] In one embodiment, the infectious disease is selected from diseases caused by coronaviruses, influenza viruses, hepatitis viruses, immunodeficiency viruses, rabies viruses, human papillomaviruses, respiratory syncytial viruses, herpesviruses, etc., as well as pneumonia, Rift Valley fever, yellow fever, etc.

[0167] Other components

[0168] 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.

[0169] 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).

[0170] 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).

[0171] 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.

[0172] 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).

[0173] 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.

[0174] 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.

[0175] 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.

[0176] For example, the compositions of this disclosure can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the compositions are administered subcutaneously.

[0177] 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.

[0178] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0179] All reagents and raw materials used in this disclosure are commercially available.

[0180] The positive and progressive effects of this disclosure are as follows: the cationic lipid compounds and lipid compositions disclosed herein can be used for the encapsulation of active pharmaceutical ingredients such as nucleic acids (e.g., mRNA). The mRNA-LNP composition prepared from the cationic lipids of this disclosure can significantly increase protein expression levels in mice, and also has significant spleen targeting, which can significantly increase protein expression levels both in vivo and in vitro. Attached Figure Description

[0181] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of this disclosure will be briefly introduced below. It should be understood that the drawings described below are only some exemplary embodiments of this disclosure, and not limitations on this disclosure.

[0182] Figure 1 The particle size of the mRNA-LNP compositions encapsulated with Fluc-mRNA prepared based on YK-1801~YK-1820, SM-102, MC3, L0533, II-8, compound 2248 and compound 1 is shown.

[0183] Figure 2 The relative fluorescence intensity of mRNA-LNP compositions encapsulated with Fluc-mRNA prepared based on YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, YK-1820, SM-102, MC3, L0533, II-8, compound 2248 and compound 1 is shown for use after cell transfection.

[0184] Figure 3This shows the mean radiation intensity in mice 6 hours after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, YK-1820, SM-102, MC3, L0533, II-8, compound 2248, and compound 1.

[0185] Figure 4 This shows the mean radiation intensity in the spleen of mice 6 hours after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, YK-1820, SM-102, MC3, L0533, II-8, compound 2248, and compound 1.

[0186] Figure 5 Imaging images of the liver and spleen 6 h after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1805, YK-1806, YK-1809, YK-1810, YK-1818 and YK-1819, respectively. Detailed Implementation

[0187] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the described embodiments of this disclosure, those skilled in the art can implement them in other specific forms without departing from the basic attributes and spirit of this disclosure. It should be understood that, without conflict, any and all embodiments of this disclosure can be combined with technical features of any other embodiment or multiple other embodiments to obtain other embodiments. This disclosure includes other embodiments obtained by such combinations.

[0188] 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.

[0189] In the following embodiments, the abbreviations have the following meanings:

[0190] NaBH4: Sodium borohydride; Boc2O: Di-tert-butyl dicarbonate; DMAP: 4-Dimethylaminopyridine; EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIEA: N,N-diisopropylethylamine; DCM: Dichloromethane; DMF: N,N-dimethylformamide; THF: Tetrahydrofuran; MeOH: Methanol; TEA: Triethylamine; ACN: Acetonitrile; TsOH·H2O: p-Toluenesulfonic acid hydrate; DMSO: Dimethyl sulfoxide; EA: Ethyl acetate; CDI: N,N'-carbonyldiimidazole.

[0191] Example 1: Synthesis of cationic lipid compounds

[0192] 1.1 Synthesis of intermediate INT-1

[0193]

[0194] Step 1: Synthesis of INT-1-PM1

[0195] (S)-1-amino-3-chloro-2-propanol hydrochloride (20.00 g, 136.98 mmol) was dissolved in dichloromethane (200 mL), and triethylamine (55.58 g, 549.28 mmol) was added. Then, di-tert-butyl dicarbonate (110.91 g, 508.18 mmol) was added dropwise. The mixture was heated to 40 °C and reacted for 24 h. The reaction was monitored by TLC until the reactants had completely reacted. Heating was stopped, and the system was cooled to room temperature. The reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0-18% ethyl acetate / n-hexane). The product was collected and concentrated to give INT-1-PM1 (20.25 g, 96.58 mmol, 70.5%). C8H 16 ClNO3, MS(ES): m / z(M+H + ) 210.1.

[0196] Step 2: Synthesis of INT-1-PM2

[0197] INT-1-PM1 (12.00 g, 57.23 mmol) was dissolved in acetonitrile (100 mL), followed by the sequential addition of N,N'-diethyl-1,3-propanediamine (2.98 g, 22.89 mmol), potassium carbonate (12.66 g, 91.57 mmol), and potassium iodide (0.76 g, 4.58 mmol). The mixture was heated to 70 °C and reacted for 14 h, monitored by TLC until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-10% methanol / dichloromethane), and the product was collected and concentrated to obtain INT-1-PM2 (8.54 g, 17.92 mmol, 78.3%). 23 H 48 N4O6, MS(ES): m / z(M+H + 477.6.

[0198] Step 3: Synthesis of INT-1

[0199] Add INT-1-PM2 (8.54 g, 17.92 mmol) and a 1,4-dioxane solution (90 mL) of hydrochloric acid, and react at room temperature for 2 h. Monitor the reaction with LC-MS until the starting material has completely reacted. Concentrate the reaction solution under reduced pressure, dissolve the residue in methanol, and then concentrate under reduced pressure again. Repeat this process twice to obtain INT-1 (5.07 g, 18.34 mmol, crude product). C 13 H 32 N4O2, MS(ES): m / z(M+H + 277.6.

[0200] 1.2 Synthesis of intermediate INT-2

[0201]

[0202] Step 1: Synthesis of INT-2-PM1

[0203] Using INT-1-PM1 (3.00 g, 14.30 mmol) as a starting material, INT-2-PM1 (2.27 g, 4.38 mmol, 91.3%) was obtained by following the synthesis method of INT-1-PM2. 26 H 54 N4O6, MS(ES): m / z(M+H + 519.5.

[0204] Step 2: Synthesis of INT-2

[0205] Using INT-2-PM1 (2.27 g, 4.38 mmol) as a starting material, INT-2 (2.10 g, 6.59 mmol, crude product) was obtained following the synthesis method of INT-1. 16 H 38 N4O2, MS(ES): m / z(M+H + 319.3.

[0206] 1.3 Synthesis of intermediate INT-3

[0207]

[0208] Step 1: Synthesis of INT-3-PM1

[0209] Using INT-1-PM1 (3.37 g, 16.08 mmol) as a starting material, INT-3-PM1 (2.40 g, 4.66 mmol, 87.2%) was obtained by following the synthesis method of INT-1-PM2. 26 H 50 N4O6, MS(ES): m / z(M+H + 515.5.

[0210] Step 2: Synthesis of INT-3

[0211] Using INT-3-PM1 (2.40 g, 4.66 mmol) as a starting material, INT-3 (2.40 g, 7.63 mmol, crude product) was obtained following the synthesis method of INT-1. 16 H 34 N4O2, MS(ES): m / z(M+H + 315.3.

[0212] 1.4 Synthesis of intermediate INT-4

[0213]

[0214] Step 1: Synthesis of INT-4-PM1

[0215] Using INT-1-PM1 (14.60 g, 69.63 mmol) as a starting material, INT-4-PM1 (6.40 g, 14.80 mmol, 63.7%) was obtained by following the synthesis method of INT-1-PM2. 20 H 40 N4O6, MS(ES): m / z(M+H + 433.3.

[0216] Step 2: Synthesis of INT-4

[0217] Using INT-4-PM1 (6.40 g, 14.80 mmol) as a starting material, INT-4 (3.86 g, 16.61 mmol, crude product) was obtained following the synthesis method of INT-1. 10 H 24 N4O2, MS(ES): m / z(M+H + ) 233.3.

[0218] 1.5 Synthesis of intermediate INT-5

[0219]

[0220] 2-Hexyldecyl alcohol (24.24 g, 99.98 mmol) was dissolved in cyclohexane (250 mL), followed by the addition of 6-bromohexanoic acid (23.40 g, 119.98 mmol) and p-toluenesulfonic acid hydrate (0.28 g, 1.49 mmol). A water separator was installed, and the reaction was carried out at 110 °C for 7 h. The reaction was monitored by TLC until the reactants had completely reacted. Heating was stopped, and the reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was separated into liquid and liquid phases. The aqueous phase was extracted once with n-hexane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give INT-5 (41.42 g, 98.74 mmol, 98.8%). 22 H 43 BrO2, MS(ES): m / z(M+H + 419.2.

[0221] 1.6 Synthesis of intermediate INT-6

[0222]

[0223] Heptadecan-9-ol (10.00 g, 38.99 mmol) was dissolved in dichloromethane (80 mL), followed by the addition of 6-bromohexanoic acid (8.37 g, 42.89 mmol), EDCI (11.21 g, 58.49 mmol), and DMAP (0.95 g, 7.80 mmol). The reaction was carried out at room temperature for 16 h, and the reaction was monitored by TLC until the reactants had completely reacted. The reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-20% ethyl acetate / n-hexane). The product was collected and concentrated to give INT-6 (15.20 g, 35.06 mmol, 89.9%). 23 H 45 BrO2, MS(ES): m / z(M+H + 433.2.

[0224] 1.7 Synthesis of intermediate INT-7

[0225]

[0226] Using 2-octyldodecyl alcohol (11.60 g, 38.85 mmol) as a starting material, INT-7 (16.50 g, 34.69 mmol, 89.3%) was obtained following the synthesis method of INT-6. 26 H 51 BrO2, MS(ES): m / z(M+H + 475.6.

[0227] 1.8 Synthesis of intermediate INT-8

[0228]

[0229] Using 2-hexyldecyl alcohol (20.00 g, 82.49 mmol) as a starting material, INT-8 (32.40 g, 72.40 mmol, 87.8%) was obtained according to the synthesis method of INT-5. C 24 H 47 BrO2, MS(ES): m / z(M+H + 447.5.

[0230] 1.9 Synthesis of YK-1801

[0231]

[0232] INT-1 (100 mg, 0.36 mmol) was dissolved in dimethyl sulfoxide (2 mL) and ethyl acetate (2 mL), followed by the sequential addition of INT-5 (1.52 g, 3.62 mmol) and potassium carbonate (300 mg, 2.17 mmol). The mixture was heated to 80 °C and reacted for 20 h, monitored by LC-MS until the reactants had completely reacted. The reaction was quenched with water, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-10% methanol / dichloromethane). The product was collected and concentrated to obtain YK-1801 (102 mg, 0.06 mmol, 17.3%). 101 H 200 N4O 10 MS(ES): m / z (M+H) + 1630.8. 1H NMR(400 MHz, CDCl3) δ 3.96 (d, J = 5.8 Hz, 8H), 3.77 - 3.67 (m, 5H), 2.98 - 2.41(m, 21H), 2.35 - 2.26 (m, 8H), 1.65 (dt, J = 23.0, 7.7 Hz, 14H), 1.39 - 1.29 (m, 15H), 1.29 - 1.20 (m, 97H), 1.14 (s, 6H), 0.92 - 0.84 (t, 24H).

[0233] 1.10 Synthesis of YK-1802

[0234]

[0235] Using INT-1 (150 mg, 0.54 mmol) as a starting material, YK-1802 (92 mg, 0.05 mmol, 10.1%) was obtained following the synthesis method of YK-1801. 105 H 208 N4O 10 MS(ES): m / z (M+H) + 1686.9. 1 H NMR (400 MHz, CDCl3)δ 4.86 (dd, J = 12.4, 6.2 Hz, 4H), 3.77 - 3.67 (m, 7H), 3.55 - 3.09 (m, 5H), 3.07 - 2.39 (m, 15H), 2.33 - 2.25 (m, 8H), 1.85 (s, 2H), 1.70-1.60 (m, 11H), 1.50 (dd, J = 12.4, 6.2 Hz, 17H), 1.34 - 1.20 (m, 109H), 1.18 - 0.96 (m, 6H), 0.92 - 0.84 (t, 24H).

[0236] 1.11 Synthesis of YK-1803

[0237]

[0238] Using INT-1 (200 mg, 0.72 mmol) as a starting material, YK-1803 (60 mg, 0.03 mmol, 4.5%) was obtained according to the synthesis method of YK-1801. C 117 H 232 N4O 10MS(ES): m / z(M+H) + 1855.1. 1 H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.8 Hz, 8H), 3.80 - 3.59 (m, 2H), 2.77 - 2.33 (m, 22H), 2.33 -2.25 (m, 10H), 1.62 (ddd, J = 20.2, 12.6, 5.8 Hz, 14H), 1.52 - 1.41 (m, 10H), 1.36 - 1.28 (m, 22H), 1.27 (m, 112H), 1.07 - 1.00 (m, 6H), 0.92 - 0.84 (t, 24H).

[0239] 1.12 Synthesis of YK-1804

[0240]

[0241] Using INT-1 (150 mg, 0.54 mmol) as a starting material, YK-1804 (120 mg, 0.07 mmol, 12.7%) was obtained according to the synthesis method of YK-1801. C 109 H 216 N4O 10 MS(ES): m / z(M+H) + 1743.1. 1 H NMR (400 MHz, CDCl3) δ4.04 (d, J = 28.8 Hz, 3H), 3.99 - 3.93 (m, 9H), 2.94 - 2.41 (m, 17H), 2.34 -2.25 (m, 8H), 1.70 - 1.46 (m, 23H), 1.36 - 1.24 (m, 124H), 1.16 - 1.02 (m,6H), 0.92 - 0.84 (t, 24H).

[0242] 1.13 Synthesis of YK-1805

[0243]

[0244]

[0245] Using INT-1 (100 mg, 0.36 mmol) and INT-9 as starting materials, YK-1805 (33 mg, 0.03 mmol, 7.2%) was obtained according to the synthesis method of YK-1801. 85 H 160 N4O2, MS(ES): m / z(M+H + 1270.3. 1 H NMR (400 MHz, CDCl3) δ 5.47 - 5.31 (m, 16H), 4.16 - 4.04 (m, 5H), 3.42 (dt, J = 14.6, 8.4Hz, 4H), 2.78 (dt, J = 11.5, 6.2 Hz, 10H), 2.65 (s, 2H), 2.13 - 2.00 (m,19H), 1.67 - 1.63 (m, 5H), 1.57 (s, 3H), 1.45 - 1.37 (m, 18H), 1.34 (m, 49H),1.31 - 1.26 (m, 9H), 1.07 (d, J = 6.8 Hz, 6H), 0.97 - 0.88 (t, 12H).

[0246] 1.14 Synthesis of YK-1806

[0247]

[0248] Step 1: Synthesis of YK-1806-PM1

[0249] YK-009 was prepared according to Example 1 of patent CN114044741A.

[0250] YK-009 (3.00 g, 4.59 mmol) was dissolved in dichloromethane (30 mL), cooled in an ice bath, and N,N'-carbonyldiimidazole (1.49 g, 9.17 mmol) was added at 0 °C. After the addition was complete, the mixture was heated to room temperature and reacted for 18 h. The reaction was monitored by TLC until the reactants had completely reacted. The reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (0-20% ethyl acetate / dichloromethane). The product was collected and concentrated to obtain YK-1806-PM1 (2.40 g, 3.21 mmol, 69.9%). 44 H 81 N3O6, MS(ES): m / z(M+H + 748.6.

[0251] Step 2: Synthesis of YK-1806

[0252] YK-1806-PM1 (650 mg, 0.87 mmol) was dissolved in N,N-dimethylformamide (6 mL), followed by the sequential addition of INT-1 (120 mg, 0.43 mmol) and potassium carbonate (240 mg, 1.74 mmol). The mixture was heated to 70 °C and reacted for 12 h, monitored by TLC until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-6% methanol / dichloromethane), and the product was collected and concentrated to obtain YK-1806 (42 mg, 0.03 mmol, 6.0%). 95 H 186 N6O 14 MS(ES): m / z(M+H) + 1636.8. 1 H NMR (400 MHz, CDCl3) δ 5.30 (s, 2H), 4.12 - 4.01 (m, 7H), 3.96 (d, J = 5.7 Hz, 4H), 3.77 (s, 2H), 3.49 - 3.20 (m, 2H), 3.22 - 2.91 (m,2H), 2.70 - 2.62 (m, 5H), 2.52 - 2.46 (m, 6H), 2.42 (dd, J = 17.1, 7.9 Hz,6H), 2.36 - 2.25 (m, 9H), 1.80 - 1.68 (m, 4H), 1.68 - 1.55 (m, 12H), 1.47 -1.39 (m, 5H), 1.36 - 1.23 (m, 94H), 1.07 - 0.99 (m, 6H), 0.92 - 0.84 (t,18H).

[0253] 1.15 Synthesis of YK-1807

[0254]

[0255] Using INT-2 (150 mg, 0.47 mmol) as a starting material, YK-1807 (120 mg, 0.07 mmol, 15.2%) was obtained according to the synthesis method of YK-1801. C 104 H 206 N4O 10 MS(ES): m / z(M+H) + 1672.9. 1H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.8 Hz, 8H), 3.77 - 3.66 (m, 2H), 2.82 - 2.37 (m, 20H), 2.34 -2.26 (m,8H), 1.67 - 1.57 (m, 13H), 1.55 - 1.41 (m, 11H), 1.36 - 1.20 (m, 112H), 1.16 - 0.95 (m, 6H), 0.92 - 0.84 (t, 24H).

[0256] 1.16 Synthesis of YK-1808

[0257]

[0258] Using INT-2 (150 mg, 0.47 mmol) as a starting material, YK-1808 (50 mg, 0.03 mmol, 6.1%) was obtained according to the synthesis method of YK-1801. C 108 H 214 N4O 10 MS(ES): m / z(M+H) + 1728.9. 1 H NMR (400 MHz, CDCl3) δ4.87 (dd, J = 12.4, 6.2 Hz, 4H), 4.22 - 4.01 (m, 3H), 3.92 - 3.66 (m, 2H), 3.61 - 3.17 (m, 4H), 3.07 (s, 1H), 2.64 (dd, J = 13.1, 6.1 Hz, 5H), 2.49 (dd,J = 20.5, 13.0 Hz, 12H), 2.33 - 2.22 (m, 9H), 1.70 - 1.59 (m, 11H), 1.53 -1.37 (m, 26H), 1.34 - 1.28 (m, 22H), 1.27 (m, 83H), 1.04 (dd, J = 16.9, 9.7Hz, 6H), 0.92 - 0.84 (t, 24H).

[0259] 1.17 Synthesis of YK-1809

[0260]

[0261] Using INT-2 (150 mg, 0.47 mmol) as a starting material, YK-1809 (100 mg, 0.05 mmol, 11.2%) was obtained according to the synthesis method of YK-1801. C 120 H 238 N4O 10 MS(ES): m / z(M+H) + 1897.2. 1 H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.7 Hz, 8H), 2.64 (s, 3H), 2.59 (s, 14H), 2.45 (d, J = 8.5 Hz,5H), 2.41 (s, 5H), 2.35 - 2.26 (m, 9H), 1.63 (dt, J = 14.8, 7.2 Hz, 14H), 1.41 (dd, J = 26.4, 7.6 Hz, 12H), 1.34 - 1.24 (m, 138H), 1.01 (d, J = 3.1 Hz, 6H), 0.92 - 0.84 (t, 24H).

[0262] 1.18 Synthesis of YK-1810

[0263]

[0264] YK-1810 (105 mg, 0.06 mmol, 13.2%) was obtained from INT-3 (150 mg, 0.48 mmol) using the same synthesis method as YK-1801. 104 H 202 N4O 10 MS(ES): m / z(M+H) + 1668.9. 1 H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.8 Hz, 8H), 3.72 (dd, J = 14.0, 7.0 Hz, 6H), 2.62 - 2.36 (m,10H), 2.30 (ddd, J = 16.2, 9.6, 5.5 Hz, 10H), 1.78 (s, 3H), 1.69 - 1.57 (m, 15H), 1.52 - 1.40 (m, 8H), 1.34 - 1.20 (m, 116H), 0.92 - 0.84 (t, 24H).

[0265] 1.19 Synthesis of YK-1811

[0266]

[0267] Using INT-3 (150 mg, 0.48 mmol) as a starting material, YK-1811 (40 mg, 0.02 mmol, 4.9%) was obtained according to the synthesis method of YK-1801. 108 H 210 N4O 10 MS(ES): m / z(M+H) + 1724.9. 1 H NMR (400 MHz, CDCl3) δ4.91 - 4.80 (m, 4H), 4.16 - 3.99 (m, 2H), 3.82 (s, 2H), 3.59 - 3.20 (m, 4H), 3.09 (dd, J = 27.7, 21.4 Hz, 4H), 2.85 (s, 2H), 2.52 (dd, J = 35.9, 26.8 Hz,6H), 2.35 - 2.22 (m, 11H), 2.00 (dd, J = 29.4, 16.7 Hz, 2H), 1.67 - 1.57 (m,11H), 1.55 - 1.36 (m, 25H), 1.34 - 1.28 (m, 20H), 1.27 (m, 90H), 0.92 - 0.84(t, 24H).

[0268] Synthesis of YK-1812 1.20

[0269]

[0270] YK-1812 (70 mg, 0.04 mmol, 7.8%) was obtained from INT-3 (150 mg, 0.48 mmol) using the same synthesis method as YK-1801. 120 H 234 N4O 10 MS(ES): m / z(M+H) + 1893.2. 1H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.8 Hz, 8H), 3.91 - 3.58 (m, 3H), 3.58 - 2.71 (m, 8H), 2.71 -2.33 (m, 9H), 2.34 - 2.26 (m, 10H), 2.01 (dd, J = 49.4, 41.5 Hz, 5H), 1.72 -1.54 (m, 20H), 1.47 - 1.36 (m, 8H), 1.34 - 1.20 (m, 139H), 0.92 - 0.84 (t,24H).

[0271] 1.21 Synthesis of YK-1813

[0272]

[0273] Using INT-3 (100 mg, 0.32 mmol) as a starting material, YK-1813 (20 mg, 0.02 mmol, 4.8%) was obtained according to the synthesis method of YK-1801. C 88 H 162 N4O2, MS(ES): m / z(M+H + 1308.3. 1 H NMR (400 MHz, CDCl3) δ5.56 - 5.18 (m, 16H), 4.52 - 4.32 (m, 2H), 4.08 (d, J = 4.7 Hz, 2H), 3.42 (m,6H), 2.92 - 2.66 (m, 8H), 2.17 - 1.92 (m, 18H), 1.81 - 1.63 (m, 12H), 1.56 -1.48 (m, 8H), 1.47 - 1.26 (m, 74H), 1.17 - 1.13 (s, 2H), 0.94 - 0.89 (t,12H).

[0274] 1.22 Synthesis of YK-1814

[0275]

[0276] Using INT-4 (180 mg, 0.77 mmol) as a starting material, YK-1814 (71 mg, 0.04 mmol, 5.4%) was obtained according to the synthesis method of YK-1801. C 106 H 208 N4O 10MS(ES): m / z(M+H) + 1699.1. 1 H NMR (400 MHz, CDCl3) δ4.09 - 4.01 (m, 1H), 3.96 (d, J = 5.7 Hz, 8H), 3.83-3.78 (m, 1H), 3.44 - 3.32(m, 1H), 3.12 - 3.07 (m, 1H), 2.80 - 2.34 (m, 20H), 2.34 - 2.25 (m, 10H), 1.65 - 1.57 (m, 20H), 1.36 - 1.25 (m, 118H), 1.25 - 1.24 (m, 4H), 0.92 - 0.84(t, 24H).

[0277] 1.23 Synthesis of YK-1815

[0278]

[0279] Using INT-4 (180 mg, 0.77 mmol) as a starting material, YK-1815 (120 mg, 0.07 mmol, 9.4%) was obtained following the synthesis method of YK-1801. 102 H 200 N4O 10 MS(ES): m / z(M+H) + 1642.9. 1 H NMR (400 MHz, CDCl3) δ4.94 - 4.84 (m, 4H), 4.29 - 3.29 (m, 8H), 3.29 - 2.43 (m, 14H), 2.39 - 2.28(m, 10H), 1.73 - 1.61 (m, 14H), 1.53 (dd, J = 12.4, 6.2 Hz, 20H), 1.37 - 1.31(m, 20H), 1.30 (d, J = 7.3 Hz, 84H), 0.91 (t, J = 6.7 Hz, 24H).

[0280] 1.24 Synthesis of YK-1816

[0281]

[0282] Using INT-4 (165 mg, 0.71 mmol) as a starting material, YK-1816 (30 mg, 0.02 mmol, 2.3%) was obtained according to the synthesis method of YK-1801.114 H 224 N4O 10 MS(ES): m / z(M+H) + 1811.1. 1 H NMR (400 MHz, CDCl3) δ3.96 (d, J = 5.7 Hz, 8H), 3.66 - 3.26 (m, 5H), 3.10 (s, 3H), 2.55 (d, J =19.5 Hz, 9H), 2.42 - 2.27 (m, 12H), 2.04 - 1.55 (m, 24H), 1.44 (d, J = 8.4Hz, 4H), 1.30 (d, J = 7.5 Hz, 16H), 1.27(s, 116H), 0.92 - 0.84 (t, 24H).

[0283] Synthesis of 1.25 YK-1817

[0284]

[0285] Using INT-4 (180 mg, 0.77 mmol) as a starting material, YK-1817 (57 mg, 0.04 mmol, 4.6%) was obtained according to the synthesis method of YK-1801. C 98 H 192 N4O 10 MS(ES): m / z(M+H) + 1586.9. 1 H NMR (400 MHz, CDCl3) δ4.00 - 3.93 (m, 8H), 2.67 (s, 20H), 2.36 - 2.28 (m, 8H), 1.72 - 1.55 (m,21H), 1.45 - 1.14 (m, 109H), 0.92 - 0.84 (t, 24H).

[0286] 1.26 Synthesis of YK-1818

[0287]

[0288] Using YK-1806-PM1 (600 mg, 0.80 mmol) as a starting material, YK-1818 (58 mg, 0.04 mmol, 4.5%) was obtained according to the synthesis method of YK-1806. C 92 H 178 N6O 14 MS(ES): m / z(M+H)+ 1592.5. 1 H NMR (400 MHz, CDCl3) δ 5.33 (dd, J = 14.1, 9.4 Hz, 2H), 4.07 (dt, J = 13.6, 6.6 Hz, 7H), 3.96 (d, J = 5.7 Hz, 4H), 3.79 (d, J = 5.5 Hz, 2H), 3.39 (dd, J = 14.4, 6.1Hz, 2H), 3.14 - 3.01 (m, 2H), 2.68 (s, 5H), 2.48 (s, 8H), 2.40 - 2.26 (m,12H), 2.02 (s, 2H), 1.79 - 1.69 (m, 4H), 1.69 - 1.53 (m, 10H), 1.47 - 1.38 (m, 4H), 1.34 - 1.20 (m, 90H), 1.11 (s, 2H), 0.92 - 0.84 (t, 18H).

[0289] 1.27 Synthesis of YK-1819

[0290]

[0291] YK-1819 (100 mg, 0.06 mmol, 12.8%) was obtained from INT-2 (150 mg, 0.47 mmol) using the same synthesis method as YK-1806. 98 H 192 N6O 14 MS(ES): m / z(M+H) + 1678.6. 1H NMR (400 MHz, CDCl3) δ 5.28 (s, 2H), 4.10 - 3.98 (m, 7H), 3.94 (d, J = 5.7 Hz, 4H), 3.75 (s, 2H), 3.47 - 3.18 (m, 2H), 3.20 - 2.88 (m, 2H), 2.69 - 2.60 (m, 5H), 2.50- 2.43 (m, 6H), 2.40 (dd, J = 17.1, 7.9 Hz, 6H), 2.33 - 2.23 (m, 9H), 1.77 -1.66 (m, 4H), 1.64 - 1.54 (m, 12H), 1.46 - 1.37 (m, 5H), 1.35 - 1.20 (m,100H), 1.05 - 0.98 (m, 6H), 0.90 - 0.83 (t, 18H).

[0292] Synthesis of 1.28 YK-1820

[0293]

[0294] YK-1820 (50 mg, 0.04 mmol, 7.8%) was obtained from INT-3 (150 mg, 0.48 mmol) using the same synthesis method as YK-1806. 98 H 188 N6O 14 MS(ES): m / z(M+H) + 1674.6. 1 H NMR (400 MHz, CDCl3)δ 5.32 (s, 2H), 4.12 - 4.00 (m, 7H), 3.96 (d, J = 5.7 Hz, 4H), 3.75 (s, 2H), 3.48 - 3.20 (m, 2H), 3.23 - 2.90 (m, 2H), 2.71 - 2.63 (m, 4H), 2.53 - 2.46(m, 7H), 2.43 (dd, J = 17.1, 7.9 Hz, 6H), 2.35 - 2.25 (m, 9H), 1.80 - 1.65(m, 6H), 1.64 - 1.56 (m, 10H), 1.49 - 1.36 (m, 7H), 1.37 - 1.23 (m, 100H), 1.07 - 1.00 (m, 6H), 0.93 - 0.86 (t, 18H).

[0295] 1.29 Synthesis of L0533

[0296]

[0297] The synthesis method of L0533 refers to paragraphs

[00250] and

[00247] of patent WO2024 / 192117. Using 2-hexyldecyl alcohol and acryloyl chloride as raw materials, the intermediate PM1 was obtained according to the synthesis method of O12B in paragraph

[00250] . Then, according to the synthesis method in paragraph

[00247] , PM1 was reacted with N-methyl-2,2-diaminodiethylamine to obtain 32 mg of L0533.

[0298] Synthesis of 1.30 II-8

[0299]

[0300] Following the synthesis method of compound 4 in CN116162071B (pages 23-24, paragraphs

[0168] -

[0176] of the specification), the starting material N,N'-diethyl-1,6-diaminohexane was replaced with N,N'-diethyl-1,3-propanediamine to synthesize 40 mg of II-8.

[0301] Synthesis of 1.31. 2248

[0302]

[0303] Following the synthesis method of 2248 in WO2023 / 091787A1 (pages 120-121 of the instruction manual), 28 mg of 2248 was synthesized.

[0304] 1.32 Synthesis of Compound 1

[0305]

[0306] Following the synthetic method of YK-1814, the starting material (S)-1-amino-3-chloro-2-propanol hydrochloride was replaced with 3-chloroprop-1-amine hydrochloride to synthesize 53 mg of compound 1.

[0307] Example 2: mRNA-LNP formulation

[0308] 2.1 Ratio of carrier (liposome) to mRNA

[0309] Step 1: Following a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5, the cationic lipids YK-1803, YK-1804, YK-1805, and YK-1807 synthesized in Example 1 were dissolved in ethanol with DSPC (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000, respectively, to obtain solution A. Solution A was rapidly added to citrate buffer (pH=4~5) using the ethanol injection method, and vortexed for 30 seconds to obtain an ethanol solution of the lipids.

[0310] Step 2: Dilute eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd., catalog number 78MRNA-1107) in citrate buffer (pH=4~5) to obtain an aqueous solution of eGFP-mRNA.

[0311] Step 3: Using a microfluidic device, the ethanol lipid solution prepared in Step 1 was mixed with the mRNA aqueous solution prepared in Step 2 at a flow rate of 10 mL / min according to the vector:mRNA mass ratios of 10:1, 15:1, 20:1, and 30:1, respectively, to prepare the corresponding liposome solutions. The liposome solutions were diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol. The solution was then brought to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP formulations encapsulated with eGFP-mRNA, with a molar ratio of cationic lipids (YK-1803, YK-1804, YK-1805, or YK-1807) / DSPC / cholesterol / DMG-PEG2000 of 49:10:39.5:1.5.

[0312] Cell transfection experiments showed that all mRNA-LNP compositions had good transfection effects, with the best transfection effect observed at a ratio of 15:1.

[0313] 2.2 Ratio of cationic lipids to neutral lipids

[0314] mRNA-LNP compositions encapsulated with eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the molar ratio of cationic lipids (YK-1803, YK-1804, YK-1805 or YK-1807) to neutral lipids DSPC was adjusted to 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1 and 15:1, respectively.

[0315] Cell transfection experiments showed that all the corresponding mRNA-LNP compositions could transfect cells, with the highest transfection efficiency being 4.9:1.

[0316] 2.3 Proportion of polymer-conjugated lipids in the carrier

[0317] mRNA-LNP compositions encapsulated with eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the cationic lipids were YK-1803, YK-1804, YK-1805 or YK-1807, and the molar percentages of the polymer conjugated lipid DMG-PEG2000 in the carrier were 0.5%, 1.5%, 2.5%, 3.5%, 5% and 10%, respectively.

[0318] Cell transfection experiments showed that the corresponding mRNA-LNP compositions could all transfect cells, with the highest transfection efficiency at 1.5%.

[0319] 2.4 Proportion of each component in the carrier

[0320] The mRNA-LNP formulation encapsulating eGFP-mRNA was prepared according to a method similar to that in 2.1, wherein the molar ratios of cationic lipids (YK-1803, YK-1804, YK-1805 or YK-1807), neutral lipid DSPC, structural lipid cholesterol, and polymer conjugated lipid DMG-PEG2000 in step 1 were 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.

[0321] Cell transfection experiments showed that transfection was achieved with molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5. Good transfection results were observed within the range of (35-49):(7.5-15):(35-55):(1-5), with the best transfection effect observed at a molar ratio of 49:10:39.5:1.5.

[0322] Example 3: Cell transfection with mRNA-LNP formulation encapsulated with eGFP-mRNA

[0323] Step 1: Cell resuscitation and passage: Resuscitate Jurkat cells and passage them in culture dishes to the required number of cells.

[0324] 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.

[0325] Step 3: Cell transfection: 1.5 μg of the mRNA-LNP preparation containing eGFP-mRNA prepared in Example 2 (the cationic lipids are YK-1803, YK-1804, YK-1805 or YK-1807, respectively) was added to the cell culture medium of a 12-well plate. After culturing for 24 hours, the transfection efficiency was assessed by fluorescence microscopy based on the fluorescence intensity.

[0326] Based on the transfection efficiency results, the following mRNA-LNP formulations were selected for the examples described below: the mass ratio of vector to mRNA was 15:1; the molar ratio of cationic lipids to neutral lipids was 4.9:1; the molar ratio of polymer-conjugated lipids to liposomes was 1.5%; and the molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids was 49:10:39.5:1.5.

[0327] Example 4: Preparation of mRNA-LNP formulation

[0328] The structures and preparation methods of YK-1801~YK-1820 are described in Example 1 of this application; the structures and preparation methods of other cationic lipid compounds are described in Table 1 below.

[0329] Table 1 Cationic lipid compounds

[0330]

[0331]

[0332] 4.1 Prepare the corresponding ethanol lipid solutions of YK-1801~YK-1820 and the cationic lipid compounds in Table 1 according to the method in step 1 of Example 2.

[0333] 4.2 Fluc-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH=4~5) to obtain the corresponding mRNA aqueous solution.

[0334] 4.3 Using a microfluidic device, the ethanol lipid solution obtained in 4.1 and the Fluc mRNA aqueous solution obtained in 4.2 were mixed at a volume ratio of 1:3 at a flow rate of 10 mL / min to prepare the corresponding liposome solution with a carrier (liposome) to mRNA mass ratio of approximately 15:1. The liposome solution was diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol. The solution was then brought to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to obtain an mRNA-LNP formulation encapsulated with Fluc-mRNA, with a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5.

[0335] Example 5: Determination of mRNA-LNP particle size, polydispersity index (PDI), and encapsulation efficiency

[0336] Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering.

[0337] 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 repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25°C;

[0338] 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 detection results are shown in Table 2 and... Figure 1 :

[0339] Table 2. Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP

[0340]

[0341] As shown in Table 2, the nanolipid particles prepared in Example 4 have a particle size between 90 and 150 nm, all of which can be used for mRNA delivery. The polydispersity index is less than 0.10, indicating good particle size uniformity. Furthermore, they exhibit high encapsulation efficiency, all exceeding 85%.

[0342] Example 6: In vitro delivery performance and toxicity of LNP

[0343] The methods for cell resuscitation, passage, and plating are the same as those in Step 1 and Step 2 of Example 3.

[0344] In step 2, an appropriate volume of Jurkat cell culture medium was added to the 96-well plate containing Jurkat 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 24-hour cultured plate. After incubating the plate in an incubator for 1 hour, the absorbance at 450 nm was measured using a microplate reader to determine cell viability. The results of relative fluorescence intensity and cell viability are shown in Table 3 and [Table data missing]. Figure 2 .

[0345] Table 3 Fluorescence detection results of Fluc-mRNA

[0346]

[0347] The relative fluorescence intensity (corresponding to mRNA translation efficiency) of the above mRNA-LNP compositions showed significant differences. The mRNA-LNP compositions prepared by YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820 had significantly higher relative fluorescence intensity than the mRNA-LNP compositions prepared by SM-102, MC3, L0533, II-8, compound 2248, and compound 1. Specifically:

[0348] 1. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820 is significantly improved compared to representative cationic lipids in the prior art. For example, the cell transfection efficiency of YK-1801 can reach 6.3 times that of SM-102 and 13.2 times that of MC3.

[0349] 2. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1801, YK-1802, YK-1803, YK-1804, YK-1808, YK-1809, YK-1810, YK-1811, YK-1814, YK-1815, YK-1816, and YK-1817 was significantly improved compared to the L0533 cationic lipid, which also has a polyamine head and a polyester tail chain structure. For example, the cell transfection efficiency of YK-1801 can reach 8.3 times that of L0533.

[0350] 3. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1806, YK-1818, YK-1819, and YK-1820 was significantly improved compared to the II-8 cationic lipid, which also has a carbamate structure. For example, the cell transfection efficiency of YK-1818 can reach 6.0 times that of II-8.

[0351] 4. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1814, YK-1815, YK-1816, YK-1817, and YK-1818 was significantly improved compared to that of compound 2248 and the cationic lipid of compound 1, which also have piperazine head structures. For example, the cell transfection efficiency of YK-1814 was 6.7 times that of compound 2248 and 7.0 times that of compound 1.

[0352] Example 7: In vivo delivery performance of LNP

[0353] 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 intraperitoneally. The mice were allowed free movement for 5 minutes, and then the mean radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the protein 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 separated. The mean radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the protein expressed by Fluc-mRNA in each mouse organ 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 4. Figure 3 , Figure 4 and Figure 5 .

[0354] Table 4. Data from mouse in vivo and organ imaging experiments.

[0355]

[0356] The mRNA-LNP compositions prepared from YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820 can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with SM-102, MC3, L0533, II-8, compound 2248, and compound 1. Specifically, compared with existing ionizable cationic lipids (SM-102, MC3, L0533, II-8, and compound 2248), the mRNA-LNP compositions prepared by YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820 of this disclosure exhibit significantly enhanced spleen mean radiation intensity and in vivo mean radiation intensity. For example, the mRNA-LNP composition prepared by YK-1801 shows a significantly enhanced in vivo mean radiation intensity. The average radiation intensity of the spleen was 3.3 times, 5.0 times, 4.8 times, 3.7 times, 6.2 times, and 6.6 times that of the mRNA-LNP compositions prepared by SM-102, MC3, L0533, II-8, Compound 2248, and Compound 1, respectively. The average radiation intensity of the liver was only 0.3 times, 0.6 times, 0.5 times, 0.4 times, 0.6 times, and 0.7 times that of the mRNA-LNP compositions prepared by SM-102, MC3, L0533, II-8, Compound 2248, and Compound 1, respectively.

[0357] Meanwhile, the mRNA-LNP compositions prepared from YK-1801, YK-1802, YK-1803, YK-1804, YK-1805, YK-1806, YK-1809, YK-1810, YK-1813, YK-1814, YK-1815, YK-1816, YK-1817, YK-1818, YK-1819, and YK-1820 disclosed herein exhibit spleen average radiation intensities that are 7.1 times, 5.7 times, 4.8 times, 4.1 times, 3.5 times, 6.2 times, 2.7 times, 3.1 times, 3.1 times, 7.3 times, 6.4 times, 7.1 times, 5.6 times, 10.1 times, 7.4 times, and 6.7 times that of the liver, respectively, demonstrating a significantly enhanced spleen-targeting effect.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: ， in: R1 is -N(R 1a )-C 1-8 Alkylene-N(R) 1b )-or ; R 1a and R 1b Independently for C 1-6 alkyl; R2 and R3 are independently C 10-20 Alkyl or C 6-16 Alkenyl groups, where R2 and R3 may be the same or different; L1 and L2 are independently C 2-8 Straight-chain alkylene; M1 is either -CH2- or -CH2NHC(O)O(CH2)2- a Terminal a is connected to terminal N; M2 and M3 are independently -CH=CH- or -C(O)O-, and M2 and M3 may be the same or different.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula (I) satisfies any one or a combination of at least two of the following conditions (1) to (3): (1) R1 is -N(R 1a )-C 3-6 Alkylene-N(R) 1b )-;R 1a and R 1b Independently for C 1-3 alkyl; (2) R2 and R3 are independently C 16-20 Branched alkyl, C 10 Straight-chain alkyl or C8 straight-chain alkenyl; (3) L1 and L2 are independently C 3-8 Straight-chain alkylene groups.

3. The compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula (I) satisfies any one or a combination of at least two of the following conditions (1) to (7): (1) R1 is , or ; (2) R2 is , , , or ; (3) R3 is , , , or ; (4) L1 is -(CH2)3-, -(CH2)5-, -(CH2)7- or -(CH2)8-; (5) L2 is -(CH2)3-, -(CH2)5-, -(CH2)7- or -(CH2)8-; (6) M2 is -CH=CH- or -C(O)O- b b end and R 2 connect; (7) M3 is -CH=CH- or -C(O)O- c c end and R 3 connect.

4. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is any one of the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or 。 5. A composition comprising a carrier, wherein the carrier comprises a cationic lipid, said cationic lipid being a compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof.

6. The composition according to claim 5, characterized in that, The carrier also contains at least one of neutral lipids, structural lipids, and polymer-conjugated lipids.

7. The composition according to claim 6, characterized in that, The composition satisfies any one or a combination of at least two of the following conditions (1) to (7): (1) The molar ratio of the cationic lipid to the carrier is 0.25:1 to 0.75:1; (2) The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1; (3) The neutral lipids are selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol; (4) The molar ratio of the structural lipid to the carrier is 0.15:1 to 0.65:1; (5) The structural lipids are selected from any one or at least two of the following groups: cholesterol, nonsterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol and corticosteroids; (6) The molar ratio of the polymer conjugated lipid to the carrier is 0.005:1 to 0.1:1; (7) The polymer conjugated lipid is selected from any one or a combination of at least two of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.

8. The composition according to claim 7, characterized in that, The composition satisfies any one or a combination of at least two of the following conditions (1) to (7): (1) The molar ratio of the cationic lipid to the carrier is 0.35:1, 0.4:1, 0.45:1, 0.49:1 or 0.65:1; (2) The molar ratio of the cationic lipid to the neutral lipid is 3:1, 3.5:1, 4:1, 4.9:1 or 10:1; (3) The neutral lipid is selected from any one 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-distearate-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl -sn-glycerol-3-phosphate choline, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate choline 1,2-Diphylanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate -rac-(1-glycerol) sodium salt, 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; (4) The molar ratio of the structural lipid to the carrier is 0.2:1, 0.25:1, 0.395:1, 0.435:1, 0.485:1 or 0.535:1; (5) The structural lipid is cholesterol; (6) The molar ratio of the polymer conjugated lipid to the carrier is 0.015:1, 0.025:1, 0.035:1 or 0.05:1; (7) The polymer conjugated lipid is selected from any one or a combination of at least two of the following: distearylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristic-sn-glycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol bistetradecylacetamide.

9. The composition according to claim 8, characterized in that, The neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine or 1,2-distearate-sn-glycerol-3-phosphate choline; Alternatively, the conjugated lipid of the polymer is 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000.

10. The composition according to claim 6, characterized in that, In the carrier, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25~75):(5~25):(15~65):(0.5~10).

11. The composition according to claim 6, characterized in that, The composition also includes one or more other ionizable lipid compounds; Alternatively, the composition may further comprise a therapeutic or preventative agent; Alternatively, the composition may also include pharmaceutically available excipients or diluents.

12. The composition according to claim 11, characterized in that, The composition satisfies any one or a combination of at least two of the following conditions (1) to (4): (1) In the carrier, the molar ratio of the cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is 49:10:39.5:1.5; (2) The composition is a nanoparticle formulation, wherein the average particle size of the nanoparticle formulation is 90 nm to 150 nm; and the polydispersity index of the nanoparticle formulation is less than 0.

10. (3) The mass ratio of the carrier to the therapeutic agent or preventive agent is 10:1 to 30:1; (4) The therapeutic agent or the preventive agent is selected from any one of the groups consisting of nucleic acids, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

13. The composition according to claim 12, characterized in that, The nucleic acid is selected from any one or a combination of at least two of the following groups: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA; Alternatively, the mass ratio of the carrier to the therapeutic or preventative agent is 15:1 to 20:1.

Images