Lipid compound and lipid nanoparticle for delivery

Abstract

Disclosed in the present application are a compound having a structural formula as represented by formula (I), and a salt and an isomer thereof. Further disclosed in the present application is a nanoparticle composition containing the compound, or the salt thereof or the isomer thereof. The nanoparticle of the present application can efficiently deliver a drug or a vaccine into cells to exert the therapeutic or preventive purpose of the drug or the vaccine.

Description

Lipid compounds and lipid nanoparticles for delivery Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to lipid compounds and lipid nanoparticle compositions for delivering effective ingredients such as drugs and vaccines. Background Art

[0002] Lipid nanoparticles (LNPs) have attracted widespread attention due to their good in vitro stability, in vivo degradation, safety and reliability, and have been widely used in mRNA vaccine research.

[0003] Lipid-containing nanoparticles, or lipid nanoparticles, liposomes, and lipid complexes have been demonstrated to be effective delivery vehicles for bioactive substances such as small molecule drugs, proteins, and nucleic acids into and / or within cells. LNPs, small vesicles formed from one or more lipid components, can effectively encapsulate and deliver a variety of nucleic acid molecules, from DNA and RNA to chromosomes and even cells. Their defined construction scheme and ease of modification with targeting ligands facilitate large-scale production.

[0004] Onpattro TM The LNP formulation consists of four main lipid components: ionizable amino lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-conjugated lipids (PEG-lipids), with molar weights of 50 / 10 / 38.5 / 1.5, respectively. TM It is still considered the gold standard for comparison of LNP-mediated efficacy studies.

[0005] Therefore, there is still a need in the art to improve the in vivo expression of nucleic acids (such as mRNA) from lipid nanoparticles. Summary of the Invention

[0006] Based on this, the present application discloses a lipid compound and a lipid nanoparticle composition comprising the compound, wherein the lipid has advantages such as high encapsulation efficiency and high expression.

[0007] Specifically, this application adopts the following technical solutions

[0008] 1. A compound of formula (I), or a salt thereof or an isomer thereof,

[0009] wherein R1 and R2 are independently C1-C6 alkyl, R3 and R4 are independently C1-C12 alkyl, and R5 and R6 are independently selected from hydrogen or C1-C12 alkyl;

[0010] X and Y are independently selected from (C=O)O or O(C=O);

[0011] m, n, and o are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0012] 2. The compound according to item 1, or a salt thereof, or an isomer thereof, wherein R1 and R2 are each independently a C1-C3 alkyl group, preferably a methyl group.

[0013] 3. The compound according to item 1 or 2, or a salt thereof, or an isomer thereof, wherein R3 and R4 are each independently a C1-C12 linear alkyl group.

[0014] 4. The compound according to any one of items 1 to 3, or a salt thereof, or an isomer thereof, wherein R3 and R4 are each independently a C5-C9 straight-chain alkyl group, preferably a C6-C8 straight-chain alkyl group, and more preferably a C6 or C8 straight-chain alkyl group.

[0015] 5. The compound according to any one of items 1 to 4, or a salt thereof, or an isomer thereof, wherein R5 is hydrogen, and R6 is a C1-C12 straight-chain alkane.

[0016] 6. The compound according to item 5, or a salt thereof, or an isomer thereof, wherein R5 is hydrogen, and R6 is a C8-C11 straight-chain alkane, preferably a C10 straight-chain alkane.

[0017] 7. The compound according to any one of items 1 to 4, or a salt thereof, or an isomer thereof, wherein R5 and R6 are each independently a C1-C12 linear alkyl group.

[0018] 8. The compound according to item 7, or a salt thereof, or an isomer thereof, wherein R5 and R6 are each independently a C5-C9 straight-chain alkyl group, preferably a C6-C8 straight-chain alkyl group.

[0019] 9. The compound according to any one of items 1 to 8, or a salt thereof, or an isomer thereof, wherein X is (C=O)O and Y is O(C=O), or X is (C=O)O and Y is (C=O)O, or X is O(C=O) and Y is O(C=O), or X is O(C=O) and Y is (C=O)O.

[0020] 10. The compound according to any one of items 1 to 9, or a salt thereof, or an isomer thereof, wherein m is selected from 3, 4, 5, 6, and 7, preferably 4, 5, and 6.

[0021] 11. The compound according to any one of items 1 to 10, or a salt thereof, or an isomer thereof, wherein n is selected from 4, 5, 6, 7, 8, and 9, preferably 5, 6, 7, and 8.

[0022] 12. The compound according to any one of items 1 to 11, or a salt thereof, or an isomer thereof, wherein o is selected from 4, 5, 6, 7, 8, and 9, preferably 5, 6, 7, and 8.

[0023] 13. The compound according to item 1, wherein the compound of (I) is selected from

[0024] 14. A lipid nanoparticle composition comprising a lipid component, wherein the lipid component comprises the compound of any one of items 1-13.

[0025] 15. The lipid nanoparticle composition of claim 14, wherein the lipid component further comprises a phospholipid.

[0026] 16. The lipid nanoparticle composition according to claim 15, wherein the phospholipid is selected from one or more of the following compounds:

[0027] Dilauroyl phosphatidylcholine (DLPC),

[0028] Dimyristoylphosphatidylcholine (DMPC),

[0029] Dioleoylphosphatidylcholine (DOPC),

[0030] Dipalmitoylphosphatidylcholine (DPPC),

[0031] Distearoylphosphatidylcholine (DSPC),

[0032] Dioleoylphosphatidylcholine (DUPC),

[0033] Palmitoyloleoylphosphatidylcholine (POPC),

[0034] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0035] 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0036] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0037] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0038] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0039] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0040] 1,2-Dihydroxytin-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),

[0041] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0042] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0043] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0044] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0045] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0046] 1,2-Diol-sn-glycero-3-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0047] 17. The nanoparticle composition of claim 15, wherein the phospholipid is DOPE.

[0048] 18. The nanoparticle composition of claim 15, wherein the phospholipid is DSPC.

[0049] 19. The nanoparticle composition of any one of items 14-18, wherein the lipid component further comprises a structural lipid.

[0050] 20. The nanoparticle composition of claim 19, wherein the structural lipid is selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol.

[0051] 21. The nanoparticle composition of any one of claim 19, wherein the structural lipid is cholesterol.

[0052] 22. The nanoparticle composition of any one of items 14-21, wherein the lipid component further comprises a PEG lipid.

[0053] 23. A nanoparticle composition according to any one of claim 22, wherein the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol.

[0054] 24. The nanoparticle composition of any one of items 14-23, wherein the lipid component further comprises cationic and / or ionizable lipids.

[0055] 25. The nanoparticle composition according to any one of items 14 to 24, further comprising a therapeutic and / or prophylactic agent selected from a vaccine or a compound capable of eliciting an immune response, a nucleic acid,

[0056] Preferably, the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

[0057] 26. The nanoparticle composition of any one of items 14-25, wherein the encapsulation efficiency of the therapeutic and / or prophylactic agent is ≥50%; or ≥80%; or ≥90%.

[0058] 27. The nanoparticle composition of any one of items 14-26, wherein the nanoparticle composition has an average particle size of 60 nm to 130 nm.

[0059] 28. The nanoparticle composition of any one of items 14-27, wherein the nanoparticle composition has a dispersibility index of 0.04-0.20.

[0060] 29. Use of a compound according to any one of items 1 to 13 in the preparation of a lipid nanoparticle composition.

[0061] 30. A pharmaceutical composition comprising the nanoparticle composition of any one of items 14-28 and a pharmaceutically acceptable carrier.

[0062] 31. A method for delivering a therapeutic and / or prophylactic agent to a mammalian cell, the method comprising administering to a subject the nanoparticle composition of any one of items 14-28 or the pharmaceutical composition of item 30, wherein the administration comprises contacting the cell with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic and / or prophylactic agent to the cell.

[0063] 32. The method of claim 31 , wherein the mammalian cell is in a mammal.

[0064] 33. The method of item 31 or 32, wherein the mammal is a human.

[0065] 34. The method of any one of items 31-33, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0066] 35. A method for producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the nanoparticle composition of any one of items 14-28 or the pharmaceutical composition of item 30 to deliver a therapeutic and / or prophylactic agent to the cell, wherein the therapeutic and / or prophylactic agent is mRNA encoding the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

[0067] 36. The method of claim 35, wherein the mammalian cell is in a mammal.

[0068] 37. The method of any one of items 35 or 36, wherein the mammalian cell is human.

[0069] 38. The method of any one of items 35-37, wherein the nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0070] 39. A method of treating a disease or condition in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the nanoparticle composition of any one of items 14-28 or the pharmaceutical composition of item 30.

[0071] 40. The method of claim 39, wherein the disease or disorder is characterized by dysfunctional or aberrant protein or polypeptide activity.

[0072] 41. The method of claim 39 or 40, wherein the disease or condition is selected from an infectious disease, cancer and proliferative disease, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, or a metabolic disease.

[0073] 42. The method of any one of items 39-41, wherein the mammal is a human.

[0074] 43. The method of any one of items 39-42, wherein the nanoparticle composition or pharmaceutical composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0075] 44. A method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising administering to the mammal the nanoparticle composition of any one of items 14-28 or the pharmaceutical composition of item 30, wherein the administration comprises contacting the mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or prophylactic agent to the organ.

[0076] 45. The method of claim 44, wherein the mammal is a human.

[0077] 46. ​​The method of item 44 or 45, wherein the nanoparticle composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0078] 47. The method of any one of items 44-46, wherein the mammal is pretreated 24 hours or less prior to the contacting or administering step.

[0079] 48. The method of any one of items 44-47, wherein the mammal is pretreated about one hour prior to the contacting or administering step.

[0080] Effects of the Invention

[0081] The compounds of the present application can be used to prepare lipid nanoparticles. Nanoparticle compositions containing the compounds provided herein can achieve the encapsulation and delivery of therapeutic / prophylactic agents, safely deliver the therapeutic / prophylactic agents to the targeted location, achieve high expression, and exert the effects of the therapeutic / prophylactic agents.

[0082] The lipid nanoparticles prepared in this application have a small average particle size, high encapsulation efficiency, and high expression, and have broad application prospects in the field of drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0084] Figure 1 shows the luciferase fluorescence intensity of different LNP formulations 24 hours after intramuscular injection. DETAILED DESCRIPTION

[0085] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0086] Terms and Definitions

[0087] As used herein, the term "alkyl" refers to a group comprising one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms), which is optionally substituted. The term "C1-C12 alkyl" refers to an optionally substituted straight or branched saturated hydrocarbon comprising 1 to 12 carbon atoms. Unless otherwise indicated, the alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0088] Unless otherwise indicated, the alkyl group may be optionally substituted. The optional substituents may be selected from, but are not limited to, a halogen atom (e.g., chloro, bromo, fluoro, or iodo), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl group, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g., -C(O)H), a carbonyl (e.g., -C(O)R, or represented by C=O), an acyl halide (e.g., -C(O)X, wherein X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy group (e.g., -OR), an acetal, a phosphate, a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfite (e.g., -S(O)R), a thiophene ... In some embodiments, the substituents include, but are not limited to, alkyl, alkyl, alkylene ... For example, a C1-6 alkyl group can be further substituted with 1, 2, 3, 4, 5, or 6 substituents as described herein.

[0089] As used herein, the term "compound" is intended to include all isomers and isotopes of the described structure. "Isotopes" refer to atoms having the same atomic number but differing in mass due to the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present application can be prepared by conventional methods by combining with solvents or water molecules to form sols and hydrates.

[0090] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in the field of biology. For example, a nanoparticle composition can be contacted with a mammalian cell placed in a mammal by a variety of routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous), and can involve a variety of amounts of the nanoparticle composition. In addition, the nanoparticle composition can contact more than one mammalian cell.

[0091] As used herein, "encapsulation efficiency" refers to the amount of therapeutic and / or prophylactic agent that becomes part of a nanoparticle composition, relative to the total amount of therapeutic and / or prophylactic agent used to prepare the nanoparticle composition. For example, if 97 mg of the therapeutic and / or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency can be 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial encapsulation, enclosure, surrounding, or encapsulation.

[0092] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0093] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (e.g., an animal, plant, or microorganism).

[0094] As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

[0095] As used herein, the term "ex vivo" refers to an event that occurs outside an organism (e.g., an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0096] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may contain one or more chiral centers and / or double bonds and, therefore, may exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis-trans isomers). This application encompasses any and all isomers of the compounds described herein. Enantiomeric and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0097] As used herein, a "lipid component" is a component of a nanoparticle composition that comprises one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, PEGylated lipids, structured lipids, or other lipids, such as phospholipids.

[0098] As used herein, "administration method" may include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering the composition to the subject. Any administration method can be selected to target delivery (e.g., specific delivery) to a specific area or system of the body.

[0099] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, the RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. "Modified" substances may also be referred to herein as "engineered" substances. Such substances can be modified or engineered chemically, structurally, or functionally. For example, a modified nucleobase species can include one or more non-naturally occurring substitutions.

[0100] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. The particle size of the nanoparticle composition is typically on the order of microns or less and may include a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0101] As used herein, "naturally occurring" means occurring in nature without human assistance.

[0102] As used herein, "patient" refers to a subject who may seek or need treatment, is in need of treatment, is currently receiving treatment, is about to receive treatment, or is being cared for by a trained professional for a particular condition.

[0103] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0104] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0105] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) that is substantially non-toxic and non-inflammatory to the patient. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compressive aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (diacid), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0106] In the present application, for convenience, the structural formula of the compound represents certain isomers, but the present application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc. It should be understood that not all isomers may have the same level of activity.

[0107] The nanoparticle compositions of the present application may also include salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glucoheptonate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmitate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, and the like.

[0108] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present application include, for example, conventional non-toxic salts of the parent compound formed by non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an alkaline or acidic part by conventional chemical methods. Typically, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two. Typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0109] As used herein, " phospholipid " is a lipid comprising a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. Phospholipid can comprise one or more multiple (such as double or triple bonds) bonds (such as one or more unsaturated bonds). Specific phospholipid can promote fusion with membrane. For example, cationic phospholipid can interact with one or more negatively charged phospholipids of membrane (such as cell membrane or intracellular membrane). The fusion of phospholipid and membrane can allow one or more elements containing lipid composition to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to the cell.

[0110] As used herein, the "polydispersity index" is a ratio that describes the uniformity of the particle size distribution of a system. A smaller value indicates a narrower particle size distribution.

[0111] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which can be produced naturally (eg, isolated or purified) or synthetically.

[0112] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, the RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. The RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, the RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, in vivo translation of the mRNA inside a mammalian cell, may produce the encoded polypeptide. The RNA may be selected from a non-limiting group including small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0113] As used herein, a "single unit dose" is a dose of any therapeutic agent that is administered in one dose / at one time / through a single route / at a single point of contact, ie, a single time.

[0114] As used herein, a "split dose" is a division of a single unit dose or total daily dose into two or more doses.

[0115] As used herein, the "total daily dose" is the amount given or prescribed over a 24-hour period. It may be administered in a single unit dose.

[0116] As used herein, "particle size" or "average particle size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0117] As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present application can be administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0118] The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as "active agents" or "active ingredients." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0119] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered that is sufficient when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0120] As used herein, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can be performed, for example, in vitro, ex vivo, or in vivo.

[0121] As used herein, the term "treat" refers to partially or completely alleviating, relieving, ameliorating, resolving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment may be performed on subjects who do not exhibit the disease, disorder, and / or condition and / or on subjects who only exhibit early signs of the disease, disorder, and / or condition in order to reduce the risk of developing a pathological condition associated with the disease, disorder, and / or condition.

[0122] The present application discloses a compound of formula (I), or a salt thereof or an isomer thereof,

[0123] wherein R1 and R2 are independently C1-C6 alkyl, R3 and R4 are independently C1-C12 alkyl, and R5 and R6 are independently selected from hydrogen or C1-C12 alkyl;

[0124] X is selected from (C=O)O or O(C=O), Y and Z are independently selected from (C=O)O or O(C=O);

[0125] m, n, and o are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0126] In a preferred embodiment, R1 is selected from C1-C6 alkyl, for example, R1 is selected from C1-C5 alkyl, C1-C4 alkyl or C1-C3 alkyl, for example, R1 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl.

[0127] In a preferred embodiment, R1 is selected from a C1-C6 straight-chain alkyl group, for example, R1 is selected from a C1-C5 straight-chain alkyl group, a C1-C4 straight-chain alkyl group, or a C1-C3 straight-chain alkyl group, for example, R1 is a C1 alkyl group, a C2 alkyl group, a C3 straight-chain alkyl group, a C4 straight-chain alkyl group, a C5 straight-chain alkyl group, or a C6 straight-chain alkyl group. In a further preferred embodiment, R1 is a methyl group or an ethyl group. In an even more preferred embodiment, R1 is a methyl group.

[0128] In a preferred embodiment, R2 is selected from C1-C6 alkyl, for example, R2 is selected from C1-C5 alkyl, C1-C4 alkyl or C1-C3 alkyl, for example, R2 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl.

[0129] In a preferred embodiment, R2 is selected from a C1-C6 straight chain alkyl group, for example, R2 is selected from a C1-C5 straight chain alkyl group, a C1-C4 straight chain alkyl group, or a C1-C3 straight chain alkyl group, for example, R2 is a C1 alkyl group, a C2 alkyl group, a C3 straight chain alkyl group, a C4 straight chain alkyl group, a C5 straight chain alkyl group, or a C6 straight chain alkyl group. In a further preferred embodiment, R2 is a methyl group or an ethyl group. In an even more preferred embodiment, R2 is a methyl group.

[0130] In a preferred embodiment, R3 is selected from C1-C12 alkyl, for example, R3 is selected from C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl, C5-C12 alkyl, C6-C12 alkyl, C6-C8 alkyl, C7-C12 alkyl, C8-C12 alkyl, C9-C12 alkyl, C10-C12 alkyl or C11-C12 alkyl, for example, R3 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl. In a further preferred embodiment, R3 is C8 alkyl or C6 alkyl.

[0131] In a preferred embodiment, R3 is selected from C1-C12 straight chain alkyl, for example, R3 is selected from C2-C12 straight chain alkyl, C3-C12 straight chain alkyl, C4-C12 straight chain alkyl, C5-C12 straight chain alkyl, C6-C12 straight chain alkyl, C6-C8 straight chain alkyl, C7-C12 straight chain alkyl, C8-C12 straight chain alkyl, C9-C12 straight chain alkyl, C10-C12 straight chain alkyl, C11-C12 straight chain alkyl, for example, R3 is C1 straight chain alkyl, C2 straight chain alkyl, C3 straight chain alkyl, C4 straight chain alkyl, C5 straight chain alkyl, C6 straight chain alkyl, C7 straight chain alkyl, C8 straight chain alkyl, C9 straight chain alkyl, C10 straight chain alkyl, C11 straight chain alkyl, C12 straight chain alkyl. In a further preferred embodiment, R3 is C8 straight chain alkyl or C6 straight chain alkyl.

[0132] In a preferred embodiment, R4 is selected from C1-C12 alkyl, for example, R4 is selected from C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl, C5-C12 alkyl, C6-C12 alkyl, C6-C8 alkyl, C7-C12 alkyl, C8-C12 alkyl, C9-C12 alkyl, C10-C12 alkyl or C11-C12 alkyl, for example, R3 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl. In a further preferred embodiment, R4 is C8 alkyl or C6 alkyl.

[0133] In a preferred embodiment, R4 is selected from C1-C12 straight chain alkyl, for example, R4 is selected from C2-C12 straight chain alkyl, C3-C12 straight chain alkyl, C4-C12 straight chain alkyl, C5-C12 straight chain alkyl, C6-C12 straight chain alkyl, C6-C8 straight chain alkyl, C7-C12 straight chain alkyl, C8-C12 straight chain alkyl, C9-C12 straight chain alkyl, C10-C12 straight chain alkyl, C11-C12 straight chain alkyl, for example, R4 is C1 straight chain alkyl, C2 straight chain alkyl, C3 straight chain alkyl, C4 straight chain alkyl, C5 straight chain alkyl, C6 straight chain alkyl, C7 straight chain alkyl, C8 straight chain alkyl, C9 straight chain alkyl, C10 straight chain alkyl, C11 straight chain alkyl, C12 straight chain alkyl. In a further preferred embodiment, R4 is C8 straight chain alkyl or C6 straight chain alkyl.

[0134] In a preferred embodiment, R5 is hydrogen and R6 is selected from C1-C12 alkyl, for example, R6 is selected from C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl, C5-C12 alkyl, C6-C12 alkyl, C6-C8 alkyl, C7-C12 alkyl, C8-C12 alkyl, C9-C12 alkyl, C10-C12 alkyl or C11-C12 alkyl, for example, R6 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl. In a further preferred embodiment, R5 is hydrogen and R6 is C9-C11 alkyl. In an even further preferred embodiment, R5 is hydrogen and R6 is C9 alkyl, C10 alkyl or C11 alkyl.

[0135] In a preferred embodiment, R5 is hydrogen, and R6 is selected from C1-C12 straight chain alkyl, for example, R6 is selected from C2-C12 straight chain alkyl, C3-C12 straight chain alkyl, C4-C12 straight chain alkyl, C5-C12 straight chain alkyl, C6-C12 straight chain alkyl, C6-C8 straight chain alkyl, C7-C12 straight chain alkyl, C8-C12 straight chain alkyl, C9-C12 straight chain alkyl, C10-C12 straight chain alkyl or C11-C12 straight chain alkyl, for example, R6 is C1 straight chain alkyl, C2 straight chain alkyl, C3 straight chain alkyl, C4 straight chain alkyl, C5 straight chain alkyl, C6 straight chain alkyl, C7 straight chain alkyl, C8 straight chain alkyl, C9 straight chain alkyl, C10 straight chain alkyl, C11 straight chain alkyl or C12 straight chain alkyl. In a further preferred embodiment, R5 is hydrogen, and R6 is C9-C11 straight chain alkyl. In a further preferred embodiment, R5 is hydrogen, and R6 is a C9 straight chain alkyl group, a C10 straight chain alkyl group, or a C11 straight chain alkyl group.

[0136] In a preferred embodiment, R5 and R6 are simultaneously independently selected from C1-C12 alkyl, for example, selected from C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl, C5-C12 alkyl, C6-C12 alkyl, C6-C8 alkyl, C7-C12 alkyl, C8-C12 alkyl, C9-C12 alkyl, C10-C12 alkyl or C11-C12 alkyl, for example, R5 and R6 are independently C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl or C12 alkyl. In a further preferred embodiment, R5 is C6 alkyl and R6 is C8 alkyl, or R6 is C6 alkyl and R5 is C8 alkyl.

[0137] In a preferred embodiment, R5 and R6 are simultaneously and independently selected from C1-C12 straight chain alkyl, for example, selected from C2-C12 straight chain alkyl, C3-C12 straight chain alkyl, C4-C12 straight chain alkyl, C5-C12 straight chain alkyl, C6-C12 straight chain alkyl, C6-C8 straight chain alkyl, C7-C12 straight chain alkyl, C8-C12 straight chain alkyl, C9-C12 straight chain alkyl, C10-C12 straight chain alkyl or C11-C12 straight chain alkyl, for example, R5 and R6 are independently C1 straight chain alkyl, C2 straight chain alkyl, C3 straight chain alkyl, C4 straight chain alkyl, C5 straight chain alkyl, C6 straight chain alkyl, C7 straight chain alkyl, C8 straight chain alkyl, C9 straight chain alkyl, C10 straight chain alkyl, C11 straight chain alkyl or C12 straight chain alkyl. In a further preferred embodiment, R5 is a C6 linear alkyl group and R6 is a C8 linear alkyl group, or R6 is a C6 linear alkyl group and R5 is a C8 linear alkyl group.

[0138] In a preferred embodiment, m is 3, 4, 5, 6, or 7. In a further preferred embodiment, m is 4, 5, or 6.

[0139] In a preferred embodiment, n is selected from 4, 5, 6, 7, 8, and 9. In a further preferred embodiment, n is 5, 6, 7, and 8.

[0140] In a preferred embodiment, o is 4, 5, 6, 7, 8, or 9. In a further preferred embodiment, o is 5, 6, 7, or 8.

[0141] In a preferred embodiment, m is 4.

[0142] In a preferred embodiment, n is 6.

[0143] In a preferred embodiment, o is 6 or 8.

[0144] In a preferred embodiment, X is (C=O)O and Y is O(C=O), or X is (C=O)O and Y is (C=O)O, or X is O(C=O) and Y is O(C=O), or X is O(C=O) and Y is (C=O)O. In a further preferred embodiment, X is O(C=O) and Y is O(C=O).

[0145] The present application further provides a nanoparticle composition comprising a lipid component, wherein the lipid component comprises a compound of formula (I) provided herein, or a salt thereof, or an isomer thereof.

[0146] In some embodiments, the nanoparticle composition has an average particle size of 60 nm to 130 nm.

[0147] The nanoparticle compositions can include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes.

[0148] The nanoparticle compositions described herein comprise a lipid component comprising at least one compound according to formula (I) or a salt thereof or an isomer thereof. For example, the lipid component of the nanoparticle composition may include one or more of the compounds of the present invention. The nanoparticle composition may also include a variety of other components. For example, in addition to the compound according to formula (I), the lipid component of the nanoparticle composition may also include one or more other lipids.

[0149] The lipid component of the nanoparticle composition may include one or more PEG or PEG-modified lipids. Such substances may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from the non-limiting group of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids, preferably DMG-PEG2000.

[0150] The lipid component of the nanoparticle composition can include one or more structural lipids.Structural lipids can be selected from, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, stigmasterol and mixtures thereof, but are not limited thereto. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone and hydrocortisone), or a combination thereof.

[0151] The lipid component of the nanoparticle composition may include one or more phospholipids. The phospholipids used in the nanoparticle composition and method may be selected from

[0152] Dilauroyl phosphatidylcholine (DLPC),

[0153] Dimyristoylphosphatidylcholine (DMPC),

[0154] Dioleoylphosphatidylcholine (DOPC),

[0155] Dipalmitoylphosphatidylcholine (DPPC),

[0156] Distearoylphosphatidylcholine (DSPC),

[0157] Dioleoylphosphatidylcholine (DUPC),

[0158] Palmitoyloleoylphosphatidylcholine (POPC),

[0159] 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC),

[0160] 1-oleoyl-2-cholesteryldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC),

[0161] l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

[0162] 1,2-Divinyl-sn-glycero-3-phosphocholine,

[0163] 1,2-Diaryl acyl-sn-glycero-3-phosphocholine,

[0164] 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE),

[0165] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

[0166] 1,2-Divinyl alcohol-sn-glycero-3-phosphoethanolamine,

[0167] 1,2-Divinyl-sn-glycero-3-phosphoethanolamine,

[0168] 1,2-Diaryl-sn-glycero-3-phosphoethanolamine,

[0169] 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine,

[0170] 1,2-Dioleoyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG) or sphingomyelin.

[0171] In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises DSPC and DOPE.

[0172] In some embodiments, the LNP comprises ionizable lipid, phospholipid, cholesterol, and PEG lipid, wherein the content of ionizable lipid is 35 mol%-65 mol%, the content of the sum of phospholipid and cholesterol is 35 mol%-65 mol%, and the content of PEG lipid is 0.5 mol%-5 mol%.

[0173] The nanoparticle composition may contain one or more therapeutic and / or prophylactic agents, wherein the therapeutic and / or prophylactic agents are selected from vaccines or compounds capable of inducing an immune response, nucleic acids, preferably the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

[0174] The present application provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing a polypeptide of interest in mammalian cells, and treating a disease or condition in a mammal in need thereof, the method comprising administering to the mammal and / or contacting the mammalian cells with a composition comprising therapeutic and / or prophylactic nanoparticles.

[0175] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any target polypeptide, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can have any size and can have any secondary structure or activity. In some embodiments, when expressed in a cell, the polypeptide encoded by the mRNA can have a therapeutic effect.

[0176] The lipid component of the nanoparticle composition may include, for example, a compound according to formula (I), a phospholipid (eg, an unsaturated lipid such as DOPE or DSPC), a PEG lipid, and a structured lipid.

[0177] The nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle compositions. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size.

[0178] The average particle size of the nanoparticle composition is 60 nm to 130 nm.

[0179] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small polydispersity index generally indicates a narrow particle size distribution.

[0180] The application further provides a method for therapeutic and / or preventative specific delivery to mammalian organs, the method comprising administering any one of the nanoparticle compositions described above to a mammal, wherein the administration comprises contacting a mammalian organ with the nanoparticle composition, thereby delivering the therapeutic and / or preventative to the organ. Therapeutic and / or preventative, for example, a protein, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, or a nucleic acid (such as RNA, for example mRNA) can be delivered to a cell or organ. In the case where therapeutic and / or preventative is mRNA, when a cell contacts the nanoparticle composition, translatable mRNA can be translated in the cell to produce a target polypeptide. However, substantially non-translatable mRNAs also can be delivered to the cell. Substantially non-translatable mRNA can be used as a vaccine and / or can isolate the translation component of a cell to reduce the expression of other species in the cell.

[0181] In some embodiments, the nanoparticle composition can be targeted to a specific type or class of cells (e.g., cells of a specific organ or system thereof). For example, a nanoparticle composition comprising a target treatment and / or prevention can be specifically delivered to the liver, kidney, spleen, gastrointestinal tract, femur, or lung of a mammal. Specific delivery to a specific class of cells, organs, or systems or groups thereof means that a higher proportion of the nanoparticle composition comprising a therapeutic and / or preventive agent, including treatment and / or prevention, is delivered to the target destination (e.g., tissue) relative to other destinations, for example, when the nanoparticle composition is administered to a mammal. In some embodiments, the target tissue is selected from the group consisting of liver, kidney, lung, spleen, femur, gastrointestinal tract, eye tissue (e.g., by intraocular, subretinal, or intravitreal injection), vascular endothelium in a blood vessel (e.g., intracoronary or intrafemoral) or kidney, and tumor tissue (e.g., by intratumoral injection).

[0182] Example

[0183] Example 1 Synthesis of Compound 1

[0184] The synthetic route is as follows:

[0185] Specifically, to a solution of compound B (15.0 g, 58.5 mmol) in dichloromethane (50 ml) were added compound A (11.2 g, 53.2 mmol), EDCI (12.2 g, 63.84 mmol), and DMAP (2.6 g, 22.0 mmol). The mixture was stirred at room temperature for 7 hours and the reaction was monitored by TLC. After the reaction was complete, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 25:1) to obtain compound C (20.5 g, 86.35%).

[0186] To a solution of compound D (18.5 g, 99.30 mmol) in dichloromethane (100 ml) were added compound A (15.0 g, 82.84 mmol), EDCI (20.0 g, 107.69 mmol), and DMAP (4.0 g, 33.13 mmol). The mixture was stirred at room temperature for 7 hours and monitored by TLC. After completion, an equal volume of saturated sodium bicarbonate solution was added to dilute the reaction solution. The layers were separated, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound E (18.9 g, 65.63%).

[0187] To a solution of compound E (5.0 g, 14.36 mmol) in DMF (30 ml) were added compound F (5.6 g, 28.72 mmol), NBu₄I (5.2 g, 14.36 mmol), and K₂CO₃ (3.9 g, 28.72 mmol). The mixture was stirred at room temperature for 12 hours and monitored by TLC. Upon completion, the reaction mixture was diluted with an equal volume of water, extracted with ethyl acetate (10 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford compound G (3.4 g, 51.12% yield).

[0188] To a solution of compound G (5.5 g, 11.91 mmol) in DMF (20 ml) were added compound C (7.5 g, 17.81 mmol), NBu₄I (4.4 g, 11.91 mmol), and Cs₂CO₃ (7.7 g, 23.70 mmol). The mixture was stirred at room temperature for 15 hours and monitored by TLC. Upon completion, the reaction mixture was diluted with an equal volume of water, extracted with ethyl acetate (20 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford compound H (5.9 g, 62.11% yield).

[0189] To a solution of compound H (5.9 g, 7.36 mmol) in dichloromethane (50 ml) was added concentrated hydrochloric acid (20 ml). The mixture was stirred at room temperature for 3 hours and the reaction was monitored by TLC. After completion of the reaction, the layers were separated and the organic layer was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound I (3.8 g, 81.19%).

[0190] Compound J (0.6 g, 5.2 mmol) and pyridinium 4-methylbenzenesulfonate (0.4 g, 1.72 mmol) were added to a solution of compound I (1.1 g, 1.72 mmol) in toluene (25 ml). A Dean-Stark apparatus was used and the mixture was heated under reflux for 20 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and diluted with an equal volume of water. The mixture was extracted with ethyl acetate (10 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound K (0.7 g, 53.85% yield).

[0191] To a solution of compound K (0.7 g, 0.93 mmol) in ultra-dry dichloromethane (10 ml) at 0°C was added methanesulfonic anhydride (0.32 g, 1.85 mmol) and anhydrous triethylamine (0.28 ml). The mixture was stirred for 12 hours and monitored by TLC. After completion of the reaction, the mixture was allowed to cool to room temperature and diluted with an equal volume of water. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound L (0.84 g, 97% yield).

[0192] To compound L (0.84 g, 1.01 mmol) was added 30 ml of dimethylamine (2 M in THF), sealed, and stirred at room temperature for 6 days. The reaction was monitored by TLC. After completion, the mixture was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to afford compound 1 (249 mg, 31.58% yield) as an oil.

[0193] 1 H NMR(600MHz, CDCl3)δ4.08–3.96(m,6H),3.42(d,J=5.8Hz,1H),2.37(t,2H),2.31(s,6H),2.29–2.24(m,3H), 1.66–1.52(m,15H),1.49–1.35(m,5H),1.34–1.17(m,46H),0.86(t,J=7.0Hz,9H).MS-ESI(m / z):781.0(M+H) + .

[0194] Example 2 Synthesis of Compound 2

[0195] The synthetic route is as follows:

[0196] To a solution of compound C (10.0 g, 21.58 mmol) in DMF (50 ml) were added compound F (1.4 g, 7.19 mmol), NBu₄I (3.0 g, 7.19 mmol), and Cs₂CO₃ (7.1 g, 21.58 mmol). The mixture was stirred at room temperature for 12 hours and monitored by TLC. After completion, the reaction mixture was diluted with an equal volume of water, extracted with ethyl acetate (50 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford compound M (5.0 g, 80.64% yield).

[0197] To a solution of compound M (5.0 g, 5.73 mmol) in dichloromethane (50 ml) was added concentrated hydrochloric acid (20 ml). The mixture was stirred at room temperature for 3 hours and the reaction was monitored by TLC. After completion of the reaction, the layers were separated and the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford compound N (3.75 g, 92.59%).

[0198] Compound J (0.6 g, 4.71 mmol) and pyridinium 4-methylbenzenesulfonate (0.6 g, 2.30 mmol) were added to a solution of compound N (1.1 g, 1.56 mmol) in toluene (25 ml). A Dean-Stark apparatus was used and the mixture was heated under reflux for 20 hours. The reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature and diluted with an equal volume of water. The mixture was extracted with ethyl acetate (10 ml x 3), concentrated, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound O (1.3 g, 91.87% yield).

[0199] To a solution of compound O (0.9 g, 1.09 mmol) in ultra-dry dichloromethane (10 ml) at 0°C, methanesulfonic anhydride (0.38 g, 2.18 mmol) and anhydrous triethylamine (0.28 ml) were added. The mixture was stirred for 12 hours and monitored by TLC. After completion of the reaction, the mixture was allowed to cool to room temperature and diluted with an equal volume of water. The layers were separated, and the organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to afford compound P (0.93 g, 95% yield).

[0200] To compound P (0.93 g, 1.04 mmol) was added 30 ml of dimethylamine (2 M in THF), sealed, and stirred at room temperature for 6 days. The reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to afford compound 2 (300 mg, 34.13% yield) as an oil.

[0201] 1 H NMR(600MHz,Chloroform-d)δ4.08–3.94(m,6H),3.42(t,J=6.2Hz,1H),2.40–2.34(m,2H),2.32–2.24(m,8H),1.63–1 .53(m,14H),1.44–1.38(m,5H),1.36–1.30(m,11H),1.24(s,44H),0.86(t,J=6.3Hz,12H).MS-ESI(m / z):851.0(M+H) + .

[0202] Example 3 Synthesis of Compound 3

[0203] The preparation method is the same as compound 1, except that 8-bromo-1-octanol is used as a raw material instead of compound A to react with compound D to obtain oily compound 3.

[0204] 1 H NMR (600MHz, CDCl3) δ4.09–3.99(m,6H),3.45–3.37(m,1H),2.33(t,J=7.6Hz,2H),2.31–2.24(m,9H),1.6 7–1.47(m,16H),1.44–1.38(m,3H),1.37–1.20(m,51H),0.87(t,J=7.0Hz,9H).MS-ESI(m / z):809.0(M+H) + .

[0205] Example 4 Synthesis of Compound 4

[0206] The preparation method is the same as compound 2, but 1,2,4-butanetriol is used instead of compound J as the raw material to obtain oily compound 4.

[0207] 1 H NMR(600MHz,Chloroform-d)δ4.11–4.02(m,6H),3.47(t,J=7.6Hz,1H),2.47–2.41(m,1H),2.37–2.33(m,1H),2.32–2.24(m,8H),1.84– 1.78(m,1H),1.72–1.65(m,1H),1.65–1.53(m,12H),1.46–1.38(m,4H),1.37–1.31(m,10H),1.30–1.21(m,42H),0.87(t,J=7.6Hz,12H).

[0208] Example 5 Synthesis of Compound 5

[0209] The preparation method is the same as compound 1, but 8-bromo-1-octanol is used instead of compound A as the raw material to obtain oily compound 5.

[0210] 1H NMR (500MHz, CDCl3) δ4.09–3.99(m,6H),3.46–3.37(m,1H),2.41(t,J=7.7Hz,2H),2.34(s,6H),2.28(t,J= 7.6Hz,3H),1.68–1.37(m,19H),1.26(d,J=18.7Hz,55H),0.87(t,J=5.9Hz,9H).MS-ESI(m / z):836.6(M+H) + .

[0211] Example 6 Synthesis of Reference Compound NTX-C16 (WO2023184038A1)

[0212] The preparation method is the same as compound 2, but 5-bromo-1-pentanol is used instead of compound A as the raw material to obtain the oily compound NTX-C16.

[0213] 1 H NMR (400MHz, CDCl3) δ4.18–4.05(m,6H),3.58–3.45(m,1H),2.51–2.32(m,4H),2.30(s,6H),1.85–1. 59(m,14H),1.47–1.39(m,8H),1.37–1.24(m,44H),0.91(t,J=6.8Hz,12H).MS-ESI(m / z):794.8(M+H) + .

[0214] Test example

[0215] Test Example 1 Lipid Nanoparticle (LNP) Encapsulation

[0216] The mRNA stock solution was dispersed in a 20 mM acetic acid solution (pH 5.3) to a final concentration of 200 μg / mL (aqueous phase). A lipid mixture (oil phase) was prepared by mixing the Example compound: cholesterol: DSPC: DMG-PEG2000 at a molar ratio of 50:38.5:10:1.5. The mRNA and lipid mixture were mixed by controlling the flow rates of the aqueous and oil phases using a T-flow mixing method to obtain LNP-encapsulated mRNA. The encapsulated LNPs were diluted with buffer and then concentrated by ultrafiltration. The diluent was then replaced, ultimately concentrating the LNPs to an mRNA concentration of 100 μg / mL. The pH of the LNPs was adjusted to approximately 7.0-8.5. Finally, the total and free mRNA content in the LNPs was determined using a Ribogreen assay kit and 10% OTG as a demulsifier, and the LNP encapsulation efficiency was calculated. The final LNP product was diluted with diluent, added to a 1 ml particle size pool, and placed on a Malvern ZetaSizer instrument to measure the particle size of the LNP. The results are shown in Table 1.

[0217] Particle size, PDI, and encapsulation efficiency are all important quality attributes of lipid nanoparticles. As shown in the table below, all tested compounds exhibited good encapsulation efficiency, a particle size suitable for mRNA delivery, and a narrow PDI.

[0218] Table 1: LNP characterization data of example compounds

[0219] Test Example 2: Test of delivery effect

[0220] 1. Luciferase mRNA was encapsulated into LNP formulations of compounds 1-3, NTX-C16, and MC3. The LNP formulation preparation method and mRNA encapsulation method were as described in Experimental Example 1;

[0221] 2. The loaded LNP formulation was injected intramuscularly into Balb / c mice (injection dose: 0.25 mg / kg intramuscularly), with 5 mice in each group.

[0222] 3. Detect the luciferase fluorescence expression intensity of each mouse at 24 hours.

[0223] 4. Fluorescence expression intensity detection: 10 minutes before the test, inject D-luciferin sodium salt (dose: 150 mg / kg) into the abdominal cavity of each mouse. Then, anesthetize the mouse with isoflurane and place it in the IVIS instrument, and select bioluminescence for detection.

[0224] 5. Use fluorescence intensity results to determine the delivery effect of the compound.

[0225] The luciferase assay is the primary method for testing mRNA expression in vivo through fluorescence intensity. As shown in Figure 1, 24 hours after intramuscular injection, the expression of compounds 1 and 3, whose tail chains have a single-di structure, was significantly better than that of compound 2, whose tail chain has a di-di structure, and the prior art compound NTX-C16.

[0226] The above results show that compounds 1 and 3 have excellent mRNA delivery performance and are superior to existing compounds.

[0227] Although the embodiments of the present application are described above, the present application is not limited to the above-mentioned specific embodiments and application fields. The above-mentioned specific embodiments are merely illustrative and instructive, and not restrictive. A person of ordinary skill in the art, under the guidance of this specification and without departing from the scope of protection of the claims of this application, can also make many forms, all of which fall within the scope of protection of this application.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein R1 and R2 are independently C1-C6 alkyl groups, R3 and R4 are each independently a straight-chain alkyl group of C5, C6, C7, C8 or C9, R5 is hydrogen and R6 is a straight-chain alkyl group of C8, C9, C10 or C11; X and Y are independently selected from (C=O)O or O(C=O); m is 4; o is independently selected from 5, 6, 7 or 8; n is independently selected from 5, 6, 7 or 8.

2. The compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein, R1 and R2 are each independently C1-C3 alkyl groups, preferably methyl groups.

3. The compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein R6 is a straight-chain alkyl group of C9.

4. The compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein R5 and R6 are each independently a straight-chain alkyl group of C6 or C8.

5. A compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein, X is (C=O)O, Y is O(C=O), or X is (C=O)O, Y is (C=O)O, or X is O(C=O), Y is O(C=O), or X is O(C=O), Y is (C=O)O, preferably X is O(C=O) and Y is O(C=O).

6. The compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein X is O(C=O) and Y is O(C=O).

7. A compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein n is selected from 6 or 8.

8. A compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein, o is selected from 6 or 8.

9. A compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of the preceding claims, wherein, n is 6.

10. The compound according to claim 1, or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein, The compound of (I) is selected from Compound 1 Compound 3 Compound 5 11. A lipid nanoparticle composition comprising a lipid component, the lipid component comprising the compound or a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of claims 1-10.

12. The lipid nanoparticle composition according to claim 11, wherein the lipid component further comprises a phospholipid, a structural lipid and / or a PEG lipid; wherein the phospholipid is preferably selected from one or more of the following compounds: dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl dimethyl succinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-divinyl-sn-glycero-3-phosphocholine, 1,2-diaryl acyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-divinyl-sn-glycero-3-phosphoethanolamine, 1,2-diaryl-sn-glycero-3-phosphoethanolamine, ​ 1,2-dithiohexenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate-(1-glycerol) sodium salt (DOPG) or sphingomyelin, For example, the phospholipid is DOPE or DSPC; The structural lipid is preferably selected from one or more of cholesterol, coprostanol, sitosterol, ergosterol, stigmasterol; for example, the structural lipid is cholesterol; and / or The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol, preferably DMG-PEG2000.

13. The nanoparticle composition according to any one of claims 11-12, further comprising a therapeutic agent and / or a prophylactic agent, the therapeutic agent and / or prophylactic agent being selected from vaccines or compounds capable of eliciting an immune response, nucleic acids, Preferably the nucleic acid is RNA, and the RNA is selected from one or more of siRNA, aiRNA, miRNA, dsRNA, shRNA or mRNA.

14. A pharmaceutical composition comprising the nanoparticle composition according to any one of claims 11-13 and a pharmaceutically acceptable carrier.

15. A method for delivering a therapeutic agent and / or a prophylactic agent to mammalian cells, the method comprising administering to a subject the nanoparticle composition according to any one of claims 11-13 or the pharmaceutical composition according to claim 14, the administration comprising contacting the cells with the nanoparticle composition or the pharmaceutical composition to deliver the therapeutic agent and / or prophylactic agent to the cells.

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