Lipid compound and pharmaceutically acceptable salt and preparation method thereof, lipid nanoparticle and nucleic acid delivery system

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to a lipid compound and pharmaceutically acceptable salt and a preparation method thereof, lipid nanoparticles and a nucleic acid delivery system, the structure of the lipid compound is as shown in formula I: R1 and R2 are respectively and independently selected from C6-C18 alkyl; r3 is selected from C1-C6 alkyl substituted by at least two hydroxyl groups; l1 and L2 are respectively and independently selected from sum; x1, X2, X3, X4 and X5 are respectively and independently selected from O, S and NR5; r4 and R5 are respectively and independently selected from H and C1-C6 alkyl; and n1 and n2 are respectively and independently selected from integers of 1-10. The lipid compound, the lipid nanoparticles and the nucleic acid delivery system have the advantages of being simple in preparation method, good in stability, high in delivery efficiency and good in safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, in particular to a lipid compound and a pharmaceutically acceptable salt and a preparation method thereof, a lipid nanoparticle and a nucleic acid delivery system. BACKGROUND

[0002] Nucleic acid drugs refer to drugs developed by using the translation or regulation function of nucleic acid molecules, mainly including mRNA drugs, antisense oligonucleotides (ASO), small interfering RNA (siRNA), nucleic acid aptamers, etc. Nucleic acid vaccines (such as mRNA vaccines and DNA vaccines) are an important branch of nucleic acid drugs, which can express antigen proteins by introducing nucleic acid sequences into the host body, so as to stimulate immune response.

[0003] Nucleic acid is a relatively unstable biological molecule, which is easily degraded by nucleases in the body. Nucleic acid delivery systems can protect nucleic acid molecules from the attack of nucleases. The commonly used nucleic acid delivery system at present includes lipid nanoparticles (Lipid Nanoparticles, LNP). Lipid nanoparticles can include various lipid components, which can combine with nucleic acids through electrostatic interaction in vitro environment, wrap nucleic acids in lipid nanoparticles, and release nucleic acids in the intracellular environment, so that they can exert corresponding biological functions.

[0004] At present, the lipid nanoparticles still have the problems of instability, complex preparation method, low delivery efficiency and many side reactions. How to develop a simple, more stable, safer and more efficient lipid nanoparticle delivery system is an urgent problem to be solved. SUMMARY

[0005] Therefore, one or more embodiments of the present application provide a lipid compound and a pharmaceutically acceptable salt and a preparation method thereof, a lipid nanoparticle and a nucleic acid delivery system. The lipid compound, the lipid nanoparticle and the nucleic acid delivery system of the present application have the advantages of simple preparation, good stability, high delivery efficiency and good safety.

[0006] The technical scheme of the present application includes the following contents:

[0007] A salt of a lipid compound, the structure of the cation in the salt of the lipid compound is shown as formula I:

[0008] I;

[0009] R1 and R2 are each independently selected from C1~C 17 alkyl;

[0010] R3 is selected from C1~C6 alkyl substituted by at least 2 hydroxyl groups;

[0011] L1and L2are each independently selected from or ;

[0012] X1, X2, X3, X4and X5are each independently selected from O, S or NR5;

[0013] R4and R5are each independently selected from H or C1-C6alkyl;

[0014] n1and n2are each independently selected from an integer from 1 to 10.

[0015] In some embodiments, the anion in the salt of the lipid compound comprises at least one of I, CI and Br.

[0016] In some embodiments, R1is selected from CHR 11 R 12 ;

[0017] R2is selected from CHR 21 R 22 ;

[0018] R 11 , R 12 , R 21 and R 22 are each independently selected from H or C1-C8alkyl.

[0019] In some embodiments, R3is selected from CHR 31 R 32 ;

[0020] R 31 is selected from hydroxyl or CHR a1 R a2 ;

[0021] R 32 is selected from hydroxyl or CHR b1 R b2 ;

[0022] R a1 , R a2 , R b1 and R b2 are each independently selected from H, hydroxyl or C1-C3alkyl, and at least one of R a1 and R a2 is hydroxyl, at least one of R b1 and R b2 is hydroxyl.

[0023] Further, R3is selected from ;

[0024] R a2 and Rb2 are each independently selected from H or C1-C3 alkyl.

[0025] Further, R3 is selected from or .

[0026] In some embodiments, R4 is selected from H or methyl.

[0027] In some embodiments, L1 and L2 are each independently selected from , , or .

[0028] A lipid compound, the structure of which is shown in Formula II:

[0029] II;

[0030] R1' and R2' are each independently selected from C1-C 17 alkyl;

[0031] R3' is selected from C1-C6 alkyl substituted with at least 2 hydroxyl groups;

[0032] L1' and L2' are each independently selected from and ;

[0033] X1', X2', X3', X4', and X5' are each independently selected from O, S, and NR5';

[0034] R5' is selected from H and C1-C6 alkyl;

[0035] n3 and n4 are each independently selected from an integer from 1 to 10.

[0036] In some embodiments, the lipid compound described above satisfies at least one of the following conditions:

[0037] (1) the definitions of R1' and R2' are each the same as the definitions of R1 and R2 described above;

[0038] (2) the definition of R3' is the same as the definition of R3 described above;

[0039] (3) the definitions of L1' and L2' are each the same as the definitions of L1 and L2 described above.

[0040] A method for preparing a lipid compound, the structure of which is shown in Formula II described above;

[0041] The method for preparing comprises the following steps:

[0042] III;

[0043] IV;

[0044] a nucleophilic substitution reaction of a compound NH2R3', a compound with a structure as shown in formula III, and a compound with a structure as shown in formula IV, to obtain a lipid compound with a structure as shown in formula II;

[0045] X6 and X7 are leaving groups.

[0046] A method for preparing a salt of a lipid compound, wherein the cation in the salt of the lipid compound has a structure as shown in formula I above;

[0047] The method for preparing comprises the following steps:

[0048] alkylating the lipid compound with a structure as shown in formula II above with a compound R4X8, to obtain a salt of a lipid compound with a cation having a structure as shown in formula I above;

[0049] X8 is a leaving group.

[0050] A lipid nanoparticle, characterized in that it comprises a cation in a salt of a lipid compound with a structure as shown in formula I above.

[0051] A nucleic acid delivery system, characterized in that it comprises a cation in a salt of a lipid compound with a structure as shown in formula I above.

[0052] The structure design of the lipid compound of the present application allows it to exhibit different charge states in different pH environments: it remains neutral at physiological pH (about 7.4), has low toxicity to normal cells, and has strong stability; it is protonated and has a positive charge in the acidic environment of the endosome (pH about 5.0), thereby promoting interaction with the endosome membrane and helping release of encapsulated components such as drugs, nucleic acids, etc.

[0053] The method for preparing the lipid compound of the present application is simple, which helps promote the development of nucleic acid drugs.

[0054] The lipid compound, nucleic acid nanoparticle, and nucleic acid delivery system of the present application, the multi-hydroxyl (at least 2 hydroxyl groups) head structure in the structure design helps improve the hydrophilicity and biocompatibility of the ionizable lipid molecule, reduces the difficulty of the preparation process of the lipid nucleic acid nanoparticle, and improves the safety of the nucleic acid delivery system. BRIEF DESCRIPTION OF DRAWINGS

[0055] In order to more clearly illustrate the technical solutions in the specific embodiments or the prior art of the present application, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0056] Figure 1 Animal fluorescence experiment results of the lipid nanoparticles of Example 1 of the present application. DETAILED DESCRIPTION

[0057] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the protection scope of the claims of the present application.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0059] Terminology

[0060] Unless otherwise defined or contradictory, the terms or phrases used herein have the following meanings:

[0061] The term "and / or" used herein is selected from two or more related listed items, and also includes any and all combinations of related listed items, which includes any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel schemes of A, B and A+B.

[0062] In the present application, "further" is used for the purpose of description, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application.

[0063] In the present application, the terms "first", "second", "third", "fourth" and the like in the description and in the claims - are used for descriptive purposes only and not to connote or imply a relative importance or a quantity. Also, "first", "second", "third", "fourth" and the like are used to merely identify and distinguish different elements in a non-exhaustive manner, and are not meant to be limiting.

[0064] In the present application, the technical features described in an open-ended manner include both the closed technical solution consisting of the listed features, and the open technical solution comprising the listed features.

[0065] In the present application, in relation to a numerical interval (i.e. a numerical range), if not otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum and maximum values) of the numerical range, and every numerical value between the two numerical end points. If not otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes both end point integers of the numerical range, and every integer between the two end points. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0066] In the present application, weight can be μg, mg, g, kg, and other mass units well known in the pharmaceutical field.

[0067] In the present application, unless otherwise specified, in relation to size, particle size, diameter, it generally refers to the average value.

[0068] The compounds provided herein can contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms, which can be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The compounds provided herein are intended to include all such possible isomers, as well as mixtures thereof, in racemic and optically pure forms. Optical active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor, or resolution of a racemic mixture (or a racemic mixture of salts or derivatives) using, for example, chiral high pressure liquid chromatography. Where the compounds provided herein contain olefinic double bonds, other geometric isomers are intended unless otherwise specified. Likewise, all tautomeric forms are also intended.

[0069] “Isomers” refer to different compounds that have the same molecular formula. “Stereoisomers” are isomers with the same molecular formula that differ only in the way the atoms are arranged in space. “Atropisomers” are stereoisomers resulting from hindered rotation about a single bond. “Enantiomers” are a pair of non-superimposable mirror images of stereoisomers. A mixture of enantiomers in any ratio can be referred to as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms that are not mirror images of each other.

[0070] “Stereoisomers” can also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds provided herein are isolated as E or Z isomers. In other embodiments, the compounds provided herein are mixtures of E and Z isomers.

[0071] “Tautomers” refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment in which the compound is found, and can vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0072] The compounds provided herein can contain unnatural proportions of atomic isotopes at one or more atoms. For example, the compounds can be radiolabeled with a radioisotope, such as tritium (3H), iodine-125 (125I), sulfur-35 (35S), or carbon-14 (14C), or can be isotopically enriched, such as deuterium (2H), carbon-13 (13C), or nitrogen-15 (15N). As used herein, an “isotopologue” is an isotopically enriched compound. The term “isotopically enriched” means that the isotopic composition of an atom differs from the natural isotopic composition of that atom. “Isotopically enriched” can also mean that the isotopic composition of at least one atom contained by a compound differs from the natural isotopic composition of that atom. The term “isotopic composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds can be useful as therapeutic agents, such as cancer therapeutic agents; research reagents, such as binding assay reagents; and diagnostic agents, such as in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, e.g., isotopologues that are deuterium, carbon-13, and / or nitrogen-15 enriched. As used herein, “deuterated” means that at least one hydrogen (H) in a compound has been replaced with deuterium (represented as D or 2H), that is, the compound is enriched in deuterium at at least one position.

[0073] In the present application, if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure shall control.

[0074] "Alkyl" refers to a saturated hydrocarbon containing a primary (normal) carbon atom, or a secondary carbon atom, or a tertiary carbon atom, or a quaternary carbon atom, or a combination thereof. Phrases containing this term, such as "C1-C8 alkyl," refer to alkyl groups containing 1 to 8 carbon atoms, and each time it appears, it can independently be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, or C8 alkyl. Suitable examples include, but are not limited to: methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2) CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3) 3-Methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-Methyl-1-butyl (-CH2CH2CH(CH3)2), 2-Methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-Hexyl (-CH2CH2CH2CH2CH2CH3), 2-Hexyl (-CH(CH3)CH2CH2CH2CH3), 3-Hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-Methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-Methyl- 2-Pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-Methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-Methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-Methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-Dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-Dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3); For example, "C6~C 18 "Alkyl" refers to an alkyl group containing 6 to 18 carbon atoms. Each time it appears, it can independently be C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, ... 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C15 alkyl, C 16 alkyl, C 17 alkyl or C 18 alkyl; suitable examples include, but are not limited to: -CH((CH2)9CH3)((CH2)6CH3), -CH((CH2)8CH3)((CH2)6CH3), -CH((CH2)9CH3)((CH2)5CH3), -CH((CH2)8CH3)((CH2)5CH3), -CH((CH2)7CH3)((CH2)5CH3), and -CH((CH2)6CH3)((CH2)6CH3).

[0075] In the present application, the term “ ” refers to the point of attachment of a group.

[0076] In one aspect of the present application, there is provided a salt of a lipid compound, the structure of the cation in the salt of the lipid compound being as shown in Formula I:

[0077] I;

[0078] R1and R2are each independently selected from C1~C 17 alkyl;

[0079] R3is selected from C1~C6alkyl substituted with at least 2 hydroxyl groups;

[0080] L1and L2are each independently selected from or ;

[0081] X1, X2, X3, X4and X5are each independently selected from O, S or NR5;

[0082] R4and R5are each independently selected from H or C1~C6alkyl;

[0083] n1and n2are each independently selected from an integer from 1 to 10. In some embodiments, the salt is a pharmaceutically acceptable salt. In the present context, the term “pharmaceutically acceptable salt” includes both acid addition salts and base addition salts.

[0084] Examples of pharmaceutically acceptable acid addition salts include, without limitation, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; and organic acids, such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like.

[0085] Examples of pharmaceutically acceptable base addition salts include, without limitation, salts prepared by the addition of inorganic or organic bases to the free acid compounds. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. In one embodiment, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines and basic ion-exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0086] In some embodiments, the anion in the salt of the lipid compound comprises at least one of I, CI, and Br.

[0087] In some embodiments, R1is selected from CHR 11 R 12 ;

[0088] R2is selected from CHR 21 R 22 ;

[0089] R 11 , R 12 , R 21 and R 22 are each independently selected from H or C1-C8alkyl.

[0090] In some embodiments, R3is selected from CHR 31 R 32 ;

[0091] R 31 is selected from hydroxyl or CHR a1 R a2 ;

[0092] R 32 is selected from hydroxyl or CHR b1 R b2 ;

[0093] R a1 , R a2 , R b1 and R b2 are each independently selected from H, hydroxyl or C1-C3alkyl, and at least one of R a1 and R a2 is hydroxyl, at least one of R b1 and R b2 is hydroxyl.

[0094] Further, R3is selected from ;

[0095] R a2 and R b2 are each independently selected from H or C1-C3alkyl.

[0096] Further, R3is selected from or .

[0097] Further, R3is selected from or .

[0098] In some embodiments, R4is selected from H or methyl.

[0099] In some embodiments, L1and L2are each independently selected from , , or .

[0100] In some embodiments, the structure of the lipid compound is selected from the following structures:

[0101] 、

[0102] 、

[0103] .

[0104] In some embodiments, the structure of the lipid compound is as follows:

[0105] .

[0106] In yet another aspect of the present application, a lipid compound is provided, the structure of the lipid compound is as shown in Formula II:

[0107] II;

[0108] R1' and R2' are each independently selected from C1-C6 alkyl; 17 alkyl;

[0109] R3' is selected from C1-C6 alkyl substituted with at least 2 hydroxyl groups;

[0110] L1' and L2' are each independently selected from and ;

[0111] X1', X2', X3', X4', and X5' are each independently selected from O, S, and NR5';

[0112] R5' is selected from H and C1-C6 alkyl;

[0113] n3 and n4 are each independently selected from an integer from 1 to 10.

[0114] In some embodiments, the definitions of R1' and R2' are the same as the definitions of R1 and R2, respectively, as described above.

[0115] In some embodiments, the definition of R3' is the same as the definition of R3 as described above.

[0116] In some embodiments, the definitions of L1' and L2' are the same as the definitions of L1 and L2, respectively, as described above.

[0117] In some embodiments, the structure of the lipid compound is selected from the following structures:

[0118] 、

[0119] 、

[0120] ,

[0121] 、

[0122] 、

[0123] 、

[0124] 、

[0125] 、

[0126] 、

[0127] 、

[0128] 、

[0129] .

[0130] In some embodiments, the structure of the lipid compound is selected from the group consisting of:

[0131] 、

[0132] 、

[0133] 、

[0134] .

[0135] In still another aspect of the present application, there is provided a method for preparing a lipid compound, the structure of which is shown in Formula II above;

[0136] The method for preparing the lipid compound comprises the following steps:

[0137] III;

[0138] IV;

[0139] subjecting the compound NH2R3', the compound having the structure shown in Formula III, and the compound having the structure shown in Formula IV to a nucleophilic substitution reaction to obtain the lipid compound having the structure shown in Formula II above;

[0140] X6 and X7 are leaving groups.

[0141] As used herein, a "leaving group" refers to an atom or group of atoms that can carry a pair of electrons away from a parent molecule in a chemical reaction, which can be displaced by a nucleophile, a base, or other reactant under certain reaction conditions, thereby facilitating the breaking of a chemical bond and the formation of a new bond. The selection of such a group is typically based on its ability to leave, and can include, but is not limited to, a halogen (e.g., -Cl, -Br, -I), a sulfonate group (e.g., tosylate (-OTs), mesylate (-OMs), triflate (-OTf), a nitro group (-NO2), a cyano group (-CN), or an activated modified hydroxyl group (e.g., phosphate, sulfate), etc.

[0142] In some embodiments, X6and X7are each independently selected from halogen.

[0143] In yet another aspect of the present application, there is provided a method for preparing a salt of a lipid compound, wherein the structure of the cation in the salt of the lipid compound is as shown in Formula I above.

[0144] The method for preparing the salt of the lipid compound comprises the following steps:

[0145] alkylating a lipid compound having a structure as shown in Formula II above with a compound R4X8to obtain a salt of a lipid compound having a cation with a structure as shown in Formula I above;

[0146] X8is a leaving group.

[0147] In some embodiments, X8is selected from halogen.

[0148] In yet another aspect of the present application, there is provided a lipid nanoparticle, which comprises a cation in a salt of a lipid compound having a structure as shown in Formula I above.

[0149] As used herein, a "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the nanometer (nm) scale (e.g., 1 to 1000 nm) that contains one or more types of lipid molecules. The LNPs provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule that is partially or completely encapsulated within a lipid shell. In particular, in some embodiments, where the payload is a negatively charged molecule (e.g., an mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is contemplated that the cationic lipid can interact with the negatively charged payload molecule and facilitate payload incorporation and / or encapsulation into the LNP during LNP formation. Other lipids that can form part of the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as sterols, polymer-conjugated lipids, and various zwitterionic lipids. In certain embodiments, the LNP according to the present disclosure comprises one or more lipids of Formula (I) (and subformulae thereof) as described herein.

[0150] A "polymer-conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol.

[0151] A "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH value range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment for which the lipid is intended for use, such as a physiological pH value. By way of non-limiting example, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(octyloxy)propyl))dimethylammonio)ethyl hydrogen phosphate (DOCP); sphingomyelin (SM); ceramides; sterols, such as cholesterols and derivatives thereof. The neutral lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0152] A "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form at a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment for which the predetermined lipid is intended to be used, such as physiological pH values. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, a sterol hemisuccinate, a dialkyi trimethylammonium-propane (e.g., DOTAP, DOTMA), a dialkyldimethylammonium propane, an ethylphosphocholine, a dimethylaminoethane carbamoyl sterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycero) sodium salt (DOPG-Na), and 1,2-dioleoyl-sn-glycero-3-phospho sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0153] In some embodiments, the lipid molecules in the composition of the lipid nanoparticle include the cation in the salt of the lipid compound of Formula I, neutral phospholipids, sterol lipids, and polyethylene glycol lipid conjugates. Optionally, the molar ratio of the salt of the lipid compound of Formula I, neutral phospholipids, sterol lipids, and polyethylene glycol lipid conjugates is 40-49: 10.0-11.8: 38.5-47.2: 1.0-2.0.

[0154] In this embodiment, the cation in the salt of the lipid compound of Formula I is used to form a coating layer for non-lipid payload molecules (such as nucleic acids), facilitating cellular uptake; the neutral phospholipid (such as DOPE or DOPC) is used to enhance lipid bilayer stability and fluidity, facilitating membrane fusion; the sterol lipid (such as cholesterol) is used to modulate membrane fluidity, enhance stability, and improve intracellular delivery efficiency; the polyethylene glycol lipid conjugate is used to reduce immunogenicity, prolong blood circulation time, and prevent particle aggregation.

[0155] In yet another aspect of the present application, a nucleic acid delivery system is provided, the composition of which includes the cation in the salt of the lipid compound of Formula I.

[0156] Nucleic acids are relatively unstable biological molecules that are susceptible to degradation by nucleases in the body, and the nucleic acid delivery system can protect the nucleic acid molecules from nucleases. Currently, lipid nanoparticles also have problems of insufficient stability, complex preparation methods, low delivery efficiency, and many side reactions. The nucleic acid delivery system of the present application has the advantages of greater stability, greater safety, simpler preparation, and higher delivery efficiency.

[0157] The nucleic acid delivery system of the present application can be used to prepare a nucleic acid drug. A nucleic acid drug is a drug developed using the translation or regulatory function of a nucleic acid molecule, which exerts a pharmacological effect through various cleavage modifications and carriers of nucleotide chains. Nucleic acid drugs can be divided into DNA-based drugs and RNA-based drugs. RNA-based drugs are further divided into mRNA drugs (used to encode proteins, such as mRNA vaccines), antisense oligonucleotides (regulate gene expression by binding to target mRNA), small interfering RNAs (silence specific genes through RNA interference mechanisms), microRNAs (regulate gene expression), and aptamers (exert function by specifically binding to target molecules). Nucleic acid drugs directly act on the gene level to regulate the transcription and expression of genes, thereby treating diseases at the root. Nucleic acid vaccines (such as mRNA vaccines and DNA vaccines) are an important branch of nucleic acid drugs, which introduce nucleic acid sequences into the host body to express antigen proteins using the host cell mechanism, thereby stimulating an immune response.

[0158] In some embodiments, the raw materials of the nucleic acid delivery system include nucleic acid, a salt of the lipid compound of Formula I, neutral phospholipid, steroidal lipid, and polyethylene glycol lipid conjugate. Alternatively, the molar ratio of the salt of the lipid compound of Formula I, neutral phospholipid, steroidal lipid, and polyethylene glycol lipid conjugate is 40-49: 10.0-11.8: 38.5-47.2: 1.0-2.0.

[0159] The following are some specific embodiments.

[0160] For experimental parameters not specified in the following specific embodiments, priority is given to the guidance provided in this application document. Reference can also be made to experimental manuals in the art or other experimental methods known in the art, or to the recommended experimental conditions of the manufacturer.

[0161] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art according to known means.

[0162] Raw materials:

[0163] 2-Aminopropane-1,3-diol was obtained from Aldrich Chemical Reagent Co., Ltd.

[0164] 6-Bromohexyl-2-hexyl decanoate was obtained from Tianjin Joinnoble Biotechnology Co., Ltd.

[0165] Ethyl diisopropylamine was obtained from China National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0166] The nucleic acid was Luciferase mRNA, which was synthesized in the laboratory. The synthesis method referred to the related methods described in CN116768987A or CN116768988A, or other commonly used mRNA preparation methods in the art.

[0167] Animals were Balb / c mice, obtained from Vantian.

[0168] Example 1

[0169] 1. Preparation of 18-(1,3-dihydroxypropan-2-yl)-9-hexyl-10-oxo-18-aza-11- oxotetracosan-24-yl-2-hexyldecanoate

[0170] To 2-aminopropane-1,3-diol (500 mg, 5.38 mmol) was added ethanol (20 mL), 6-bromohexyl-2-hexyldecanoate (4.74 g, 11.29 mmol) and ethyldiisopropylamine (1.49 g, 11.29 mmol) respectively. The reaction was stirred at 65 °C in an oil bath for 48 h. The reaction was monitored by TLC and concentrated. Silica gel was added and the product was isolated by column chromatography (methanol / dichloromethane = 0-5%). 18-(1,3-dihydroxypropan-2-yl)-9-hexyl-10-oxo-18-aza-11-oxotetracosan-24-yl-2- hexyldecanoate (670 mg, 0.872 mmol, 16.2%) was obtained as a light yellow oily liquid.

[0171] 2. Preparation of (1,3-dihydroxypropan-2-yl)(bis{6-[ (2-hexyl-1-oxodecyl)oxy]hexyl})(methyl)ammonium iodide (hereinafter also referred to as ionizable lipid molecule)

[0172] To 18-(1,3-dihydroxypropan-2-yl)-9-hexyl-10-oxo-18-aza-11-oxotetracosan-24-yl-2- hexyldecanoate (100 mg, 0.130 mmol) was added acetonitrile (2 mL) and iodomethane (142 mg, 0.521 mmol) respectively. The reaction was stirred at 70 °C in an oil bath overnight. The reaction was monitored by TLC and there was a spot of the target product but a small amount of starting material remained. After additional iodomethane (142 mg, 0.521 mmol) was added, the reaction was continued at 70 °C in an oil bath overnight. The reaction was monitored by TLC and concentrated. Silica gel was added and the product was isolated by column chromatography (methanol / dichloromethane = 0-5%). The target product (1,3-dihydroxypropan-2-yl)(bis{6-[ (2-hexyl-1-oxodecyl)oxy]hexyl})(methyl)ammonium iodide (64.0 mg, 70.0 μmol, 54.0%) was obtained as a light yellow oily liquid.

[0173] 3. Preparation of lipid nanoparticles

[0174] 3.1 Preparation of lipid stock solution

[0175] (1) Preparation of experimental group lipid working solution

[0176] The experimental group lipid raw materials were weighed and dissolved in anhydrous ethanol to prepare the experimental group lipid working solution. The molar percentages of ionizable lipid molecules, DSPC, cholesterol (Chol), and DMG-PEG2000 were 47.5 mol%, 10 mol%, 40.7 mol%, and 1.8 mol%, respectively.

[0177] (2) The control group lipid raw materials were weighed and dissolved in anhydrous ethanol to prepare the control group lipid working solution. The molar percentages of ALC-0315, DSPC, cholesterol, and ALC-0159 were 47.5 mol%, 10 mol%, 40.7 mol%, and 1.8 mol%, respectively.

[0178] 3.2 Preparation of luciferase mRNA solution

[0179] The mRNA solution was diluted to 0.2 mg / mL with 50 mM citrate buffer pH 4.0 to obtain the luciferase mRNA solution.

[0180] 3.3 Preparation of lipid nanoparticles

[0181] (1) The lipid nanoparticles were prepared using the Maianna microfluidic device and the microfluidic chip of Maianna. The steps were as follows: the microfluidic chip channel was cleaned with anhydrous ethanol and enzyme-free water and emptied, 1 mL of the experimental group lipid working solution prepared in the "3.1 Preparation of lipid working solution" subsection or the control group lipid working solution was loaded into the left side of the device using a syringe, and 3 mL of the luciferase mRNA solution prepared in the "3.2 Preparation of luciferase mRNA solution" subsection was loaded into the right side of the device using a syringe. The software parameters of the microfluidic device were set as follows: total volume 4.000 mL, total flow rate 12 mL / min, left-right flow rate ratio 1:3, initial waste volume 0.500 mL, and end waste volume 0.100 mL. The set parameters were repeated several times, and the lipid nanoparticle solutions obtained each time were collected and diluted with DPBS solution to reduce the ethanol proportion in the solution.

[0182] (2) The solution was exchanged using the Reprocell KR2i TFF system and hollow fiber column. The lipid nanoparticle solution was first concentrated to about 0.5 mg / mL, then ultrafiltration was performed using DPBS, the exchange volume was 8-10 DV, the ethanol component in the lipid nanoparticle solution was removed, the lipid nanoparticles were dispersed in DPBS, then concentration treatment was performed, 1.2 M sucrose solution was added, and the final sucrose concentration was 0.3 M to obtain the purified lipid nanoparticle solution.

[0183] The experimental group lipid nanoparticles and the control group lipid nanoparticles were obtained using the above steps, respectively.

[0184] 3.4 Lipid nanoparticle characterization results

[0185] The purified lipid nanoparticle solution was stored at -80°C. The encapsulation efficiency of the product was tested according to the Ribogreen kit instructions; the particle size and polydispersity coefficient (PDI) were tested using standard detection methods on a Zetasizer nano instrument from Malvern, and the Zeta potential was analyzed. The encapsulation efficiency was higher than 96%, and the particle size range was 69-100 nm, and the PDI was between 0.01 and 0.08.

[0186] The characterization results of the lipid nanoparticles in the experimental group and the control group are shown in Table 1.

[0187] Table 1 Particle size, PDI value and encapsulation efficiency of lipid nanoparticles

[0188]

[0189] 4. Biological test results of the compound

[0190] The lipid nanoparticles in the control group and the experimental group were injected and imaged by the following steps.

[0191] 4.1 Mouse muscle injection of LNP

[0192] Female Balb / c mice (6-8 weeks, purchased from Zhejiang Vintoon Lihua Experimental Animal Technology Co., Ltd.) were raised under experimental conditions of 22±2°C and relative humidity of 45%-75%. mRNA encoding luciferase was used as a reporter gene (luciferase mRNA), and in the presence of ATP, magnesium ions, oxygen and substrate (D-Luciferin), the luciferase was oxidized and decarboxylated to emit blue-green bioluminescence at a wavelength of about 560 nm. After 15 minutes of reaction, the bioluminescence signal in the mouse body was detected by a live imaging instrument, and the luciferase fluorescence signal intensity was used to evaluate the in vivo transfection efficiency of the lipid nanoparticle preparation. A dose of 5 μg / mouse mRNA was administered to the left leg muscle of the mouse, and at 4h and 24h after administration, the mouse was injected intraperitoneally with a quantitative luciferin potassium salt (15 mg / mL, 200 μL). After 15 minutes, the luciferase fluorescence signal in the mouse body and muscle was detected by a small animal live imaging instrument.

[0193] (1) The mice were randomly divided into groups (experimental group, control group, blank group), 3 mice per group, and the weight difference of each group of mice was not more than 20%, and the mice were numbered.

[0194] (2) Preparation of test solution: The lipid nanoparticle solution sample stored at -80°C was taken out, thawed at room temperature, and shaken uniformly. After the test solution was completely thawed, the required volume was taken with a pipette, and the sample was diluted to the required concentration with normal saline according to the test solution table. The mixture was mixed uniformly and placed on ice for standby.

[0195] (3) 100 μL of the test solution mixture was taken with a 1.0 mL insulin syringe, and the syringe was lightly tapped to expel the air in the syringe. After the syringe was covered with a protective cover, it was placed on ice for standby.

[0196] (4) The mouse was taken out of the cage, the tail was lifted, and the mouse was placed on the stainless steel mesh cover of the IVC cage or other rough surface. The state of the mouse was observed.

[0197] (5) After the mouse was in a normal state, one person pinched the mouse's back skin and fixed the mouse on the rough surface.

[0198] (6) The other person fixed the mouse's left hind leg with the left hand, wiped the calf muscle with alcohol, found the best injection site, and injected the test solution mixture at an angle with the left hind leg. After no backflow was drawn back, the injection was completed. In the experimental group, the mice were injected with the experimental group lipid nanoparticle solution, the control group mice were injected with the control group lipid nanoparticle solution, and the blank group mice were injected with the same volume of sucrose solution.

[0199] (7) The syringe was pulled out and thrown into the sharpener box. After confirming that the mouse was in a normal state, the mouse was returned to the cage, and the next mouse was operated repeatedly. After all the injections were completed, the cage was placed back on the feeding rack;

[0200] (8) After the injection was completed, the entire operation table was sprayed with 75% alcohol for cleaning and disinfection, and the garbage was thrown into the medical waste garbage bag.

[0201] (9) After 4 hours and 24 hours of administration on the same day, all the mice were taken out and injected with luciferin potassium salt in the abdominal cavity. The mice were imaged and photographed in vivo to observe the bioluminescence signal intensity of the left leg muscle of the mice, and the experimental data was photographed and saved.

[0202] 4.2 In vivo imaging of mice

[0203] D-luciferin luciferin potassium salt preparation: The D-luciferin luciferin potassium salt was taken out of the refrigerator, equilibrated to room temperature, dissolved in PBS, prepared into a D-luciferin luciferin potassium salt solution with a concentration of 15 mg / mL, filtered with a 0.22 μm filter membrane, transferred to a 15 mL centrifuge tube, and stored in a tin paper for light protection standby.

[0204] (1) Take 200 uL of D-luciferin potassium salt solution with a concentration of 15 mg / mL with a 1.0 mL insulin syringe, and gently shake the syringe to remove the air in the syringe. Then, cover the syringe with a protective cover and place it on ice in the dark for standby.

[0205] (2) Take off the cage, open the cage box, and hold the mouse tail to lift it to the stainless steel mesh cover of the IVC cage box or other rough surface.

[0206] (3) Hold the mouse back skin with one hand, fix the mouse, find the abdominal cavity position of the mouse, and keep the mouse head downward and the abdominal cavity upward.

[0207] (4) Wipe the mouse abdominal cavity with alcohol with the other hand, and inject the D-luciferin potassium salt solution into the abdominal cavity at an angle with the syringe.

[0208] (5) After the injection is completed, place the mouse back into the cage box, wait for 10 min, and at the same time, turn on the anesthetizing machine, and place the mouse in the anesthetizing machine for anesthesia. After 3 min of anesthesia, take out the mouse and place it in the dark box of the mouse live imaging instrument. Keep the mouse on its side, and the left hind limb of the mouse upward to facilitate the collection of luciferase fluorescent signals at the injection site.

[0209] (6) Adjust the computer instrument interface, set the parameter group information, and take a photo and save the experimental data immediately after 15 min of D-luciferin potassium salt injection.

[0210] The results of the animal fluorescence imaging are shown in Figure 1 , and the vertical coordinate is the total light intensity (photons / second). According to Figure 1 , it can be found that the in vivo transfection effect of the experimental group is equivalent to that of the delivery system of the control group, and the experimental group and the control group mice are significantly different from the blank group.

[0211] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is incorporated by reference individually. Unless and to the extent that the documents mentioned in the present application conflict with the application purpose and / or technical scheme of the present application, the documents mentioned in the present application are incorporated by reference in the present application in the whole content and purpose. When the present application refers to the documents mentioned in the present application, the definition of the related technical features, terms, names, phrases, etc. in the documents mentioned in the present application are also incorporated by reference. When the present application refers to the documents mentioned in the present application, the examples, preferred modes of the related technical features mentioned in the documents mentioned in the present application are also incorporated by reference in the present application, but are limited to the implementation of the present application. It should be understood that when the content of the documents mentioned in the present application conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application adaptively.

[0212] Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner, and, for the sake of brevity, not all possible combinations are described, but it is to be understood that the scope of the present specification includes all possible combinations.

[0213] The above-described embodiments are merely illustrative of several embodiments of the present application and do not limit the scope of the patent application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these are all within the scope of the present application. Furthermore, it should be understood that, after reading the above teachings of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms are also within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, and these are all within the scope of the claims of the present application. Therefore, the scope of the patent application of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A salt of a lipid compound, the structure of the cation in the salt of the lipid compound being represented by Formula I: ###0001### Formula I wherein: R1 and R2 are independently selected from H, C1-C6 alkyl, or C1-C6 alkyl substituted with at least one hydroxyl group; R3 is selected from C1-C6 alkyl substituted with at least two hydroxyl groups; X1, X2, X3, X4, and X5 are independently selected from O, S, or NR5; R4 and R5 are independently selected from H or C1-C6 alkyl; and n1 and n2 are independently selected from an integer from 1 to 10. I； R1 and R2 are independently selected from C1 to C2, respectively. 17 alkyl; The anion in the salt of the lipid compound comprises at least one of I", CI", and Br". L1and L2are each independently selected from or ; R4 is selected from H or methyl.

8. A lipid compound, the structure of the lipid compound being represented by Formula II: ###0002### Formula II wherein: R1' and R2' are independently selected from H, C1-C6 alkyl, or C1-C6 alkyl substituted with at least one hydroxyl group; R3' is selected from C1-C6 alkyl substituted with at least two hydroxyl groups; X1', X2', X3', X4', and X5' are independently selected from O, S, and NR5'; R5' is selected from H and C1-C6 alkyl; n3 and n4 are independently selected from an integer from 1 to 10. At least one of the following conditions is met:

2. The salt of a lipid compound according to claim 1, characterized by, (1) R1' and R2' are defined the same as R1 and R2, respectively, as described in claim 3; 3. The salt of a lipid compound according to claim 1, characterized by, R1is selected from CHR 11 R 12 ; R2is selected from CHR 21 R 22 ; R 11 , R 12 , R 21 and R 22 are each independently selected from H or C1-C8alkyl.

4. The salt of a lipid compound according to claim 1, characterized by, R3is selected from CHR 31 R 32 ; R 31 selected from hydroxyl or CHR a1 R a2 ; R 32 selected from hydroxyl or CHR b1 R b2 ; R a1 , R a2 , R b1 and R b2 are each independently selected from H, hydroxyl or C1-C3alkyl, and at least one of R a1 and R a2 is hydroxyl, at least one of R b1 and R b2 is hydroxyl.

5. The salt of a lipid compound according to claim 4, characterized by, R3is selected from ; R a2 and R b2 are each independently selected from H or C1-C3alkyl.

6. The salt of a lipid compound according to claim 1, characterized by, (2) R3' is defined the same as R3 as described in any one of claims 4 to 6; 7. The salt of the lipid compound according to claim 1, characterized by, L1and L2are each independently selected from , , or . (3) L1' and L2' are defined the same as L1 and L2, respectively, as described in claim 7. Ⅱ； R1' and R2' are independently selected from C1 to C2, respectively. 17 alkyl; The structure of the lipid compound is represented by Formula II as described in claim 8 or 9; L1and L2are each independently selected from and ; The method of preparation comprises the following steps: performing a nucleophilic substitution reaction on a compound NH2R3', a compound having a structure represented by Formula III, and a compound having a structure represented by Formula IV to obtain the lipid compound having a structure represented by Formula II as described; X6 and X7 are leaving groups.

9. The lipid compound according to claim 8, characterized by The structure of the cation in the salt of the lipid compound is represented by Formula I as described in any one of claims 1 to 7; The method of preparation comprises the following steps: performing an alkylation reaction on the lipid compound having a structure represented by Formula II as described in claim 8 or 9 and a compound R4X8 to obtain the salt of the lipid compound having a structure represented by Formula I as described, wherein the structure of the cation in the salt of the lipid compound is represented by Formula I as described in any one of claims 1 to 7; X8 is a leaving group.

10. A method for producing a lipid compound, characterized by, The composition comprises the cation in the salt of the lipid compound having a structure represented by Formula I as described in any one of claims 1 to 7. The composition comprises the cation in the salt of the lipid compound having a structure represented by Formula I as described in any one of claims 1 to 7. Ⅲ； Ⅳ； ​ ​ 11. A method for producing a salt of a lipid compound, characterized by, ​ ​ ​ ​ 12. A lipid nanoparticle characterized in that, ​ 13. A nucleic acid delivery system, comprising: ​

Images