Ionizable lipid compound and preparation method and application thereof

Abstract

The present specification provides a compound represented by formula (I), or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof, wherein the definitions of the groups in the formula are detailed in the specification. Also provided are lipid nanoparticles and pharmaceutical compositions comprising the compound, and the use of the compound, lipid nanoparticles and compositions in the delivery of nucleic acids. The compound can be used to deliver various biologically active substances, and has the characteristics of high encapsulation rate and high delivery efficiency.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an ionizable lipid compound and a preparation method and application thereof. Background Art

[0002] Nucleic acid vaccines are vaccines that directly introduce exogenous genes (DNA or RNA) encoding specific antigen proteins into host cells, express the antigen proteins in the host cells, and induce the host to produce an immune response to the antigen proteins to achieve the purpose of preventing and treating diseases.

[0003] The nucleic acid vaccine developed by Moderna and BioNTech uses lipid nanoparticles (LNP) as a delivery system. The main components of LNP include cationic lipid molecules (Cationic Lipid), cholesterol, neutral lipids and polyethylene glycol-conjugated lipids. Among them, cationic lipid molecules are the core of the LNP delivery system, and their molecular structure plays a decisive role in the delivery efficiency, targeting, and formulation stability of the entire liposome nanoparticle.

[0004] Since the delivery of different types of nucleic acid substances and the specific delivery of different targets have different requirements for the delivery system, in order to meet the different needs of gene therapy, new lipid molecules need to be further developed. Summary of the invention

[0005] The purpose of the present invention is to provide an ionizable lipid compound with a novel structure and its application, which can be used to deliver various bioactive substances, has the characteristics of high encapsulation rate, high delivery efficiency and good biocompatibility, and provides more options for the delivery of disease treatment or prevention agents.

[0006] In a first aspect of the present invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof:

[0007]

[0008] in,

[0009] R1, R2, R3 are each independently selected from hydrogen or C 1-8 alkyl;

[0010] L1 is selected from C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne;

[0011] G is phenylene;

[0012] R4 and R5 are each independently selected from C 1-30 Alkyl, C 2-30 Alkenyl or C2-30 Alkynyl.

[0013] In one embodiment, G is selected from

[0014] In one embodiment, the compound of formula (I) has the structure shown in formula (II):

[0015]

[0016] Among them, R1, R2, R3, R4, R5, and L1 are defined as before.

[0017] In one embodiment, the total length of R1, the total length of R2, and the total length of R3 are each independently 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0018] In one embodiment, R1, R2, and R3 are each independently selected from hydrogen, methyl, ethyl, or propyl.

[0019] In one embodiment, R1 is hydrogen; in another embodiment, R1 is C1 alkyl; in another embodiment, R1 is C2 alkyl; in another embodiment, R1 is C3 alkyl; in another embodiment, R1 is C4 alkyl; in another embodiment, R1 is C5 alkyl; in another embodiment, R1 is C6 alkyl; in another embodiment, R1 is C7 alkyl; in another embodiment, R1 is C8 alkyl.

[0020] In one embodiment, R2 is hydrogen; in another embodiment, R2 is C1 alkyl; in another embodiment, R2 is C2 alkyl; in another embodiment, R2 is C3 alkyl; in another embodiment, R2 is C4 alkyl; in another embodiment, R2 is C5 alkyl; in another embodiment, R2 is C6 alkyl; in another embodiment, R2 is C7 alkyl; in another embodiment, R2 is C8 alkyl.

[0021] In one embodiment, R3 is hydrogen; in another embodiment, R3 is C1 alkyl; in another embodiment, R3 is C2 alkyl; in another embodiment, R3 is C3 alkyl; in another embodiment, R3 is C4 alkyl; in another embodiment, R3 is C5 alkyl; in another embodiment, R3 is C6 alkyl; in another embodiment, R3 is C7 alkyl; in another embodiment, R3 is C8 alkyl.

[0022] In one embodiment, L1 is selected from C 1-10 In another embodiment, L1 is selected from C 2-10 In another embodiment, L1 is selected from C 2-10 Alkyne group.

[0023] In one embodiment, L1 has a total length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0024] In one embodiment, L1 is selected from C 1-6 In another embodiment, L1 is selected from C 1-4 In another embodiment, L1 is selected from C 1-2 Alkylene.

[0025] In one embodiment, L1 is selected from -CH2-, -(CH2)2-, -(CH2)3- or -(CH2)4-.

[0026] In one embodiment, L1 is -CH2-; in another embodiment, L1 is -(CH2)2-; in another embodiment, L1 is -(CH2)3-; in another embodiment, L1 is -(CH2)4-.

[0027] In one embodiment, G is

[0028] In one embodiment, R4 is selected from C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl.

[0029] In one embodiment, R4 has a total length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

[0030] In one embodiment, R4 is selected from -(CH2) m1 CH=CH(CH2) n1 CH=CH(CH2) m2 CH3;

[0031] m1 and m2 are each independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11;

[0032] n1 is selected from 0, 1, 2 or 3.

[0033] In one embodiment, R4 is selected from -(CH2)7CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)8CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)9CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)7CH=CH(CH2)2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)8CH=CH(CH2)2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)9CH=CH(CH2)2CH=CH(CH2)4CH3.

[0034] In one embodiment, R5 is selected from C 1-30 Alkyl, C 2-30 Alkenyl or C 2-30 Alkynyl.

[0035] In one embodiment, R5 has a total length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

[0036] In one embodiment, R5 is selected from -(CH2) m1 CH=CH(CH2) n1 CH=CH(CH2) m2 CH3;

[0037] m1 and m2 are each independently selected from 6, 7, 8, 9, 10 or 11;

[0038] n1 is selected from 0, 1, 2 or 3.

[0039] In one embodiment, R5 is selected from -(CH2)7CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R4 is selected from -(CH2)8CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R5 is selected from -(CH2)9CH=CHCH2CH=CH(CH2)4CH3; in another embodiment, R5 is selected from -(CH2)7CH=CH(CH2)2CH=CH(CH2)4CH3; in another embodiment, R5 is selected from -(CH2)8CH=CH(CH2)2CH=CH(CH2)4CH3; in another embodiment, R5 is selected from -(CH2)9CH=CH(CH2)2CH=CH(CH2)4CH3.

[0040] The first aspect of the present invention provides the following compounds:

[0041]

[0042] The second aspect of the present invention provides a method for preparing the compound of formula (II) above, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, comprising:

[0043]

[0044] The compound of formula (IIa) reacts with the compound of formula (IIb) to obtain a compound of formula (II);

[0045] Wherein, X1 is selected from halogen; R1, R2, R3, R4, R5, and L1 are each as defined above.

[0046] The third aspect of the present invention provides a method for preparing compound 1, comprising:

[0047] React linolenic acid (compound 1-1) and carbon tetrabromide in the presence of triphenylphosphine to obtain compound 1-2;

[0048]

[0049] The p-toluenesulfonylmethyl isocyanide (TosMIC) is reacted with compound 1-2 to obtain compound 1-3; further, the reaction is carried out under strong alkaline conditions; further, the reaction is carried out in the presence of sodium hydride;

[0050]

[0051] Hydrolyzing compound 1-3 to obtain compound 1-4; further, hydrolyzing under acidic conditions; further, hydrolyzing in the presence of hydrochloric acid;

[0052]

[0053] Compound 1-4 is reduced to obtain compound 1-5; further, compound 1-4 is reduced with sodium borohydride to obtain compound 1-5;

[0054]

[0055] Compound 1-5 is reacted with 4-fluorobenzoic acid to obtain compound 1-6; further, compound 1-5 and 4-fluorobenzoic acid are reacted in the presence of EDCI and DMAP (4-dimethylaminopyridine) to obtain compound 1-6;

[0056]

[0057] Compound 1-6 is reacted with N,N-dimethylethylenediamine to obtain compound 1; further, compound 1-6 is reacted with N,N-dimethylethylenediamine in the presence of DIEA to obtain a compound represented by formula 1;

[0058]

[0059] In one embodiment, the preparation method of compound 1 comprises:

[0060]

[0061] S1: reacting linolenic acid and carbon tetrabromide in the presence of triphenylphosphine to obtain compound 1-2;

[0062] S2: reacting p-toluenesulfonylmethyl isocyanide with compound 1-2 under strong alkaline conditions to obtain compound 1-3;

[0063] S3: hydrolyzing compound 1-3 under acidic conditions to obtain compound 1-4;

[0064] S4: reducing compound 1-4 with NaBH4 to obtain compound 1-5;

[0065] S5: reacting compound 1-5 and 4-fluorobenzoic acid in the presence of EDCI and DMAP to obtain compound 1-6;

[0066] S6: Compound 1-6 is reacted with N,N-dimethylethylenediamine in the presence of DIEA to obtain compound 1.

[0067] The fourth aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0068] A fifth aspect of the present invention provides a lipid nanoparticle comprising a lipid component and optionally an active ingredient;

[0069] The lipid components include: ionizable cationic lipids, structural lipids, neutral lipids and polymer lipids;

[0070] The ionizable cationic lipid is the compound described in the first aspect, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

[0071] In one embodiment, the neutral lipid is selected from one or more of DSPC, DMPC, DOPC, DPPC, POPC, DOPE, DMPE, POPE or DPPE.

[0072] In one embodiment, the structured lipid is selected from one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

[0073] In one embodiment, the polymer lipid is a pegylated lipid.

[0074] In one embodiment, the PEGylated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0075] In one embodiment, the pegylated lipid is selected from one or more of DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, DMG-PEG2000, Ceramide-PEG2000, DMPE-PEG2000, DPPE-PEG2000, DSPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, and DSPE-PEG5000.

[0076] A sixth aspect of the present invention provides a method for preparing lipid nanoparticles, comprising:

[0077] (1) dissolving and mixing an ionizable lipid compound, cholesterol, phospholipid and polyethylene glycol lipid in ethanol in order of (molar percentage) 50%, 38.5%, 10% and 1.5% respectively;

[0078] (2) Dissolve the mRNA active ingredient in 25 mM sodium acetate solution (pH = 4.5);

[0079] (3) using an automated high-throughput microfluidic system to mix the organic phase containing the lipid mixture and the aqueous phase containing the mRNA component at a flow rate ratio ranging from 1:1 to 1:4, with the mixing speed being 10-18 mL / min;

[0080] (4) the prepared lipid nanoparticles (N / P=6) were diluted with phosphate buffered saline, and then the nanoparticle solution was ultrafiltered to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (purchased from Millipore);

[0081] (5) The obtained nanoparticles were sterilized by filtration through a 0.2 μm sterile filter membrane and then stored in a sealed glass bottle at low temperature.

[0082] The seventh aspect of the present invention provides the use of the compound described in the first aspect, or its pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer, or the pharmaceutical composition described in the fourth aspect, or the lipid nanoparticles described in the fifth aspect in the preparation of a drug for treating, diagnosing or preventing a disease.

[0083] In the eighth aspect of the present invention, there is provided use of the compound described in the first aspect, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or the pharmaceutical composition described in the fourth aspect, or the lipid nanoparticles described in the fifth aspect in the preparation of a drug for delivering a load, wherein the load is selected from one or more of a therapeutic agent, a preventive agent or a diagnostic agent.

[0084] The ninth aspect of the present invention provides a method for treating, diagnosing or preventing a disease in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described in the fourth aspect, or the lipid nanoparticles described in the fifth aspect.

[0085] In one embodiment, the therapeutic agent, preventive agent or diagnostic agent is selected from one or more of nucleic acids, small molecule compounds, polypeptides or proteins.

[0086] In one embodiment, the nucleic acid is selected from one or more of RNA or DNA.

[0087] In one embodiment, the RNA is selected from one or more of small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long noncoding RNA (lncRNA), micro RNA (miRNA), small activating RNA (saRNA), polycoding nucleic acid (MCNA), polycoding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA) or ribozyme.

[0088] In one embodiment, the RNA is mRNA.

[0089] In one embodiment, the RNA is a modified mRNA. DETAILED DESCRIPTION

[0090] I. Definitions

[0091] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0092] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0093] Herein, the compounds are named using standard nomenclature. For compounds with asymmetric centers, it should be understood that (unless otherwise specified) all optical isomers and mixtures thereof are included. In addition, unless otherwise specified, all isomeric compounds included in the present invention may appear in the form of Z and E with carbon-carbon double bonds. Compounds that exist in different tautomeric forms, a compound is not limited to any specific tautomer, but is intended to cover all tautomeric forms.

[0094] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0095] The compounds of the present invention may exist in the form of tautomers. Tautomers are functional group isomers resulting from the rapid movement of an atom in a molecule between two positions. Tautomers are a special type of functional group isomers, a pair of tautomers can be converted into each other, but usually a relatively stable isomer is the main form of existence. The most important examples are enol and keto tautomers.

[0096] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described by formula (I), formula (IA) or formula (IB), but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P.35 S. 18 F and 36 Cl. Compounds of the present invention containing the above isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which a radioactive isotope (e.g. 3 H and 14 C) can be used for drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because higher metabolic stability can provide therapeutic benefits, such as prolonged half-life in vivo or reduced dosage requirements, and thus may be preferred in some cases. Isotope-labeled compounds of formula (I), formula (IA) or formula (IB) of the present invention and their prodrugs can generally be obtained by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents during the compound preparation process.

[0097] The term "substituted" or "substitution" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent.

[0098] When a numerical range is listed, every value and sub-range within the stated range is intended to be included.

[0099] The term "alkyl" refers to a straight chain or branched saturated hydrocarbon moiety. In one embodiment, the alkyl group is a straight chain saturated hydrocarbon. 1-30 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 30 carbon atoms. 1-30 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0100] The term "alkenyl" refers to a straight or branched chain hydrocarbon moiety having one or more carbon-carbon double bonds. 2-30 "Alkenyl" refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-20 Alkenyl, C 15-20 Alkenyl, C 17-20 Alkenyl, C 18 Alkenyl, C 19 Alkenyl, C 20 Alkenyl is preferred. The term "C 2-30"Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0101] The term "alkynyl" refers to a straight or branched chain hydrocarbon moiety having one or more carbon-carbon triple bonds. 2-30 "Alkynyl" refers to a straight or branched hydrocarbon group having 2 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-20 Alkenyl, Alkenyl C 15-20 Alkynyl, C 17-20 Alkynyl, C 18 Alkynyl, C 19 Alkynyl, C 20 Alkynyl is preferred. The term "C 2-30 "Alkynyl" also includes heteroalkynyl groups, in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0102] The term "alkylene" refers to a divalent group formed by removing another hydrogen from an alkyl group, which may be substituted or unsubstituted. 1-10 "Alkylene" refers to the removal of C 1-10 The divalent group is formed by the other hydrogen of the alkyl group, and may be substituted or unsubstituted. In some embodiments, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-) are preferred.

[0103] The term "alkenylene" refers to a divalent group formed by removing another hydrogen from an alkenyl group, which may be substituted or unsubstituted. 2-10 "Alkenylene" means removing the C 2-10 The other hydrogen atom of the alkenyl group forms a divalent group, and may be substituted or unsubstituted.

[0104] The term "alkynylene" refers to a divalent radical formed by removing another hydrogen from an alkynyl group, which may be substituted or unsubstituted. 2-10 "Alkynylidene" refers to the removal of C 2-10 The other hydrogen of the alkynyl group forms a divalent group and may be substituted or unsubstituted.

[0105] The term "the total length of variable A and variable B is x carbon atoms" means that the number of carbon atoms in the main chain of the group represented by variable A and the number of carbon atoms in the main chain of the group represented by variable B are x.

[0106] "Optionally substituted..." means that the group may be substituted by a designated substituent or may be unsubstituted.

[0107] "Nucleic acid" refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and their hybrid molecules. Examples of nucleic acid molecules include, but are not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), self-amplifying RNA (saRNA), and antisense oligonucleotides (ASOs). Nucleic acids can be further chemically modified, and the chemical modification is selected from one of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5-methylcytosine, or a combination thereof. The mRNA molecule contains a protein coding region and may further contain an expression regulatory sequence, and typical expression regulatory sequences include, but are not limited to, a 5' cap (5' cap), a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a polyadenylic acid sequence (Poly A), and a miRNA binding site.

[0108] "Cationic lipid" refers to a lipid molecule that is capable of being positively charged under physiological pH conditions.

[0109] "Neutral lipids" refer to lipid molecules that are uncharged under specific pH conditions, such as physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0110] "Structured lipids" refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids, such as steroids. Steroids are compounds with a cyclopentane pyrophenanthrene carbon skeleton. In a preferred embodiment, the steroid is selected from cholesterol, sitosterol, coprostanol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

[0111] "Polymer lipid" refers to a molecule containing a polymer portion and a lipid portion. In some embodiments, the polymer lipid is a polyethylene glycol (PEG) lipid. Other lipids that can reduce aggregation, such as compounds with uncharged, hydrophilic, steric barrier moieties coupled to lipids, can also be used.

[0112] "Lipid nanoparticles" refer to particles containing lipid components and having nanometer dimensions.

[0113] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0114] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable acid addition salts or base addition salts, including salts formed between a compound and an inorganic acid or an organic acid, and salts formed between a compound and an inorganic base or an organic base.

[0115] The term "pharmaceutically acceptable excipient" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art. Pharmaceutically acceptable excipients used in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the composition of the present invention include (but are not limited to) ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0116] Typically, physiological saline can be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.9% saline, 0.3% glycine, etc., including glycoproteins for improving stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other salts containing carriers, it is preferred that the carrier is added after the lipid particles are formed. Therefore, after the lipid-nucleic acid composition is formed, the composition can be diluted in a pharmaceutically acceptable carrier such as physiological saline.

[0117] The term "treat" relates to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such a disorder or condition. The noun "treat" as used herein relates to the action of the verb treat, the latter being as just defined.

[0118] The term "subject" includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0119] The term "effective amount" refers to an amount sufficient to cause a target biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of the pharmaceutical composition and lipid nanoparticles of the present invention may vary depending on factors such as biological targets, pharmaceutical compositions, pharmacokinetics of lipid nanoparticles, diseases to be treated, modes of administration, and age, health conditions, and symptoms of the subject. An effective amount includes a therapeutically effective amount and a prophylactically effective amount.

[0120] Unless otherwise specified, a "therapeutically effective amount" as used herein is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a pharmaceutical composition or lipid nanoparticle refers to an amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a disease, disorder or condition when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effect of other therapeutic agents.

[0121] Unless otherwise specified, a "prophylactically effective amount" of a pharmaceutical composition or lipid nanoparticle as used herein is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a pharmaceutical composition or lipid nanoparticle refers to an amount of a therapeutic agent that provides a prophylactic benefit in the process of preventing a disease, disorder, or condition when used alone or in combination with other agents. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

[0122] II. Embodiment

[0123] In order to make the technical solution of the present invention clearer and more specific, the present invention is further described in detail by the following examples. The following examples are only used to illustrate the specific embodiments of the present invention so that those skilled in the art can understand the present invention, but are not used to limit the scope of protection of the present invention. In the specific embodiments of the present invention, the technical means or methods not specifically described are conventional technical means or methods in the art. The materials, reagents, etc. used in the examples, if not specifically stated, can be obtained from commercial sources.

[0124] Table 1

[0125] English or abbreviation Chinese THF Tetrahydrofuran DCM Dichloromethane MeOH Methanol NMP N-Methylpyrrolidone EtOAc Ethyl acetate <![CDATA[CDCl3]]> Deuterated chloroform TosMIC p-Toluenesulfonylmethyl isocyanide DMAP 4-Dimethylaminopyridine EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide <![CDATA[NaBH4]]> Sodium borohydride NaH Sodium hydride <![CDATA[Na2SO4]]> Sodium sulfate

[0126] Example 1: Synthesis of Compound 1

[0127]

[0128] Linolenic alcohol 1-1 (20 g, 75.0 mmol, 1.0 eq.), carbon tetrabromide (37.3 g, 112.5 mmol, 1.5 eq.), and triphenylphosphine (29.5 g, 112.5 mmol, 1.5 eq.) were dissolved in dichloromethane (200 mL) in a 250 mL round-bottom flask at room temperature. The reaction system was stirred overnight at room temperature. The reaction was monitored by LCMS. After the reaction was completed, water (250 mL) was added to the reaction system to quench it, and dichloromethane (100 mL) was used to extract 3 times. The organic phases were combined, washed with brine (200 mL), and the organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain 14 g of oily compound 1-2.

[0129] TosMIC (3.85 g, 19.7 mmol, 0.5 eq.) was dissolved in anhydrous DMF (30 mL) under ice bath conditions, and then sodium hydride (60%, 2.36 g, 59.1 mmol, 1.5 eq.) was added to the reaction system in batches, and the mixture was heated to room temperature and reacted for 0.5 hours, and then compound 1-2 (13.0 g, 39.4 mmol, 1.0 eq.) was added. After the addition was completed, the reaction was continued at room temperature for 12 hours. After the reaction was completed, water (200 mL) was added to the reaction system to quench, and ethyl acetate (70 mL) was used to extract 3 times, the organic phases were combined, and the organic phases were washed 3 times with brine (200 mL). The organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain 6.9 g of oily compound 1-3.

[0130] Compound 1-3 (4.9 g, 7.1 mmol, 1.0 eq.) was dissolved in tetrahydrofuran (30 mL) in a 100 mL round-bottom flask at room temperature, hydrochloric acid (6 M, 5.0 mL) was added to the reaction system, and then the reaction system was stirred at room temperature for 2 hours. The reaction system was adjusted to a neutral pH with a saturated sodium bicarbonate aqueous solution, extracted 3 times with ethyl acetate (30 mL), the organic phases were combined, washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column to obtain 3.0 g of compound 1-4.

[0131] Compound 1-4 (1.5 g, 2.8 mmol, 1.0 eq.) was dissolved in 10 mL methanol in a 25 mL round-bottom flask, and sodium borohydride (158.9 mg, 4.2 mmol, 1.5 eq.) was added and reacted at room temperature for 3 hours. After the reaction was complete, 15 mL of water was added to quench, and the mixture was extracted with ethyl acetate (20 mL) for 3 times. The organic phases were combined, washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to obtain 1.2 g of oily compound 1-5, which was directly used in the next step without purification.

[0132] In a 50 mL round-bottom flask, 4-fluorobenzoic acid (300 mg, 2.1 mmol, 1.0 eq.), compound 1-5 (1.1 g, 2.1 mmol, 1.0 eq.), EDCI (1.2 g, 6.3 mmol, 3.0 eq.), and DMAP (256.6 mg, 2.1 mmol, 1.0 eq.) were dissolved in 20 mL of dichloromethane. The reaction was allowed to react overnight at room temperature. The reaction was monitored by LCMS. After the reaction was completed, 20 mL of water was added to quench the reaction, and the mixture was extracted with dichloromethane (20 mL) three times. The organic phases were combined, washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the crude product was purified by silica gel column to obtain 410 mg of oily compound 1-6.

[0133] Compound 1-6 (300 mg, 0.46 mmol, 1.0 eq.) and N,N-dimethylethylenediamine (405.5 mg, 4.6 mmol, 10.0 eq.) were dissolved in DIEA (5 mL) and NMP (5 mL) in a 20 mL sealed tube, and the reaction system was heated to 150°C and stirred for 6 hours. The reaction system was cooled to room temperature and poured into 30 mL of water for quenching, and extracted with ethyl acetate (30 mL) 3 times, the organic phases were combined, washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, concentrated, and the crude product was purified by prep-TLC to obtain 80.02 mg of oily compound 1.

[0134] 1H NMR (400MHz, CDCl3): δppm 0.89(t,J=6.8Hz,6H),1.19-1.36(m,36H),1.55-1.64(m,4H),2.03-2.08(m,8H),2.74-2.79(m,4H),2.87(s,6H),3.29(t,J=5.2Hz,2H ),3.62(t,J=5.2Hz,2H),5.08(p,J=6.4Hz,1H),5.32-5.42(m,8H),6.60(d,J=8.4Hz,2H),7.90(d,J=8.4Hz,2H),13.20(s,1H); ESI-MS m / z:719.7[M+H] + .

[0135] Pharmacological experiments

[0136] Experimental Example 1: Nanoparticle Preparation

[0137] The materials used for lipid nanoparticle assembly are: (1) ionizable lipid compounds: such as the ionizable lipids designed and synthesized in the present invention or DLin-MC3-DMA (MC3, purchased from AVT) as a control group; (2) structural lipids: such as cholesterol (purchased from Sigma-Aldrich); (3) phospholipids: such as 1,2-distearoyl-SN-glycero-3-phosphocholine (DSPC, purchased from AVT); (4) polyethylene glycol lipid compounds: such as dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000, purchased from AVT); (5) nucleic acid fragment active ingredients: such as luciferase (Luciferase mRNA), siRNA, CRISPRCas 9 mRNA, etc. The names and structural formulas of lipid nanoparticle components are detailed in Table 2.

[0138] Table 2

[0139]

[0140]

[0141] Preparation method of lipid nanoparticles: (1) dissolving and mixing ionizable lipid compounds, cholesterol, phospholipids and polyethylene glycol lipids in ethanol in molar percentages of 50%, 38.5%, 10% and 1.5% respectively; (2) dissolving mRNA effective components in 25mM sodium acetate solution (pH=4.5); (3) using an automated high-throughput microfluidic system to mix an organic phase containing a lipid mixture and an aqueous phase containing mRNA components at a flow rate ratio of 1:1 to 1:4, with a mixing speed of 10-18mL / min; (4) diluting the prepared lipid nanoparticles (N / P of 6) with a phosphate buffered saline solution, and then ultrafiltering the nanoparticle solution to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 30kDa (purchased from Millipore); (5) filtering and sterilizing the obtained nanoparticles through a 0.2μm sterile filter membrane and storing them at low temperature in a sealed glass bottle.

[0142] Methods for preparing lipid nanoparticles include, but are not limited to, microfluidic mixing systems and also include T-type mixers and ethanol injection methods.

[0143] Experimental Example 2: Characterization of Physical Properties of Lipid Nanoparticles

[0144] The particle size and particle size dispersion index (PDI) of the prepared lipid nanoparticles were measured using Zetasizer Pro (purchased from Malvern Instruments Ltd) and DynaPro NanoStar (purchased from Wyatt) dynamic light scattering instruments. The degree of RNA encapsulation of lipid nanoparticles is characterized by the encapsulation efficiency (Encapsulation Efficiency%), which reflects the degree of binding between lipid nanoparticles and RNA fragments. This coefficient is measured by Quant-it TM The lipid nanoparticle sample was diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 7.5), and a portion of the sample solution was added with 0.5% Triton X-100 and then allowed to stand at 37°C for 30 minutes. The reaction solution was immediately washed with a Varioskan LUX multifunctional microplate reader (purchased from Thermofisher) to read the fluorescence value under the conditions of an absorption light band of 485 nm and an emission light band of 528 nm to obtain the encapsulation efficiency value.

[0145] Experimental Example 3: Animal Experiment

[0146] Evaluation of the delivery effect and safety of nanoparticles containing luciferase mRNA (purchased from Trilink, L-7202) in mice. The experimental mice were SPF-grade C57BL / 6 mice, female, 6-8 weeks old, weighing 18-22g, purchased from Beijing Sibeifu Biotechnology Co., Ltd. All animals were adaptively raised for more than 7 days before the experiment, and had free access to food and water during the experiment. The light was alternating between light and dark for 12 / 12h, the indoor temperature was 20-26℃, and the humidity was 40-70%. The mice were randomly divided into groups.

[0147] The lipid nanoparticles loaded with luciferase mRNA prepared above were injected into mice by intravenous administration at a single dose of 0.5 mg / kg mRNA, and the mice were subjected to in vivo bioluminescence detection using a small animal in vivo imaging system (IVIS LUMINAIII, purchased from PerkinElmer) 6 hours after administration. The specific operation steps of the detection are as follows: a 15 mg / mL concentration of D-luciferin solution was prepared with normal saline, and each mouse was given the substrate by intraperitoneal injection. After 10 minutes of the substrate administration, the mice were placed in an anesthesia box and anesthetized with 2.5% isoflurane. The anesthetized mice were placed in IVIS for fluorescence imaging and data collection and data analysis were performed on the parts where the fluorescence was concentrated.

[0148] The in vivo delivery efficiency of the lipid nanoparticle carrier is represented by the average value of the fluorescence intensity and the total photon number of different animals in the same test group, as shown in Table 3. The higher the values ​​of the fluorescence intensity and the total photon number, the higher the in vivo delivery efficiency of the lipid nanoparticle for the mRNA fragment. The lipid nanoparticles containing the ionizable lipids of the present invention have good in vivo delivery efficiency.

[0149] Table 3

[0150]

[0151] Example 4: In vitro delivery efficiency and safety assessment

[0152] The delivery effect and safety of nanoparticles loaded with luciferase mRNA (purchased from Trilink, L-7202) at the in vitro cellular level were evaluated. The cells used in the experiment were human embryonic kidney cells 293 (HEK293T cells), cultured in DMEM Dulbecco's modified Eagle medium (purchased from Thermofisher) containing 10% fetal bovine serum and 5% penicillin-streptomycin double antibody, and the culture environment was 37°C at room temperature and 5% carbon dioxide concentration. The cells were evenly broken up and flattened in a 48-well plate, and the lipid nanoparticle solution loaded with luciferin mRNA was added after 24 hours of static culture in the incubator. After incubation for 24 hours, the cells were broken, and the intensity and relative light unit (RLU) of luciferase mediated by each lipid nanoparticle in the cell were detected with luciferase detection reagent (purchased from Promega). The higher the intensity of expression, the higher the delivery efficiency of the lipid material at the cellular level. At the same time, after 24 hours, the cell group treated with parallel lipid nanoparticles was tested for cytotoxicity using CCK-8 reagent (purchased from DOJINDO). In the experiment, the cell group to which only PBS was added was used as a negative control. The specific steps are as follows: After adding CCK-8 solution to the cells, the cells were placed in a cell culture incubator at 37°C for 4 hours, and the absorbance was read using a multifunctional microplate reader under the condition that the absorption band was 450nm. The ratio of the absorbance value read for cells treated with nanoparticles to the absorbance value of the negative control was used as a parameter characterizing cell survival rate. The delivery effect and toxicity data of nanoparticles at the in vitro cellular level are shown in Table 4. The lipid nanoparticles containing the ionizable lipid compound of the present invention have good in vitro delivery efficiency and low toxicity.

[0153] Table 4

[0154]

[0155] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A compound of formula (II), or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof: (II); in, R1, R2, R3 are each independently selected from hydrogen, methyl, ethyl or propyl; L1 is selected from -CH2- or -(CH2)2-; R4 and R5 are each independently selected from -(CH2) m1 CH=CH(CH2) n1 CH=CH(CH2) m2 CH3; m1 and m2 are each independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; n1 is selected from 0, 1, 2 or 3.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein: The compound has the following structure: 。 3. A method for preparing a compound of formula (II) according to claim 1, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, comprising: ； The compound of formula (IIa) reacts with the compound of formula (IIb) to obtain a compound of formula (II); Wherein, X1 is selected from halogen; R1, R2, R3, R4, R5, and L1 are each defined as in claim 1.

4. A pharmaceutical composition comprising the compound of claim 1 or 2, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, and a pharmaceutically acceptable excipient.

5. Lipid nanoparticles comprising a lipid component and optionally an active ingredient; The lipid components include: ionizable cationic lipids, structural lipids, neutral lipids and polymer lipids; The ionizable cationic lipid is a compound according to claim 1 or 2, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof.

6. Use of a compound according to claim 1 or 2, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, or a pharmaceutical composition according to claim 4, or a lipid nanoparticle according to claim 5 in the preparation of a drug for delivering a load selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent.

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