Ionizable lipid, lipid carrier prepared from ionizable lipid and application of ionizable lipid

Abstract

The invention discloses an ionizable lipid, a lipid carrier prepared from the ionizable lipid and application of the ionizable lipid. Specifically, the invention discloses an ionizable lipid compound as shown in a formula (I), and a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof. The compound provided by the invention is a novel lipid molecule with both a phosphate group and a cation head group, and has the advantages of simple production process, high transfection efficiency, low cytotoxicity, wide application range and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an ionizable lipid, a lipid carrier prepared therefrom and use thereof. BACKGROUND

[0002] With the rapid development of gene therapy, mRNA vaccines and RNA interference technology, safe and efficient nucleic acid delivery systems have become a key bottleneck restricting their clinical translation. Nucleic acid molecules (such as DNA, mRNA, siRNA) have hydrophilic, negative and easily degradable properties, and are difficult to spontaneously penetrate the cell membrane barrier, and therefore need to be designed with targeting, protective and membrane fusion capabilities.

[0003] Cationic liposomes have become the core carrier of non-viral delivery systems due to their ability to efficiently condense nucleic acids, promote cell uptake and endosome escape through electrostatic interactions. Traditional cationic liposomes usually use a multi-component complex system (such as a "four-component system"), which includes: (1) cationic lipids (such as DLin-MC3-DMA): providing pH-dependent membrane fusion ability; (2) auxiliary phospholipids (such as DOPE / DSPC): stabilizing the lipid bilayer structure and enhancing endosome escape; (3) cholesterol: regulating membrane fluidity and in vivo stability; (4) PEGylated lipids (such as DMG-PEG2000): reducing immune clearance and prolonging circulation half-life.

[0004] Although this system has been successful in some drugs (such as ), it still has the following significant defects: 1) system complexity: the precise ratio optimization of the four components is difficult, the production process is complex, and batch consistency is difficult to guarantee; 2) toxicity risk: cationic lipids can easily cause cell membrane damage, inflammatory response and organ toxicity (such as liver toxicity) at high doses; 3) transfection efficiency bottleneck: the efficiency is insufficient in difficult-to-transfect cells (such as primary cells, stem cells), and the in vivo targeting is limited.

[0005] Existing research generally believes that cationic liposomes must rely on the synergistic action of multiple components to form stable nucleic acid complexes (Lipid Nanoparticles, LNPs). Auxiliary phospholipids and cholesterol are essential for maintaining the integrity of the lipid bilayer, and PEG-based lipids are indispensable for providing colloidal stability. This "multi-component necessity" paradigm severely limits the flexibility of carrier design and the potential for large-scale application.

[0006] Phosphate groups, as hydrophilic head groups of natural cell membrane components (such as phosphatidylcholine), possess excellent biocompatibility and membrane integration capabilities. Previous studies have attempted to introduce phosphate groups into lipid molecules, but these have mostly focused on the design of neutral or anionic phospholipids. Their application in cationic lipids faces the following challenges: 1) The negative charge of the phosphate group and the electrostatic conflict with the cationic head group may lead to the failure of molecular self-assembly; 2) Traditional synthetic strategies struggle to balance the stability of the phosphate group with the nucleic acid condensation ability of liposomes; 3) Existing phosphate-containing liposomes still heavily rely on multi-component support, failing to overcome the trade-off between stability and efficiency.

[0007] Therefore, there is an urgent need in this field for a lipid molecule that has a simple production process, high transfection efficiency, high biosafety, and wide application. Summary of the Invention

[0008] To overcome the series of problems associated with liposomes, this invention provides a cationic compound, its preparation method, and its application. Specifically, this invention provides a novel lipid molecule with both phosphate groups and cationic head groups, a lipid carrier prepared therefrom, and its uses.

[0009] In a first aspect of the invention, an ionizable lipid compound of formula (I), its stereoisomers, tautomers, or pharmaceutically acceptable salts are provided.

[0010]

[0011] Each R1 is independently C 8-18 alkyl;

[0012] Each R2 is independently C 1-4 alkyl;

[0013] Z is an N-containing group containing 2-10 atoms.

[0014] In another preferred embodiment, all R1 are C 8-12 Alkyl groups, preferably, all of the R1 groups are n-octyl, n-nonyl, or n-decyl.

[0015] In another preferred embodiment, all R2 are C 1-2 Alkyl groups, preferably, all of the R2 groups are methyl or ethyl.

[0016] In a preferred embodiment, Z is -(CH2). m -NR a -(CH2) n -NR b -(CH2) p -、-(CH2) m -NR a -(CH2) n-or-(CH2) m -4-7 membered nitrogen-containing heterocyclic group -(CH2) n -;

[0017] Where m, n and p are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0018] R a and R b Each is independently selected from the following groups: H, C 1-6 Alkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups;

[0019] Preferably, Z is selected from the group consisting of: -(CH2)2-NH-(CH2)4-NH-(CH2)2-, -(CH2)3-NH-(CH2)2-, -CH2-N(CH3)-CH2-.

[0020] In a preferred embodiment, the ionizable lipid compound has the following structure:

[0021]

[0022] A second aspect of the present invention provides a method for preparing an ionizable lipid compound as described in the first aspect of the present invention, the method comprising the following steps:

[0023] (i) In the presence of organic solvent A and basic catalyst, 3,4-dihydroxybenzaldehyde and R1Br react to generate intermediate compound (II);

[0024]

[0025] (ii) In the presence of a catalyst, intermediate compound (II), phosphite (OR2)2P(O)H and NH2-CH2-Z-CH2-NH2 are reacted to obtain an ionizable lipid compound as described in the first aspect of the present invention.

[0026]

[0027] R1, R2, and Z are defined as above.

[0028] In another preferred embodiment, the preparation method includes the following steps:

[0029] (i) In the presence of organic solvent A and basic catalyst, 3,4-dihydroxybenzaldehyde and bromo-n-nonane react to generate 3,4-di(nonoxy)benzaldehyde.

[0030] Methyl(1,16-bis[3,4-bis(nonoxy)phenyl]-2,6,11,15-tetraazahexadecane-1,16-diyl)bisphosphonate;

[0031]

[0032] In a preferred embodiment, the NH2-CH2-Z-CH2-NH2 is selected from the group consisting of spermine (H2N-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2), spermidine (H2N-(CH2)4-NH-(CH2)3-NH2), and 1,4-piperazine diethylamine.

[0033] N-Methyl-2,2-diaminodiethylamine (H2N-CH2-CH2-N(CH3)-CH2-CH2-NH2).

[0034] In another preferred embodiment, in step (i), the organic solution A is N,N-dimethylformamide.

[0035] In another preferred embodiment, in step (i), the alkaline catalyst is selected from the group consisting of potassium carbonate, potassium iodide, or combinations thereof.

[0036] In another preferred embodiment, in step (i), the reaction temperature is 60–100°C, preferably 70–90°C, and more preferably 70–80°C.

[0037] In another preferred embodiment, in step (i), the reaction time is 10-16 hours, preferably 10-12 hours.

[0038] In another preferred embodiment, in step (ii), the reaction is carried out under solvent-free conditions.

[0039] In another preferred embodiment, in step (ii), the catalyst is magnesium perchlorate.

[0040] In another preferred embodiment, in step (ii), the reaction temperature is 60–100°C, preferably 70–90°C, and more preferably 70–80°C.

[0041] In another preferred embodiment, in step (ii), the reaction time is 0.5-6 hours, preferably 0.5-3 hours, and more preferably 0.5-1 hour.

[0042] In a third aspect of the invention, the use of an ionizable lipid compound as described in the first aspect of the invention in the preparation of a lipid composition is provided.

[0043] In another preferred embodiment, the lipid composition is a drug delivery carrier.

[0044] In another preferred embodiment, the drug delivery carrier is a lipid nanoparticle (LNP).

[0045] In another preferred embodiment, the drug is a small molecule, nucleic acid, protein, or a combination thereof.

[0046] In another preferred embodiment, the nucleic acid is DNA or RNA, and preferably, the DNA is linear DNA or circular DNA.

[0047] In another preferred embodiment, the circular DNA is a plasmid.

[0048] In another preferred embodiment, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA, or shRNA, preferably siRNA or mRNA.

[0049] In a fourth aspect of the invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising an ionizable lipid compound as described in the first aspect of the invention.

[0050] In another preferred embodiment, the lipid nanoparticles have an average particle size of 100–250 nm, more preferably 100–180 nm, and even more preferably 105–165 nm.

[0051] In another preferred embodiment, the PDI of the lipid nanoparticles is 0.1 to 0.3, preferably 0.15 to 0.25, and more preferably 0.15 to 0.23.

[0052] In another preferred embodiment, the zeta potential of the lipid nanoparticles is 1.5–15 mV, preferably 2–12 mV, and more preferably 2.5–12 mV.

[0053] In a preferred embodiment, the lipid nanoparticles further include other lipids;

[0054] The other lipids mentioned are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

[0055] In another preferred embodiment, the lipid nanoparticles further include an active pharmaceutical ingredient.

[0056] In another preferred embodiment, the phospholipid is DOPE (dioleoylphosphatidylcholine).

[0057] In another preferred embodiment, the steroid lipid is cholesterol.

[0058] In another preferred embodiment, the PEG lipid is DMG PEG, preferably DMG PEG 2000.

[0059] In another preferred embodiment, the ionizable lipid compound accounts for 20-80% of the total molar amount of the lipid nanoparticles, preferably 30-70%, more preferably 40-70%, and most preferably 50-70%.

[0060] In another preferred embodiment, the phospholipid accounts for 20-90% of the total molar amount of the lipid nanoparticles, preferably 30-80%, and more preferably 35-80%.

[0061] In another preferred embodiment, the sterol lipids account for 20-80% of the total molar amount of the lipid nanoparticles, preferably 30-75%, and more preferably 35-70%.

[0062] In another preferred embodiment, the PEG-type lipid accounts for 0.5 to 2.5% of the total molar amount of the lipid nanoparticles, preferably 1 to 2%, and more preferably 1.2 to 1.8%.

[0063] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the phospholipid is (20-80):(18.5-88.5), preferably (20-75):(25-85), more preferably (30-60):(38.5-78.5), and most preferably (50-70):(28.5-48.5).

[0064] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the sterol lipid is (20-80):(18.5-78.5), preferably (25-75):(25-75), more preferably (30-60):(38.5-68.5), and most preferably (50-70):(28.5-48.5).

[0065] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the PEG lipid is (20-80):(0.5-2.5), preferably (25-75):(1-2), more preferably (20-70):1.5, most preferably (30-60):1.5, and most preferably (50-70):1.5.

[0066] In another preferred embodiment, the molar ratio of the compound of formula (I), phospholipid and PEG lipid is (20-80):(18.5-88.5):(0.5-2.5), preferably (20-70):(28.5-78.5):(1-2) or (30-60):(38.5-78.5):1.5.

[0067] In another preferred embodiment, the molar ratio of the compound of formula (I), the sterol lipid and the PEG lipid is (20-80):(18.5-78.5):(0.5-2.5), preferably (25-75):(25-75):(1-2) or (30-60):(38.5-68.5):1.5.

[0068] A fifth aspect of the present invention provides a method for preparing lipid nanoparticles as described in the fourth aspect of the present invention, the method comprising the following steps:

[0069] Step (1): Dissolve the ionizable lipid compound and other lipids as described in the first aspect of the present invention in an organic solvent to obtain an organic phase solution;

[0070] Step (2): Mix the buffer solution with an optional aqueous medium to obtain an aqueous solution;

[0071] Step (3): In a microfluidic system, the organic phase solution and the aqueous phase solution are mixed at a volume ratio of 1:(1-5), a flow rate ratio of 1:(1-5), and a total flow rate of 9-15 ml / min to obtain lipid nanoparticles as described in this invention.

[0072] In another preferred embodiment, in step (1), the organic solvent is an alcohol-based organic solvent, preferably ethanol.

[0073] In another preferred embodiment, in step (2), the aqueous medium is absent or contains an active pharmaceutical ingredient.

[0074] In another preferred embodiment, the active pharmaceutical ingredient includes a bioactive agent.

[0075] In another preferred embodiment, in step (3), the buffer solution is PBS.

[0076] In another preferred embodiment, in step (3), the volume ratio of the organic phase solution to the aqueous phase solution is 1:3.

[0077] In another preferred embodiment, in step (3), the total flow rate is 9 to 12 ml / min.

[0078] In another preferred embodiment, in step (3), the flow rate ratio of the organic phase solution to the aqueous phase solution is (1-2):(2-3), preferably 1:(2-4), and more preferably 1:3.

[0079] In another preferred embodiment, in step (3), the molar concentration of the lipid nanoparticles is 1 to 8 mM, preferably 1 to 5 mM, and more preferably 2 to 4 mM.

[0080] A sixth aspect of the invention provides the use of an ionizable lipid compound, its stereoisomers, tautomers, or pharmaceutically acceptable salt as described in the first aspect of the invention, or lipid nanoparticles as described in the first aspect of the invention, said use being selected from the group consisting of:

[0081] (1) Encapsulated therapeutic and / or preventative agents;

[0082] (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventative agents; and / or

[0083] (3) Prepare the transfection kit.

[0084] A seventh aspect of the present invention provides a pharmaceutical composition, characterized in that the composition comprises lipid nanoparticles as described in the fourth aspect of the present invention, a bioactive agent encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier, excipient, or excipient.

[0085] In another preferred embodiment, the mass ratio of the lipid nanoparticles to the bioactive agent is (1-30):1, preferably (2-20):1, more preferably (2-10):1, and most preferably (2-4):1.

[0086] In another preferred embodiment, the N / P ratio of the lipid nanoparticles to the nucleic acid is 1:(1-30), preferably 1:(1.5-25), more preferably 1:(2-20), and most preferably 1:(5-10).

[0087] In another preferred embodiment, the N / P ratio of the lipid nanoparticles to the nucleic acid is 1:(5-25), preferably 1:(9-24).

[0088] In another preferred embodiment, the bioactive agent is selected from the group consisting of nucleic acids, proteins, small molecules, or combinations thereof.

[0089] In another preferred embodiment, the nucleic acid is DNA or RNA.

[0090] In another preferred embodiment, the nucleic acid is pDNA or mRNA.

[0091] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical medications.

[0092] In another preferred embodiment, the pharmaceutical composition is administered by injection, inhalation, or transdermal administration.

[0093] In another preferred embodiment, the pharmaceutical composition is administered by injection, preferably by intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal injection.

[0094] In another preferred embodiment, the pharmaceutical composition is administered by inhalation, preferably intranasal administration.

[0095] In another preferred embodiment, the pharmaceutical composition is administered transdermally, preferably by topical application or electrode delivery.

[0096] An eighth aspect of the present invention provides the use of a pharmaceutical composition as described in the seventh aspect of the present invention for the preparation of a medicament for the prevention and / or treatment of inflammatory, infectious, cancer, proliferative, genetic, autoimmune, or metabolic diseases.

[0097] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0098] Figure 1 It is compound 1 1 H NMR spectrum.

[0099] Figure 2 It is compound 2. 1 H NMR spectrum.

[0100] Figure 3 This is the particle size distribution diagram of LNP with added cationic lipid compound 2.

[0101] Figure 4 This is the potential distribution diagram of LNP with added cationic lipid compound 2.

[0102] Figure 5 This describes the expression of eGFP in cells after incubation of liposomes with eGFP-pDNA following in vitro delivery.

[0103] Figure 6 This describes the expression of eGFP in cells after incubation of prescription 2 liposomes with eGFP-pDNA.

[0104] Figure 7 This describes the expression of eGFP in cells after incubation of prescription 3 liposomes with eGFP-pDNA.

[0105] Figure 8 This describes the expression of eGFP in cells after incubation of prescription 4 liposomes with eGFP-pDNA.

[0106] Figure 9 This describes the expression of eGFP in cells after incubation of Prescription 6 liposomes with eGFP-pDNA.

[0107] Figure 10 This describes the expression of eGFP in cells after incubation of prescription 7 liposomes with eGFP-pDNA.

[0108] Figure 11 This describes the expression of eGFP in cells after incubation of Prescription 8 liposomes with eGFP-pDNA in vitro.

[0109] Figure 12 This describes the expression of eGFP in cells after incubation of Prescription 9 liposomes with eGFP-pDNA.

[0110] Figure 13 This describes the expression of eGFP in cells after incubation of Prescription 10 liposomes with eGFP-pDNA.

[0111] Figure 14 The expression of eGFP was observed 48 hours after incubation of LNP containing cholesterol components with eGFP-pDNA and subsequent in vitro delivery to cells, with the fluorescence intensity representing the ratio of the fluorescence intensity to that of the lipo8000 transfection group.

[0112] Figure 15 This represents the expression of eGFP 48 hours after incubation of LNP containing DOPE components with eGFP-pDNA and subsequent in vitro delivery to cells, and the ratio of fluorescence intensity to that of the lipo8000 transfection group.

[0113] Figure 16 This describes the expression of eGFP in cells after incubation of liposomes with eGFP-mRNA following in vitro delivery.

[0114] Figure 17 The expression of eGFP was observed 48 hours after incubation of liposome 1 with eGFP-mRNA and subsequent in vitro delivery to cells, and the fluorescence intensity was compared to that of the lipo8000 transfection group. Detailed Implementation

[0115] Through long-term and in-depth research and extensive screening, the inventors have developed a novel type of lipid molecule that combines phosphate groups and cationic head groups. By regulating intramolecular charge and optimizing spatial conformation, they have achieved advantages such as simple production process, high transfection efficiency, low cytotoxicity, and wide applicability, demonstrating significant application value. Based on this, the inventors completed this invention.

[0116] the term

[0117] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0118] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0119] As used herein, the term "alkyl" refers to a monovalent, straight-chain or branched saturated hydrocarbon group consisting of carbon and hydrogen atoms, for example, "C 1-6 "Alkyl" means an alkyl group having 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6). Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. In this application, alkyl is also intended to include deuterated alkyl groups, examples of which include, but are not limited to, CD3, CD2CD3, and CD2CD2CD3.

[0120] As used herein, the term "cycloalkyl" refers to a monovalent saturated carbocyclic group consisting of carbon and hydrogen atoms, such as "C". 3-8 "Cycloalkyl" refers to a cycloalkyl group containing 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C14. 3-6 Cycloalkyl groups. Cycloalkyl groups can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or similar groups, or bicyclic, such as fused rings, bridged rings or spirocyclic rings.

[0121] As used herein, the term "heterocyclic group" refers to a fully or partially saturated monocyclic or polycyclic cyclic group with one or more (e.g., 1, 2, 3, or 4) heteroatoms selected from N, S, or O on its ring backbone. For example, "4-7 membered heterocyclic group" refers to a group having 4-7 (e.g., 4, 5, 6, or 7) ring members. The nitrogen or sulfur atom may be oxidized, or the nitrogen atom may be quaternized. The heterocyclic group may be attached to any heteroatom or carbon residue in a ring or ring system molecule. Monocyclic heterocyclic groups include, but are not limited to: nitrogen-containing heterocyclic butyl groups, pyrrolyl groups, oxocyclic butyl groups, pyrazolinyl groups, imidazolinyl groups, imidazoalkyl groups, oxazolinyl groups, isoxazolinyl groups, thiazoalkyl groups, isothiazolinyl groups, tetrahydrofuranyl groups, piperidinyl groups, piperazinyl groups, 2-oxopiperidinyl groups, 2-oxopiperidinyl groups, 2-oxopiperidinyl groups, hexahydroacoxaneyl groups, 4-piperidinoneyl groups, tetrahydropyranyl groups, morpholinyl groups, thiomorpholinyl groups, thiomorpholinyl sulfoxide groups, thiomorpholinyl sulfone groups, 1,3-dioxylyl groups, and tetrahydro-1,1-dioxothiophene groups, etc. Polycyclic heterocyclic groups include, but are not limited to, heterocyclic groups of spiroheterocyclic, fused heterocyclic, and bridged heterocyclic groups.

[0122] As used in this article, the term "N / P" refers to the molar ratio of nitrogen in ionizable lipids to molar phosphorus in nucleic acids, reflecting the charge ratio of lipids to nucleic acids.

[0123] In this document, “optionally” means that the event or condition described below may, but is not required to, occur, and the description includes both the possibility that the event or condition occurs and the possibility that the event or condition does not occur.

[0124] In this article, the term "multiple" refers to 2, 3, 4, 5, or a positive integer greater than 5.

[0125] Ionizable lipids

[0126] As used herein, the terms “compound of the present invention,” “ionizable lipid,” “ionizable lipid compound,” “ionizable cationic lipid,” “cationic lipid,” and “cationic lipid compound” are used interchangeably and refer to a compound having the structure of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0127] Ionizable lipids protonate and transform into cationic lipids at low pH values, while at normal physiological pH values ​​they transform into helper phospholipids. Helper phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are endocytosed by cells, the pH value within the endosomes is low, causing the lipids to protonate and become positively charged. This reduces or even disrupts the membrane structure, facilitating the escape of lipid nanoparticle endosomes. In summary, the pH-sensitive nature of lipids is beneficial for the in vivo delivery of lipid nanoparticles carrying bioactive components (such as mRNA molecules).

[0128] Unless otherwise specified, the structural formulas described in this invention are intended to include all stereoisomers (such as cis-trans isomers, enantiomers, diastereomers, and conformational isomers): R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, cis-trans isomers of cycloalkanes, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or conformational isomers, is within the scope of this invention.

[0129] The compounds of this invention may contain cis-trans isomers, one or more chiral carbon atoms, and thus can produce cis-trans isomers, chiral isomers, enantiomers, diastereomers, and other combinations of stereoisomers. Cis-trans isomerism refers to the diastereomeric phenomenon in which different functional groups in a compound molecule are arranged differently in space due to a restrictive factor that limits free rotation. This restrictive factor is generally caused by non-rotating functional groups in the structure of organic compounds, such as C=C double bonds, C=N double bonds, C=S double bonds, N=N double bonds, heterocycles, or cycloalkanes. Organic molecules containing such isomers, such as alkenes, azo compounds, and cycloalkanes, are considered cis-trans isomers. Cis refers to the same ligands being in adjacent positions, generally denoted by "cis" or "cis-"; trans refers to the same ligands being in diagonal positions, generally denoted by "trans" or "trans-". Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry. This invention aims to include all possible isomers, their racemic and optically pure forms. The compounds of this invention can be prepared using racemic, cis-trans, chiral, diastereomer, or enantiomers as starting materials or intermediates. Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0130] Conventional techniques for preparing / separating individual optical isomers (i.e., cis-trans isomers and chiral isomers) include chiral synthesis from suitable cis-trans precursors or optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography.

[0131] To design the synthesis of a specific stereoisomer of the compound of this invention, it can be prepared asymmetrically or derivatized with a chiral auxiliary. The resulting stereo mixture is then separated, and the chiral auxiliary is removed to obtain pure cis-trans monomers, chiral monomers, or mixed stereoisomers. If the molecule contains a cis-trans isomer center, it can be purified by column chromatography (normal-phase silica gel column or reverse-phase high-performance liquid chromatography) to obtain pure cis or trans products. Alternatively, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, it can be formed with a suitable optically active acid or base to form a diastereomeric salt, which is then separated by conventional methods such as separation crystallization or chromatography to obtain pure enantiomers.

[0132] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0133] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared using methods known in this field.

[0134] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, 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, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0135] Preparation method of ionizable lipid compounds

[0136] The method for preparing the ionizable lipid compound of the present invention includes the following steps:

[0137] (i) In the presence of organic solvent A and basic catalyst, 3,4-dihydroxybenzaldehyde and R1Br react to generate intermediate compound (II);

[0138]

[0139] (ii) In the presence of a catalyst, intermediate compound (II), phosphite (OR2)2P(O)H and NH2-CH2-Z-CH2-NH2 react to generate the ionizable lipid compound as described above;

[0140]

[0141] R1, R2, and Z are as described above.

[0142] In one embodiment, the method includes using an amino compound With intermediate compound (II) and phosphite The steps for a one-pot reaction of the reactants; wherein R1 is selected from C8-18 alkyl groups.

[0143] In one embodiment, the method includes the following steps:

[0144] S1: Synthesis of 3,4-di(nonoxy)benzaldehyde;

[0145]

[0146] S2: Synthesis of tetramethyl(1,16-bis[3,4-bis(nonoxy)phenyl]-2,6,11,15-tetraazahexadecane-1,16-diyl)bisphosphonate;

[0147]

[0148] More preferably, step S1 involves dissolving 3,4-dihydroxybenzaldehyde and n-bromononane in anhydrous N,N-dimethylformamide, adding potassium carbonate and potassium iodide, and after the addition is complete, heating to 80°C and reacting for 10-16 hours. The reaction process is monitored by TLC throughout the entire reaction.

[0149] After the reaction was complete, N,N-dimethylformamide was removed using a rotary evaporator;

[0150] Dichloromethane was added, and the resulting mixture was washed three times with deionized water. The organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified to obtain 3,4-di(nonoxy)benzaldehyde.

[0151] More preferably, in step S2, the 3,4-di(nonoxy)benzaldehyde, dimethyl phosphite, spermine and magnesium perchlorate obtained in step S1 are mixed, heated to 80°C, and stirred after the mixture becomes liquid. After reacting for 0.5 h, TLC is used for detection. The reaction is stopped after the starting material spot disappears.

[0152] Add deionized water to disperse the mixture in water, and dialyze to remove unreacted dimethyl phosphite and magnesium perchlorate catalyst. Concentrate and purify the dialyzed reaction solution to obtain tetramethyl(1,16-bis[3,4-bis(nonoxy)phenyl]-2,6,11,15-tetraazahexadecane-1,16-diyl)bisphosphonate.

[0153] In one embodiment, the cationic lipid compound is used as an emulsifier, suspending agent, dispersant, solubilizer, lubricant, thickener, antibacterial agent, and preservative.

[0154] Applications of ionizable lipid compounds

[0155] In another aspect, the present invention provides the use of cationic lipid compounds as described in any embodiment herein in the preparation of lipid compositions.

[0156] In one embodiment, the lipid composition is used as a drug delivery carrier, preferably, the drug delivery carrier is a lipid nanoparticle (LNP).

[0157] In one embodiment, the drug is a small molecule drug, a nucleic acid, or a protein. More preferably, the drug is a nucleic acid, which is DNA or RNA. More preferably, the DNA is linear DNA or circular DNA. More preferably, the circular DNA is a plasmid. More preferably, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA, or shRNA. More preferably, the RNA is siRNA or mRNA.

[0158] In another aspect, the present invention provides a lipid composition comprising cationic lipids as described in any embodiment herein;

[0159] Preferably, the lipid composition further includes other lipids selected from the group consisting of phospholipids, sterols (e.g., steroidal compounds), PEG-like lipids, or combinations thereof.

[0160] In another preferred embodiment, the lipid composition further includes therapeutic and / or preventative agents (i.e., active pharmaceutical ingredients).

[0161] In another preferred embodiment, the active pharmaceutical ingredient includes a bioactive agent.

[0162] In one embodiment, the molar ratio of the cationic lipid to other lipid components is 20:80 to 80:20, preferably 30:70 to 70:30, and more preferably 50:50 to 60:40.

[0163] In one embodiment, the molar ratio of the cationic lipid, phospholipid or sterol compound (e.g., sterol compound) and PEG lipid is 20%–80%:18.5%–78.5%:1.5%, preferably 30%–70%:28.5%–68.5%:1.5%, and most preferably 50%–70%:28.5%–48.5%:1.5, for example 50:48.5:1.5, 60:38.5:1.5, 70:28.5:1.5.

[0164] In another aspect, the present invention provides use of the cationic lipid as described above or the lipid composition as described above, said use being selected from the group consisting of:

[0165] (1) Encapsulated therapeutic and / or preventative agents;

[0166] (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventative agents; and / or

[0167] (3) Prepare the transfection kit.

[0168] In another aspect, the present invention provides a pharmaceutical composition comprising a lipid composition, a therapeutic and / or preventive agent, and a pharmaceutically acceptable carrier, excipient, or excipient as described in any embodiment herein.

[0169] In one embodiment, the mass ratio of the lipid composition to the active ingredient (i.e., the therapeutic agent and / or preventive agent) is 1:1 to 30:1, preferably 2:1 to 20:1.

[0170] In one embodiment, the therapeutic agent and / or preventive agent is selected from one or more combinations of small molecule drugs, nucleic acids, and proteins.

[0171] Preferably, the therapeutic and / or preventative agent is a nucleic acid. More preferably, the drug is a nucleic acid, wherein the nucleic acid is DNA or RNA. Further preferably, the DNA is linear DNA or circular DNA. Further preferably, the circular DNA is a plasmid. Further preferably, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA, or shRNA. Further preferably, the RNA is siRNA or mRNA.

[0172] In another aspect, the present invention provides the use of the pharmaceutical composition described above in the preparation of a medicament for the prevention or treatment of inflammation, infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, or metabolic diseases.

[0173] lipid nanoparticles

[0174] The lipid nanoparticles of the present invention include the ionizable lipid compounds of the present invention as described in formula (I) and other lipids, wherein the other lipids are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

[0175] Cholesterol plays a crucial role in the self-assembly of membrane structures. It has been shown to be an effective substance for increasing the fluidity of lipid membranes while maintaining their stability. Cholesterol analogues include: vitamin D2, vitamin D3, β-sitosterol, brassosterol, and ergosterol.

[0176] Cholesterol is known to be (3β)-cholesterol-5-en-3-ol, an essential component of cell membranes in higher animals. As used herein, "cholesterol compounds" include cholesterol, cholesterol derivatives, and cholesterol analogs. Cholesterol derivatives refer to natural or synthetic cholesterol derivatives, such as acyl cholesterol as an ester body with a fatty acid partially bonded to the hydroxyl group. Cholesterol analogs refer to natural or synthetic cholesterol analogs, such as phytosterols derived from plant cells (e.g., sitosterol, stigmasterol, fucosterol, spinasterol, campesterol, and brassicerol) and ergosterol derived from fungi.

[0177] Cholesterol, cholesterol derivatives, and cholesterol analogs are all classified as steroids, belonging to the subgroup known as sterols (steroidal alcohols).

[0178] The PEG-based lipids described in this invention generally refer to conjugates formed by chemically linking PEG (polyethylene glycol) and lipid molecules. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy-polyethylene glycol bis(tetradecylacetamide).

[0179] In one embodiment, the PEG lipids include, but are not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearate phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, and polyethylene glycol diglycerol. The following are combinations of one or more of the following: PEG-DMA, PEG distearate glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearate, PEG diacyl glycine, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl ethylenedimyristyloxypropyl-3-amine, PEG oxypropyl alcoholamine, 1,2-distearate oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol bis(tetradecyl acetamide) (ALC0159).

[0180] In one embodiment, the PEG lipid is DMG PEG.

[0181] In one embodiment, the weight-average molecular weight of PEG in the PEG lipid is 1000 to 10000, for example, 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.

[0182] Pharmaceutical Composition

[0183] The lipid compositions of the present invention comprise, as described above, a bioactive agent encapsulated within the lipid nanoparticles, and a pharmaceutically acceptable carrier, excipient, or excipient.

[0184] In one embodiment, the bioactive agent is selected from the group consisting of nucleic acids, peptides, proteins, and small molecules, or combinations thereof.

[0185] The "nucleic acid" described in this invention can be a nucleotide polymer of any length. This includes, but is not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), miRNA, siRNA, telomerase RNA, small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNAmimics, miRNAagomir, miRNAantagomir), antisense oligonucleotides (ASO), ribozymes, asymmetric interfering RNA (aiRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholine antisense oligonucleotides, morpholine-substituted oligonucleotides, or bio-customized oligonucleotides, or one or more combinations thereof.

[0186] In one embodiment, the nucleic acid is mRNA. mRNA is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template, carrying genetic information and directing protein synthesis. mRNA can encode one protein or multiple proteins simultaneously. Preferably, mRNA is obtained through in vitro transcription synthesis.

[0187] The term "small molecule" as used in this invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecules of therapeutic and / or preventative agents, such as antibiotics, anti-inflammatory drugs, anticancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungal drugs, antiparasitic drugs, anticonvulsants, antidepressants, anti-anxiety drugs, antipsychotics, lipid-lowering drugs, hypoglycemic drugs, weight-loss drugs, etc.

[0188] The term "protein" as used in this invention refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals interactions. Additionally, or alternatively, proteins may include disulfide bonds, for example, between thiol groups of cysteine ​​residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, glycoproteins, lipoproteins, ribosomal proteins, chemically modified proteins, etc.

[0189] In one embodiment, the nucleic acid is DNA or RNA.

[0190] In one embodiment, the nucleic acid is pDNA or mRNA.

[0191] The term "pharmaceutical acceptable" as used in this invention means that when the drug is properly administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions.

[0192] "Pharmaceuticalally acceptable excipients" should be compatible with the active ingredient, meaning they can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; colorants; flavoring agents; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, etc., and combinations thereof. These substances are used, as needed, to improve the stability of the formulation or to help improve its activity or bioavailability, or to produce an acceptable taste or odor when taken orally.

[0193] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0194] In one embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical medications.

[0195] The administration methods of the pharmaceutical compositions of the present invention include, but are not limited to, injection, inhalation, and transdermal administration.

[0196] In one embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal injection.

[0197] In one embodiment, the pharmaceutical composition is administered by inhalation, such as intranasal administration.

[0198] In one embodiment, the pharmaceutical composition is administered transdermally, such as by topical application or electrode delivery.

[0199] In one embodiment, the pharmaceutical composition is delivered to target cells; preferably, the target cells are eukaryotic cells, and more preferably, the target cells are one or more of immune cells, stem cells, or nerve cells.

[0200] In one embodiment, the immune cells include, but are not limited to, one or more of T cells, macrophages, dendritic cells, natural killer cells, monocytes, bone marrow cells, B cells, lymphocytes, and tumor-infiltrating lymphocytes; and / or, the stem cells include, but are not limited to, one or more of induced pluripotent stem cells (iPSCs), human embryonic stem cells, adult stem cells, mesenchymal stem cells, and hematopoietic stem cells; and / or, the nerve cells include, but are not limited to, one or more of neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, and neuroblasts.

[0201] In one embodiment, the pharmaceutical composition is delivered to target cells for cell therapy to treat cancer.

[0202] The cell therapies include, but are not limited to, one or more combinations of chimeric antigen receptor T cell (CAR-T cell) therapy, T-cell receptor engineered T cell (TCR-T) therapy, chimeric antigen receptor natural killer cell (CAR-NK cell) therapy, chimeric antigen receptor natural killer T cell (CAR-NKT cell) therapy, chimeric antigen receptor macrophage cell (CAR-M cell) therapy, and tumor-infiltrating lymphocytes cell (TIL cell) therapy. The cancers include, but are not limited to, one or more of leukemia, lymphoma, melanoma, cervical cancer, and sarcoma. The leukemias include, but are not limited to, one or more of childhood acute lymphoblastic leukemia and chronic lymphocytic leukemia. The lymphomas include, but are not limited to, one or more of adult advanced lymphoma and relapsed or refractory non-Hodgkin's lymphoma.

[0203] The main advantages of this invention are:

[0204] 1. Single-component liposome stabilization: Breaking the traditional paradigm of "four essential components" and significantly simplifying the production process.

[0205] 2. High transfection efficiency: Phosphate groups enhance membrane fusion and endosome escape, improving nucleic acid release efficiency.

[0206] 3. Low cytotoxicity: The biomimetic structure of the phosphate group reduces the risk of membrane damage and significantly improves biocompatibility.

[0207] 4. Broad applicability: Suitable for delivery of various nucleic acid drugs such as pDNA, mRNA, and siRNA.

[0208] 5. The compound of this invention has a novel structure, and its chemical structure is different from that of cationic lipids disclosed in the prior art, making it a completely new compound.

[0209] 6. The cationic lipid compounds of the present invention introduce uncharged phosphate groups, which provide suitable charge-binding small molecule drugs while stabilizing LNPs.

[0210] 7. The LNP prepared by adding the cationic lipid (20 mol% to 80 mol%) of the present invention has a good particle size (~150 nm) and uniform particle size (PDI ~0.2).

[0211] 8. A phosphate group is introduced into the hydrophilic amino head of the compound of the present invention. While carrying a positive charge, it stabilizes the LNP stability brought by phospholipids and enhances endosome escape, breaking the limitation of the traditional four-component liposome.

[0212] 9. The compounds of the present invention, when used to prepare LNPs, achieve excellent nucleic acid delivery effects both in vivo and in vitro, while exhibiting low toxicity.

[0213] 10. Under the same experimental conditions, compared with commercially available transfection reagents, the transfection efficiency of the compounds of the present invention is significantly improved, and the toxicity is significantly reduced.

[0214] 11. Common methods for delivering gene drugs using cationic lipids (such as ALC-0315 and SM-102) involve preparing mRNA-LNPs, where the gene is encapsulated within the LNP during LNP preparation. The delivery method used in this application is novel; the gene drug is incubated with the LNP after preparation, unlike traditional methods. The advantages of this method are significant savings in gene drug, reduced losses due to differences in storage conditions between the LNP and the gene drug, and lower costs.

[0215] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, were generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0216] Example 1: Synthesis of intermediate compound 1

[0217] The specific synthetic route for 3,4-di(nonoxy)benzaldehyde (compound 1) is as follows:

[0218]

[0219] 3,4-Dihydroxybenzaldehyde (0.4144 g, 3 mmol) and n-nonane bromide (1.2429 g, 6 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 mL), followed by the addition of potassium carbonate (0.9951 g, 7.2 mmol) and potassium iodide (0.17 g, 1.0 mmol). After the addition was complete, the mixture was heated to 80 °C and reacted for 12 h. Once TLC indicated that the reactants had completely reacted, the N,N-dimethylformamide was removed, and 20 mL of dichloromethane was added. The mixture was washed three times with 5 mL of deionized water, and the organic phase was collected. After drying with anhydrous sodium sulfate, the liquid was filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (100% dichloromethane) to give a white powdery compound (Compound 1, 1.0436 g, yield 89.06%).

[0220] The proton NMR spectrum of compound 1 is as follows: Figure 1 As shown, its 1 The characteristics of H NMR are: 1 HNMR(400MHz,Chloroform-d)9.82(s,1H),7.42-7.38(m,2H),6.94(d,J=8.1Hz1H)4.06(dt,J =10.9,6.6Hz,4H),1.84(h,J=6.9Hz,4H),1.52-1.43(m,4H),1.28(s,20H),0.90-0.85(m.6H).

[0221] Example 2: Synthesis of Compound 2

[0222] The synthetic route for tetramethyl(1,16-bis[3,4-bis(nonoxy)phenyl]-2,6,11,15-tetraazahexadecane-1,16-diyl)bisphosphonate (compound 2) is as follows:

[0223]

[0224] Intermediate compound 1 (0.1953 g, 0.5 mmol) and magnesium perchlorate (0.0056 g, 0.025 mmol) were mixed thoroughly, and then spermine (0.0506 g, 0.25 mmol) and dimethyl phosphite (0.0550 g, 0.5 mmol) were added. The mixture was heated to 80 °C. After the mixture melted, it was stirred for 0.5 h under solvent-free conditions. Heating was stopped after TLC indicated that the reactants had reacted completely. 50 mL of deionized water was added, and the mixture was sonicated to disperse it evenly in the water to form stable micelles. The mixture was then transferred to a 3.5 kD dialysis bag and dialyzed for 12 h to remove unreacted water-soluble reactants. After dialysis, the mixture was transferred to a 500 mL round-bottom flask, and the solvent water was removed by rotary evaporation to obtain a yellow viscous crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 10:1, v / v), a pale yellow viscous substance (compound 2) was obtained.

[0225] The proton NMR spectrum of compound 2 is as follows: Figure 2 As shown, its 1 The characteristics of H NMR are: 1 H NMR(400MHz,Chloroform-d)δ7.06-6.96(m,4H),6.86(d,J=8.3Hz,2H).5.30(s,1H)4.95(d,J=10.1Hz,2H),4.04-3.95 (m,8H),3.74-3.62(m,12H),1.81(t,J=7.5Hz,8H).1.46(t,J=7.6Hz,11H),1.31-1.23(m,44H).0.89(dJ=6.4Hz.12H).

[0226] Example 3: Preparation and parameter characterization of cationic LNPs

[0227] Cationic lipid (compound 2), cholesterol or DOPE, and DMG-PEG 2000 were dissolved in anhydrous ethanol to obtain an organic phase, wherein the formulation components are compound 2, cholesterol or DOPE, and DMG-PEG 2000, and the molar ratios are shown in Table 1 below.

[0228] 0.7 ml organic phase, 2.1 ml aqueous phase (PBS), 6.3 μmol total molar amount of compound 2, cholesterol or DOPE, and DMG-PEG2000, microfluidic organic phase to aqueous phase flow rate ratio 1:3, total flow rate 12 ml / min.

[0229] The resulting cation LNP molar concentration was 3 mM.

[0230] Table 1: LNP formulations with different components and molar ratios

[0231] Formulation Compound 2 (mol%) Cholesterol / DOPE (mol%) DMG-PEG 2000 (mol%) 1 60 38.5 (Cholesterol) 1.5 2 50 48.5 (Cholesterol) 1.5 3 40 58.5 (Cholesterol) 1.5 4 30 68.5 (Cholesterol) 1.5 5 20 78.5 (Cholesterol) 1.5 6 60 38.5 (DOPE) 1.5 7 50 48.5 (DOPE) 1.5 8 40 58.5 (DOPE) 1.5 9 30 68.5 (DOPE) 1.5 10 20 78.5 (DOPE) 1.5

[0232] Ten groups of LNPs, as shown in Table 1, were prepared under the above microfluidic conditions. Ethanol was removed by rotary evaporation under a 40°C water bath. Deionized water was added to make up the liquid volume to 2.1 ml, and the liquid was homogenized by ultrasonication three times under 40% power.

[0233] Example 4: Parameter characteristics of LNP prepared from cationic lipid (compound 2)

[0234] The average particle size, PDI, and zeta potential of the lipid nanoparticles containing cationic lipids (compound 2) prepared in Example 2 were determined by dynamic light scattering using an Omec NS-90Z Plus (purchased from Omec). The results are shown in Table 2 below. Figure 3 and 4 As shown.

[0235] Table 2: Characterization of LNP parameters for different components and different molar ratios

[0236] Formulation Average particle size (nm) PDI Zeta (mv) 1 133.5 0.1761 11.93 2 159.5 0.1808 9.186 3 160.1 0.179 6.593 4 147.5 0.2133 2.816 5 261.6 0.3742 1.398 6 124.9 0.1832 10.61 7 121.8 0.1837 8.489 8 108.8 0.1940 4.779 9 121.9 0.1585 4.926 10 141.8 0.1617 3.891

[0237] From Table 2, Figure 3 and 4 It is known that when preparing LNP containing cationic lipids (compound 2), the LNP formed when the cholesterol component is 78.5% (i.e., formulation 5) is unstable, while the LNP formed by other components has a good particle size (~150nm) and uniform particle size (PDI~0.2).

[0238] Example 5: Detection of the in vitro gene delivery effect of LNP prepared with cationic lipids (compound 2)

[0239] The LNP used in this embodiment is the LNP prepared in Example 3.

[0240] 5.1 The transfected nucleic acid is pDNA

[0241] The pDNA used here is pCMV-C-EGFP (purchased from Beyotime Biotechnology Co., Ltd., catalog number D2626).

[0242] 293T cells were seeded into 24-well plates, and DMEM medium was added to a final volume of 500 μl. The cell density was ensured to reach approximately 60%-80% of the plate area before transfection the following day. 1 μg of pDNA (1 μg / μl) was added to different LNP formulations (concentrations shown below) and lipo8000 at varying volume ratios, and incubated in 50 μl of Opti-MEM medium for 20-30 min. The lipo8000 used (purchased from Beyotime Biotechnology Co., Ltd., catalog number D0533) was administered according to the recommended dosage in the technical instructions. The cells were then ready for transfection. 48 h after transfection, observation and recording were performed using a Mateo FL digital inverted fluorescence microscope (purchased from Leica-Microsystems), and eGFP fluorescence intensity was detected using a Cytation1 cell imaging microplate reader (purchased from Beijing Boteng Instrument Co., Ltd.). Transfection results are shown below. Figures 5-15 As shown.

[0243] The concentrations of prescriptions 1 through 10 were calculated to be: ① 2.63 mg / ml; ② 2.40 mg / ml; ③ 2.17 mg / ml; ④ 1.97 mg / ml; ⑤ 1.70 mg / ml; ⑥ 3.17 mg / ml; ⑦ 2.93 mg / ml; ⑧ 2.83 mg / ml; ⑨ 2.67 mg / ml; ⑩ 2.57 mg / ml.

[0244] Figures 5-13 This represents the expression of eGFP in cells after incubation of liposomes with eGFP-pDNA according to prescriptions 1-10. Here, n-1 represents the volume ratio of LNP to pDNA as 2; n-2 represents the volume ratio of LNP to pDNA as 4; n-3 represents the volume ratio of LNP to pDNA as 6; n-4 represents the volume ratio of LNP to pDNA as 8; n-5 represents the volume ratio of LNP to pDNA as 10; and n represents the prescription number.

[0245] The conversion between the N / P ratio of LNP:pDNA and the volume ratio of LNP:pDNA is as follows:

[0246] The plasmid used was 5010 bp in length, and the negative charge it carried was the number of phosphate groups in the gene. The average molecular weight of the nucleotides was:

[0247] DNA: ~330 g / mol (single nucleotide); RNA: ~340 g / mol

[0248] Charge / nucleotide = 1 (as described above)

[0249] Example: 1 μg of 5010 bp dsDNA

[0250] Molecular weight ≈ 5010bp × 660g / mol (double strand) = 3306600g / mol

[0251] Number of moles = 1 × 10 -6 g / 3306600g / mol≈3.02×10 -13 mol

[0252] Total negative charge = 1.52 × 10 -12 mol × 10020 charge / mol = 3.03 × 10 -9 mol

[0253] *(Note: 5010bp dsDNA = 10020 phosphate groups)*

[0254] The cationic lipids described in this article contain four ionizable amino groups per lipid molecule. The number of ionizable amino groups can be obtained depending on the proportion of cationic lipids in each formulation, and the positive charge they carry is the number of amino groups.

[0255] Example: In a 60% cationic formulation (CHOL-HP component), i.e., formulation 1, the total lipid concentration is 3 mM, the ionizable lipid concentration is 1.8 mM, and the ionizable amino concentration is 7.2 mM; therefore, 2 μl of LNP contains 1.44 × 10⁻⁶ moles of nitrogen. -8 Therefore, when the LNP:pDNA volume ratio is 2:1, N / P is 1.44 × 10⁻⁶ mol. -8 mol / 3.03×10 -9 mol≈4.8.

[0256] Specifically, Figure 5 This describes the expression of eGFP in cells after incubation of liposomes with eGFP-pDNA following in vitro delivery.

[0257] Among them, 1-1 has an N / P ratio of 4.8 for LNP:pDNA; 1-2 has an N / P ratio of 9.6 for LNP:pDNA; 1-3 has an N / P ratio of 14.4 for LNP:pDNA; 1-4 has an N / P ratio of 19.2 for LNP:pDNA; and 1-5 has an N / P ratio of 24 for LNP:pDNA.

[0258] Figure 6 This describes the expression of eGFP in cells after incubation of prescription 2 liposomes with eGFP-pDNA.

[0259] Among them, 2-1 has an N / P ratio of 4 for LNP:pDNA; 2-2 has an N / P ratio of 8 for LNP:pDNA; 2-3 has an N / P ratio of 12 for LNP:pDNA; 2-4 has an N / P ratio of 16 for LNP:pDNA; and 2-5 has an N / P ratio of 20 for LNP:pDNA.

[0260] Figure 7This describes the expression of eGFP in cells after incubation of prescription 3 liposomes with eGFP-pDNA.

[0261] Among them, 3-1 has an N / P ratio of 3.2 for LNP:pDNA; 3-2 has an N / P ratio of 6.4 for LNP:pDNA; 3-3 has an N / P ratio of 9.6 for LNP:pDNA; 3-4 has an N / P ratio of 12.8 for LNP:pDNA; and 3-5 has an N / P ratio of 16 for LNP:pDNA.

[0262] Figure 8 This describes the expression of eGFP in cells after incubation of prescription 4 liposomes with eGFP-pDNA.

[0263] Among them, 4-1 has an N / P ratio of 2.4 for LNP:pDNA; 4-2 has an N / P ratio of 4.8 for LNP:pDNA; 4-3 has an N / P ratio of 7.2 for LNP:pDNA; 4-4 has an N / P ratio of 9.6 for LNP:pDNA; and 4-5 has an N / P ratio of 12 for LNP:pDNA.

[0264] Figure 9 This describes the expression of eGFP in cells after incubation of Prescription 6 liposomes with eGFP-pDNA.

[0265] Among them, 6-1 has an N / P ratio of 4.8 for LNP:pDNA; 6-2 has an N / P ratio of 9.6 for LNP:pDNA; 6-3 has an N / P ratio of 14.4 for LNP:pDNA; 6-4 has an N / P ratio of 8 for LNP:pDNA; and 6-5 has an N / P ratio of 10 for LNP:pDNA.

[0266] Figure 10 This describes the expression of eGFP in cells after incubation of prescription 7 liposomes with eGFP-pDNA.

[0267] Among them, 7-1 has an N / P ratio of 4 for LNP:pDNA; 7-2 has an N / P ratio of 8 for LNP:pDNA; 7-3 has an N / P ratio of 12 for LNP:pDNA; 7-4 has an N / P ratio of 16 for LNP:pDNA; and 7-5 has an N / P ratio of 20 for LNP:pDNA.

[0268] Figure 11 This describes the expression of eGFP in cells after incubation of Prescription 8 liposomes with eGFP-pDNA in vitro.

[0269] Among them, 8-1 has an N / P ratio of 3.2 for LNP:pDNA; 8-2 has an N / P ratio of 6.4 for LNP:pDNA; 8-3 has an N / P ratio of 9.6 for LNP:pDNA; 8-4 has an N / P ratio of 12.8 for LNP:pDNA; and 8-5 has an N / P ratio of 16 for LNP:pDNA.

[0270] Figure 12 This describes the expression of eGFP in cells after incubation of Prescription 9 liposomes with eGFP-pDNA.

[0271] Among them, 9-1 has an N / P ratio of 2.4 for LNP:pDNA; 9-2 has an N / P ratio of 4.8 for LNP:pDNA; 9-3 has an N / P ratio of 7.2 for LNP:pDNA; 9-4 has an N / P ratio of 9.6 for LNP:pDNA; and 9-5 has an N / P ratio of 12 for LNP:pDNA.

[0272] Figure 13 This describes the expression of eGFP in cells after incubation of Prescription 10 liposomes with eGFP-pDNA.

[0273] Among them, 10⁻¹ is LNP:pDNA with an N / P ratio of 1.6; 10⁻² is LNP:pDNA with an N / P ratio of 3.2; 10⁻³ is LNP:pDNA with an N / P ratio of 4.8; 10⁻⁴ is LNP:pDNA with an N / P ratio of 6.4; and 10⁻⁵ is LNP:pDNA with an N / P ratio of 8.

[0274] Figure 14 and 15 The results show the expression of eGFP 48 hours after incubation of LNP containing cholesterol and DOPE components with eGFP-pDNA and in vitro delivery to cells, and the ratio of fluorescence intensity to that of the lipo8000 transfection group.

[0275] Experimental results show that in LNP containing DOPE components, when the cationic lipid (compound 2) component is 60% (formulation 6), it exhibits excellent delivery performance, which is better than the results achieved by delivery with lipo8000 at the same transfection concentration.

[0276] In LNP containing cholesterol components, when the molar ratio of cationic lipids (compound 2) was 60% (Formula 1), the fluorescence intensity of transfections with different N / P ratios (9.6–24) was more than 1.95 times stronger than that of the lipo8000 group.

[0277] Therefore, a cationic lipid (compound 2) molar ratio of about 60% LNP is preferred.

[0278] 5.2 The transfected nucleic acid is mRNA

[0279] The mRNA used here was prepared using the T7 Co-transcription RNA Synthesis Kit (purchased from Jiangsu Shenji Biotechnology Co., Ltd., catalog number C3011).

[0280] 293T cells were seeded into 24-well plates, and DMEM medium was added to a final volume of 500 μl. The cell density was ensured to reach approximately 60%-80% of the plate area before transfection the following day. 1 μg of mRNA (1 μg / μl) was added to LNP (2.63 mg / ml) and lipo8000 according to different volume ratios, and incubated in 50 μl of Opti-MEM medium for 20-30 min. The lipo8000 used (purchased from Beyotime Biotechnology Co., Ltd., catalog number D0533) was administered according to the recommended dosage in the technical instructions. The cells were then ready for transfection. 48 h after transfection, observation and recording were performed using a Mateo FL digital inverted fluorescence microscope (purchased from Leica-Microsystems), and eGFP fluorescence intensity was detected using a Cytation1 cell imaging microplate reader (purchased from Beijing Boteng Instrument Co., Ltd.). The transfection results are shown below. Figure 16 , Figure 17 As shown.

[0281] Figure 16 The results show the expression of eGFP in cells after incubation of liposomes with eGFP-mRNA following in vitro delivery. In case 1-1, the N / P ratio of LNP:mRNA was 4.8; in case 1-2, the N / P ratio of LNP:mRNA was 9.6; in case 1-3, the N / P ratio of LNP:mRNA was 14.4; in case 1-4, the N / P ratio of LNP:mRNA was 19.2; and in case 1-5, the N / P ratio of LNP:mRNA was 24.

[0282] Depend on Figure 16 and Figure 17 It can be seen that when the transfected nucleic acid is mRNA, in LNP containing cholesterol components, when the molar ratio of cationic lipid (compound 2) is 60% (Formula 1), the fluorescence intensity of transfection by different N / P ratios (2:1 to 10:1) is more than 2.44 times stronger than that of the lipo8000 group, and the fluorescence intensity is the highest, about 5.45 times, when the volume ratio is 4:1.

[0283] In summary, common methods for delivering gene drugs using cationic lipids (such as ALC-0315 and SM-102) involve preparing mRNA-LNPs, i.e., loading the gene into the LNP during LNP preparation. The delivery method used in this application is novel, involving incubation with the gene drug after LNP preparation, unlike traditional methods. The advantages of this method are significant savings in gene drug, reduced losses due to differences in storage conditions between LNPs and gene drugs, and cost reduction.

[0284] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An ionizable lipid compound of formula (I), its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof. in, Each R1 is independently C 8-18 alkyl; Each R2 is independently C 1-4 alkyl; Z is an N-containing group containing 2-10 atoms.

2. The ionizable lipid compound, its stereoisomer, tautomer, or pharmaceutically acceptable salt as described in claim 1, characterized in that, The Z is -(CH2). m -NR a -(CH2) n -NR b -(CH2) p -、-(CH2) m -NR a -(CH2) n -or-(CH2) m -4-7 membered nitrogen-containing heterocyclic group -(CH2) n -; Where m, n and p are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R a and R b Each is independently selected from the following groups: H, C 1-6 Alkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; Preferably, Z is selected from the group consisting of: -(CH2)2-NH-(CH2)4-NH-(CH2)2-, -(CH2)3-NH-(CH2)2-, -CH2-N(CH3)-CH2-.

3. The ionizable lipid compound, its stereoisomer, tautomer, or pharmaceutically acceptable salt as described in claim 1, characterized in that, The ionizable lipid compound has the following structure:

4. A method for preparing the ionizable lipid compound as described in claim 1, characterized in that, The preparation method includes the following steps: (i) In the presence of organic solvent A and basic catalyst, 3,4-dihydroxybenzaldehyde and R1Br react to generate intermediate compound (II); (ii) In the presence of a catalyst, intermediate compound (II), phosphite (OR2)2P(O)H and NH2-CH2-Z-CH2-NH2 are reacted to obtain the ionizable lipid compound as described in claim 1; R1, R2 and Z are as defined in claim 1.

5. The preparation method according to claim 4, characterized in that, The NH2-CH2-Z-CH2-NH2 is selected from the group consisting of spermine (H2N-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2), spermidine (H2N-(CH2)4-NH-(CH2)3-NH2), and 1,4-piperazine diethylamine. N-Methyl-2,2-diaminodiethylamine (H2N-CH2-CH2-N(CH3)-CH2-CH2-NH2).

6. A lipid nanoparticle, characterized in that, The lipid nanoparticles include the ionizable lipid compounds as described in claim 1.

7. The lipid nanoparticles as described in claim 6, characterized in that, The lipid nanoparticles also include other lipids; The other lipids mentioned are selected from the group consisting of phospholipids, sterol lipids, PEG lipids, or combinations thereof.

8. Use of an ionizable lipid compound as claimed in claim 1, its stereoisomers, tautomers, or pharmaceutically acceptable salts, or lipid nanoparticles as claimed in claim 6, characterized in that, The intended use is selected from the following group: (1) Encapsulated therapeutic and / or preventative agents; (2) Preparation of in vivo delivery systems for therapeutic agents and / or preventative agents; and / or (3) Prepare the transfection kit.

9. A pharmaceutical composition, characterized in that, The composition comprises the lipid nanoparticles as described in claim 6, a bioactive agent encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier, excipient, or excipient.

10. Use of the pharmaceutical composition as claimed in claim 9, characterized in that, Drugs used to prepare for the prevention and / or treatment of inflammatory, infectious, cancer, proliferative, genetic, autoimmune, or metabolic diseases.

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