A lipid compound and lipid nanoparticles

Abstract

The present invention relates to a lipid compound and lipid nanoparticles, wherein the lipid is a cationic lipid compound having a structure as shown in formula (I) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, and the compound comprises one or more biodegradable groups. The cationic lipid compound is doped into lipid nanoparticles and can be used to deliver therapeutic agents or preventive agents such as nucleic acids to animal cells or organs to achieve therapeutic and preventive effects.

Description

Technical Field

[0001] The present invention relates to an ionizable lipid compound. Lipid nanoparticles containing the lipid compound can deliver nucleic acid drugs into animal cells. Background Art

[0002] Nucleic acid drugs, including mRNA, siRNA, ASO, etc., are delivered to human target cells through certain means, and achieve therapeutic and preventive effects in the human body by translating target proteins or silencing target mRNA. Delivery methods include viral vectors and non-viral vectors. The application of viral vectors is limited due to their own toxicity and immunogenicity. Non-viral vectors, especially lipid nanoparticles, have been commercialized by encapsulating and delivering nucleic acid drugs to target cells through liposome molecular assembly. The first siRNA drug (Onpattro) approved by the FDA from Alnylam and the two mRNA new crown vaccines approved in 2020 both use nanoparticle delivery systems containing ionizable cationic lipid molecules.

[0003] These lipid nanoparticles generally comprise one or more cationic lipids, neutral phospholipids, sterols, polyethylene glycol lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Scientists have developed several cationic lipids to deliver nucleic acid drugs, but still need more structurally rich cationic lipids to realize functionalized lipid nanoparticles. Summary of the invention

[0004] In one aspect, the present invention provides a cationic lipid compound having a structure as shown in formula (I):

[0005]

[0006] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein

[0007] L 1 ,L 2 ,L 3 ,L 4 ,L 5 are the same as or different from each other and are independently selected from: not present, C1-C 24 Straight chain alkyl or branched chain alkyl, C2-C 24 alkenylene or branched alkenyl;

[0008] M 1 and M 2 Each independently selected from -OC(O)-, -C(O)O- or -NHC=N(CN)NH-;

[0009] G 1and G 2 Each independently selected from H, C5-C 10 aryl or 5- to 10-membered heteroaryl, and C5-C 10 The hydrogen atoms on the aryl or 5- to 10-membered heteroaryl group may be independently optionally replaced by n -XR 1 substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 1 C4-C 20 Straight or branched alkyl, C4-C 20 Straight-chain or branched alkenyl.

[0010] In another aspect, the present invention provides a lipid nanoparticle comprising the cationic lipid compound or an acceptable salt or stereoisomer thereof as described above.

[0011] In yet another aspect, the present invention provides a pharmaceutical composition comprising the lipid compound or nanoparticle composition as described above, and optionally a pharmaceutically acceptable excipient.

[0012] In another aspect, the present invention provides a method for producing a target polypeptide in a subject's cells, the method comprising contacting the cells with a nanoparticle composition as described above comprising mRNA encoding the target polypeptide, whereby the mRNA can be translated in the cells to produce the target polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The luciferase protein expression level was detected after lipid nanoparticles prepared with different lipid compounds were delivered to B16F10 cells and HSKMC cells.

[0014] Figure 2 Lipid nanoparticles prepared with different lipid compounds were injected intramuscularly into mice. The fluorescence intensity of the injection site and liver of the mice was measured 12 hours later. DETAILED DESCRIPTION

[0015] Definition of terms

[0016] The term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms.

[0017] The term "alkoxy" refers to an alkyl group as described herein that is attached to the remainder of the molecule through an oxygen atom.

[0018] The term "alkylene" refers to a divalent group formed by the loss of a hydrogen atom from a corresponding alkyl group.

[0019] The term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. The alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds.

[0020] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system.

[0021] The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Heteroaryl can have 5-10 ring atoms (5-10 membered heteroaryl), including 5, 6, 7, 8, 9 or 10 members, especially 5 or 6 membered heteroaryl.

[0022] The term "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0023] The term "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. The RNA may be selected from: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0024] The term "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component can include one or more cationic / ionizable lipids, pegylated lipids, structured lipids, or other lipids, such as phospholipids.

[0025] The term "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system.

[0026] The term "particle size" refers to the average diameter of a nanoparticle composition.

[0027] The term "zeta potential" refers to the surface potential of lipids in, for example, a nanoparticle composition.

[0028] The term "encapsulation efficiency" refers to the ratio of the amount of therapeutic or prophylactic agent that becomes part of the nanoparticle composition to the initial total amount of therapeutic or prophylactic agent used to prepare the nanoparticle composition.

[0029] The term "delivery" refers to providing an entity to a target. For example, delivering a therapeutic or prophylactic agent to a subject may involve administering a nanoparticle composition comprising the therapeutic or prophylactic agent to the subject (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

[0030] The term "target cell" refers to one or a group of target cells. These cells can be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal.

[0031] The term "expression" refers to the translation of mRNA into polypeptides or proteins and / or polypeptides or proteins.

[0032] The term "subject" refers to a target to which a treatment is intended, including but not limited to humans, other primates and other mammals, such as cows, pigs, horses, sheep, cats, dogs, mice or rats. Preferably, the subject can be a mammal, particularly a human.

[0033] Lipid compounds

[0034] In one aspect, the present invention provides a cationic lipid compound having a structure as shown in formula (I):

[0035]

[0036] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein

[0037] L 1 ,L 2 ,L3 ,L 4 ,L 5 are the same as or different from each other and are independently selected from: not present, C1-C 24 Straight chain alkyl or branched chain alkyl, C2-C 24 alkenylene or branched alkenyl;

[0038] M 1 and M 2 Each independently selected from -OC(O)-, -C(O)O- or -NHC=N(CN)NH-;

[0039] G 1 and G 2 Each independently selected from H, C5-C 10 aryl or 5- to 10-membered heteroaryl, and C5-C 10 The hydrogen atoms on the aryl or 5- to 10-membered heteroaryl group may be independently optionally replaced by n -XR 1 substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 1 C4-C 20 Straight or branched alkyl, C4-C 20 Straight-chain or branched alkenyl.

[0040] In one embodiment, L 1 Selected from C2-C5 alkyl, L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl, L 4 ,L 5 Each independently selected from C4-C 20 Straight chain or branched chain alkyl.

[0041] In one embodiment, G 1 and G 2 are each independently selected from H, or C5-C 10 Aryl. Among them, C5-C 10 The hydrogen atoms on the aryl group may be independently optionally replaced by n -XR 1 substituted, n is an integer of 1-2, X is nitrogen, oxygen, R 3 C4-C 20 Straight or branched chain alkyl;

[0042] In certain embodiments, Formula (I) comprises the structure described by Formula (II):

[0043] Where L 1 ,L 2 ,L 3 ,L 4 ,L 5, M 1 , M 2 As defined herein;

[0044] In one embodiment, L 1 is a C2-C3 alkyl group;

[0045] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0046] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0047] M 1 , M 2 Each is independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-; in certain embodiments, formula (I) comprises the structure of formula (III):

[0048]

[0049] Where L 1 ,L 2 ,L 3 ,L 4 ,L 5 , M 1 , M 2 As defined herein;

[0050] In one embodiment, L 1 is a C2-C3 alkyl group;

[0051] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0052] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0053] M 1 , M 2 are each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-; R 2 C4-C 20 Straight chain alkyl and alkoxy groups or branched chain alkyl and alkoxy groups;

[0054] In certain embodiments, Formula (I) comprises the structure described by Formula (IV):

[0055]

[0056] Where L 1 ,L 2 ,L 3 ,L 4 ,L 5 , M 1 , M 2 As defined herein;

[0057] In one embodiment, L 1 is a C2-C3 alkyl group;

[0058] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0059] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0060] M 1 , M 2 are each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-; R 3 and R 4 C4-C 20 Straight chain alkyl and alkoxy or branched chain alkyl and alkoxy; In a specific embodiment, the lipid compound of the present application comprises:

[0061]

[0062] or a pharmaceutically acceptable salt, prodrug, stereoisomer thereof.

[0063] The lipid compounds of the present invention (including lipid compounds of formula (I), (II), (III), (IV)) are ionizable cationic compounds, wherein the tertiary amine portion can be protonated at less than physiological pH. The lipid is also a zwitterionic compound. Such zwitterionic compounds are encompassed within the scope of the present invention, whether charged or not.

[0064] Nanoparticle composition

[0065] The present invention relates to a lipid nanoparticle, which comprises the lipid compound of the present invention and may further comprise one or more other lipids.

[0066] Cationic lipids

[0067] In addition to the lipids of the present invention (such as lipids of formula (I), (II), (III), (IV)), the nanoparticle composition may also contain one or more cationic and / or ionizable lipids. Including but not limited to DLinDMA, DODMA, DOTAP, DOTMA, DLin-KC2-DMA, DC-Chol, etc.

[0068] Anionic lipids

[0069] The lipid component of the nanoparticle composition may also include one or more anionic lipids, including but not limited to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, DOPG, DOPS, etc.

[0070] PEG lipids

[0071] The lipid component of nanoparticle composition can also comprise one or more PEG or PEG-modified lipids. Including but not limited to PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.

[0072] Structural lipids

[0073] The lipid component of the nanoparticle composition can also include one or more structured lipids. The structured lipids can include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol.

[0074] Phospholipids

[0075] The lipid component of the nanoparticle composition may also include one or more phospholipids, including but not limited to: 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphatidylcholine (DMPC), 1,2 -Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycerol-3-phosphatidylcholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphatidylcholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycerol-3-phosphatidylcholine, 1,2-diarachidonoyl-sn-glycerol-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycerol-3-phosphatidylcholine, 1,2-diphytanoyl-sn-glycerol-3-phosphatidylethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl Oleylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof. In some embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0076] Effects of the Invention

[0077] The present invention provides a series of novel cationic lipid compounds, which are combined with other lipid compounds to prepare lipid carriers, with controllable particle size and uniform distribution, and exhibit excellent encapsulation efficiency and expression efficiency for drugs with negative charges. Moreover, the compounds of the present invention with specific structures can be selected according to the organs that the drugs need to reach. The effects are significantly better than those of the prior art, and have broad and excellent application prospects.

[0078] Example

[0079] This article uses the following abbreviations:

[0080] THF: Tetrahydrofuran

[0081] MeCN: Acetonitrile

[0082] LAH: Lithium Aluminum Hydride

[0083] DCM: Dichloromethane

[0084] DMAP: 4-dimethylaminopyridine

[0085] LDA: Lithium diisopropylamide

[0086] rt: room temperature

[0087] DME: 1,2-dimethoxyethane

[0088] n-BuLi: n-butyllithium

[0089] CPME: Cyclopentyl Methyl Ether

[0090] EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

[0091] DIEA: N,N-Diisopropylethylamine

[0092] PE: Petroleum ether

[0093] EA: Ethyl acetate

[0094] Example 1: Synthesis of Compound CY-1

[0095] Synthesis of intermediate 1-3

[0096]

[0097] Under nitrogen protection, compound 1-2 (4.20 g, 12.0 mmol, 1.30 eq) and sodium carbonate (2.94 g, 27.7 mmol, 3.00 eq) were added to a solution of isopropanol (25 mL) containing compound 1-1 (5.00 g, 9.24 mmol, 1.00 eq.) and stirred at 80-85 ° C for 20 hours. The reaction mixture was concentrated under reduced pressure to remove isopropanol, the residue was diluted with 200 mL of water and extracted with ethyl acetate (200 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0-0 / 1) to obtain 6.11 g of yellow oily compound 1-3.

[0098] 1 H NMR: (400MHz, CDCl3) δ5.03-4.99`(m,1H),4.90-4.84`(m,1H),4.06(t,J=6.4Hz,2H),3.16-3.06(m,2H),2.51-2.40(m,5H),2. 29(td,J1=7.6,J2=5.2Hz,5H),1.62(t,J=6.8Hz,6H),1.55-1.48(m,5H),1.45(s,11H),1.31-1.26(m,49H),0.90-0.86(m,9H).

[0099] ESI-LCMS: m / z, [M+H] + =809.9.

[0100] Synthesis of intermediates 1-4

[0101]

[0102] To a solution of compound 1-3 (5.50 g, 6.80 mmol, 1.00 eq.) in dichloromethane (15 mL) was added hydrochloric acid / ethyl acetate (4.00 M, 17.0 mL, 10.0 eq.). The mixture was stirred at 20-30 ° C for 0.5 hours. LCMS showed that compound 1-3 was completely consumed. The reaction mixture was diluted with 40 mL of water and adjusted to pH 7-8 with saturated sodium bicarbonate. The mixture was extracted with dichloromethane (20 mL × 3). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue to obtain 4.78 g of a yellow oily compound.

[0103] 1H NMR (400MHz, CDCl3) δ4.90-4.83(m,1H),4.06(t,J=6.8Hz,2H),2.77(t,J=6.0Hz,2H),2.51(t,J=6. 0Hz,4H),2.42(t,J=7.6Hz,4H),2.29-2.27`(m,4H),1.62(t,J=6.8Hz,6H),1.51-1.50(m,4H),1.45 -1.40(m,4H),1.31-1.26(m,48H),0.90-0.86(m,9H).

[0104] ESI-LCMS: m / z, [M+H] + =709.8.

[0105] Synthesis of Compound CY-1

[0106]

[0107] At 20-30°C, N,N-carbonyldiimidazole (1.42 g, 8.76 mmol, 1.30 eq) was added dropwise to a tetrahydrofuran (20 mL) solution containing compound 1-4 (4.78 g, 6.74 mmol, 1 eq). After addition, the mixture was stirred at 20-30°C for 1 hour, and then N,N-diisopropylethylamine (1.74 g, 13.48 mmol, 2.35 mL, 2.00 eq.) and hydroxylamine hydrochloride (562.06 mg, 8.09 mmol, 1.20 eq.) were added dropwise. The resulting mixture was stirred at 20-30°C for 20 hours. LCMS showed that compound 1-4 was completely consumed. The reaction mixture was diluted with 30 mL of water and extracted with ethyl acetate (20 mL×3). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain 2.25 g of compound CY-1 as a yellow oil.

[0108] 1 H NMR (400 MHz, CDCl 3)δ6.65(s,1H),6.51(s,1H),4.90-4.84`(m,1H),4.06(t,J=6.8Hz,2H),3.36-3.32`(m,2H),2.57(t,J=5.6Hz,2 H),2.43(t,J=6.0Hz,4H),2.42(t,J=7.2Hz,4H),2.32-2.27`(m,4H),1.62-1.61(m,6H),1.51-1.50(m,4H),1.45 -1.43(m,4H),1.31-1.26(m,48H),0.90-0.86(m,9H).

[0109] ESI-LCMS: m / z, [M+H] + =768.8.

[0110] Example 2: Synthesis of lipid compound CY-2

[0111] Synthesis of intermediate 2-2

[0112]

[0113] A mixture containing compound 2-1 (18.0 g, 89.5 mmol, 12.5 mL, 1.00 eq.), n-hexyne (22.1 g, 268.58 mmol, 30.2 mL, 3.00 eq.), cuprous iodide (3.41 g, 17.9 mmol, 0.20 eq.), dichlorobis(triphenylphosphine palladium) (12.6 g, 17.9 mmol, 0.20 eq.), triethylamine (400 mL) was protected with nitrogen, and then the mixture was stirred at 80-90 ° C for 32 hours. TLC showed that compound 2-1 was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure to remove triethylamine. The residue was diluted with 300 mL of water and extracted with ethyl acetate (100 mL×3). The combined organic layer was washed with water (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain 11.2 g of compound 2-2 as a yellow oil.

[0114] 1 H NMR (400MHz, CDCl3) δ7.35(d,J=7.6Hz,2H),7.15(d,J=7.2Hz,2H),3.82(t,J=6.4Hz,2H) ,2.84(t,J=6.4Hz,2H),2.41(t,J=6.4Hz,2H),1.62-1.46(m,5H),0.96(t,J=6.8Hz,3H).

[0115] Synthesis of intermediate 2-3

[0116]

[0117] Under N2 protection, palladium / carbon catalyst (5.60 g, 10% purity) was added to a methanol (50 mL) solution of compound 2-2 (11.2 g, 55.4 mmol, 1.00 eq.). The suspension was degassed and purged with hydrogen three times. The mixture was stirred at 60-70 ° C for 24 hours under hydrogen (50 Psi) protection. TLC showed that the reactant 2-2 was completely consumed. The mixture was filtered and concentrated under reduced pressure to obtain a residue to obtain 10.4 g of yellow oily compound 2-3.

[0118] 1 H NMR (400MHz, CDCl3) δ7.14 (s, 4H), 3.86 (t, J = 6.4Hz, 2H), 2.85 (t, J = 6.8Hz, 2H), 2.63-2.57(m,2H),1.63-1.57(m,2H),1.36-1.31(m,6H),0.90(t,J=6.8Hz,3H).

[0119] Synthesis of intermediate 2-4

[0120]

[0121] 4-Dimethylaminopyridine (613 mg, 5.02 mmol, 0.10 eq.) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (10.6 g, 55.2 mmol, 1.10 eq.) were added to a dichloromethane (50 mL) solution containing compound 2-3 (10.4 g, 50.2 mmol, 1.00 eq.) and 8-bromooctanoic acid (12.3 g, 55.2 mmol, 1.10 eq.). The mixture was stirred at 20-30 ° C for 16 hours. TLC showed that compound 2-3 was completely consumed, and the mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0-0 / 1) to obtain 15.6 g of yellow oily compound 2-4.

[0122] 1H NMR (400MHz, CDCl3) δ7.13(s,4H),4.29(t,J=7.2Hz,2H),3.41(t,J=6.8Hz,2H),2.92(t,J=6.8Hz,2H),2.59(t,J=7.6Hz,2H) ,2.30(t,J=7.2Hz,2H),1.88-1.84(m,2H),1.63-1.61(m,4H),1.48-1.40(m,2H),1.38-1.32(m,10H),0.90(t,J=6.4Hz,3H).

[0123] Synthesis of intermediate 2-5

[0124]

[0125] To a solution of compound 2-4 (15.6 g, 37.9 mmol, 1.00 eq.) in isopropanol (75 mL) was added 1-1 (22.6 g, 41.7 mmol, 1.10 eq.), sodium carbonate (12.1 g, 0.114 mol, 3.00 eq.), and the mixture was stirred at 80-85 ° C for 12 hours under nitrogen protection. LCMS showed that compound 2-4 was still present, and the reaction mixture was concentrated under reduced pressure to remove isopropanol. The residue was diluted with 200 mL of water and extracted with ethyl acetate (200 mL×3), and the combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0- / 1) to obtain 18.1 g of yellow oily compound 2-5.

[0126] 1 H NMR (400MHz, CDCl3) δ7.12(s,4H),4.98(s,1H),4.90-4.84(m,1H),4.27(t,J=7.2Hz,2H ),3.15-3.14(m,2H),2.90(t,J=7.2Hz,2H),2.58(t,J=7.6Hz,2H),2.49(t,J=5.6Hz,2H) ,2.38(t,J=6.8Hz,4H),2.30-2.29(m,4H),1.61-1.60(m,7H),1.63-1.61(m,4H),1.52- 1.50(m,5H),1.45(s,9H),1.39-1.38(m,5H),1.30-1.26(m,41H),0.88(t,J=6.4Hz,9H).

[0127] ESI-LCMS: m / z, [M+H] + =871.9

[0128] Synthesis of intermediate 2-6

[0129]

[0130] Hydrochloric acid / ethyl acetate (4.00M, 30mL, 5.77eq.) was added to a dichloromethane (60mL) solution containing compound 2-5 (18.1g, 20.8mmol, 1.00eq.). The mixture was stirred at 20-30°C for 1 hour. LCMS showed that compound 2-6 was completely consumed. The reaction mixture was diluted with 20mL of water and adjusted to pH 7-8 with saturated sodium bicarbonate. The mixture was extracted with dichloromethane (20mL×3). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. 15.5g of compound 2-6 was obtained as a yellow oil.

[0131] 1H NMR: EW43818-10-P1B (400MHz, CDCl3) δ7.12 (s, 4H), 4.89-4.82 (m, 1H), 4.2 6(t,J=7.2Hz,2H),3.47(s,4H),2.96-2.88(m,6H),2.57(t,J=7.6Hz,2H),2 .31-2.26(m,4H),1.72(s,4H),1.61-1.60(m,6H),1.51-1.50(m,4H),1.35- 1.31(m,13H),1.31-1.30(m,7H),1.30-1.26(m,22H),0.88(t,J=6.4Hz,9H).

[0132] ESI-LCMS: m / z, [M+H]+=771.7.

[0133] Synthesis of Compound CY-2

[0134]

[0135] At 20-30°C, N,N-carbonyldiimidazole (2.73 g, 16.9 mmol, 1.30 eq.) was added dropwise to a tetrahydrofuran (50 mL) solution containing compound 2-6 (10.0 g, 12.9 mmol, 1.00 eq.). After addition, the mixture was stirred at 20-30°C for 1 hour, and then N,N-diisopropylethylamine (3.35 g, 25.9 mmol, 4.52 mL, 2.00 eq.) and hydroxylamine hydrochloride (1.08 g, 15.6 mmol, 1.20 eq.) were added. The resulting mixture was stirred at 20-30°C for 12 hours. LCMS showed that compound 2-6 was completely consumed. The reaction mixture was diluted with 50 mL of water and extracted with ethyl acetate (50 mL*3). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain 6.37 g of compound CY-3 as a yellow oil.

[0136] 1 H NMR (400MHz, CDCl3) δ7.12(s,4H),6.65(s,1H),6.52(s,1H),4.90-4.84(m,1 H),4.27(t,J=7.2Hz,2H),3.36(d,J=4.8Hz,2H),2.90(t,J=7.2Hz,2H),2.60- 2.58(m,4H),2.46(t,J=7.2Hz,4H),2.32-2.28(m,4H),1.61-1.58(m,6H),1.5 2-1.50(m,4H),1.50-1.45(m,4H),1.30-1.26(m,42H),0.88(t,J=6.4Hz,9H).

[0137] ESI-LCMS: m / z, [M+H] + =830.7.

[0138] Example 3: Synthesis of compounds CY-3 to CY-6

[0139] The synthesis process of CY-3~CY-6 is the same as that of CY-1 / 2, and only the relevant intermediates need to be replaced.

[0140] Example 4: Preparation of lipid nanoparticles

[0141] Preparation of lipid solution: Dissolve ionizable lipid: DSPC: cholesterol: mPEG2000-DMG in ethanol solution at a molar ratio of 50:10:38.5:1.5;

[0142] Preparation of mRNA solution: Dissolve a certain amount of luciferase mRNA in 20 mM citric acid buffer solution at pH = 4.0;

[0143] Preparation of lipid nanoparticles: 1 mL of luciferase mRNA and 3 mL of lipid solution were drawn into a syringe, respectively, and inserted into the microfluidic chip. The parameters were set as follows: Volume: 4.0 mL; Flowrate ratio: 3:1, Total flow rate: 18 mL / min, and the lipid nanoparticle solution was obtained by mixing.

[0144] Solution replacement: The lipid nanoparticle solution is added to an ultrafiltration tube for centrifugal ultrafiltration, and the phosphate buffer is replaced multiple times to obtain a finished product.

[0145] Example 5: Characterization of lipid nanoparticles

[0146] The particle size, polydispersity index (PDI), and zeta potential of nanoparticle compositions can be measured using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). The particle size is measured in 1× PBS and the zeta potential is measured in 15 mM PBS. For nanoparticle compositions comprising RNA, the QUANT-IT TM RNA assay (Invitrogen Corporation Carlsbad, CA) evaluates the encapsulation of RNA by nanoparticle compositions. The sample was diluted to a concentration of about 5 μg / mL in TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted sample was transferred to a polystyrene 96-well plate and 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution was added to each well. The plate was incubated at 37°C for 15 minutes. The RIBOGREENR reagent was diluted 1:100 in TE buffer and 100 μL of the solution was added to each well. Fluorescence intensity can be measured using a fluorescent plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA) at, for example, an excitation wavelength of about 480 nm and an emission wavelength of about 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and the percentage of free RNA was determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0147] Table 1. Physicochemical characterization of lipid nanoparticles prepared from lipid compounds synthesized in the examples of the present invention

[0148] Serial number Cationic lipids Size(nm) PDI Potential (mV) Encapsulation rate (%) 1 MC3 91±1.1 0.13±0.02 -0.71±0.13 89.2 2 CY-1 109±0.1 0.05±0.01 21.1±0.91 92.2 3 CY-2 84±0.9 0.17±0.01 21.5±1.32 95.0 4 CY-3 83±0.3 0.14±0.01 20.0±1.13 97.2 5 CY-4 87±0.2 0.15±0.01 17.8±0.42 90.2 6 CY-5 73±1. 0.19±0.03 13.1±0.71 93.2 7 CY-6 87±0.06 0.13±0.02 13.8±0.42 95.5

[0149] Example 6: In vitro cell expression detection

[0150] In vitro cell expression evaluation method: B16F10 cells and HSKMC cells were cultured and plated in 96-well plates at 100,000 cells per well. 100 ng of LNP sample was taken and mixed with cells for 24 hours (n=3). 24 hours after administration, the protein expression was detected using a luciferase quantitative kit. The positive control of the present invention was the commercially available cationic lipid MC3, and the negative control was PBS. Figure 1 It can be seen that the lipid nanoparticles prepared from each compound have a certain degree of in vitro cellular protein expression.

[0151] Example 7: Protein expression study in mice

[0152] After the lipid nanoparticles are injected into the mouse through intravenous injection, intramuscular injection, etc., the time course of protein expression can be measured and evaluated by enzyme-linked immunosorbent assay (ELISA), bioluminescence imaging or other methods. By comparing with the positive control and comparing the amount of translated protein, the delivery performance of the lipid molecule can be evaluated. Specifically, each lipid nanoparticle sample containing 3 μg of luciferase mRNA was taken and injected intramuscularly into Balb / C mice. Six hours after administration, the mice were intraperitoneally injected with 0.15 mg / kg of D-luciferin substrate. Within 15 minutes after the substrate injection, the mice were placed under a small animal in vivo imaging device to detect the fluorescence intensity of the liver and the injection site. Figure 2 As shown, obvious fluorescence signals can be observed for all lipid nanoparticles, and the fluorescence signals of many compounds are stronger than those of commercially available MC3 lipids.

[0153] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and apply the present application. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to pay creative labor. Therefore, the present application is not limited to the embodiments herein, and improvements and modifications made by those skilled in the art based on the contents disclosed in the present application without departing from the scope and spirit of the present application are within the scope of the present application.

Claims

1. A lipid compound, characterized in that The lipid compound is a cationic lipid compound having a structure as shown in formula (III) or formula (IV) or a pharmaceutically acceptable salt or stereoisomer thereof: in, L 1 is C2-C3 alkylene; L 2 ,L 3 Each independently selected from C4-C9 straight chain alkylene; L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkylene; M 1 and M 2 Each independently selected from -OC(O)-, -C(O)O- or -NHC=N(CN)NH-; R 2 C4-C 20 Straight chain alkyl or branched chain alkyl or R 2 C4-C 20 Alkoxy; R 3 C4-C 20 Straight chain alkyl or branched chain alkyl or R 3 C4-C 20 Alkoxy; R 4 C4-C 20 Straight chain alkyl or branched chain alkyl or R 4 C4-C 20 Alkoxy; G 2 Selected from H or C5-C 10 Aryl, wherein C5-C 10 Each hydrogen atom on the aryl group is independently optionally replaced by n -XR 1 substituted, n is an integer of 1-2, X is nitrogen, oxygen, R 1 C4-C 20 Straight chain or branched chain alkyl.

2. The lipid compound according to claim 1, characterized in that It has the following structure:

3. A lipid nanoparticle, characterized in that: The lipid nanoparticles comprise the lipid compound according to any one of claims 1 or 2.

4. The lipid nanoparticle according to claim 3, characterized in that The lipid nanoparticles contain neutral lipids, polyethylene glycol lipids, sterol lipids and one or more bioactive agents.

5. The lipid nanoparticle according to claim 4, characterized in that The neutral lipid is one of DSPC, DOPE, DPPC or POPC, the polyethylene glycol lipid is PEG2000-DMG, the sterol lipid is cholesterol, and the bioactive agent is RNA, mRNA, siRNA, ASO, DNA, tRNA, rRNA, miRNA, plasmid or snRNA.

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