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 contains 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 by certain means, and achieve therapeutic and preventive effects by translating target proteins or silencing target mRNA in the human body. Delivery methods include viral vectors and non-viral vectors. Viral vectors are limited in their application due to their own toxicity and immunogenicity. Non-viral vectors, especially lipid nanoparticles, encapsulate nucleic acid drugs and deliver them to target cells by assembling liposome molecules, and have achieved commercial application.

[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] R 1 Yes-CR 1a R 1b R 1c , R 1a R 1b R 1c are the same as or different from each other and are independently selected from H, halogen, hydroxy, alkyl, alkoxy, amino, aminoalkyl, diaminoalkyl;

[0009] R 2It is halogen, or -CR 2a R 2b R 2c , R 2a R 2b R 2c are the same as or different from each other, and are each independently selected from H, halogen, hydroxy, alkyl, alkoxy, amino, aminoalkyl, diaminoalkyl;

[0010] M 1 and M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0011] 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 3 substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 3 C4-C 20 Straight or branched alkyl, C4-C 20 Straight or branched alkenyl;

[0012] On the other hand, the present invention provides a lipid nanoparticle comprising the cationic lipid compound or an acceptable salt or stereoisomer thereof according to any one of claims 1 to 10.

[0013] 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.

[0014] 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

[0015] 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.

[0016] 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

[0017] Definition of terms

[0018] The term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms.

[0019] The term "alkoxy" refers to an alkyl group as described herein that is attached to the remainder of the molecule through an oxygen atom.

[0020] The term "alkylene" refers to a divalent group formed by the loss of a hydrogen atom from a corresponding alkyl group.

[0021] 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.

[0022] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The term "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system.

[0028] The term "particle size" refers to the average diameter of a nanoparticle composition.

[0029] The term "zeta potential" refers to the surface potential of lipids in, for example, a nanoparticle composition.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The term "expression" refers to the translation of mRNA into polypeptides or proteins and / or polypeptides or proteins.

[0034] 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.

[0035] Lipid compounds

[0036] In one aspect, the present invention provides a cationic lipid compound having a structure as shown in formula (I):

[0037]

[0038] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein

[0039] 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;

[0040] R 1 Yes-CR 1a R 1b R 1c , R 1a R 1b R 1care the same as or different from each other and are independently selected from H, halogen, hydroxy, alkyl, alkoxy, amino, aminoalkyl, diaminoalkyl;

[0041] R 2 It is halogen, or -CR 2a R 2b R 2c , R 2a R 2b R 2c are the same as or different from each other, and are each independently selected from H, halogen, hydroxy, alkyl, alkoxy, amino, aminoalkyl, diaminoalkyl;

[0042] M 1 and M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0043] 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 3 substituted, n is an integer from 1 to 3, X is absent, nitrogen, oxygen, sulfur, selenium, R 3 C4-C 20 Straight or branched alkyl, C4-C 20 Straight or branched alkenyl;

[0044] 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.

[0045] In one embodiment, R 1 is an alkyl group;

[0046] In one embodiment, R 2 is halogen or alkyl;

[0047] 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 3 substituted, n is an integer of 1-2, X is nitrogen, oxygen, R3 C4-C 20 Straight or branched chain alkyl;

[0048] In certain embodiments, Formula (I) comprises the structure described by Formula (II):

[0049]

[0050] Where R 1 , R 2 , L 1 ,L 2 ,L 3 ,L 4 ,L 5 , M 1 , M 2 As defined herein;

[0051] In one embodiment, R 1 is a C1-C3 alkyl group;

[0052] R 2 is a halogen;

[0053] L 1 is a C2-C3 alkyl group;

[0054] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0055] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0056] M 1 , M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0057] In certain embodiments, Formula (I) comprises the structure described by Formula (III):

[0058]

[0059] Where R 2 , L 1 ,L 2 ,L 3 ,L 4 ,L 5 , M 1 , M 2 As defined herein;

[0060] In one embodiment, R 2 is a halogen;

[0061] L 1 is a C2-C3 alkyl group;

[0062] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0063] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0064] M 1 , M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0065] In certain embodiments, Formula (I) comprises the structure described by Formula (IV):

[0066]

[0067] Where L 1 ,L 2 ,L 3 ,L 4 ,L 5 , M 1 , M 2 As defined in any of the clauses herein;

[0068] In one embodiment, L 1 is a C2-C3 alkyl group;

[0069] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0070] L 4 ,L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0071] M 1 , M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0072] In certain embodiments, Formula (I) comprises the structure described by Formula (V):

[0073]

[0074] Where L 1 ,L 2 ,L 3 ,L 4,L 5 , M 1 , M 2 As defined in any of the clauses herein;

[0075] In one embodiment, L 1 is a C2-C3 alkyl group;

[0076] L 2 ,L 3 Each independently selected from C4-C9 straight chain alkyl;

[0077] L 5 Each independently selected from C4-C 20 Straight or branched chain alkyl;

[0078] M 1 , M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-;

[0079] R 3 C4-C 20 Straight or branched chain alkyl;

[0080] In a specific embodiment, the lipid compound of the present application comprises:

[0081]

[0082]

[0083] or a pharmaceutically acceptable salt, prodrug, stereoisomer thereof.

[0084] The lipid compounds of the present invention (including lipid compounds of formula (I), (II), (III), (IV) or (V)) are ionizable cationic compounds, wherein the tertiary amine moiety can be protonated at less than physiological pH. The lipids are also zwitterionic compounds. Such zwitterionic forms of compounds are encompassed within the scope of the present invention, whether charged or not.

[0085] Nanoparticle composition

[0086] 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.

[0087] Cationic lipids

[0088] In addition to the lipids of the present invention (such as lipids of formula (I), (II), (III), (IV) or (V)), 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.

[0089] Anionic lipids

[0090] 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.

[0091] PEG lipids

[0092] The lipid component of the nanoparticle composition can also include 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.

[0093] Structural lipids

[0094] 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.

[0095] Phospholipids

[0096] The lipid component of the nanoparticle composition may also include one or more phospholipids, which may include but are 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- Glycerol-phosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1,2-diundecanoyl-sn-glycerol-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-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-Dilinolenoyl-sn-glycerol-3-phosphatidylcholine, 1,2-Diarachidonoyl-sn-glycerol-3-phosphatidylcholine, 1,2-Bisdocosahexaenoyl-sn-glycerol-3-phosphatidylcholine, 1,2-Diphytanoyl-sn-glycerol-3-phosphatidylethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-Dilinoleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-Dilinolenoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-Diarachidonoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-Bisdocosahexaenoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (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, nanoparticle compositions comprise both DSPC and DOPE.

[0097] Effects of the Invention

[0098] 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.

[0099] Example

[0100] This article uses the following abbreviations:

[0101]

[0102] Example 1: Synthesis of lipid compound SY-1

[0103]

[0104] Synthesis of intermediate 1-12

[0105]

[0106] Potassium acetate (15 g, 153 mmol, 2.5 eq.) was added to an acetic acid solution (100 mL) containing compound 1-11 (10 g, 60 mmol, 1 eq.) and methylamine hydrochloride (10 g, 143 mmol, 2.3 eq.), and the mixture was stirred at 110 ° C for 3 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 5 / 1) showed that a new main point was observed. Saturated NaHCO3 solution (50 mL) was added to the reaction mixture, extracted with ethyl acetate (50 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with petroleum ether / ethyl acetate (1 / 0-4:1, v / v) to obtain white solid compound 1-12 (8 g, 66%).

[0107] Synthesis of intermediate 1-3

[0108]

[0109] To a dichloromethane solution (100 mL) containing compound 1-1 (5 g, 21 mmol, 1 eq.) and compound 1-2 (4 g, 32 mmol, 1.5 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8 g, 45 mmol, 2 eq.), 4-dimethylaminopyridine (1 g, 89 mmol, 0.4 eq.) were added, and then diisopropylethylamine (12 g, 89.7 mmol, 4 eq.) was added. The reaction mixture was stirred overnight at room temperature under nitrogen protection, and the reaction mixture was quenched with aqueous solution. The pH was adjusted to 5-6 with 1M HCl, and extracted with ethyl acetate (300 mL×3). The organic layer was washed with saturated brine (300 mL×2), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 8, v / v) to give compound 1-3 (3.75 g, 50%) as a yellow oil.

[0110] Synthesis of intermediates 1-4

[0111]

[0112] To a dichloromethane solution (200 mL) containing compound 1-1 (10 g, 44.8 mmol, 1 eq.) and compound 1-2B (17.2 g, 67.3 mmol, 1.5 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16 g, 90 mmol, 2 eq.), 4-dimethylaminopyridine (2 g, 19.8 mmol, 0.4 eq.) and then diisopropylethylamine (24 g, 180 mmol, 4 eq.) were added. The reaction mixture was stirred overnight at room temperature under nitrogen protection, and the reaction mixture was quenched with aqueous solution. The pH was adjusted to 5-6 with 1M HCl and extracted with ethyl acetate (300 mL×3). The organic layer was washed with saturated brine (300 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 8, v / v) to give yellow oily compound 1-3 (10.3 g, 50%).

[0113] Synthesis of intermediates 1-6

[0114]

[0115] An ethanol solution (5 mL) containing compound 1-4 (1 g, 2.18 mmol, 1.0 eq.) and compound 1-5 (5.0 g, 66.00 mmol, 30 eq.) was stirred at 60 ° C for 16 hours under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed that compound 1-4 was consumed and a new major spot was observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with ethyl acetate (150 mL), washed with water (2×40 mL), and then washed with brine (2×30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give a yellow oil compound 1-5 (610 mg, 60%).

[0116] Synthesis of intermediates 1-7

[0117]

[0118] Potassium carbonate (510 mg, 3.42 mmol, 5 eq.), potassium iodide (220 mg, 1.41 mmol, 2 eq.) and cyclopentyl methyl ether (3.0 mL) were added to an acetonitrile solution (3.0 mL) containing compound 1-6 (300 mg, 0.66 mmol, 1.0 eq.) and compound 1-3 (320 mg, 1.01 mmol, 1.5 eq.). The reaction mixture was stirred at 90 ° C overnight under nitrogen protection. TLC (DCM / MeOH=10 / 1) showed that the product was consumed and a new major spot was observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL), washed with water (2×20 mL), and then washed with saturated brine (2×20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 10% MeOH / DCM to give compound 1-7 (391 mg, 91%) as a yellow oil.

[0119] Synthesis of intermediates 1-8

[0120]

[0121] At 0°C and under nitrogen protection, a solution of compound 1-7 (370 mg, 0.54 mmol, 1 eq.) and triethylamine (220 mg, 2.11 mmol, 4 eq.) in dichloromethane (4.0 mL) was stirred at room temperature for 10 minutes, and then methanesulfonyl chloride (180 mg, 1.62 mmol, 3 eq.) was added dropwise. The reaction mixture was stirred at 25°C for 16 hours under nitrogen protection. TLC (MeOH / DCM=1 / 15) showed that the reaction was complete and new major spots were observed. The mixture was quenched with water (50 mL) and extracted with dichloromethane (40 mL×3). The combined organic layer was washed with saturated brine (2×20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude compound 1-8 (400 mg) as a yellow oil, and the residue was used directly in the next step.

[0122] Synthesis of intermediates 1-9

[0123]

[0124] Under nitrogen protection, sodium azide (180 mg, 2.71 mmol, 5 eq.) was added to a dimethyl sulfoxide solution (5 mL) containing compound 1-8 (400 mg, 0.52 mmol, 1 eq.). After addition, the reaction mixture was stirred at 100 ° C for 16 hours under nitrogen protection. TLC (MeOH / DCM=1 / 15) showed that compound 8 was consumed and a new major spot was observed. The mixture was diluted with ethyl acetate (100 mL) and washed with water (30 mL×3) and brine (30 mL×2). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH / DCM=1 / 15) to give a light yellow oil compound 1-9 (280 mg, 98%)

[0125] Synthesis of Intermediates 1-10

[0126]

[0127] Pd / C (100 mg) was added to an ethanol solution (3 mL) containing compound 1-9 (280 mg, 0.45 mmol, 1 eq.), and then stirred at room temperature for 16 hours under a hydrogen atmosphere. TLC (DCM / MeOH=10 / 1) showed that compound 1-9 was consumed and new major spots were observed. The mixture was filtered and washed with ethanol (20 mL×2). The filtrate was concentrated under reduced pressure to obtain a pale yellow oily compound 1-10 crude product (210 mg, 81%).

[0128] Synthesis of SY-1

[0129]

[0130] Sodium carbonate (200 mg, 2.0 mmol, 3.0 eq.) was added to a tetrahydrofuran solution (5 mL) containing compound 1-10 (200 mg, 0.5 qmmol, 1.0 eq.) and compound 1-12 (200 mg, 1.24 mmol, 2.0 eq.). The mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 4 / 1) showed that compound 1-10 was consumed and a new main spot was observed. Water (15 mL) was added to the reaction mixture and extracted with ethyl acetate (3 × 20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0-4: 1, v / v) to give a yellow oily compound SY-1 (100 mg, 44.3%).

[0131] LCMS:Rt:2.231min;MS m / z(ELSD):852.6[M+H] + ;

[0132] HPLC: 97.81% purity at ELSD; RT=10.911min.

[0133] 1 H NMR (400MHz, CDCl3) δ7.76–7.66(m,1H),4.92–4.81(m,1H),4.05(t,J= 8.0Hz,2H),3.80–3.70(m,2H),2.99(s,3H),2.62–2.45(m,2H),2.43–2.36(m,4H) ,2.31–2.22(m,4H),1.92–1.75(m,2H),1.72–1.58(m,6H),1.57–1.43(m,9H),1.42 –1.22(m,45H),0.94–0.81(m,9H).

[0134] 13C NMR (100MHz, CDCl3) δ174.91,174.61,168.20,164.55,140.22,131.91,129.82,77.37,77.13,76.70,74.32,67.34,53.16,53 .01,42.88,35.61,33.33,31.10,31.01,30.68,29.77,29.54,29.50,29.39,29.31,29.25,29.24,29.20,28.76,27.54,27.50, 26.48,25.95,25.61,25.34,25.23,24.98,24.42,23.67,20.19,14.21.

[0135] Example 2: Synthesis of lipid compound SY-2

[0136]

[0137] Synthesis of intermediate 2-3

[0138]

[0139] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.8 g, 9 mmol, 2 eq.) and 4-dimethylaminopyridine (200 mg, 1.64 mmol, 0.4 eq.) were added to dichloromethane (15 mL) containing compound 2-1 (1 g, 4.5 mmol, 1 eq.) and compound 2-2 (600 mg, 6.72 mmol, 1.5 eq.), and then diisopropylethylamine (2.4 g, 19 mmol, 4 eq.) was added. The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. The reaction mixture was quenched with aqueous solution. 1M HCl was used to adjust the pH to 5-6, and extracted with ethyl acetate (90 mL×3). The organic layer was washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 8, v / v) to give yellow oily compound 2-3 (800 mg, 57%).

[0140] Synthesis of intermediate 2-6

[0141]

[0142] Potassium carbonate (900 mg, 6.60 mmol, 5 eq.), potassium iodide (450 mg, 2.7 mmol, 2 eq.) and cyclopentyl methyl ether (6.0 mL) were added to a solution of acetonitrile (5 mL) containing compound 1-6 (600 mg, 1.32 mmol, 1.0 eq.) and compound 2-5 (580 mg, 2 mmol, 1.5 eq.). The reaction mixture was stirred at 90 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that compound 1-6 was consumed and a new major spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate (80 mL) and washed with water (2×40 mL) and saturated brine (2×40 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH / DCM=1 / 20) to give a yellow oily compound 2-6 (680 mg, 80%).

[0143] Synthesis of intermediate 2-7

[0144]

[0145] A dichloromethane solution (20 mL) containing compound 2-6 (680 mg, 1 mmol, 1 eq.) and triethylamine (400 mg, 4.0 mmol, 4 eq.) was stirred at room temperature for 10 minutes, and MsCl (300 mg, 2.55 mmol, 2.5 eq.) was added dropwise at 0 ° C. and under nitrogen protection. The reaction mixture was stirred at 25 ° C. for 5 hours under nitrogen protection. TLC (MeOH / DCM=1 / 15) showed that the reaction was complete and new major spots were observed. The mixture was quenched with water (50 mL) and extracted with dichloromethane (40 mL×3). The combined organic layer was washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with (MeOH / DCM=1 / 20) to obtain a yellow oily crude compound 2-7 (700 mg).

[0146] Synthesis of Intermediate 2-8

[0147]

[0148] Under nitrogen protection, sodium azide (150 mg, 2.3 mmol, 5 eq.) was added to DMF (5 mL) containing compound 2-7 (350 mg, 0.50 mmol, 1.05 eq.). After addition, the reaction mixture was stirred at 100 ° C for 16 hours under nitrogen protection. TLC (MeOH / DCM=1 / 15) showed that compound 2-7 was consumed and a new major spot was observed. The mixture was diluted with ethyl acetate (50 mL) and washed with water (30 mL×3) and saturated brine (30 mL×2). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with (MeOH / DCM=1 / 20) to obtain a light yellow oily compound 2-8 (250 mg, 78%).

[0149] Synthesis of Intermediate 2-9

[0150]

[0151] Pd / C (100 mg) was added to an ethanol solution (3 mL) containing compound 2-8 (250 mg, 0.35 mmol, 1 eq.), and then stirred at room temperature for 16 h under hydrogen protection. TLC (DCM / MeOH=15 / 1) showed that compound 2-8 was consumed and new major spots were observed. The mixture was filtered and washed with ethanol (20 mL×2), and concentrated under reduced pressure to obtain a pale yellow oily compound 2-9 crude product (200 mg, 85.6%).

[0152] Synthesis of lipid compound SY-2

[0153]

[0154] The solution in ethanol solution (3mL) containing compound 2-9 (200mg, 0.31mmol, 1eq.) and compound 1-12 (100mg, 0.52mmol, 1.5eq) was stirred overnight at 25°C under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (using DCM / MeOH=1 / 0-20 / 1, v / v) to obtain a brown oily product (70mg, 30%).

[0155] LCMS:Rt:1.481min; MS m / z(ELSD):810.5[M+H] + ;

[0156] HPLC: 93.5% purity at ELSD; RT=11.917min.

[0157] 1 H NMR (400MHz, CDCl3) δ7.68 (s, 1H), 4.91–4.88 (m, 1H), 4.02 (t, J = 6.2Hz, 2H),3.73(d,J=5.2Hz,2H),2.92(s,3H),2.62(s,2H),2.50–2.38(m,4H),2.22(m,J =7.6,4.4Hz,4H),1.79(d,J=5.2Hz,2H),1.62(m,J=6.8,2.8Hz,6H),1.50(d,J=5.2Hz,8H),1.34–1.18(m,40H),0.92(m,J=13.2,4.6Hz,9H).

[0158] Example 3: Synthesis of lipid compound SY-3

[0159]

[0160] Synthesis of intermediate 3-3

[0161]

[0162] Compound 3-2 (800 mg, 5.4 mmol, 1.2 eq.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.8 g, 9.6 mmol, 2.0 eq.), diisopropylethylamine (2.4 g, 18.0 mmol, 4.0 eq.) and 4-dimethylaminopyridine (220 mg, 1.8 mmol, 0.4 eq.) were added to a dichloromethane solution (10 mL) containing compound 3-1 (1 g, 4.48 mmol, 1.0 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen. TLC (PE / EA=30 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with 1M HCl and adjusted to pH=4-6, and extracted with DCM (50 mL×3). The combined organic layer was washed with brine (2×50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography

[0163] (PE / EA=30 / 1) to obtain yellow oily compound 3-3 (1.3 g, 89%).

[0164] Synthesis of intermediate 3-5

[0165]

[0166] Potassium carbonate (900 mg, 6.58 mmol, 5.0 eq) was added to an acetonitrile solution (2 mL) containing compound 3-4 (600 mg, 1.32 mmol, 1 eq.) and compound 3-3 (500 mg, 1.58 mmol, 1.2 eq), potassium iodide (440 mg, 2.64 mmol, 2.0 eq) and cyclopentyl methyl ether (2 mL). The reaction mixture was stirred overnight at 90 ° C under nitrogen protection. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to obtain a light yellow oily compound 3-5 (350 mg, 42%).

[0167] Synthesis of intermediate 3-6

[0168]

[0169] At 0 ° C, triethylamine (100 mg, 0.93 mmol, 4.0 eq.) and methanesulfonyl chloride (120 mg, 1.1 mmol, 2.5 eq.) were added to a dichloromethane solution (5 mL) containing compound 3-5 (300 mg, 0.45 mmol, 1.0 eq.). After addition, the mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (PE / EA=3 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layer was washed with brine (2×30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a yellow oily crude compound 3-6 (300 mg), and the residue was used directly in the next step.

[0170] Synthesis of intermediate 3-7

[0171]

[0172] A dimethylformamide solution (5 mL) containing compound 3-6 (300 mg, 0.21 mmol, 1.0 eq.) and sodium azide (130 mg, 2 mmol, 5.0 eq) was stirred at 100 ° C for 16 h under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that compound 3-6 was consumed and a new main spot was observed. The reaction was quenched with water (20 ml) and extracted with ethyl acetate (20×3 mL). The combined organic layer was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-15 / 1, v / v) to obtain a yellow oily compound 3-7 (240 mg, 80%).

[0173] Synthesis of intermediate 3-8

[0174]

[0175] Pd / C (80 mg) was added to an ethanol solution (3 mL) containing compound 3-7 (240 mg, 0.24 mmol, 1 eq.). The reaction mixture was stirred overnight at 25 ° C under hydrogen conditions. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and new major spots were observed. The reaction was filtered and the filter cake was washed with ethanol (20 mL x 2). The combined organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (1 / 0-10 / 1, v / v) to obtain yellow oily compound 8 (150 mg, 66%).

[0176] Synthesis of compound SY-3

[0177]

[0178] Potassium carbonate (120 mg, 0.9 mmol, 3.0 eq) and compound 1-12 (50 mg, 0.3 mmol, 2.0 eq) were added to an acetonitrile solution (4 mL) containing compound 3-8 (150 mg, 0.25 mmol, 1 eq.). The reaction mixture was stirred at 25 ° C for 4 hours. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched with water (40 ml) and extracted with ethyl acetate (40×3 mL). The combined organic layer was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-15 / 1, v / v) to give a yellow oily compound SY-3 (100 mg, 62%).

[0179] LCMS:Rt:1.435min;MS m / z(ELSD):824.5[M+H] + ;

[0180] HPLC:95% purity at ELSD; RT=7.916min.

[0181] 1H NMR (400MHz, CDCl3) δ7.42 (d, J = 17.2Hz, 1H), 4.76 (p, J = 6.4Hz, 1H), 3.96(t,J=6.8Hz,2H),3.72(d,J=5.4Hz,2H),2.97(s,3H),2.54(d,J=17.6Hz,2H), 2.42(s,4H),2.21(dd,J=15.6,7.8Hz,4H),1.72(s,2H),1.60–1.50(m,6H),1.40(d, J=5.4Hz,7H),1.30–1.00(m,43H),0.80(dd,J=7.6,6.1Hz,9H).

[0182] Example 4: Synthesis of lipid compound SY-4

[0183]

[0184] Synthesis of intermediate 4-3

[0185]

[0186] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8g, 40mmol, 2eq.), 4-dimethylaminopyridine (200mg, 4.1mmol, 0.2eq.) and diisopropylethylamine (10g, 82mmol, 4eq.) were added to a dichloromethane solution (20mL) containing compound 4-1 (4g, 20.52mmol, 1eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 30 / 1) showed that compound 4-1 was consumed and the desired product was formed. The pH was adjusted to 6-7 with 1M HCl, and then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=1 / 0-20 / 1) to give compound 4-3 (4 g, 58%) as a colorless oil.

[0187] Synthesis of intermediate 4-6

[0188]

[0189] An ethanol solution (5 mL) containing compound 4-4 (5 g, 10.87 mmol, 1.0 eq.) and compound 4-5 (25 g, 330 mmol, 30 eq.) was stirred at 80 ° C for 16 hours. TLC (DCM / MeOH=10 / 1) showed that a new main spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-10:1, v / v) to give yellow oily compound 4-6 (3.9 g, 79%).

[0190] Synthesis of intermediate 4-7

[0191]

[0192] Potassium carbonate (1 g, 8 mmol, 6.0 eq.) and potassium iodide (400 mg, 2.5 mmol, 2 eq.) were added to an acetonitrile solution (10 mL) containing compound 4-6 (600 mg, 1.30 mmol, 1 eq.) and compound 4-3 (400 mg, 1.30 mmol, 1 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen protection. TLC (DCM / MeOH=10 / 1) showed that a new main spot was observed. The mixture was diluted with water and extracted 3 times with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-10:1, v / v) to give a yellow oily compound 4-7 (400 mg, 65%).

[0193] Synthesis of intermediate 4-8

[0194]

[0195] Under nitrogen protection, methylsulfonyl chloride (0.04 mL, 0.56 mmol, 1.25 eq.) was added dropwise to dichloromethane (5 mL) containing compound 4-7 (300 mg, 0.45 mmol, 1 eq.) and triethylamine (0.08 mL, 0.58 mmol, 1.3 eq.) at 0 ° C. After addition, the reaction mixture was stirred overnight at 25 ° C under nitrogen protection. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water and extracted with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a yellow oily compound 4-8 (500 mg).

[0196] Synthesis of intermediate 4-9

[0197]

[0198] Sodium azide (220 mg, 3.4 mmol, 5 eq.) was added to a dimethylformamide solution (5 mL) containing compound 4-8 (500 mg, 0.66 mmol, 1 eq.). After addition, the reaction mixture was stirred at 100 ° C for 16 hours under nitrogen protection. TLC (PE / EA=2 / 1) showed that a new major spot was observed. The mixture was diluted with ethyl acetate and washed three times with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain yellow oily compound 4-9 (240 mg, 65%).

[0199] Synthesis of intermediate 4-10

[0200]

[0201] Pd / C (25 mg, 10% wt) was added to ethanol (5 mL) containing compound 4-9 (240 mg, 0.34 mmol, 1 eq.). After addition, the reaction mixture was stirred at 25 ° C for 3 hours under hydrogen conditions (30 psi). TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was filtered through diatomaceous earth and the filter cake was washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure to obtain a yellow oily compound 4-10 (240 mg).

[0202] Synthesis of Compound SY-4

[0203]

[0204] Compound 1-12 (100 mg, 0.52 mmol, 1.8 eq.) and sodium carbonate (100 mg, 0.88 mmol, 2.5 eq.) were added to a tetrahydrofuran (5 mL) solution containing compound 4-10 (240 mg, 0.33 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to give a yellow oily compound SY-4 (80 mg, 30%).

[0205] LCMS:Rt:1.222min; MS m / z(ELSD):796.5[M+H] + ;

[0206] HPLC: 95.2% purity at ELSD; RT=11.027min.

[0207] 1 H NMR (400MHz, CDCl3) δ4.92–4.80 (m, 1H), 4.02 (t, J = 7.2Hz, 2H), 3.77 (d,J=5.2Hz,2H),2.69(s,3H),2.57(d,J=60.2Hz,5H),2.32–2.21(m,4H),1.81(s ,2H),1.70–1.40(m,15H),1.30(dd,J=24.9,18.7Hz,39H),0.86(td,J=6.8,3.2Hz, 9H).

[0208] 13C NMR (100MHz, CDCl3) δ173.64,168.09,165.53,141.09,77.30,77.04, 76.72,74.14,64.50,34.65,34.16,31.79,29.51,29.19,28.91,28.65,27.31,26.98,25.79,25.31,25.05,24.78,24.04,22.61,14.08.

[0209] Example 5: Synthesis of lipid compound SY-5

[0210]

[0211]

[0212] The synthesis of intermediate 5-1 refers to the aforementioned synthesis route.

[0213] Sodium carbonate (120 mg, 1.11 mmol, 2.5 eq.) was added to a tetrahydrofuran solution (3 mL) containing compound 5-1 (300 mg, 0.44 mmol, 1 eq.) and compound 1-12 (120 mg, 0.66 mmol, 1.5 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (1 / 0-10 / 1, v / v) to give a light yellow compound SY-5 (280 mg, 75%).

[0214] LCMS:Rt:1.271min;MS m / z(ELSD):852.5[M+H]+ ;

[0215] HPLC: 96.5% purity at ELSD; RT=6.134min.

[0216] 1H NMR (400MHz, CDCl3) δ4.86 (s, 1H), 4.05 (t, J=6.8Hz, 2H), 3.77 (d, J= 5.1Hz,2H),2.99(d,J=5.3Hz,3H),2.59(s,2H),2.43(s,3H),2.28(td,J=7.5,5.8Hz ,4H),1.77(s,2H),1.70–1.39(m,15H),1.39–1.21(m,47H),0.88(td,J=6.8,3.1Hz, 9H).

[0217] 13C NMR (100MHz, CDCl3) δ173.85,173.61,165.58,77.29,77.03,76.71,74.12,64.45,34.68,34.20,3 1.79,29.52,29.42–28.81,28.66,27.33,27.20–26.86,25.80,25.32,25.02,24.02,22.62,14.08.

[0218] Example 6: Synthesis of lipid compound SY-6

[0219]

[0220] The synthesis of intermediate 5-1 refers to the aforementioned synthesis route.

[0221] Sodium carbonate (100 mg, 0.86 mmol, 2.5 eq.) was added to a tetrahydrofuran (3 mL) solution containing compound 6-1 (240 mg, 0.35 mmol, 1 eq.) and compound 1-12 (90 mg, 0.50 mmol, 1.5 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH = 15 / 1) showed that the reaction was complete and a new major spot was observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH = 1 / 0-10 / 1, v / v) to give a light yellow compound SY-6 (81 mg, 30%).

[0222] LCMS:Rt:1.340min;MS m / z(ELSD):838.5[M+H] + ;

[0223] HPLC: 98.3% purity at ELSD; RT=7.87min.

[0224] 1H NMR (400MHz, CDCl3) δ4.91–4.82 (m, 1H), 4.05 (t, J = 6.8Hz, 2H), 3.77 (d,J=5.8Hz,2H),2.99(s,3H),2.57(s,2H),2.41(s,2H),2.28(dd,J=13.9,7. 3Hz, 4H), 1.68–1.45 (m, 19H), 1.38–1.19 (m, 44H), 0.88 (dd, J = 7.7, 6.0Hz, 9H).

[0225] 13C NMR (100MHz, CDCl3) δ171.88,173.63,77.34,77.03,76.71,74.14,64.47,34.69,34.20 ,31.87,29.53,29.25,28.66,27.42,25.93,25.68,25.33,25.02,24.04,22.67,14.12.

[0226] Example 7: Synthesis of lipid compound SY-7

[0227]

[0228] The synthesis of compound 7-1 was carried out according to the aforementioned steps.

[0229] Potassium carbonate (160 mg, 1.17 mmol, 4.0 eq) was added to an acetonitrile solution (2 mL) containing compound 7-1 (200 mg, 0.28 mmol, 1 eq.) and compound 1-12 (50 mg, 0.28 mmol, 1.0 eq), potassium iodide (70 mg, 0.43 mmol, 1.5 eq) and cyclopentyl methyl ether (2 mL). The reaction mixture was protected by nitrogen at 90 ° C and stirred overnight. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The reaction was quenched with water (50 mL) and extracted with ethyl acetate (50 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-15 / 1, v / v) to give a light yellow oily product (100 mg, 37%).

[0230] LCMS:Rt:2.111min;MS m / z(ELSD):824.5[M+H] + ;

[0231] HPLC: 96% purity at ELSD; RT=8.110min.

[0232] 1 H NMR (400MHz, CDCl3) δ6.07 (s, 1H), 4.80 (dd, J = 12.4, 6.2Hz, 1H), 3.98 (t, J=6.4Hz,2H),3.59(d,J=3.6Hz,2H),2.93(d,J=6.8Hz,3H),2.60(s,2H),2.38(s, 4H),2.21(td,J=7.6,4.5Hz,4H),1.53(dd,J=14.0,6.9Hz,6H),1.47–1.34(m,8H), 1.31–1.12(m,44H),0.81(dd,J=7.6,6.0Hz,9H).

[0233] 13 C NMR (100MHz, CDCl3) δ171.18,170.59,167.94,165.45,140.73,218.33–76.95,76.73,74.10,64.40,53.76,53.41,52.71, 40.04,34.66,34.23,31.85,29.62–29.33,29.33–28.91,28.65,27.22,26.88,25.92,25.31,25.01,24.06,22.65,14.09.

[0234] Example 8: Synthesis of lipid compound SY-8

[0235]

[0236] Synthesis of intermediate 8-3

[0237]

[0238] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.4 g, 12.28 mmol, 2 eq.) and p-dimethylaminopyridine (100 mg, 0.15 mmol, 0.3 eq.) were added to dichloromethane (10 mL) containing compound 8-1 (1.2 g, 6.2 mmol, 1 eq.) and compound 8-2 (1.38 g, 6.2 mmol, 1 eq.), and then diisopropylethylamine (3.2 g, 24.8 mmol, 4 eq.) was added. The reaction mixture was stirred at room temperature under nitrogen protection for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) generated a new product spot. The reaction mixture was extracted with ethyl acetate and washed with water, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (with petroleum ether / ethyl acetate = 1 / 0-10 / 1) to obtain a colorless oil compound 8-3 (1.2 g, 52%).

[0239] Synthesis of intermediate 8-5

[0240]

[0241] Potassium carbonate (2.4 g, 18 mmol, 6 eq.) and potassium iodide (1.0 g, 8 mmol, 2 eq.) were added to a mixture of CPME / CH3CN (5 mL / 5 mL) containing a mixed solution of compound 8-3 (1.3 g, 1.65 mmol, 1.1 eq.) and compound 8-4 (1.2 mg, 1.5 mmol, 1 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen protection. TLC (dichloromethane / methanol = 10 / 1) showed that the reaction was complete and new major spots were observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with (dichloromethane / methanol = 10 / 1) to obtain compound 8-5 (1.2 g, 64%) as a yellow oil.

[0242] Synthesis of intermediate 8-6

[0243]

[0244] Under nitrogen protection, methylsulfonyl chloride (0.2 mL, 2.5 mmol, 2.5 eq.) was added dropwise to dichloromethane (10 mL) containing compound 8-5 (760 mg, 1 mmol, 1 eq.) and triethylamine (0.2 mL, 1.2 mmol, 1.2 eq.) at 0 ° C. After addition, the reaction mixture was stirred overnight at 25 ° C under nitrogen protection. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water and extracted with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain yellow oily compound 8-6 (850 mg), and the crude product was used directly in the next step.

[0245] Synthesis of intermediate 8-7

[0246]

[0247] Sodium azide (220 mg, 3.4 mmol, 3.4 eq.) was added to a dimethylformamide solution (5 mL) containing compound 8-6 (840 mg, 1 mmol, 1 eq.). After addition, the reaction mixture was stirred at 100 ° C for 16 hours under nitrogen protection. TLC (PE / EA=2 / 1) showed that a new major spot was observed. The mixture was diluted with ethyl acetate and washed three times with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain yellow oily compound 8-7 (800 mg, 65%), which was used directly in the next step.

[0248] Synthesis of intermediate 8-8

[0249]

[0250] Pd / C (50 mg, 10% wt) was added to ethanol (5 mL) containing compound 8-7 (780 mg, 0.34 mmol, 1 eq.). After addition, the reaction mixture was stirred at 25 ° C for 3 hours under hydrogen conditions (30 psi). TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and new major spots were observed. The mixture was filtered through diatomaceous earth and the filter cake was washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure to obtain yellow oily compound 8-8 (380 mg), and the crude product was directly used in the next step.

[0251] Synthesis of Compound SY-8

[0252]

[0253] Compound 1-12 (200 mg, 1.04 mmol, 2 eq.) and sodium carbonate (200 mg, 1.7 mmol, 3.2 eq.) were added to a tetrahydrofuran (5 mL) solution containing compound 8-8 (380 mg, 0.5 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to obtain a yellow oily compound SY-8 (225 mg, 25%).

[0254] LCMS:Rt:1.223min;MS m / z(ELSD):902.5[M+H] + ;

[0255] HPLC: 94.3% purity at ELSD; RT=9.866min.

[0256] 1H NMR (400MHz, CDCl3) δ7.14 (t, J = 3.4Hz, 2H), 6.90–6.79 (m, 2H), 5.01 (s,2H),4.87–4.76(m,1H),3.93(t,J=6.6Hz,2H),3.54(s,2H),3.21(s,3H),2.66( s,2H),2.60(s,4H),2.28(dt,J=16.4,7.5Hz,4H),1.80–1.72(m,2H),1.64–1.17(m, 52H),0.89(dt,J=13.7,7.1Hz,9H).

[0257] 13 C NMR (100MHz, CDCl3) δ173.61,130.12,114.52,77.25,77.14,76.71,74.11,68.13,65.88,219.45–24.95,57.85,56.06,5 3.97,40.61,31.97,33.74,40.61,29.35,40.61,28.30,30.63,40.61,26.09,40.61,25.43,24.82,22.67,22.46,14.07.

[0258] Example 9: Synthesis of lipid compound SY-9

[0259]

[0260] The synthesis of 9-1 refers to the above steps.

[0261] Compound 1-12 (102 mg, 0.57 mmol, 2 eq.) and sodium carbonate (80 mg, 0.76 mmol, 2 eq.) were added to a tetrahydrofuran (5 mL) solution containing compound 9-1 (300 mg, 0.38 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to obtain a yellow oily compound SY-9 (71 mg, 20%).

[0262] LCMS:Rt:1.152min;MS m / z(ELSD):930.6[M+H] + ;

[0263] HPLC: 94.2% purity at ELSD; RT=3.976min.

[0264] 1 H NMR (400MHz, CDCl3) δ7.11 (d, J = 8.4Hz, 2H), 6.83 (d, J = 8.4Hz, 2H), 4.86(s,2H),4.24(t,J=7.2Hz,2H),3.90(t,J=6.8Hz,2H),3.82–3.72(m,2H),3.2 3(s,3H),2.88(dt,J=14.4,6.0Hz,4H),2.82–2.67(m,4H),2.28(t,J=7.2Hz,4H), 1.79(q,J=7.2Hz,2H),1.68–1.44(m,12H),1.37–1.21(m,42H),1.03(t,J=7.2Hz, 3H), 0.88 (t, J = 6.8 Hz, 6H).

[0265] 13C NMR(101MHz,Chloroform-d)δ173.68,173.57,157.85,129.81,129.64,114.52,74.19,69.52,65.03,57.34,56.14,53.99,34 .58,34.27,34.18,34.13,31.86,29.53,29.50,29.24,29.01,28.97,28.91,26.91,26.85,25.32,25.08,24.97,24.74,22.67, 22.61,14.12,10.54.

[0266] Example 10: Synthesis of Compound SY-10

[0267]

[0268] The synthesis of 10-1 was carried out according to the aforementioned steps.

[0269] Compound 1-12 (200 mg, 1.14 mmol, 2.28 eq.) and sodium carbonate (160 mg, 1.52 mmol, 3 eq.) were added to a tetrahydrofuran (8 mL) solution containing compound 10-1 (400 mg, 0.5 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to obtain a yellow oily compound SY-10 (117 mg, 25%).

[0270] LCMS:Rt:1.212min;MS m / z(ELSD):958.6[M+H] + ;

[0271] HPLC: 96.65% purity at ELSD; RT=4.113min.

[0272] 1H NMR (400MHz, CDCl3) δ7.13 (d, J = 8.4Hz, 2H), 6.84 (d, J = 8.4Hz, 2H), 4.84(s,2H),4.22(m,2H),3.88(m,2H),3.80–3.70(m,2H),3.23(s,3H),2.89m,4H) ,2.80–2.64(m,4H),2.26(m,4H),1.76(q,J=7.2Hz,2H),1.66–1.41(m,16H),1.36– 1.20(m,42H),1.00(t,J=7.2Hz,3H),0.91(t,J=6.8Hz,6H).

[0273] Example 11: Synthesis of Compound SY-11

[0274]

[0275] The synthesis of 11-1 was carried out according to the above steps.

[0276] Compound 1-12 (100 mg, 0.57 mmol, 2.1 eq.) and sodium carbonate (80 mg, 0.76 mmol, 2.8 eq.) were added to a tetrahydrofuran (4 mL) solution containing compound 11-1 (200 mg, 0.27 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was eluted by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to obtain a yellow oily compound SY-11 (50 mg, 21%).

[0277] LCMS:Rt:1.323min; MS m / z(ELSD):888.5[M+H] + ;

[0278] HPLC: 99.4% purity at ELSD; RT=8.229min.

[0279] 1H NMR (400MHz, CDCl3) δ7.27 (dd, J=4.8, 3.8Hz, 2H), 6.88 (d, J=8.6Hz, 2H),5.04(s,2H),4.64(dt,J=12.4,6.2Hz,4H),3.92(t,J=6.6Hz,2H),3.75–3.61( m,2H),2.68(dd,J=31.5,24.8Hz,6H),2.30(dt,J=16.6,7.5Hz,4H),1.87–1.73(m, 2H),1.70–1.09(m,48H),1.03(t,J=7.4Hz,3H),0.88(t,J=6.7Hz,6H).

[0280] 13 C NMR (101MHz, CDCl3) δ172.5 (d, J = 7.7Hz), 158.2, 129.0, 127.0, 113.5, 76.2,76.8,76.7,74.2,69.6,65.9,57.7,56.0,53.9,34.6,34.2,31.8, 29.5,29.3–28.7,27.0,25.7,25.32,25.02,24.80,22.61,14.11,10.50.

[0281] Example 12: Synthesis of Compound SY-12

[0282]

[0283] The synthesis of 12-1 was carried out according to the aforementioned steps.

[0284] Compound 1-12 (150 mg, 0.87 mmol, 2.1 eq.) and sodium carbonate (120 mg, 1.14 mmol, 2.8 eq.) were added to a tetrahydrofuran (6 mL) solution containing compound 12-1 (300 mg, 0.38 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and new major spots were observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to give a yellow oily compound SY-12 (50 mg, 15%).

[0285] LCMS:Rt:1.223min;MS m / z(ELSD):730.5[M+H] + ;

[0286] HPLC: 98.8% purity at ELSD; RT=4.105min.

[0287] 1 H NMR (400MHz, CDCl3) δ7.11 (d, J = 8.4Hz, 2H), 6.83 (d, J = 8.4Hz, 2H), 4.86(s,2H),4.24(m,5H),3.90(m,5H),3.82–3.72(m,5H),2.88(dt,J=14.4,6.0H z,4H),2.82–2.67(m,4H),2.28(t,J=7.2Hz,4H),1.79(q,J=7.2Hz,2H),1.68–1.44 (m,12H),1.37–1.21(m,36H),1.03(t,J=7.2Hz,3H),0.88(t,J=6.8Hz,6H).

[0288] Example 13: Synthesis of Compound SY-13

[0289]

[0290] The synthesis of 13-1 was carried out according to the aforementioned steps.

[0291] Compound 1-12 (100 mg, 0.56 mmol, 4.3 eq.) and sodium carbonate (120 mg, 1.14 mmol, 10 eq.) were added to a tetrahydrofuran (5 mL) solution containing compound 13-1 (100 mg, 0.13 mmol, 1 eq.). After addition, the mixture was stirred at 25 ° C for 16 hours under nitrogen protection. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The reaction mixture was extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH=1 / 0-10 / 1, v / v) to give a yellow oily compound SY-13 (20 mg, 17%). LCMS: Rt: 1.554 min; MS m / z (ELSD): 918.5 [M+H] + ;

[0292] HPLC: 93.6% purity at ELSD; RT=5.988min.

[0293] 1H NMR(400MHz, CDCl3)δ7.55(s,1H),7.19–6.98(m,3H),4.97(s,2H),4.07 (m,4H),3.98(t,J=6.8Hz,2H),3.70(d,J=6.8Hz,2H),2.90(s,3H),2.52(m,6H), 2.36(m,4H),2.23(m,4H),1.70(m,2H),1.62–1.35(m,14H),1.34–1.08(m,32H), 0.81(m,9H).

[0294] 13 C NMR (101MHz, CDCl3) δ171.8,171.6,163.5,140.0,139.8,138.7,135.2,129.3,129.1,126.7,69.2,68.4,66.2,63.4,52.9,52.3,4 2.7,33.3,33.3,30.8,30.2,30.1,30.0,28.7,28.7,28.4,28.2,28.1,28.0,27.6,26.3,26.2,25.3,24.9,24.7,23.9,23.8,23.0, 21.6,13.0.

[0295] Example 14: Preparation of lipid nanoparticles

[0296] 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;

[0297] Preparation of mRNA solution: Dissolve a certain amount of luciferase mRNA in 20 mM citric acid buffer solution at pH = 4.0;

[0298] 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.

[0299] 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.

[0300] Example 15: Characterization of lipid nanoparticles

[0301] 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 RIBOGREENR 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 (WallacVictor 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).

[0302] Table 1. Physicochemical characterization of lipid nanoparticles prepared from lipid compounds synthesized in the examples of the present invention

[0303]

[0304]

[0305] Example 16: In vitro cell expression detection

[0306] 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 1It can be seen that the lipid nanoparticles prepared from each compound have a certain degree of in vitro cellular protein expression.

[0307] Example 17: Protein expression study in mice

[0308] After the lipid nanoparticles are injected into the mouse through intravenous or intramuscular injection, 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.

[0309] 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 (V) or a pharmaceutically acceptable salt or stereoisomer thereof: L 1 is a C2-C3 alkyl group; 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 or branched chain alkyl; M 1 , M 2 Each independently selected from -OC(O)-, -C(O)O-, -NHC=N(CN)NH-; R 3 C4-C 20 A straight chain or branched chain alkyl group; n is an integer of 1-2.

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 1-2.

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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