Ionizable lipid compound and application thereof

Abstract

The invention provides an ionizable lipid compound and application thereof, the ionizable lipid compound has a structure as shown in a formula (I), a liposome prepared from the lipid compound provided by the invention is uniform in particle size distribution, carries negative charges on the surface, is low in cytotoxicity, and has better encapsulation efficiency and better cell or in-vivo transfection rate. The active substances can be specifically delivered to cells, tissues or organs in a targeted manner, so that the targeted delivery of the active substances is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal compounds and relates to an ionizable lipid compound and application thereof. Background Art

[0002] Nanoparticles are granular dispersions or solid particles with a particle size ranging from 10 to 1000 nm. Their use for delivery can achieve enhanced permeability and retention (EPR) effects. Their high electrical potential and specific surface area allow for a larger gene payload, while also protecting nucleic acid molecules from enzymatic degradation and immune recognition, resulting in higher transcellular membrane transport efficiency compared to other vectors. Furthermore, nanocarriers generally exhibit good biocompatibility and are even biodegradable, resulting in minimal impact on cell growth and metabolism.

[0003] In the treatment of various diseases, successful patient recovery depends on the physician's ability to deliver drugs to specific sites. Current treatments are unable to localize drugs at their site of action, resulting in the drug molecules simply distributing and diffusing throughout the body, which can easily cause adverse side effects and lead to significant drug waste. Long-term drug delivery research has revealed that nanoparticles have emerged as suitable carriers to overcome the limitations of conventional drug formulations and their associated pharmacokinetic properties. For example, liposomes have been shown to offer advantages in solubilizing therapeutic drugs, enabling controlled, long-term release and significantly extending their circulation life. However, biological barriers in the drug delivery process prevent the accumulation of nanoparticles at the diseased site, thereby limiting effective drug response. Despite extensive research efforts aimed at incorporating multiple functionalities into overall nanoparticle design, many of these strategies fail to adequately address these obstacles, such as nonspecific distribution and insufficient accumulation of therapeutic agents, which remain significant challenges for drug developers. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an ionizable lipid compound and its application. The ionizable lipid compound provided by the present invention is used to prepare lipid nanoparticles for effective nucleic acid delivery, achieving targeted delivery to specific organs.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides an ionizable lipid compound having a structure represented by formula (I):

[0007]

[0008] M is selected from -(CH2) m1 -、-(CH2) n1 C(O)(CH2) p、-(CH2) n1 C(O)O(CH2) p -, m1 is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), n1 is an integer from 1 to 3 (e.g., 1, 2, or 3), and p is an integer from 1 to 3 (e.g., 1, 2, or 3);

[0009] R1 and R2 are the same or different and are selected from C 1-6 Alkyl, C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl, benzoheterocyclyl or heterocyclyl, the above cycloalkyl, heterocyclyl, aryl, heteroaryl, benzoheterocyclyl or heterocyclyl groups are optionally substituted by a group selected from the following: halogen atoms, -(CH2) 1-6 CX3, -OH, -(CH2) 1-3 OH, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkyl hydroxyl, C 1-6 Alkylamino; wherein X is selected from halogen.

[0010] Preferably, R1 and R2 are independently selected from thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, trifluoromethylphenyl, 3,5-difluorobromophenyl, benzothiazolyl, 1-phenylpyrrolidinyl, pyridinyl, oxazolyl or 2,2,2-trifluoroethyl.

[0011] R' and R" are the same or different and are selected from -(CH) q -、-(CH) q CH=CH(CH) s -、-(CH2) q NH(CH2) s 、-(CH) q C(O)(CH) s -, wherein q and s are independently an integer from 1 to 3 (eg, 1, 2 or 3).

[0012] R3 and R4 may be the same or different and are selected from a linear or branched C 3-30 Alkyl, hydroxy-substituted or unsubstituted straight or branched chain C 3-30 Alkenyl, hydroxy-substituted or unsubstituted straight or branched C 3-30 Alkynyl, Wherein Rc and Rd may be the same or different and are selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20wherein n and m are selected from integers of 1 to 15 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), and the wavy line represents the site of attachment of the group.

[0013] Preferably, said R3 and R4 are selected from

[0014] in Indicates the attachment site of a group.

[0015] In the present invention, the ionizable lipid compound contains two hydrophilic head groups and two hydrophobic tail chains.

[0016] Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas A1-A12:

[0017]

[0018]

[0019] M, R3, R4, and R" have the same ranges as those of M, R3, R4, and R" described in formula (I).

[0020] In the present invention, the ionizable lipid compound has any one of the structures shown in the following formulas B1-B15:

[0021]

[0022]

[0023]

[0024] R3, R4, and R" have the same ranges as those of R3, R4, and R" described in formula (I).

[0025] In the present invention, the ionizable lipid compound is selected from any one of the compounds in Table 1 below:

[0026] Table 1

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] In a second aspect, the present invention provides a liposome, wherein the liposome comprises the ionizable lipid compound as described in the first aspect.

[0037] Preferably, the liposome further comprises a helper lipid.

[0038] Preferably, the helper lipid comprises a phospholipid.

[0039] Preferably, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (1,2-dioleo ... PC), 1-oleoyl-2-cholesteryl hemisuccinyl sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Any one or a combination of at least two of phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0040] Preferably, the helper lipids further comprise structural lipids.

[0041] Preferably, the structured lipid comprises at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol, or a combination of at least two thereof.

[0042] Preferably, the helper lipid further comprises a PEGylated lipid.

[0043] Preferably, the PEGylated lipid comprises any one or a combination of at least two of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide or PEG-modified dialkylamine.

[0044] Preferably, the liposomes include ionizable lipid compounds, phospholipids, structural lipids and PEGylated lipids.

[0045] Preferably, the liposomes comprise, by mole percentage, 20%-80% (for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, etc.) of ionizable lipid compounds, 5%-30% (for example, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%, 30%, etc.) of phospholipids, 10%-50% (for example, 10%, 20%, 30%, 40%, 50%, etc.) of structural lipids, and 1%-10% (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.) of pegylated lipids.

[0046] In a third aspect, the present invention provides a lipid complex comprising the liposome according to the second aspect and an active substance;

[0047] Preferably, the active substance includes any one of small molecule compounds, nucleic acid molecules or protein molecules, or a combination of at least two of them.

[0048] Preferably, the nucleic acid molecule comprises any one or a combination of at least two of DNA, mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA or lncRNA.

[0049] Preferably, the protein molecule comprises any one or a combination of at least two of an enzyme, an antibody, a short peptide, a polypeptide or a recombinant protein.

[0050] Preferably, the method for preparing the lipid complex comprises the following steps: mixing liposomes and active substances by a mixer to obtain the lipid complex.

[0051] In a fourth aspect, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the liposome described in the second aspect or the lipid complex described in the third aspect.

[0052] Preferably, the particle size of the lipid nanoparticles is 40-300 nm (for example, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, etc.).

[0053] In a fifth aspect, the present invention provides a reagent, a kit, a preparation or a pharmaceutical composition, each of which independently comprises any one of the ionizable lipid compound described in the first aspect, the liposome described in the second aspect, the lipid complex described in the third aspect or the lipid nanoparticles described in the fourth aspect, or a combination of at least two thereof.

[0054] In a sixth aspect, the present invention provides a use of the ionizable lipid compound according to the first aspect, the liposome according to the second aspect, the lipid complex according to the third aspect, the lipid nanoparticle according to the fourth aspect, or the reagent, kit, preparation or pharmaceutical composition according to the fifth aspect in the preparation of a product having any one of the following functions (1) to (4):

[0055] (1) Encapsulation of active substances;

[0056] (2) delivering the encapsulated active substance to cells, tissues, or organs;

[0057] (3) enabling the delivered active substance to exert its active effect in cells, tissues or organs;

[0058] (4) Prevent, diagnose or treat diseases.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention provides a series of novel ionizable lipid compounds. Liposomes prepared using these lipid compounds exhibit uniform particle size distribution, negative surface charge, low cytotoxicity, good encapsulation efficiency, and enhanced cellular or in vivo transfection efficiency. The lipid nanoparticles provided by the present invention can specifically deliver active substances (such as small molecules, siRNA, mRNA, and proteins) to cells, tissues, or organs, achieving targeted delivery of active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Figure 2 shows the encapsulation efficiency test results of the lipid nanoparticles prepared by the present invention;

[0062] Figure 2This is a graph showing the relative luminescence intensity measurement results of the lipid nanoparticles prepared in the present invention in 293T cells;

[0063] Figure 3 This is a graph showing the results of measuring the cell viability of 293T cells at different concentrations of LNP using the CCK-8 reagent prepared in the present invention;

[0064] Figure 4 This is a diagram showing the organ luminescence of I-50 and I-64, which have better in vivo delivery effects in the tail vein injection screening experiment of the present invention (Figure A) and a comparison diagram of the total fluorescence values in various organs (Figure B).

[0065] Figure 5 Particle size, PDI (Figure A), and encapsulation efficiency (Figure B) of LNPs with 12 randomly generated lipid molar ratios using the Design Expert software;

[0066] Figure 6 Figure 2 shows the particle size, PDI (Figure A), and encapsulation efficiency of I-50-mLuc-LNPs with different mRNA-to-total lipid mass ratios (Figure B).

[0067] Figure 7 When the mRNA-to-total lipid mass ratio was 3.9%, 5.9% and 7.9, respectively, the in vivo imaging images of mice of the three LNPs (Figure A), the fluorescence luminescence images of important organs (lymph, heart, liver, spleen, lung, kidney and muscle) (Figure B), the pie chart of the distribution ratio of total fluorescence values of organs (Figure C) and the fluorescence intensity per unit area of organs (Figure D). DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] Example 1 Synthesis of ionizable lipid I-25

[0070]

[0071] (1) Synthesis of linolenic acid a-2: Compound a-1 (5.32 g, 20.0 mmol) was dissolved in DCM (100 ml, 0.20 M), and then DMP (5.086 g, 24.0 mmol) was added and stirred at room temperature for 20 min. After TLC monitoring, the reaction was completed and quenched by adding saturated Na2S2O3 solution and saturated NaHCO3 solution. The mixture was stirred vigorously for 20 min until the solution became clear. The mixture was extracted with DCM (80 ml × 3), and the organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA volume ratio = 80:1) to obtain compound a-2 as a light yellow oily liquid (3.684 g, 13.95 mmol, 70%).

[0072] MS (ESI) m / z: [M+H] + 265.45. 1 H NMR (500MHz, CDCl3, ppm) δ9.75 (s, 1H), 5.42–5.28 (m, 4H), 2.76 (t, J=6.4Hz, 2H), 2.41 (t, J= 7.3Hz, 2H), 2.04–1.97 (m, 4H), 1.63–1.61 (m, 2H), 1.34–1.30 (m, 14H), 0.88 (t, J = 6.7Hz, 3H).

[0073] (2) Synthesis of compound a-4: First, compound a-3 (1.04 g, 6.5 mmol) was dissolved in DCM (65 ml, 0.10 M), and then compound a-2 (3.432 g, 13.0 mmol) was added and stirred for 10 min. Finally, NaBH(OAc)3 (4.133 g, 19.5 mmol) was added and stirred at room temperature for 3 h. After TLC monitoring, the reaction was quenched by adding saturated NaHCO3 solution (60 ml), extracted with DCM (60 ml × 3), and the organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA=10:1) to obtain compound a-4 as a light yellow viscous liquid (2.334 g, 3.558 mmol, 55%).

[0074] MS (ESI) m / z: [M+H] + 657.62. 1H NMR (500MHz, CDCl3, ppm) δ5.41-5.29(m,8H),3.15(s,2H),2.77(t,J=6.2Hz,4H),2.49(s,2H),2.40-2.37 (m, 4H), 2.04 (dd, J = 13.1, 6.4Hz, 8H), 1.44 (s, 9H), 1.33 (dd, J = 21.1, 14.7Hz, 36H), 0.88 (t, J = 6.2Hz, 6H).

[0075] (3) Synthesis of compound a-5: Compound a-4 (1.968 g, 3.0 mmol) was first dissolved in EA (15.0 ml, 0.20 M), and then a 4 M hydrogen chloride solution in ethyl acetate (6.0 ml, 24.0 mmol) was added and stirred at room temperature for 4 h. After completion of the reaction, the mixture was quenched by adding a saturated NaHCO 3 solution (30 ml), and extracted with EA (30 ml × 4). The organic phases were combined and dried over anhydrous Na 2 SO 4 . After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH=10:1) to obtain compound a-5 as a light yellow viscous liquid (1.601 g, 2.88 mmol, 96%).

[0076] MS (ESI) m / z: [M+H] + 557.32. 1 H NMR (400MHz, CDCl3, ppm) δ5.41–5.29(m,8H),2.78–2.71(m,6H),2.47(t,J=5.9Hz,2H),2. 41–2.38(m,4H),2.04(dd,J=13.4,6.6Hz,8H),1.41–1.27(m,36H),0.88(t,J=6.6Hz,6H).

[0077] (4) Synthesis of I-25: First, compound a-5 (111.2 mg, 0.2 mmol) was dissolved in DCM (4 ml, 0.05 M), and then compound a-6 (39.6 mg, 0.4 mmol) was added and stirred for 10 min. Finally, NaBH(OAc)3 (131.1 mg, 0.6 mmol) was added and stirred at room temperature for 12 h. The reaction was monitored by TLC. After completion, saturated NaHCO3 solution (6 ml) was added to quench the reaction. The mixture was extracted with DCM (6 ml × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 5:1) to obtain compound I-25 as a colorless viscous liquid (86.6 mg, 0.1209 mmol, 60%).

[0078] MS (ESI) m / z: [M+H]+ 717.58. 1 H NMR (500MHz, CDCl3, ppm) δ7.45 (s, 4H), 5.35–5.28 (m, 8H), 3.53 (s, 4H), 2.74 (d, J = 6.2Hz, 6H) ,2.62(s,2H),2.52(s,4H),2.03(d,J=5.5Hz,8H),1.33–1.24(m,36H),0.87(t,J=6.1Hz,6H).

[0079] Example 2 Synthesis of ionizable lipid I-47

[0080]

[0081] (1) Synthesis of I-47: Referring to the method of step (4) of Example 1, compound I-47 was synthesized using a5 and a-7 as starting materials as a colorless viscous liquid (78.8 mg, 0.110 mmol, 55%).

[0082] MS (ESI) m / z: [M+H] + 717.44. 1 H NMR (500MHz, CDCl3, ppm) δ7.65(s,2H,),6.98(s,2H),5.35–5.28(m,8H),3.53(s,4H),2.74(d,J=6.2 Hz, 6H), 2.62 (s, 2H), 2.52 (s, 4H), 2.03 (d, J = 5.5Hz, 8H), 1.33–1.24 (m, 36H), 0.87 (t, J = 6.1Hz, 6H).

[0083] Example 3 Synthesis of ionizable lipid I-40

[0084]

[0085] (1) Synthesis of I-40: Referring to the method of step (4) of Example 1, compound I-40 was synthesized using a-5 and a-10 as starting materials as a colorless viscous liquid (88.4 mg, 0.091 mmol, 55%).

[0086] MS (ESI) m / z: [M+H] + 965.36. 1 H NMR (500MHz, CDCl3) δ7.02 (d, J=6.7Hz, 4H), 5.33–

[0087] 5.29(m,8H),3.63(s,4H),2.75(t,J=6.8Hz,4H),2.48(s,4H),2.27(d,J= 7.6Hz, 4H), 2.02–1.96 (m, 8H), 1.37–1.03 (m, 36H), 0.86 (t, J = 6.8Hz, 6H).

[0088] Example 4 Synthesis of ionizable lipid I-41

[0089]

[0090] (1) Synthesis of I-41: Referring to the method of step (4) of Example 1, a-14 and a-5 were used as starting materials to synthesize compound I-41, which was a colorless viscous liquid (108.4 mg, 0.123 mmol, 65%).

[0091] MS (ESI) m / z: [M+H] + 875.38. 1 H NMR (500MHz, CDCl3) δ7.15 (dd, J=23.0, 8.2Hz, 4H),

[0092] 6.50(d,J=8.1Hz,4H),5.34–5.29(m,8H),3.48(d,J=5.0Hz,4H),3.25(d,J=6.3Hz,8H),2.77(t,J=6.8Hz,4H),2.61(d,J=7.2Hz,2H) ,2.54(d,J=7.3Hz,2H),2.36(t,J=7.8Hz,4H),2.04(t,J=7.4Hz,8H),1.97–1.94(m,8H),1.35–1.20(m,36H),0.88(t,J=7.1Hz,6H).

[0093] Example 5 Synthesis of ionizable lipid I-42

[0094]

[0095] (1) Synthesis of I-42: Referring to the method of step (4) of Example 1, compound I-42 was synthesized using a-5 and a-9 as starting materials as a colorless viscous liquid (113.4 mg, 0.151 mmol, 47%).

[0096] MS (ESI) m / z: [M+H] + 749.74. 1H NMR (500MHz, CDCl3) δ7.22–7.18(m,2H),6.91(d,J=3.3Hz,4H),5.39–5.29(m,8H),3.84(s,4H),2.75(t,J= 6.7Hz,8H),2.54(s,4H),2.02–1.98(m,8H),1.52–1.41(m,4H),1.32–1.21(m,32H),0.86(t,J=6.7Hz,6H).

[0097] Example 6 Synthesis of ionizable lipid I-43

[0098]

[0099] (1) Synthesis of I-43: Referring to the method of step (4) of Example 1, a-5 and a-11 were used as starting materials to synthesize compound I-43, which was a colorless viscous liquid (116.4 mg, 0.133 mmol, 57%).

[0100] MS (ESI) m / z: [M+H] + 873.48. 1 H NMR (500MHz, CDCl3) δ7.55(dd,J=13.7,7.9Hz,4H),7.44(t,J=8.0Hz,4H),5.33–5.30(m,8H),3.65(d,J=20.0Hz,4H),2.74(t ,J=6.7Hz,2H),2.45–2.41(m,6H),2.02(d,J=7.2Hz,8H),1.41(d,J=15.1Hz,4H),1.32–1.16(m,36H),0.86(t,J=6.8Hz,6H).

[0101] Example 7 Synthesis of ionizable lipid I-44

[0102]

[0103] (1) Synthesis of I-44: Referring to the method of step (4) of Example 1, compound I-44 was synthesized using a-5 and a-14 as starting materials as a colorless viscous liquid (156.4 mg, 0.211 mmol, 74%).

[0104] MS (ESI) m / z: [M+H] + 739.65. 1H NMR (500MHz, CDCl3) δ8.47(d,J=4.8Hz,2H),7.59(t,J=7.7Hz,2H),7.50(d,J=7.9Hz,2H),7.09(t,J=6.2Hz,2H),5.31–5.29(m,8 H),3.81(s,4H),2.73(t,J=6.8Hz,4H),2.61(s,4H),2.30(s,4H),2.00–1.97(m,8H),1.38–1.15(m,36H),0.84(t,J=6.8Hz,6H).

[0105] Example 8 Synthesis of ionizable lipid I-45

[0106]

[0107] (1) Synthesis of I-45: Referring to the method of step (4) of Example 1, a-5 and a-15 were used as starting materials to synthesize compound I-45, which was a colorless viscous liquid (166.4 mg, 0.221 mmol, 56%).

[0108] MS (ESI) m / z: [M+H] + 751.78. 1 H NMR (500MHz, CDCl3) δ7.70 (s, 2H), 7.28 (s, 2H), 5.30–5.27 (m, 4.5Hz, 8H), 4.13 (d, J = 13.8Hz, 4H), 3.03–2.81 (m, 4H), 2.73 (t,J=6.8Hz,4H),2.70–2.41(m,4H),2.00(t,J=7.1Hz,8H),1.52–1.49(m,4H),1.30–1.18(m,32H),0.84(t,J=7.1Hz,6H).

[0109] Example 9 Synthesis of ionizable lipid I-46

[0110]

[0111] (1) Synthesis of I-46: Referring to the method of step (4) of Example 1, compound I-46 was synthesized using a-5 and a-12 as starting materials as a colorless viscous liquid (135.4 mg, 0.16 mmol, 47%).

[0112] MS (ESI) m / z: [M+H] + 851.48. 1 H NMR (500MHz, CDCl3) δ8.91 (s, 2H), 8.04 (d, J = 8.4Hz,

[0113] 2H),7.92(s,2H),7.48(d,J=8.4Hz,2H),5.38–5.27(m,8H),3.76(s,4H),2.72(t,J=6.7 Hz, 8H), 2.49 (s, 4H), 1.99 (t, J = 6.8Hz, 8H), 1.40–1.06 (m, 36H), 0.83 (t, J = 6.8Hz, 6H).

[0114] Example 10 Synthesis of ionizable lipid I-52

[0115]

[0116] (1) Synthesis of I-52: Referring to the method of step (4) of Example 1, compound I-52 was synthesized using a-5 and a-17 as starting materials as a colorless viscous liquid (127.7 mg, 0.171 mmol, 47%).

[0117] MS (ESI) m / z: [M+H] + 741.65. 1 H NMR (500MHz, CDCl3) δ8.65 (s, 2H), 8.45 (d, J = 15.3

[0118] Hz,4H),5.29–5.27(m,8H),3.92(s,4H),2.93(d,J=33.7Hz,4H),2.79–2.58(m,8H), 1.99(t,J=7.1Hz,8H),1.61–1.40(m,4H),1.36–1.20(m,32H),0.83(t,J=7.1Hz,6H).

[0119] Example 11 Synthesis of ionizable lipid I-53

[0120]

[0121] (1) Synthesis of I-53: Referring to the method of step (4) of Example 1, compound I-53 was synthesized using a-5 and a-8 as starting materials as a colorless viscous liquid (137.7 mg, 0.183 mmol, 47%).

[0122] MS (ESI) m / z: [M+H] + 749.87. 1 H NMR(500MHz, CDCl3)δ5.42–5.23(m,8H),2.74(t,J=

[0123] 6.9Hz,4H),2.70(t,J=7.7Hz,4H),2.48(d,J=9.7Hz,4H),2.37(t,J=7.6Hz,4H), 2.22–2.19(m,4H),2.02–1.97(m,8H),1.36–1.22(m,36H),0.86(t,J=6.7Hz,6H).

[0124] Example 12 Synthesis of ionizable lipid I-11

[0125]

[0126] (1) Synthesis of compound b-2: Referring to the method of step (2) of Example 1, compound b-2 was synthesized using b-1 and a-3 as starting materials as a light yellow viscous liquid (1.747 g, 3.972 mmol, 61.1%).

[0127] MS (ESI) m / z: [M+H] + 441.73. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26(s,28H),0.88(t,J=6.8Hz,6H).

[0128] (2) Synthesis of compound b-3: Referring to the method of step (3) of Example 1, compound b-3 was synthesized using b-2 as the starting material as a light yellow viscous liquid (0.997 g, 2.93 mmol, 97.4%).

[0129] MS (ESI) m / z: [M+H] + 341.63. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0130] J=6.1Hz, 2H), 2.45 (t, J=7.6Hz, 4H), 1.43 (s, 4H), 1.27 (s, 28H), 0.89 (t, J= 6.9Hz, 6H).

[0131] (3) Synthesis of Compound I-11: Referring to the method of step (4) of Example 1, compound I-11 was synthesized using b-3 as the starting material as a colorless viscous liquid (56.1 mg, 0.112 mmol, 56%).

[0132] MS (ESI) m / z: [M+H]+ 501.82. 1 H NMR (500MHz, CDCl3) δ7.48(s,4H),3.56(s,4H),2.66(d,J=8.1Hz,2H),2.59(d,J=8.1H z, 2H), 2.45 (t, J = 7.8Hz, 4H), 1.43–1.40 (m, 4H), 1.26 (s, 28H), 0.88 (t, J = 6.8Hz, 6H).

[0133] Example 13 Synthesis of ionizable lipid I-12

[0134]

[0135] (1) Synthesis of Compound I-12: Referring to the method of step (4) of Example 1, compound I-12 was synthesized using b-3 and a-8 as starting materials as a colorless viscous liquid (59.59 mg, 0.112 mmol, 53.3%).

[0136] MS (ESI) m / z: [M+H] + 533.74. 1 H NMR (500MHz, CDCl3) δ2.66 (t, J = 5.9Hz, 4H), 2.54 (s,

[0137] 2H), 2.40 (s, 4H), 2.32 (d, J = 3.9Hz, 2H), 1.41–1.36 (m, 4H), 1.28–1.24 (m, 32H), 0.88 (s, 6H).

[0138] Example 14 Synthesis of ionizable lipid I-18

[0139]

[0140] (1) Synthesis of Compound I-18: Referring to the method of step (4) of Example 1, compound I-18 was synthesized using b-3 and a-9 as starting materials as a colorless viscous liquid (86.6 mg, 0.1209 mmol, 60%).

[0141] MS (ESI) m / z: [M+H] + 533.56. 1 H NMR (500MHz, CDCl3) δ7.25 (d, J=4.9Hz, 2H), 6.96

[0142] (d,J=6.4Hz,4H),3.90(s,4H),2.65(s,4H),2.40(t,J=7.7Hz,4H),1.29–1.25(m,32H),0.92(t,J=6.8Hz,6H).

[0143] Example 15 Synthesis of ionizable lipid I-1

[0144]

[0145] (1) Synthesis of compound c-2: Referring to the method of step (2) of Example 1, compound c-2 was synthesized using c-1 and a-3 as starting materials as a light yellow viscous liquid (1.454 g, 3.529 mmol, 54.3%).

[0146] MS (ESI) m / z: [M+H] + 413.4. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.23(m,24H),0.88(t,J=6.8Hz,6H).

[0147] (2) Synthesis of compound c-3: Referring to the method of step (3) of Example 1, compound c-3 was synthesized using c-2 as the starting material as a light yellow viscous liquid (0.920 g, 2.95 mmol, 98.3%).

[0148] MS (ESI) m / z: [M+H] + 313.62. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0149] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.22(m,24H),0.89(t,J=6.9Hz,6H).

[0150] (3) Synthesis of Compound I-1: Referring to the method of step (4) of Example 1, compound I-1 was synthesized using c3 and a13 as starting materials as a colorless viscous liquid (83.8 mg, 0.1164 mmol, 58.2%).

[0151] MS (ESI) m / z: [M+H] + 721.83. 1H NMR (500MHz, CDCl3) δ7.07(s,4H),3.69(s,4H),2.53(s,4H),2.30(s,4H),1.29–1.23(m,28H),0.91(s,6H).

[0152] Example 16 Synthesis of ionizable lipid I-3

[0153]

[0154] (1) Synthesis of Compound I-3: Referring to the method of step (4) of Example 1, compound I-3 was synthesized using c-3 and a-9 as starting materials as a colorless viscous liquid (86.6 mg, 0.1209 mmol, 60%).

[0155] MS (ESI) m / z: [M+H] + 505.36. 1 H NMR (500MHz, CDCl3) δ7.25 (d, J=4.9Hz, 2H), 6.96

[0156] (d,J=6.4Hz,4H),3.90(s,4H),2.65(s,4H),2.40(t,J=7.7Hz,4H),1.29–1.23(m,28H),0.92(t,J=6.8Hz,6H).

[0157] Example 17 Synthesis of ionizable lipid I-5

[0158]

[0159] (1) Synthesis of Compound I-5: Referring to the method of step (4) of Example 1, compound I-5 was synthesized using c-3 and a-11 as starting materials as a colorless viscous liquid (96.6 mg, 0.15 mmol, 60%).

[0160] MS (ESI) m / z: [M+H] + 629.78. 1 H NMR (500MHz, CDCl3) δ7.56 (s, 4H), 7.50 (s, 4H), 3.66 (s, 4H), 2.57 (s, 4H), 2.32 (t, J = 7.5Hz, 4H), 1.25–1.23 (m, 28H), 0.89 (t, J = 7.0Hz, 6H).

[0161] Example 18 Synthesis of ionizable lipid I-13

[0162]

[0163] (1) Synthesis of compound d-2: Referring to the method of step (2) of Example 1, compound d-2 was synthesized using d-1 and a-3 as starting materials as a light yellow viscous liquid (1.844 g, 3.718 mmol, 57.2%).

[0164] MS (ESI) m / z: [M+H] + 497.51. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.21(m,36H),0.88(t,J=6.8Hz,6H).

[0165] (2) Synthesis of compound d-3: Referring to the method of step (3) of Example 1, compound d-3 was synthesized using d-2 as the starting material as a light yellow viscous liquid (1.130 g, 2.856 mmol, 95.2%).

[0166] MS (ESI) m / z: [M+H] + 397.44. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0167] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.21(m,36H),0.89(t,J=6.9Hz,6H).

[0168] (3) Synthesis of Compound I-13: Referring to the method of step (4) of Example 1, compound I-13 was synthesized using d-3 and a-6 as starting materials as a colorless viscous liquid (53.70 mg, 0.0966 mmol, 48.3%).

[0169] MS (ESI) m / z: [M+H] + 557.52. 1 H NMR (500MHz, CDCl3) δ7.48 (s, 4H), 3.56 (s, 4H), 2.66 (d, J = 8.1Hz, 2H), 2.59 (d, J = 8.1Hz, 2H), 2.45(t,J=7.8Hz,4H), 1.43–1.37(m,4H), 1.26–1.22(m,36H), 0.88(t,J=6.8Hz,6H).

[0170] Example 19 Synthesis of ionizable lipid I-14

[0171]

[0172] (1) Synthesis of Compound I-14: Referring to the method of step (4) of Example 1, compound I-14 was synthesized using d-3 and a-8 as starting materials as a colorless viscous liquid (72.73 mg, 0.1236 mmol, 61.8%).

[0173] MS (ESI) m / z: [M+H] + 589.13. 1 H NMR (500MHz, CDCl3) δ2.86 (s, 4H), 2.66 (t, J = 5.9Hz,

[0174] 2H), 2.40 (s, 4H), 2.32 (d, J = 3.9Hz, 2H), 1.41–1.37 (m, 4H), 1.28–1.22 (m, 40H), 0.88 (s, 6H).

[0175] Example 20 Synthesis of ionizable lipid I-2

[0176]

[0177] (1) Synthesis of Compound I-2: Referring to the method of step (4) of Example 1, compound I-2 was synthesized using d-3 and a-10 as starting materials as a colorless viscous liquid (93.1 mg, 0.1158 mmol, 57.9%).

[0178] MS (ESI) m / z: [M+H] + 805.75. 1 H NMR (500MHz, CDCl3) δ7.07(s,4H),3.69(s,4H),2.53(s,4H),2.30(s,4H),1.29–1.22(m,40H),0.91(s,6H).

[0179] Example 21 Synthesis of ionizable lipid I-4

[0180]

[0181] (1) Synthesis of Compound I-4: Referring to the method of step (4) of Example 1, compound I-4 was synthesized using d-3 and a-9 as starting materials as a colorless viscous liquid (74.85 mg, 0.1272 mmol, 63.6%).

[0182] MS (ESI) m / z: [M+H] +589.47. 1 H NMR (500MHz, CDCl3) δ7.25 (d, J=4.9Hz, 2H), 6.96

[0183] (d,J=6.4Hz,4H),3.90(s,4H),2.65(s,4H),2.40(t,J=7.7Hz,4H),1.29–1.22(m,40H),0.92(t,J=6.8Hz,6H).

[0184] Example 22 Synthesis of ionizable lipid I-6

[0185]

[0186] (1) Synthesis of Compound I-6: Referring to the method of step (4) of Example 1, compound I-6 was synthesized using d-3 and a-11 as starting materials as a colorless viscous liquid (94.62 mg, 0.1328 mmol, 66.4%).

[0187] MS (ESI) m / z: [M+H] + 713.51. 1 H NMR (500MHz, CDCl3) δ7.60 (d, J=7.9Hz, 4H), 7.52

[0188] (d,J=7.8Hz,4H),3.70(s,4H),2.67–2.52(m,4H),2.35(t,J=7.6Hz,4H),1.30–1.22(m,40H),0.92(t,J=6.8Hz,6H).

[0189] Example 23 Synthesis of ionizable lipid I-15

[0190]

[0191] (1) Synthesis of compound e-2: Referring to the method of step (1) of Example 1, compound e-2 was synthesized using e-1 as the starting material as a light yellow oily liquid (3.607 g, 17 mmol, 68%).

[0192] MS (ESI) m / z: [M+H] + 213.34. 1 H NMR (500MHz, CDCl3, ppm) δ9.75 (s, 1H), 2.41 (t, J=

[0193] 7.3Hz, 2H) 1.63–1.61 (m, 2H), 1.34–1.26 (m, 20H), 0.88 (t, J = 6.7Hz, 3H).

[0194] (2) Synthesis of compound e-4: Referring to the method of step (2) of Example 1, compound e-4 was synthesized using e-2 and a-3 as starting materials as a light yellow viscous liquid (2.519 g, 4.56 mmol, 57%).

[0195] MS (ESI) m / z: [M+H] + 553.56. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.22(m,44H),0.88(t,J=6.8Hz,6H).

[0196] (3) Synthesis of compound e-5: Referring to the method of step (3) of Example 1, compound e-5 was synthesized using e-4 as the starting material as a light yellow viscous liquid (1.719 g, 3.80 mmol, 95%).

[0197] MS (ESI) m / z: [M+H] + 453.67. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0198] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.21(m,44H),0.89(t,J=6.9Hz,6H).

[0199] (4) Synthesis of Compound I-15: Referring to the method of step (4) of Example 1, using e-5 and a-6 as starting materials, compound I-15 was obtained as a colorless viscous liquid (59.6 mg, 0.097 mmol, 48.7%).

[0200] MS (ESI) m / z: [M+H] + 613.04. 1H NMR (500MHz, CDCl3) δ7.48 (s, 4H), 3.56 (s, 4H), 2.66 (d, J = 8.1Hz, 2H), 2.59 (d, J = 8.1Hz, 2H), 2.45(t,J=7.8Hz,4H),1.43–1.37(m,4H),1.26–1.21(m,44H),0.88(t,J=6.8Hz,6H).

[0201] Example 24 Synthesis of ionizable lipid I-16

[0202]

[0203] (1) Synthesis of Compound I-16: Referring to the method of step (4) of Example 1, e-5 and a-8 were used as starting materials to obtain Compound I-16 as a colorless viscous liquid (80.3 mg, 0.1246 mmol, 62.3%).

[0204] MS (ESI) m / z: [M+H] + 645.54. 1 H NMR (500MHz, CDCl3) δ2.86 (s, 4H), 2.66 (t, J = 5.9Hz,

[0205] 2H), 2.40 (s, 4H), 2.32 (d, J = 3.9Hz, 2H), 1.41–1.35 (m, 4H), 1.28–1.22 (m, 48H), 0.88 (s, 6H).

[0206] Example 25 Synthesis of ionizable lipid I-10

[0207]

[0208] (1) Synthesis of compound f-2: Referring to the method of step (1) of Example 1, compound f-2 was synthesized using f-1 as the starting material as a light yellow oily liquid (3.607 g, 17 mmol, 68%).

[0209] MS (ESI) m / z: [M+H] + 241.34. 1 H NMR (500MHz, CDCl3, ppm) δ9.75 (s, 1H), 2.41 (t, J=

[0210] 7.3Hz,2H)1.63–1.61(m,2H),1.34–1.28(m,24H),0.88(t,J=6.7Hz,3H).

[0211] (2) Synthesis of compound f-4: Referring to the method of step (2) of Example 1, compound f-4 was synthesized using f-2 and a-3 as starting materials as a light yellow viscous liquid (2.772 g, 4.56 mmol, 57%).

[0212] MS (ESI) m / z: [M+H] + 609.62. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.22(m,52H),0.88(t,J=6.8Hz,6H).

[0213] (3) Synthesis of compound f-5: Referring to the method of step (3) of Example 1, compound f-5 was synthesized using f-4 as the starting material as a light yellow viscous liquid (1.961 g, 3.80 mmol, 96.5%).

[0214] MS (ESI) m / z: [M+H] + 509.52. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0215] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.21(m,52H),0.89(t,J=6.9Hz,6H).

[0216] (4) Synthesis of Compound I-10: Referring to the method of step (4) of Example 1, using f-5 and a-6 as starting materials, compound I-10 was obtained as a colorless viscous liquid (59.6 mg, 0.097 mmol, 48.7%).

[0217] MS (ESI) m / z: [M+H] + 669.64. 1 H NMR (500MHz, CDCl3) δ7.48 (s, 4H), 3.56 (s, 4H), 2.66 (d, J = 8.1Hz, 2H), 2.59 (d, J = 8.1Hz, 2H), 2.45(t,J=7.8Hz,4H), 1.43–1.38(m,4H), 1.26–1.99(m,52H), 0.88(t,J=6.8Hz,6H).

[0218] Example 26 Synthesis of ionizable lipid I-17

[0219]

[0220] (1) Synthesis of Compound I-17: Referring to the method of step (4) of Example 1, compound I-17 was obtained as a colorless viscous liquid (78.26 mg, 0.112 mmol, 55.9%) using f-5 and a-9 as starting materials.

[0221] MS (ESI) m / z: [M+H] + 701.26. 1 H NMR (500MHz, CDCl3) δ7.25 (d, J=4.9Hz, 2H), 6.96

[0222] (d,J=6.4Hz,4H),3.90(s,4H),2.65(s,4H),2.40(t,J=7.7Hz,4H),1.29–1.23(m,56H),0.92(t,J=6.8Hz,6H).

[0223] Example 27 Synthesis of ionizable lipid I-9

[0224]

[0225] (1) Synthesis of Compound I-9: Referring to the method of step (4) of Example 1, compound I-9 was obtained as a colorless viscous liquid (82.1 mg, 0.1172 mmol, 58.6%) using f-5 and a-8 as starting materials.

[0226] MS (ESI) m / z: [M+H] + 701.56. 1 H NMR (500MHz, CDCl3) δ2.86 (s, 4H), 2.66 (t, J = 5.9Hz,

[0227] 2H), 2.40 (s, 4H), 2.32 (d, J = 3.9Hz, 2H), 1.41–1.37 (m, 4H), 1.28–1.22 (m, 56H), 0.88 (s, 6H).

[0228] Example 28 Synthesis of ionizable lipid I-8

[0229]

[0230] (1) Synthesis of Compound I-8: Referring to the method of step (4) of Example 1, compound I-8 was obtained as a colorless viscous liquid (100.3 mg, 0.1248 mmol, 62.4%) using f-5 and a-12 as starting materials.

[0231] MS (ESI) m / z: [M+H] + 803.35. 1 H NMR (500MHz, CDCl3) δ8.95 (s, 2H), 8.09 (d, J = 8.4Hz,

[0232] 2H),7.99(s,2H),7.56(d,J=8.4Hz,2H),3.79(s,4H),2.65(s,4H),2.31(t,J=7.6Hz,4H),1.26–1.21(m,56H),0.90(t,J=6.8Hz,6H).

[0233] Example 29 Synthesis of ionizable lipid I-7

[0234]

[0235] (1) Synthesis of Compound I-7: Referring to the method of step (4) of Example 1, compound I-7 was obtained as a colorless viscous liquid (110.9 mg, 0.1346 mmol, 67.3%) using f-5 and a-13 as starting materials.

[0236] MS (ESI) m / z: [M+H] + 825.21. 1 H NMR (500MHz, CDCl3) δ7.64 (s, 2H), 7.56 (d, J = 7.7Hz,

[0237] 2H),7.51(d,J=7.8Hz,2H),7.44(t,J=7.7Hz,2H),3.68(s,4H),2.58(s,4H),2.33(s,4H),1.28–1.21(m,56H),0.90(t,J=6.8Hz,6H).

[0238] Example 30 Synthesis of ionizable lipid I-20

[0239]

[0240] (1) Synthesis of compound g-2: Referring to the method of step (2) of Example 1, compound g-2 was synthesized using g-1 and a-3 as starting materials as a light yellow viscous liquid (1.544 g, 4.108 mmol, 63.2%).

[0241] MS (ESI) m / z: [M+H] + 377.56. 1H NMR (500MHz, CDCl3, ppm) δ6.20 (dd, J=15.2,10.4

[0242] Hz,2H),6.08(dd,J=14.9,10.5Hz,2H),5.70–5.64(m,2H),5.60–5.56(m,2H),3.16(d,J=7.1Hz,4H),2 .73(t,J=7.7Hz,4H),2.62–2.52(m,4H),2.33–2.27(m,4H),1.44(s,9H),0.93(dd,J=8.0,6.6Hz,6H).

[0243] (2) Synthesis of compound g-3: Referring to the method of step (3) of Example 1, compound g-3 was synthesized using g-2 as the starting material as a light yellow viscous liquid (0.81 g, 2.934 mmol, 97.8%).

[0244] MS (ESI) m / z: [M+H] + 277.45. 1 H NMR (400MHz, CDCl3, ppm) δ6.20 (dd, J=15.2,10.4

[0245] Hz,2H),6.08(dd,J=14.9,10.5Hz,2H),5.70–5.65(m,2H),5.60–5.54(m,2H),3.16(d,J=7.1 Hz, 4H), 2.73 (t, J = 7.7 Hz, 4H), 2.33–2.28 (m, 4H), 1.44–1.41 (m, 4H), 0.88 (t, J = 6.6 Hz, 6H).

[0246] (3) Synthesis of Compound I-20: Referring to the method of step (4) of Example 1, compound I-20 was synthesized using g-3 and a-6 as starting materials as a colorless viscous liquid (57.9 mg, 0.1328 mmol, 66.4%).

[0247] MS (ESI) m / z: [M+H] + 437.56. 1 H NMR (500MHz, Methanol-d4) δ7.64 (s, 4H), 6.23 (dd, J=

[0248] 15.0,10.3Hz,2H),6.09(dd,J=15.1,10.3Hz,2H),5.78–5.72(m,2H),5.55–5.51(m,2H),3.70(s,4H),3.30(d,J=7 .3Hz,4H),2.87(t,J=6.9Hz,2H),2.68(t,J=6.8Hz,2H),2.09–1.97(m,4H),1.46–1.41(m,4H),0.94–0.91(m,6H).

[0249] Example 31 Synthesis of Ionizable Lipid I-21

[0250]

[0251] (1) Synthesis of Compound I-21: Referring to the method of step (4) of Example 1, compound I-21 was synthesized using g-3 and a-8 as starting materials as a colorless viscous liquid (54.9 mg, 0.1174 mmol, 58.7%).

[0252] MS (ESI) m / z: [M+H] + 467.57. 1 H NMR(500MHz,Methanol-d4)δ6.20(dd,J=15.2,10.4

[0253] Hz,2H),6.08(dd,J=14.9,10.5Hz,2H),5.70–5.63(m,2H),5.60–5.56(m,2H),3.16(d,J=7.1Hz,4H),2.73(t,J=7. 7Hz,4H),2.62–2.52(m,4H),2.33–2.28(m,4H),2.08–1.96(m,4H),1.44–1.36(m,4H),0.93(dd,J=8.0,6.6Hz,6H).

[0254] Example 32 Synthesis of ionizable lipid I-22

[0255] (1) Synthesis of intermediate r-6

[0256]

[0257] 1) Synthesis of Intermediate r-5: Compound a-3-1 (2.61 g, 15 mmol) was dissolved in DCM (150 ml, 0.10 M), followed by the addition of compound a-6 (3.6 g, 37.5 mmol) and stirring for 10 min. Finally, NaBH(OAc)3 (9.537 g, 45 mmol) was added and stirred at room temperature for 3 h. The reaction was monitored by TLC and quenched by the addition of saturated NaHCO3 solution (80 ml). The mixture was extracted with DCM (60 × 3 ml), and the organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound r-5 as a light yellow viscous liquid (2.167 g, 6.495 mmol, 43.3%).

[0258] MS (ESI) m / z: [M+H] + 335.45. 1 H NMR (500MHz, Methanol-d4) δ8.21 (s, 4H), 4.41 (s, 4H), 3.17 (s, 2H), 2.98 (d, J = 7.5Hz, 2H), 2.22 (s, 2H), 1.45 (s, 9H).

[0259] 2) Synthesis of Intermediate r-6: Compound r-5 (2.167 g, 6.495 mmol) was dissolved in EA (15.0 ml, 0.20 M), and a 4 M solution of hydrogen chloride in ethyl acetate (13.0 ml, 51.96 mmol) was added. The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. The mixture was quenched by the addition of saturated NaHCO₃ solution (60 ml), and extracted with EA (60 ml x 3). The organic phases were combined and used without purification to obtain compound r-6 (1.49 g, 6.4 mmol, 99%).

[0260] MS (ESI) m / z: [M+H] + 235.14. 1 H NMR (500MHz, Methanol-d4) δ 8.21 (s, 4H), 4.41 (s, 4H), 3.17 (s, 2H), 2.98 (d, J = 7.5Hz, 2H), 2.22 (s, 2H).

[0261] (2) Synthesis of Compound I-22

[0262]

[0263] 1) Referring to the method of step (4) of Example 1, compound I-22 was synthesized using b-1 and r-6 as starting materials as a colorless viscous liquid (108.3 mg, 0.21 mmol, 65.2%).

[0264] MS (ESI) m / z: [M+H] + 515.79. 1 H NMR (500MHz, Methanol-d4) δ7.55(s,4H),3.57(s,4H),2.49(m,J=10.4,3.4,2.4Hz,6H),2.42(t ,J=7.3Hz,2H),1.77–1.68(m,2H),1.50–1.44(m,4H),1.30–1.23(m,28H),0.90(t,J=6.8Hz,6H).

[0265] Example 33 Synthesis of ionizable lipid I-19

[0266]

[0267] (1) Synthesis of Compound I-19: Referring to the method of step (4) of Example 1, compound I-19 was obtained as a colorless viscous liquid (110.9 mg, 0.1346 mmol, 67.3%) using f-2 and r-6 as starting materials.

[0268] MS (ESI) m / z: [M+H] + 683.34. 1 H NMR (500MHz, Methanol-d4) δ7.55 (s, 4H), 3.57 (s, 4H), 2.49 (m, J = 10.4, 3.4, 2.4Hz, 6H), 2.42 (t, J =7.3Hz,2H),1.77–1.68(m,2H),1.50–1.44(m,4H),1.30(d,J=10.0Hz,52H),0.90(t,J=6.8Hz,6H).

[0269] Example 34 Synthesis of ionizable lipid I-23

[0270]

[0271] (1) Synthesis of Compound I-23: Referring to the method of step (4) of Example 1, compound I-23 was synthesized using b-1 and r-6 as starting materials as a colorless viscous liquid (128.3 mg, 0.2 mmol, 63.2%).

[0272] MS (ESI) m / z: [M+H] + 627.17. 1H NMR(500MHz,Methanol-d4)δ7.56(s,4H),3.57(s,4H),2.51–2.47(m,6H),2.42(t,J=7.3 Hz,2H),1.76–1.69(m,2H),1.49–1.43(m,4H),1.29–1.23(m,44H),0.90(t,J=6.9Hz,6H).

[0273] Example 35 Synthesis of ionizable lipid I-24

[0274]

[0275] (1) Synthesis of Compound I-24: Referring to the method of step (4) of Example 1, compound I-24 was synthesized using d-1 and r-6 as starting materials as a colorless viscous liquid (113.3 mg, 0.198 mmol, 60.5%).

[0276] MS (ESI) m / z: [M+H] + 571.72. 1 H NMR(500MHz, Methanol-d4)δ7.58(s,4H),3.60(s,4H),2.74–2.68(m,6H),2.48(t,J=6.9 Hz,2H),1.82–1.73(m,2H),1.54–1.51(m,4H),1.34–1.25(m,36H),0.90(t,J=6.9Hz,6H).

[0277] Example 36 Synthesis of Ionizable Lipid I-26

[0278] (1) Synthesis of intermediate r-2

[0279]

[0280] 1) Synthesis of intermediate r-1: Referring to the method of step (1) 1) of Example 32, compound r-1 was synthesized using a-3 and a-6 as starting materials as a light yellow viscous liquid (2.078 g, 6.495 mmol, 43.3%).

[0281] MS (ESI) m / z: [M+H] + 321.45. 1 H NMR (500MHz, Methanol-d4) δ7.60 (s, 4H), 3.61 (s, 4H), 3.22 (t, J = 6.7Hz, 2H), 2.51 (t, J = 6.8Hz, 2H), 1.45 (s, 9H).

[0282] 2) Synthesis of intermediate r-2: Referring to the method of step (1) 2) of Example 1, compound r-2 was synthesized using r-1 as the starting material as a light yellow viscous liquid (1.66 g, 6.4 mmol, 100%).

[0283] MS (ESI) m / z: [M+H] + 221.21. 1 H NMR (500MHz, Methanol-d4) δ8.21 (s, 4H), 4.41 (s, 4H), 3.17 (s, 2H), 2.98 (d, J = 7.5Hz, 2H).

[0284] (2) Synthesis of Compound I-26:

[0285]

[0286] 1) Compound r-2 (66.3 mg, 0.3 mmol) was dissolved in a mixed solvent of MeCN (3 ml, 0.1 M) and THF (3 ml, 0.1 M), and Br6 Acid was added. 10 (207.6 mg, 0.6 mmol), K2CO3 (124.2 mg, 0.9 mmol), and KI (9.96 mg, 0.06 mmol) were added to the mixture and heated to reflux with stirring for 6 h. The reaction was monitored for completion by TLC and quenched by the addition of saturated NH4Cl (10 ml). The mixture was extracted with EA (30 ml x 4). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 7:1) to obtain compound ZM-26 as a light yellow viscous liquid (139.6 mg, 0.186 mmol, 61%).

[0287] MS (ESI) m / z: [M+H] + 753.13. 1 H NMR(500MHz,Methanol-d4)δ7.58(s,4H),5.80–5.75(m,2H),5.03–4.91(m,4H),4.07(t,J=6.6Hz,4H),3.60(s,4H),2.61(dd,J=9.5,5.6 Hz,2H),2.52(dd,J=9.5,5.4Hz,2H),2.43–2.37(m,4H),2.30(t,J=7.4Hz,4H),2.04–1.97(m,4H),1.64–1.58(m,8H),1.42–1.28(m,32H).

[0288] Example 37 Synthesis of ionizable lipid I-28

[0289]

[0290] (1) Synthesis of Compound I-28: Referring to the method of step (2) of Example 36, r-2 and Br6Acid 11 As the starting material, compound Ⅰ-28 was synthesized as a colorless viscous liquid (104.4 mg, 0.1332 mmol, 66.6%).

[0291] MS (ESI) m / z: [M+H] + 785.6. 1 H NMR (500MHz, Methanol-d4) δ7.60(s,4H),4.09(t,J=6.5Hz,4H),3.61(s,4H),2.63(dd,J=9.6,5.5Hz,2H),2.53(dd,J=9 .5,5.4Hz,2H),2.44–2.39(m,4H),2.32(t,J=7.4Hz,4H),1.67–1.60(m,8H),1.43–1.25(m,44H),0.92(t,J=6.9Hz,6H).

[0292] Example 38 Synthesis of Ionizable Lipid I-48

[0293]

[0294] (1) Synthesis of Compound I-48: Compound r-2 (105.5 mg, 0.5 mmol) was dissolved in EtOH (5 ml, 0.10 M), and compound a-16 (184 mg, 1.0 mmol) was added. The temperature was raised to 80°C and stirred for 4 h. The reaction was monitored by TLC and concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH = 15:1) to obtain compound I-48 as a light yellow viscous liquid (133.6 mg, 0.23 mmol, 28.4%).

[0295] MS (ESI) m / z: [M+H] + 589.59. 1 H NMR (500MHz, Methanol-d4) δ7.64 (s, 4H), 3.75–3.57 (m, 6H), 2.99–2.38 (m, 8H), 1.44–1.28 (m, 36H), 0.90 (t, J = 6.9Hz, 6H).

[0296] Example 39 Synthesis of Ionizable Lipid I-29

[0297]

[0298] (1) Synthesis of Compound I-29: Referring to the method of step (2) of Example 36, r-3 and Br6Acid 15 As the starting material, compound Ⅰ-29 was synthesized as a colorless viscous liquid (92.8 mg, 0.111 mmol, 55.5%).

[0299] MS (ESI) m / z: [M+H] + 897.43. 1 H NMR (500MHz, Methanol-d4) δ7.60(s,4H),4.09(t,J=6.5Hz,4H),3.62(s,4H),2.67(t,J=7.5Hz,2H),2.55(dd,J=9.2,5 .6Hz,2H),2.45(t,J=7.9Hz,4H),2.32(t,J=7.4Hz,4H),1.66–1.59(m,8H),1.46–1.21(m,60H),0.92(t,J=6.9Hz,6H).

[0300] Example 40 Synthesis of ionizable lipid I-30

[0301]

[0302] (1) Synthesis of Compound I-30: Referring to the method of step (2) of Example 36, r-2 and Br6Acid 17 As the starting material, compound Ⅰ-30 was synthesized as a colorless viscous liquid (92.8 mg, 0.111 mmol, 55.5%).

[0303] MS (ESI) m / z: [M+H] + 953.54. 1 H NMR(500MHz,Methanol-d4)δ7.62(s,4H),4.09(t,J=6.5Hz,4H),3.65(s,4H),2.80(s,2H),2.5 9(s,6H),2.32(t,J=7.4Hz,4H),1.67–1.60(m,8H),1.50–1.28(m,68H),0.92(t,J=6.9Hz,6H).

[0304] Example 41 Synthesis of Ionizable Lipid I-31

[0305]

[0306] (1) Synthesis of Compound I-31: Referring to the method of step (2) of Example 36, compound I-31 was synthesized using r-2 and Br6Acid5 as starting materials as a colorless viscous liquid (73.67 mg, 0.111 mmol, 59.0%).

[0307] MS (ESI) m / z: [M+H] + 617.67. 1 1H NMR (500MHz, Methanol-d4) δ7.60(s,4H),4.08(t,J=6.6Hz,4H),3.62(s,4H),2.65(dd,J=9.3,5.7Hz,2H),2.54(dd,J=9 .4,5.5Hz,2H),2.46–2.40(m,4H),2.32(t,J=7.4Hz,4H),1.63–1.57(m,8H),1.46–1.27(m,20H),0.93(t,J=7.1Hz,6H).

[0308] Example 42 Synthesis of Ionizable Lipid I-32

[0309]

[0310] (1) Synthesis of Compound I-32: Referring to the method of step (2) of Example 36, compound I-32 was synthesized using r-2 and Br6Acid6 as starting materials as a colorless viscous liquid (62.59 mg, 0.097 mmol, 48.6%).

[0311] MS (ESI) m / z: [M+H] + 645.5. 1 H NMR (500MHz, Methanol-d4) δ7.60 (s, 4H), 4.08 (t, J=

[0312] 6.6Hz,4H),3.61(s,4H),2.62(dd,J=9.5,5.4Hz,2H),2.53(dd,J=9.4,5.4Hz,2H),2.45–2.39 (m,4H),2.32(t,J=7.4Hz,4H),1.66–1.59(m,8H),1.49–1.26(m,24H),0.92(t,J=6.7Hz,6H).

[0313] Example 43 Synthesis of ionizable lipid I-33

[0314]

[0315] (1) Synthesis of Compound I-33: Referring to the method of step (2) of Example 36, compound I-33 was synthesized using r-2 and Br6Acid7 as starting materials as a colorless viscous liquid (68.95 mg, 0.103 mmol, 51.3%).

[0316] MS (ESI) m / z: [M+H] + 673.7. 1 H NMR (500MHz, Methanol-d4) δ7.61 (s, 4H), 4.09 (t, J=

[0317] 6.6Hz,4H),3.63(s,4H),2.77–2.66(m,2H),2.57(dd,J=8.8,5.7Hz,2H),2.50(t,J=7.9H z, 4H), 2.32 (t, J = 7.4Hz, 4H), 1.63–1.57 (m, 8H), 1.46–1.29 (m, 28H), 0.94–0.90 (m, 6H).

[0318] Example 44 Synthesis of Ionizable Lipid I-34

[0319]

[0320] (1) Synthesis of Compound I-34: Referring to the method of step (2) of Example 36, compound I-34 was synthesized using r-2 and Br6Acid8 as starting materials as a colorless viscous liquid (60.34 mg, 0.086 mmol, 43.1%).

[0321] MS (ESI) m / z: [M+H] + 701.76. 1 H NMR (500MHz, Methanol-d4) δ7.60 (s, 4H), 4.08 (t, J=

[0322] 6.6Hz,4H),3.63(s,4H),2.68(t,J=7.4Hz,2H),2.55(dd,J=9.1,5.6Hz,2H),2.51–2.41(m ,4H),2.32(t,J=7.4Hz,4H),1.67–1.59(m,8H),1.45–1.28(m,32H),0.92(t,J=6.8Hz,6H).

[0323] Example 45 Synthesis of ionizable lipid I-54

[0324]

[0325] (1) Synthesis of Compound I-54: Referring to the method of step (2) of Example 36, r-2 and Br6Acid 10-1 As the starting material, compound Ⅰ-54 was synthesized as a colorless viscous liquid (68.34 mg, 0.09 mmol, 46.1%).

[0326] MS (ESI) m / z: [M+H] + 757.80. 1 H NMR (500MHz, Methanol-d4) δ7.60 (s, 4H), 4.09 (t, J=

[0327] 6.5Hz,4H),3.61(s,4H),2.63(dd,J=9.6,5.5Hz,2H),2.53(dd,J=9.5,5.4Hz,2H),2.44–2.39 (m,4H),2.32(t,J=7.4Hz,4H),1.67–1.60(m,8H),1.43–1.25(m,40H),0.92(t,J=6.9Hz,6H).

[0328] Example 46 Synthesis of Ionizable Lipid I-55

[0329]

[0330] (1) Synthesis of Compound I-55: Referring to the method of step (2) of Example 36, r-6 and Br6Acid 11 As the starting material, compound Ⅰ-55 was synthesized as a colorless viscous liquid (68.34 mg, 0.09 mmol, 46.1%).

[0331] MS (ESI) m / z: [M+H] + 799.41. 1 H NMR (500MHz, Methanol-d4) δ7.61 (s, 4H), 4.08 (t, J=

[0332] 6.6Hz,4H),3.64(s,4H),2.97–2.85(m,6H),2.54(t,J=6.8Hz,2H),2.30(t,J=7.4H z,4H),1.90–1.82(m,2H),1.62(m,12H),1.44–1.28(m,40H),0.90(t,J=6.9Hz,6H).

[0333] Example 47 Synthesis of Ionizable Lipid I-36

[0334] (1) Synthesis of intermediate r-2

[0335]

[0336] 1) Synthesis of intermediate r-3: Referring to the method of step (1) 1) of Example 32, compound r-3 was synthesized using a-3 and a-7 as starting materials as a light yellow viscous liquid (2.078 g, 6.495 mmol, 43.3%).

[0337] MS (ESI) m / z: [M+H] + 321.45. 1 H NMR (500MHz, Methanol-d4) δ 8.96 (s, 2H), 7.67 (s, 2H), 3.61 (s, 4H), 3.22 (t, J = 6.7Hz, 2H), 2.51 (t, J = 6.8Hz, 2H), 1.45 (s, 9H).

[0338] 2) Synthesis of intermediate r-4: Referring to the method of step (1) 2) of Example 32, compound r-4 was synthesized using r-3 as a starting material as a light yellow viscous liquid (1.66 g, 6.4 mmol, 100%).

[0339] MS (ESI) m / z: [M-2HCl+H] + 221.32. 1 H NMR (500MHz, Methanol-d4) δ8.96(s,2H),7.67(s,2H),3.92(s,4H),3.21(s,2H),2.90(s,2H).

[0340] (2) Synthesis of Compound I-36

[0341]

[0342] 1) Referring to the method of step (2) of Example 36, r-4 and Br6Acid 11 As the starting material, compound Ⅰ-36 was synthesized as a colorless viscous liquid (95.14 mg, 0.1214 mmol, 60.7%).

[0343] MS:m / z(M+H + )785.22. 1H NMR (500MHz, Methanol-d4) δ7.66(s,2H),6.99(s,2H),4.08(t,J=6.6Hz,4H),3.66(s,4H),2.58(tt,J=8.4,4.2Hz ,4H),2.45–2.38(m,4H),2.32(t,J=7.4Hz,4H),1.63(q,J=7.2Hz,8H),1.44–1.27(m,44H),0.92(t,J=6.9Hz,6H).

[0344] Example 48 Synthesis of Ionizable Lipid I-27

[0345]

[0346] (1) Synthesis of Compound I-27: Referring to the method of step (2) of Example 36, r-4 and Br6Acid 10 As the starting material, compound Ⅰ-27 was synthesized as a colorless viscous liquid (93.1 mg, 0.1238 mmol, 61.9%).

[0347] MS:m / z(M+H + )753.45. 1 H NMR(500MHz,Methanol-d4)δ7.65(s,2H),6.98(s,2H),5.88–5.71(m,2H),4.91(s,4H),4.07(t,J=6.6Hz,4H),3.65(s,4H) ,2.57(d,J=3.9Hz,4H),2.41–2.37(m,4H),2.30(d,J=7.3Hz,4H),2.05–1.96(m,4H),1.62–1.57(m,8H),1.43–1.25(m,32).

[0348] Example 49 Synthesis of Ionizable Lipid I-37

[0349]

[0350] (1) Synthesis of Compound I-37: Referring to the method of step (2) of Example 36, compound I-37 was synthesized using r-4 and Br6Acid6 as starting materials as a colorless viscous liquid (73.1 mg, 0.113 mmol, 62.5%).

[0351] MS (ESI) m / z: [M+H] + 645.65. 1H NMR (500MHz, Methanol-d4) δ7.64 (d, J = 1.1Hz, 2H),

[0352] 6.98(s,2H),4.06(t,J=6.6Hz,4H),3.65(s,4H),2.56–2.53(m,4H),2.41–2.37(m,4H) ,2.30(d,J=7.4Hz,4H),1.63–1.58(m,8H),1.39–1.26(m,24H),0.90(t,J=7.1Hz,6H).

[0353] Example 50 Synthesis of ionizable lipid I-38

[0354]

[0355] (1) Synthesis of Compound I-38: Referring to the method of step (2) of Example 36, compound I-38 was synthesized using r-4 and Br6Acid5 as starting materials as a colorless viscous liquid (93.1 mg, 0.15 mmol, 68.5%).

[0356] MS (ESI) m / z: [M+H] + 617.63. 1 H NMR (500MHz, Methanol-d4) δ7.64 (d, J = 1.1Hz, 2H),

[0357] 6.98(s,2H),4.06(t,J=6.6Hz,4H),3.64(s,4H),2.56–2.52(m,4H),2.41–2.36(m,4H) ,2.30(t,J=7.4Hz,4H),1.65–1.58(m,8H),1.42–1.26(m,20H),0.91(t,J=7.0Hz,6H).

[0358] Example 51 Synthesis of Ionizable Lipid I-39

[0359]

[0360] (1) Synthesis of Compound I-39: Referring to the method of step (2) of Example 36, r-4 and Br6Acid 15 As the starting material, compound Ⅰ-39 was synthesized as a colorless viscous liquid (133.1 mg, 0.148 mmol, 68.5%).

[0361] MS (ESI) m / z: [M+H] + 897.78. 1H NMR (500MHz, Methanol-d4) δ7.64 (d, J = 1.1Hz, 2H),

[0362] 6.97(s,2H),4.07(t,J=6.5Hz,4H),3.65(s,4H),2.58–2.53(m,4H),2.44–2.36(m,4H) ,2.30(t,J=7.4Hz,4H),1.61–1.58(m,8H),1.44–1.24(m,60H),0.90(t,J=6.9Hz,6H).

[0363] Example 52 Synthesis of ionizable lipid I-35

[0364]

[0365] (1) Synthesis of Compound I-35: Referring to the method of step (2) of Example 36, compound I-35 was synthesized using r-4 and Br6Acid8 as starting materials as a colorless viscous liquid (69.86 mg, 0.099 mmol, 49.9%).

[0366] MS (ESI) m / z: [M+H] + 701.52. 1 H NMR (500MHz, Methanol-d4) δ7.67 (d, J = 1.1Hz, 2H),

[0367] 7.00(s,2H),4.08(t,J=6.6Hz,4H),3.67(s,4H),2.66–2.62(m,2H),2.59(dd,J=8.4,4.9Hz,2H),2 .47–2.43(m,4H),2.32(t,J=7.4Hz,4H),1.63–1.57(m,8H),1.48–1.27(m,32H),0.93–0.90(m,6H).

[0368] Example 53 Synthesis of Ionizable Lipid I-49

[0369]

[0370] (1) Synthesis of Compound I-49: Referring to the method of step (2) of Example 36, r-2 and Br8OL 17 As the starting material, compound Ⅰ-35 was synthesized as a colorless viscous liquid (2.014 g, 2.357 mmol, 71%).

[0371] MS (ESI) m / z: [M+H] + 891.98.1 H NMR(500MHz,Methanol-d4)δ7.52(s,4H),4.93–4.87(m,2H),3.58(s,4H),2.66–2.57(m,2H),2.57–2.50(m,2H),2.41(t,J=7.5Hz,4H),2 .31(t,J=7.5Hz,4H),1.63(t,J=7.2Hz,4H),1.53–1.47(m,8H),1.40(t,J=7.7Hz,4H),1.28–1.22(m,60H),0.89–0.82(t,J=6.9Hz,12H).

[0372] Example 54 Synthesis of Ionizable Lipid I-50

[0373]

[0374] (1) Synthesis of Compound I-50: Referring to the method of step (2) of Example 36, r-2 and Br6Acid 15 As the starting material, compound Ⅰ-50 was synthesized as a colorless viscous liquid (69.86 mg, 0.099 mmol, 49.9%).

[0375] MS (ESI) m / z: [M+H] + 897.78. 1 H NMR (500MHz, Methanol-d4) δ7.53(s,4H),4.08(t,J=6.8Hz,4H),3.58(s,4H),2.63(d,J=7.2Hz,2H),2.56(d,J=8.0Hz,2H),2 .44(s,4H),2.33(dd,J=8.9,5.3,3.5Hz,2H),1.61–1.56(m,8H),1.44–1.38(m,8H),1.27–1.22(m,48H),0.91–0.87(m,12H).

[0376] Example 55 Synthesis of ionizable lipid I-57

[0377]

[0378] (1) Synthesis of Compound I-57: Referring to the method of step (2) of Example 36, r-4 and Br6Acid 15 As the starting material, compound Ⅰ-57 was synthesized as a colorless viscous liquid (69.86 mg, 0.099 mmol, 49.9%).

[0379] MS (ESI) m / z: [M+H]+ 898.03. 1 H NMR (500MHz, Methanol-d4) δ7.68(s,2H),7.00(s,2H),4.10(t,J=6.4Hz,4H),3.66(s,4H),2.70(dd,J=8.4,5.7Hz,2H),2.62(dd,J=8 .5,5.6Hz,2H),2.56–2.45(m,4H),2.35–2.32(m,2H),1.76–1.54(m,8H),1.47–1.44(m,8H),1.44–1.23(m,48H),0.92–0.86(m,12H).

[0380] Example 56 Synthesis of Ionizable Lipid I-51

[0381]

[0382] (1) Synthesis of Compound I-51: Referring to the method of step (2) of Example 36, r-2 and Br8Acid 15 As the starting material, compound Ⅰ-51 was synthesized as a colorless viscous liquid (77.3 mg, 0.081 mmol, 33.2%).

[0383] MS (ESI) m / z: [M+H] + 954.06. 1 H NMR (500MHz, Methanol-d4) δ7.56 (s, 4H), 4.09 (t, J=

[0384] 6.7Hz,4H),3.60(s,4H),2.82–2.74(m,2H),2.69(s,2H),2.60(s,4H),2.36–2.31(m,2 H),1.65–1.58(m,8H),1.53–1.43(m,8H),1.33–1.24(m,56H),0.89(t,J=6.8Hz,12H).

[0385] Example 57 Synthesis of Ionizable Lipid I-60

[0386]

[0387] (1) Synthesis of Compound I-60: Referring to the method of step (2) of Example 36, compound I-60 was synthesized using r-2 and Br9Acid8 as starting materials as a colorless viscous liquid (169.86 mg, 0.22 mmol, 58.9%).

[0388] MS (ESI) m / z: [M+H]+ 785.86. 1 H NMR (500MHz, Methanol-d4) δ7.59(s,4H),4.82–4.76(m,2H),3.60(s,4H),2.67(dd,J=9.1,5.7Hz,2H),2.54(dd,J=9.1,5.6Hz,2 H),2.44(t,J=7.9Hz,4H),2.31(t,J=7.3Hz,4H),1.63–1.52(m,12H),1.45–1.39(m,4H),1.33–1.27(m,32H),0.91–0.79(m,12H).

[0389] Example 58 Synthesis of Ionizable Lipid I-63

[0390]

[0391] (1) Synthesis of Compound I-63: Referring to the method of step (2) of Example 36, compound I-63 was synthesized using r-2 and Br7Acid8 as starting materials as a colorless viscous liquid (104.86 mg, 0.144 mmol, 58.9%).

[0392] MS (ESI) m / z: [M+H] + 729.73. 1 H NMR (500MHz, Methanol-d4) δ7.58(s,4H),4.82–4.75(m,2H),3.60(s,4H),2.64(dd,J=9.3,5.6Hz,2H),2.53(dd,J=9.3,5 .6Hz,2H),2.47–2.37(m,4H),2.31(t,J=7.3Hz,4H),1.63–1.51(m,12H),1.47–1.22(m,28H),0.89(m,J=9.7,6.9Hz,12H).

[0393] Example 59 Synthesis of Ionizable Lipid I-64

[0394]

[0395] (1) Synthesis of Compound I-64: Referring to the method of step (2) of Example 36, r-2 and Br6Acid 15 As the starting material, compound Ⅰ-64 was synthesized as a colorless viscous liquid (110.86 mg, 0.123 mmol, 49.9%).

[0396] MS (ESI) m / z: [M+H] +897.90. 1 H NMR (500MHz, Methanol-d4) δ7.53(s,4H),4.08(t,J=6.8Hz,4H),3.58(s,4H),2.63(d,J=7.2Hz,2H),2.56(d,J=8.0Hz,2H),2. 44(s,4H),2.33(dd,J=8.9,5.3,3.5Hz,2H),1.61–1.57(m,8H),1.44–1.38(m,8H),1.27(d,J=5.0Hz,48H),0.91–0.87(m,12H).

[0397] Example 60 Synthesis of Ionizable Lipid I-58

[0398] (1) Synthesis of intermediate r-8

[0399]

[0400] 1) Synthesis of intermediate r-7: Referring to the method of step (1) 1) of Example 32, compound r-7 was synthesized using a-3 and a-18 as starting materials as a light yellow viscous liquid (2.078 g, 6.453 mmol, 43.3%).

[0401] MS (ESI) m / z: [M+H] + 323.45. 1 H NMR (500MHz, Methanol-d4) δ8.36 (d, J = 7.1Hz, 4H), 4.54 (s, 4H), 3.63 (s, 4H), 1.45 (s, 9H).

[0402] 2) Synthesis of intermediate r-8: Referring to the method of step (1) 2) of Example 32, compound r-8 was synthesized using r-7 as the starting material as a light yellow viscous liquid (1.42 g, 6.4 mmol, 100%).

[0403] MS (ESI) m / z: [M+H] + 223.32. 1 H NMR (500MHz, Methanol-d4) δ8.36 (d, J = 7.1Hz, 4H), 4.54 (s, 4H), 3.63 (s, 4H).

[0404] (2) Synthesis of Compound I-58

[0405]

[0406] 1) Synthesis of Compound I-58: Referring to the method of step (2) of Example 36, r-8 and Br6Acid 15 As the starting material, compound Ⅰ-58 was synthesized as a colorless viscous liquid (69.86 mg, 0.078 mmol, 50%).

[0407] MS (ESI) m / z: [M+H] + 900.03. 1 H NMR (500MHz, Methanol-d4) δ8.23(s,2H),7.94(s,2H),4.11(t,J=6.5Hz,4H),3.70(s,4H),3.04(d,J=23.2Hz,2H),2. 95–2.72(m,6H),2.36–2.32(m,2H),1.71–1.59(m,12H),1.52–1.40(m,12H),1.37–1.23(m,40H),0.92–0.88(m,12H).

[0408] Example 61 Synthesis of Ionizable Lipid I-59

[0409]

[0410] (1) Synthesis of Compound I-59: Referring to the method of step (2) of Example 36, compound I-59 was synthesized using r-8 and Br9Acid8 as starting materials as a colorless viscous liquid (69.86 mg, 0.099 mmol, 49.9%).

[0411] MS (ESI) m / z: [M+H] + 787.80. 1 H NMR (500MHz, Methanol-d4) δ8.19(s,2H),7.91(s,2H),4.84–4.74(m,2H),3.67(s,4H),2.80(d,J=8.4Hz,2H),2.70(t,J=7.0Hz, 2H), 2.57 (d, J = 9.0Hz, 4H), 2.31 (t, J = 7.3Hz, 4H), 1.68–1.55 (m, 8H), 1.54–1.46 (m, 8H), 1.31–1.28 (m, 32H), 0.89–0.86 (m, 12H).

[0412] Example 62 Synthesis of Ionizable Lipid I-61

[0413]

[0414] (1) Synthesis of Compound I-61: Referring to the method of step (2) of Example 36, r-8 and Br7Acid 17 As the starting material, compound Ⅰ-61 was synthesized as a colorless viscous liquid (114.86 mg, 0.12 mmol, 69.9%).

[0415] MS (ESI) m / z: [M+H] + 983.36. 1 H NMR(500MHz, Methanol-d4)δ8.20(s,2H),7.91(s,2H),4.86(d,J=7.3Hz,2H),3.67(s,4H),3.32–3.30(m,8H),2.92(s,2H),2.73 (d,J=31.3Hz,6H),2.31(t,J=7.2Hz,4H),1.62(t,J=7.2Hz,4H),1.53–1.50(m,12H),1.35–1.25(m,52H),0.89(d,J=7.1Hz,12H).

[0416] Example 63 Synthesis of Ionizable Lipid I-62

[0417]

[0418] (1) Synthesis of Compound I-62: Referring to the method of step (2) of Example 36, compound I-62 was synthesized using r-8 and Br7Acid8 as starting materials as a colorless viscous liquid (119.86 mg, 0.164 mmol, 49.9%).

[0419] MS (ESI) m / z: [M+H] + 731.70. 1 H NMR(500MHz,Methanol-d4)δ8.20(s,2H),7.91(s,2H),4.85–4.75(m,2H),3.68(s,4H),2.92(s,2H),2.82–2.59 (m,6H),2.32(t,J=7.3Hz,4H),1.65–1.61(m,4H),1.59–1.49(m,12H),1.38–1.26(m,24H),0.91–0.83(m,12H).

[0420] Example 64 Synthesis of Ionizable Lipid I-56

[0421]

[0422] (1) Synthesis of compound h-1: Referring to the method of step (2) of Example 36, Br6Acid 15 and r-2 as starting materials, compound h-1 was synthesized as a light yellow viscous liquid (83 mg, 0.25 mmol, 7.8%).

[0423] MS (ESI) m / z: [M+H] + 559.37. 1 H NMR (500MHz, Methanol-d4) δ7.43 (s, 4H), 4.05 (t, J=

[0424] 6.7Hz,2H),3.53(s,4H),2.78(d,J=6.2Hz,2H),2.61–2.58(m,4H),2.33 –2.25(m,1H),1.61–1.58(m,4H),1.36–1.21(m,28H),0.91–0.84(m,6H).

[0425] (2) Synthesis of Compound I-56: Referring to the method of step (4) of Example 1, h-1 and AL-10 were used as starting materials to synthesize Compound I-56, which was a light yellow viscous liquid (30 mg, 0.036 mmol, 14.5%).

[0426] MS (ESI) m / z: [M+H] + 827.74. 1 H NMR(500MHz,Methanol-d4)δ7.55(s,4H),4.07–4.02(m,4H),3.60(s,4H),2.71(s,2H),2.66–2.59(m,2H),2.53(s,4H),2 .32–2.28(m,3H),1.61–1.56(m,8H),1.46(dd,J=12.8,6.6Hz,6H),1.33–1.25(m,42H),0.89–0.84(m,J=6.9,2.4Hz,9H).

[0427] Example 65 Synthesis of Ionizable Lipid I-65

[0428]

[0429] (1) Synthesis of Compound I-65: Referring to the method of step (4) of Example 1, h-1 and AL-11 were used as starting materials to synthesize Compound I-65, which was a light yellow viscous liquid (45 mg, 0.053 mmol, 16.5%).

[0430] MS (ESI) m / z: [M+H]+ 841.87. 1 H NMR(500MHz,Methanol-d4)δ7.60(s,4H),4.08–3.94(m,4H),3.63(s,4H),2.80(t,J=7.0Hz,2H),2 .58–2.52(m,6H),2.39–2.28(m,3H),1.60–1.56(m,8H),1.47–1.22(m,50H),0.90(t,J=6.8Hz,9H).

[0431] Example 66 Synthesis of Ionizable Lipid I-66

[0432]

[0433] (1) Synthesis of Compound I-66: Referring to the method of step (4) of Example 1, h-2 and AL-11 were used as starting materials to synthesize Compound I-66, which was a light yellow viscous liquid (65 mg, 0.076 mmol, 17.5%).

[0434] MS (ESI) m / z: [M+H] + 855.95. 1 H NMR (500MHz, Methanol-d4) δ7.67–7.47(m,4H),4.08–3.94(m,4H),3.57(s,4H),2.47(t,J=7.7Hz,6H),2.41(t,J=7.4Hz, 2H),2.35–2.28(m,3H),1.76–1.68(m,2H),1.65–1.54(m,8H),1.48–1.40(m,6H),1.30–1.24(m,44H),0.90–0.84(m,9H).

[0435] Example 67 Preparation of Lipid Nanoparticles by Encapsulating mRNA with Ionizable Lipids

[0436] The materials used for assembling lipid nanoparticles include: (1) ionizable lipid compounds, such as the ionizable lipids designed and synthesized in the present invention; (2) structural lipids, such as cholesterol (purchased from Sigma-Aldrich); (3) phospholipids, such as DSPC, which is 1,2-distearoyl-SN-glycero-3-phosphocholine (distearoylphosphatidylcholine, purchased from AVT); (4) PEGylated lipid compounds, such as DMG-PEG2000, which is dimyristoylglycerol-polyethylene glycol 2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, purchased from AVT); and (5) nucleic acid fragment active ingredients, such as Luciferase mRNA (self-made).

[0437] Preparation method of lipid nanoparticles: ionizable lipid: phospholipid: cholesterol: PEGylated lipid are mixed in anhydrous ethanol in a ratio of 48.5:10:40:1.5 (molar percentage) to form an alcohol phase, and DLin-MC3-DMA (purchased from AVT) is used as a positive control.

[0438] Luciferase mRNA was then dissolved in 50mM sodium acetate solution (pH=5) to form an aqueous phase, in which the mass ratio of mRNA to ionizable lipids was 1:10, and the volume ratio of the alcohol phase to the aqueous phase was 1:3. LNPs were then prepared using an automated high-throughput microfluidic system: the mixing speed of the LNPs was set to 16mL / min, and the mixing flow rate ratio of the alcohol phase and the aqueous phase was 1:3. The prepared LNPs (N / P ratio of 6) were diluted with phosphate buffered saline (PBS) and then placed in an ultrafiltration tube with a molecular weight cutoff of 30kDa. The tubes were centrifuged at 4°C and 3000rpm for 30 minutes, and the elution was repeated twice to remove ethanol and sodium acetate as much as possible. Finally, the concentrated LNPs were obtained and stored in a 4°C refrigerator for later use.

[0439] Methods for preparing lipid nanoparticles include, but are not limited to, microfluidic mixing systems and also include T-type mixers and ethanol injection methods.

[0440] Example 68 Particle Size and PDI of Lipid Nanoparticles

[0441] After the freshly prepared LNP sample was equilibrated at room temperature, it was diluted with PBS at a ratio of 1:100 and then placed in a sample cell. The particle size and PDI size were measured using a dynamic light scattering instrument (Malvern Zetasizer Nano-ZS). The results are shown in Table 2.

[0442] Table 2 Particle size and PDI of lipid nanoparticles

[0443]

[0444] Table 2 Particle size and PDI of lipid nanoparticles

[0445]

[0446]

[0447] Table 2 Particle size and PDI of lipid nanoparticles

[0448]

[0449] As shown in Table 2, the lipid nanoparticles prepared by the present invention mostly have a particle size between 40 and 200 nm, with the exception of I-59, which has a particle size greater than 200 nm. Furthermore, the PDI values of all lipid nanoparticles are less than 0.5. We found that ionizable lipids with benzene rings (trifluoromethylphenyl, 3,5-difluorobromobenzene, benzothiazole, benzothiazole, and 1-phenylpyrrolidine) and pyrimidine attached to their heads produced lipid nanoparticles with particle sizes ranging from approximately 70 to 150 nm and PDI values ranging from approximately 0.25 to 0.4. Ionizable lipids with pyrazole and oxazole in their heads produced lipid nanoparticles with particle sizes ranging from approximately 60 to 150 nm and PDI values less than 0.3. Furthermore, ionizable lipids with imidazole incorporated into their heads produced lipid nanoparticles with larger particle sizes, ranging from approximately 100 to 200 nm, and also exhibited relatively high PDI values, ranging from approximately 0.25 to 0.5.

[0450] The size of lipid nanoparticles has a significant impact on their cellular uptake. Normally, a particle size between 50-200 nm is conducive to efficient uptake and transfection.

[0451] Example 69 Encapsulation Efficiency of Lipid Nanoparticles

[0452] The lipid nanoparticle encapsulation efficiency was determined using Quant-iT RiboGreen RNA Assay (purchased from Invitrogen). This coefficient reflects the degree of binding between lipid nanoparticles and mRNA fragments. The lipid nanoparticle sample was diluted in TE buffer (10mM Tris HCl, 1mM EDTA, pH = 7.5), and a portion of the sample solution was added with 0.5% Triton X100 and allowed to stand at 37°C for 30 minutes. Then, RiboGreen reagent was added to each sample, and the fluorescence value was read on a microplate reader (ex / em = 480 / 520nm). Encapsulation efficiency (%) = (total mRNA - free mRNA) / total mRNA × 100%

[0453] The encapsulation efficiency test results are as follows Figure 1 As shown in the figure, it was found that the LNPs of ionizable lipids with thiophene, trifluoromethylphenyl, benzothiazole, 3,5-difluorobromobenzene and 2,2,2-trifluoroethyl attached to the head tertiary amine were less than 60%; the LNPs of ionizable lipids with pyrazole, thiazole and oxazole introduced into the head had good mRNA encapsulation ability, and the encapsulation efficiency was greater than 60%; it was also found that the LNP encapsulation efficiency of ionizable lipids with imidazole attached to the head tertiary amine was greatly affected by the length of its tail chain. For example, for I-35 and I-36, when the number of carbon atoms attached to the ester end was 8, the encapsulation efficiency was above 90%. When the number of carbon atoms attached to the ester end was 5 (I-38) and 6 (I-37), the encapsulation efficiency would drop severely, both less than 50%.

[0454] Example 70 Transfection of lipid nanoparticles with 293T cells

[0455] 293T cells were seeded in 96-well plates at a density of 2 × 10 4 Cells were plated at 100 μL per well and incubated at 37°C, 5% CO2, and 90% humidity for 12 hours. Lipid nanoparticles containing different mRNA concentrations (2 μg / mL and 1 μg / mL) were added to 96-well plates in duplicate, and incubated at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0456] After 24 h, the protein expression level was detected using a multifunctional microplate reader (Thermo Scientific, model: Thermo Varioskan LUX).

[0457] like Figure 2 As shown, I-10, I-50 and I-64 have higher transfection efficiency, and the I-64-LNP with the highest transfection efficiency is 1.86 times that of the commercial ionizable lipid Dlin-MC3-DMA.

[0458] Combined with the chemical structure of ionizable lipids, it can be found that the ionizable lipids with pyrazole, imidazole and oxazole introduced into the head have relatively high transfection efficiency of lipid nanoparticles in 293T cells, among which the ionizable lipids with pyrazole introduced into the head have the best transfection ability. In addition, it can be seen from I-10, I-13, I-15 and I-47 that when the chain length of the two tail chains of the ionizable lipid containing a pyrazole ring is greater than 16, it has a higher transfection efficiency; from I-58, I-60 and I-61, it can be seen that in the ionizable lipid containing an oxazole ring, the chain length of the tail chain carbonyl oxygen end should not be too short, and when it is greater than or equal to 6, it has a higher transfection efficiency; from I-57 and I-58, it can be seen that the ionizable lipid with pyrazole introduced into the head has a higher transfection efficiency in lipid nanoparticles than the ionizable lipid with oxazole connected to the head; from I-64 and I-66, it can be seen that when the ionizable lipid tail chain is two branched tail chains containing ester groups, its lipid nanoparticle transfection efficiency is higher than that of the ionizable lipid containing one branched tail chain; from I-65 and I-66, when the ionizable lipid tail chain contains one branched tail chain, extending the connecting fragment will lead to a decrease in the cell transfection efficiency of its lipid nanoparticles.

[0459] Example 71 Lipid Nanoparticle Cytotoxicity Analysis

[0460] 293T cells in the logarithmic growth phase were seeded in 96-well plates at a seeding density of 3×10 4 Cells were added to each well (100 μL per well) and incubated at 37°C, 5% CO2, and 90% humidity for 12 hours. Lipid nanoparticles were incubated with 293T cells at different mRNA concentrations (4 μg / mL, 2 μg / mL, and 1 μg / mL) for 24 hours, and cell viability was measured using a CCK-8 assay.

[0461] like Figure 3 It can be seen that the cell viability of I-22, I-24, I-25 and I-29 is less than 50%, and the cell viability of I-23 and I-27 is between 50% and 60%, indicating that the above-mentioned ionizable lipids have greater cytotoxicity; the remaining lipid nanoparticles generally show less cytotoxicity (when administered at high concentrations, the cell viability is greater than 60%), indicating that most of the ionizable lipids prepared by the present invention have good biocompatibility and potential application value.

[0462] It can be seen from I-10, I-13, I-15 and that when the connecting segment of the ionizable lipid with a pyrazole ring at the head is extended, the LNP cell survival rate will decrease; it can be seen from I-26 and I-28 that when an unsaturated bond is added to the end of the ionizable lipid tail chain, the cell survival rate increases; it can be seen from I-28 and I-29 that the extension of the saturated carbon chain at the end of the ester group in the ionizable lipid tail chain will lead to a decrease in cell survival rate.

[0463] Example 72 In vivo screening of lipid nanoparticles

[0464] The experimental mice were SPF-grade female BALB / c mice, 6-8 weeks old, weighing 20-22 g, purchased from Guangdong Yaokang Biotechnology Co., Ltd. All animals were acclimated for at least 7 days before the experiment and had free access to food and water during the experiment. The experimental period was maintained under a 12 / 12 h light / dark cycle, with a room temperature of 25°C and a humidity of 50%. The mice were randomly divided into groups.

[0465] 1) Intramuscular injection screening

[0466] Lipid nanoparticles were administered by in situ intramuscular injection, with each mouse receiving 10 μg of mRNA in a 100 μL injection volume. Six hours after administration, the mouse lymph, heart, liver, spleen, lung, kidney, and muscle were harvested and bioluminescence was detected in vivo using an IVIS imaging system. The organ specificity of lipid nanoparticles after in situ intramuscular administration is shown in Table 2 (n=3). The fluorescence distribution ratio of lipid nanoparticles in mouse organs was calculated as follows: fluorescence value of (a specific organ) / total fluorescence value of (lymph, heart, liver, spleen, lung, kidney, and muscle) × 100%. The experimental results are expressed as the average ratio of mice within the same test group, as shown in Table 3.

[0467] Table 3 Fluorescence distribution in vivo after intramuscular injection

[0468]

[0469] The higher the total fluorescence value of a single organ accounts for the total fluorescence value of all organs (lymph, heart, liver, spleen, lung, kidney and muscle), the stronger the targeting of lipid nanoparticles to the organ.

[0470] Table 3 shows that I-26 and I-28 differ in their in vivo targeting. Combined with the structures in Table 1, it can be seen that the structural differences between I-26 and I-28 primarily lie in the degree of carbon chain saturation at the tail end. When the tail end contains an unsaturated bond, the lipid nanoparticles are distributed to a certain extent in the liver, spleen, and muscle. However, when the ionizable lipid tail end is saturated, the lipid nanoparticles are primarily distributed in muscle after in situ intramuscular injection. As shown by I-26 and I-27, when the ionizable lipid head end contains an imidazole group, the lipid nanoparticles accumulate less in the liver. As shown by I-30, I-31, and I-32, the length of the ionizable lipid tail can affect in vivo distribution. The longer the saturated carbon chain at the tail end, the stronger the muscle targeting ability of the lipid nanoparticles after intramuscular injection. As shown in I-50, I-54, I-55, and I-56, when the ionizable lipid head group is a tertiary amine with two pyrazol groups, the greater the degree of branching of the ester-containing tail chain, the better the muscle delivery ability of the lipid nanoparticle. As shown in I-50, I-57, and I-58, the ionizable lipid head group can also affect the in vivo targeting of the lipid nanoparticle: when the ionizable lipid head group is pyrazole or oxazole, the lipid nanoparticle is mainly expressed in muscle after in situ intramuscular injection; however, when the ionizable lipid head group is imidazole, the lipid nanoparticle is distributed to the liver, spleen, and muscle to a certain extent after in situ intramuscular injection.

[0471] 2) Tail vein injection screening

[0472] To further explore the effect of ionizable lipid structures on in vivo biodistribution, we continued to administer the drug by tail vein injection. Each mouse was injected with a dose of 10 μg mRNA in a volume of 100 μL. Six hours after administration, the heart, liver, spleen, lung, kidney, and muscle of the mice were collected and in vivo bioluminescence detection was performed using an IVIS imaging system. The organ specificity of lipid nanoparticles after tail vein injection is shown in Table 3 (n=3). The fluorescence distribution ratio of lipid nanoparticles in mouse organs was calculated as follows: (a certain organ) fluorescence value / (heart, liver, spleen, lung, kidney, and muscle) total fluorescence value × 100%. The experimental results are expressed as the average ratio value of mice in the same test group, and the results are shown in Table 4.

[0473] Table 4 Fluorescence distribution in vivo after tail vein injection

[0474]

[0475] As shown in Table 4, the aforementioned lipid nanoparticles were primarily distributed in the liver and spleen after tail vein injection. Among them, we found that ionizable lipids with a 2,2,2-trifluoroethyl and imidazole head group were primarily distributed in the liver, such as I-9 (78.93%) and I-27 (54.17%), while I-10, I-15, I-25, I-47, and I-64 were primarily distributed in the spleen. Considering the chemical structure of the ionizable lipids, it was found that the head group of the ionizable lipids targeting the spleen was a tertiary amine connected to two pyrazole rings, suggesting that this structure may play a dominant role in spleen targeting. Although the head group of I-26 is a tertiary amine connected to two pyrazole rings, the introduction of an unsaturated bond at the end of the tail chain reduces the distribution of the lipid nanoparticle in the spleen and instead increases its accumulation in the lungs. It can be seen from I-25, I-47, I-50 and I-64 that extending the ionizable lipid linker segment will reduce the targeted distribution ratio of their lipid nanoparticles in the spleen.

[0476] In this tail vein injection screening, the organ luminescence images of I-50 and I-64 with better in vivo delivery effects are shown below: Figure 4 As shown. Figure 4 -A and 4-B show that the extension of the ionizable lipid I-50 linker fragment will lead to a significant decrease in the distribution of its lipid nanoparticles in the liver, while increasing its distribution in the spleen (I-64); and the total fluorescence signal of the lipid nanoparticles of the ionizable lipid with extended linker fragments in the spleen is higher than that of the positive control SM102, which indicates that the linker fragment of the lipid molecule has a significant effect on the in vivo targeting of lipid nanoparticles.

[0477] Based on the in vivo screening results, we selected the ionizable lipid I-50 with the strongest muscle delivery ability for further optimization.

[0478] Example 73: Formulation Optimization of I-50-LNP

[0479] 1) To further improve the in vivo delivery efficiency of I-50-LNP, we used a design-of-experiment (DOE) approach to optimize the lipid molar ratio of I-50-LNP. Note: Lipid nanoparticles (LNPs) are abbreviated as LNPs. I-50-LNPs specifically refer to lipid nanoparticles prepared with I-50 as the ionizable lipid in combination with several other lipids (DSPC, cholesterol, and PEGylated lipids); SM102-LNPs specifically refer to lipid nanoparticles prepared with SM102 as the ionizable lipid in combination with several other lipids (DSPC, cholesterol, and PEGylated lipids).

[0480] According to Example 67 of 12 lipid molar ratios randomly generated by the Design Expert software, lipid nanoparticles I-50-LNP were prepared; commercial ionizable lipid SM102 was used as a positive control for this experiment and was prepared according to the lipid molar ratio (classic ratio) of formula number 12 in Table 5.

[0481] Table 5 12 formulas with different lipid molar ratios randomly generated by Design Expert software

[0482]

[0483] Particle size, PDI, zeta potential, and encapsulation efficiency were measured in Examples 68 and 69. Lipid nanoparticles were administered intramuscularly in situ, with 10 μg of mRNA per mouse in a 100 μL injection volume. Six hours after administration, lymph nodes, heart, liver, spleen, lung, kidney, and muscle were harvested and bioluminescence was measured in vivo using an IVIS imaging system.

[0484] From Figure 5 -A shows that after adjusting the lipid molar ratio, the particle size of LNP is approximately distributed between 75nm and 115nm, and the PDI is less than 0.3; and the encapsulation efficiency of the 12 LNP samples is good, all above 90% ( Figure 5 -B).

[0485] Table 6 Summary of total muscle fluorescence values of 12 different LNP formulations and their ratios to the positive control SM102 fluorescence values

[0486]

[0487]

[0488] Table 7 In vivo distribution of positive control SM102

[0489]

[0490] As shown in Table 6, the lipid nanoparticles prepared according to the lipid molar ratio of Formula 1 had the highest fluorescence value in mouse muscle after in situ intramuscular injection, indicating that the lipid nanoparticles prepared with this lipid molar ratio had the best delivery efficiency in this optimization. In addition, the fluorescence proportion of lipid nanoparticles in muscle of the 12 different lipid molar ratio formulas can reach more than 60%, among which Formula 1 has the highest total fluorescence value, which is approximately 2.4 times that of Formula 9 (the lowest total fluorescence value). This shows that simply adjusting the lipid molar ratio of LNP has a huge impact on the delivery efficiency of LNP in vivo.

[0491] The LNP prepared by formula 1 was slightly higher than the positive control SM102-LNP (fluorescence intensity value 2.30×10 10) Intramuscular delivery efficiency. By comparing the total fluorescence values of I-50-LNP and SM102-LNP in muscle, it was found that after in situ intramuscular injection, the total fluorescence values of I-50-LNP with 12 different lipid molar ratios in muscle remained between 38.64% and 102.48% of the total fluorescence value of SM102-LNP.

[0492] 2) To explore whether the in vivo delivery efficiency of LNP can be improved by adjusting the mRNA-to-total lipid mass ratio of I-50-LNP, we selected formula 1, which had the highest muscle expression, as the LNP lipid molar ratio and optimized the mRNA-to-total lipid mass ratio.

[0493] Depend on Figure 6 As shown in Figure 1, the particle sizes of the three I-50-LNPs prepared at mRNA-to-total lipid ratios of 3.9%, 5.9%, and 7.9% ranged from 60 nm to 90 nm. This suggests that varying the mRNA-to-total lipid ratio has little effect on LNP particle size, but a greater impact on the PDI. When the mRNA-to-total lipid ratio was 5.9%, the overall PDI of the prepared LNPs was relatively low. Specifically, the PDI of the I-50-LNP at a mRNA-to-total lipid ratio of 5.9% was 0.123. Furthermore, the encapsulation efficiencies of all three I-50-LNPs were greater than 90%. These results demonstrate that by adjusting the mRNA-to-total lipid ratio of I-50-LNPs, we were able to produce LNPs with smaller particle size, more uniform dispersion, and better encapsulation efficiency, which is suitable for subsequent in vivo experiments.

[0494] From the mouse live body and organ imaging, it can be found that: a group of mice injected with the positive control SM102 in situ had a strong fluorescent signal in their liver, which indicates that most of the LNPs were transferred from the muscle to the liver after intramuscular injection ( Figure 7 -A, 7-B and 7-C). However, in the three groups of mice injected with I-50-LNP, obvious fluorescence signals were only observed in the muscles of the injected side of the hind limb of the mice, and no corresponding fluorescence signals were found in other organs, especially the liver. This indicates that the I-50-LNP with three mRNA-to-total lipid mass ratios has a high targeting effect on the muscles after in situ injection ( Figure 7 -A, 7-B, and 7-C). Among them, SM102-LNP with an mRNA-to-total lipid mass ratio of 5.9% showed the strongest fluorescence signal in the muscle, with a targeted distribution to the muscle reaching 94.75%, and the total fluorescence value reached 1.74 times that of the positive control (( Figure 7 -A, 7-B, 7-C and 7-D), indicating that this mRNA-to-total lipid mass ratio is the best ratio for I-50-LNP expression efficiency in muscle.

[0495] In summary, the relationship between the mRNA-to-total lipid mass ratio in LNPs and protein expression is nonlinear, indicating that a critical mass ratio exists for LNPs that influences their physical properties and expression. This critical mass ratio is not always the same for ionizable lipids with different chemical properties and may depend on the chemical structure of the ionizable lipid. The mRNA-to-total lipid mass ratio may influence expression levels through factors such as cellular uptake, transfected cell type, and endosomal escape efficiency; further investigation of this hypothesis is needed.

[0496] From the above, it can be seen that compared with the commercialized SM102-LNP, I-50-LNP can improve the muscle delivery efficiency after intramuscular injection while reducing the accumulation in the liver, and has certain application potential for local muscle delivery.

[0497] The applicant states that while the present invention illustrates the ionizable lipid compounds and their applications through the aforementioned embodiments, the present invention is not limited to these embodiments, and does not necessarily rely on these embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. An ionizable lipid compound, characterized in that The ionizable lipid compound has a structure shown in formula (I): M is selected from -(CH2) m1 -、-(CH2) n1 C(O)(CH2) p 、-(CH2) n1 C(O)O(CH2) p -, m1 is an integer from 1 to 6, n1 is an integer from 1 to 3, and p is an integer from 1 to 3; R1 and R2 are the same or different and are selected from C 1-6 Alkyl, C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl, benzoheterocyclyl or heterocyclyl, the above cycloalkyl, heterocyclyl, aryl, heteroaryl, benzoheterocyclyl or heterocyclyl groups are optionally substituted by a group selected from the following: halogen atoms, -(CH2) 1-6 CX3, -OH, -(CH2) 1-3 OH, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkyl hydroxyl, C 1-6 Alkylamino; wherein X is selected from halogen; R' and R" are the same or different and are selected from -(CH) q -、-(CH) q CH=CH(CH) s -、-(CH2) q NH(CH2) s 、-(CH) q C(O)(CH) s -, wherein q and s are independently integers from 1 to 3; R3 and R4 may be the same or different and are selected from a linear or branched C 3-30 Alkyl, hydroxy-substituted or unsubstituted straight or branched chain C 3-30 Alkenyl, hydroxy-substituted or unsubstituted straight or branched C 3-30 Alkynyl, Wherein Rc and Rd may be the same or different and are selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 wherein n and m are integers selected from 1 to 15, and the wavy line represents the attachment site of the group.

2. The ionizable lipid compound according to claim 1, characterized in that Said R1 and R2 are independently selected from thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, trifluoromethylphenyl, 3,5-difluorobromophenyl, benzothiazolyl, 1-phenylpyrrolidinyl, pyridinyl, oxazolyl or 2,2,2-trifluoroethyl; Preferably, said R3 and R4 are selected from in Indicates the attachment site of a group.

3. The ionizable lipid compound according to claim 1 or 2, characterized in that The ionizable lipid compound has any one of the structures shown in the following formulas A1-A13: M, R3, R4, and R" have the same ranges as those of M, R3, R4, and R" in claim 1.

4. The ionizable lipid compound according to any one of claims 1 to 3, characterized in that The ionizable lipid compound has any one of the structures shown in the following formulas B1-B15: R3, R4, and R" have the same ranges as those of R3, R4, and R" described in formula (I).

5. The ionizable lipid compound according to any one of claims 1 to 4, characterized in that The ionizable lipid compound is selected from any one of the following compounds:

6. A liposome, characterized in that The liposome comprises an ionizable lipid compound according to any one of claims 1 to 5; Preferably, the liposome further comprises a helper lipid; Preferably, the helper lipid comprises a phospholipid; Preferably, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2 -Di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphyton Alkyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl Any one or a combination of at least two of palmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine; Preferably, the helper lipids further comprise structural lipids; Preferably, the structured lipid comprises at least one or a combination of at least two of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol; Preferably, the helper lipid further comprises a PEGylated lipid; Preferably, the PEGylated lipid comprises any one or a combination of at least two of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide or PEG-modified dialkylamine; Preferably, the liposomes comprise ionizable lipid compounds, phospholipids, structural lipids and PEGylated lipids; Preferably, the liposomes comprise, by molar percentage, 20%-80% of ionizable lipid compounds, 5%-30% of phospholipids, 10%-50% of structural lipids, and 1%-10% of PEGylated lipids.

7. A lipid complex, characterized in that The lipid complex comprises the liposome according to claim 6 and an active substance; Preferably, the active substance comprises any one or a combination of at least two of a small molecule compound, a nucleic acid molecule or a protein molecule; Preferably, the nucleic acid molecule comprises any one or a combination of at least two of DNA, mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA or lncRNA; Preferably, the protein molecule comprises any one or a combination of at least two of an enzyme, an antibody, a short peptide, a polypeptide or a recombinant protein; Preferably, the method for preparing the lipid complex comprises the following steps: mixing liposomes and active substances by a mixer to obtain the lipid complex.

8. A lipid nanoparticle, characterized in that The lipid nanoparticles include the liposome according to claim 6 or the lipid complex according to claim 7; Preferably, the particle size of the lipid nanoparticles is 40-300 nm.

9. A reagent, kit, preparation or pharmaceutical composition, characterized in that: The reagent, kit, preparation or pharmaceutical composition each independently comprises any one or a combination of at least two of the ionizable lipid compound according to any one of claims 1 to 5, the liposome according to claim 6, the lipid complex according to claim 7 or the lipid nanoparticle according to claim 8.

10. Use of the ionizable lipid compound according to any one of claims 1 to 5, the liposome according to claim 6, the lipid complex according to claim 7, the lipid nanoparticle according to claim 8, or the reagent, kit, preparation or pharmaceutical composition according to claim 9 in the preparation of a product having any one of the following functions (1) to (4): (1) Encapsulation of active substances; (2) delivering the encapsulated active substance to cells, tissues, or organs; (3) enabling the delivered active substance to exert its active effect in cells, tissues or organs; (4) Prevent, diagnose or treat diseases.

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