AMINO LIPID COMPOUND AND LIPID NANOPARTICLE FOR DELIVERING BIOACTIVE INGREDIENT

Abstract

Provided are an amino lipid compound for preparing a lipid nanoparticle for delivering an active ingredient and a preparation method therefor, a lipid nanoparticle and a pharmaceutical composition containing the amino lipid compound, and the use thereof.

Claims

1-106. (canceled)

107. An amino lipid compound having a structure of formula (Va): ##STR00961## or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: A.sub.1, A.sub.2, and A.sub.3 are one of the following: (1) when the dashed line connecting A.sub.1 and A.sub.2 and the dashed line connecting A.sub.1 and A.sub.3 are absent, A.sub.1 is C.sub.1-C.sub.5 hydrocarbyl or C.sub.1-C.sub.5 heterohydrocarbyl, A.sub.2 is C.sub.1-C.sub.5 hydrocarbyl or C.sub.1-C.sub.5 heterohydrocarbyl, and A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene or a bond; (2) when the dashed line connecting A.sub.1 and A.sub.2 is a bond and the dashed line connecting A.sub.1 and A.sub.3 is absent, A.sub.1 is C.sub.1-C.sub.3 hydrocarbylene or C.sub.1-C.sub.3 heterohydrocarbylene, A.sub.2 is C.sub.1-C.sub.3 hydrocarbylene or C.sub.1-C.sub.3 heterohydrocarbylene, A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene or a bond, and A.sub.1 and A.sub.2, together with the nitrogen atom to which they are both attached, form a four-, five-, or six-membered ring; or (3) when the dashed line connecting A.sub.1 and A.sub.3 is a bond and the dashed line connecting A.sub.1 and A.sub.2 is absent, A.sub.1 is C.sub.1-C.sub.3 hydrocarbylene or C.sub.1-C.sub.3 heterohydrocarbylene, A.sub.2 is C.sub.1-C.sub.5 hydrocarbyl or C.sub.1-C.sub.5 heterohydrocarbyl, A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene, and A.sub.1 and A.sub.3, together with the nitrogen atom to which they are both attached, form a four-, five-, or six-membered ring; A.sub.4 is C.sub.1-C.sub.5 hydrocarbylene or a bond; A.sub.5 is C.sub.1-C.sub.16 hydrocarbylene or a bond; A.sub.6 is C.sub.1-C.sub.16 hydrocarbylene or a bond; A.sub.2 is C.sub.1-C.sub.18 hydrocarbylene or a bond; A.sub.8 is C.sub.1-C.sub.18 hydrocarbylene or a bond; R.sub.9 is C.sub.1-C.sub.18 hydrocarbyl, or C.sub.1-C.sub.18 heterohydrocarbyl containing O or S; and R.sub.11 is C.sub.1-C.sub.18 hydrocarbyl, or C.sub.1-C.sub.18 heterohydrocarbyl containing O or S.

108. The amino lipid compound according to claim 107, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: the dashed line connecting A.sub.1 and A.sub.2 and the dashed line connecting A.sub.1 and A.sub.3 are absent, A.sub.1 is C.sub.1-C.sub.5alkyl, A.sub.2 is C.sub.1-C.sub.5alkyl, and A.sub.3 is C.sub.1-C.sub.5 alkylene; A.sub.4 is a bond; A.sub.5 is C.sub.1-C.sub.16 alkylene; A.sub.6 is C.sub.1-C.sub.16 alkylene; A.sub.7 is C.sub.1-C.sub.18 alkylene; A.sub.8 is C.sub.1-C.sub.18 alkylene; R.sub.9 is C.sub.1-C.sub.18 alkyl; and R.sub.11 is C.sub.1-C.sub.18 alkyl.

109. The amino lipid compound according to claim 107, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein A.sub.5 is C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, or C.sub.16 hydrocarbylene; and/or A.sub.6 is C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, or C.sub.16 hydrocarbylene; and/or A.sub.7 is C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, or C.sub.18 hydrocarbylene; and/or A.sub.8 is C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, or C.sub.18 hydrocarbylene; and/or R.sub.9 is a straight C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, or C.sub.18 alkyl; and/or R.sub.11 is a straight C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, or C.sub.18 alkyl.

110. An amino lipid compound having a structure of formula (II-Va): ##STR00962## or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: A.sub.1, A.sub.2, and A.sub.3 are one of the following: (1) when the dashed line connecting A.sub.1 and A.sub.2 and the dashed line connecting A.sub.1 and A.sub.3 are absent, A.sub.1 is H, C.sub.1-C.sub.5 hydrocarbyl, or C.sub.1-C.sub.5 heterohydrocarbyl, A.sub.2 is H, C.sub.1-C.sub.5 hydrocarbyl, or C.sub.1-C.sub.5 heterohydrocarbyl, and A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene or a bond; (2) when the dashed line connecting A.sub.1 and A.sub.2 is a bond and the dashed line connecting A.sub.1 and A.sub.3 is absent, A.sub.1 is C.sub.1-C.sub.5 hydrocarbylene or C.sub.1-C.sub.5 heterohydrocarbylene, A.sub.2 is C.sub.1-C.sub.5 hydrocarbylene or C.sub.1-C.sub.5 heterohydrocarbylene, A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene or a bond, and A.sub.1 and A.sub.2, together with the nitrogen atom to which they are both attached, form an N-containing heterocyclic ring; (3) when the dashed line connecting A.sub.1 and A.sub.3 is a bond and the dashed line connecting A.sub.1 and A.sub.2 is absent, A.sub.1 is C.sub.1-C.sub.5 hydrocarbylene or C.sub.1-C.sub.5 heterohydrocarbylene, A.sub.2 is H, C.sub.1-C.sub.5 hydrocarbyl, or C.sub.1-C.sub.5 heterohydrocarbyl, A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene, and A.sub.1 and A.sub.3, together with the nitrogen atom to which they are both attached, form an N-containing heterocyclic ring; or (4) when the dashed line connecting A.sub.1 and A.sub.2 and the dashed line connecting A.sub.1 and A.sub.3 are a bond, A.sub.1 is C.sub.1-C.sub.5 hydrocarbylene or C.sub.1-C.sub.5 heterohydrocarbylene, A.sub.2 is C.sub.1-C.sub.5 hydrocarbylene or C.sub.1-C.sub.5 heterohydrocarbylene, A.sub.3 is C.sub.1-C.sub.5 hydrocarbylene, and A.sub.1, A.sub.2 and A.sub.3, together with the nitrogen atom to which they are all attached, form an N-containing spiro-, fused, or bridged heterocyclic ring; A.sub.4 is a bond; A.sub.5 and A.sub.6 are each independently C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkenylene; or each independently C.sub.7, C.sub.8, C.sub.9, or C.sub.10 alkenylene; A.sub.7 and A.sub.8 are each independently C.sub.1-C.sub.18 hydrocarbylene or a bond; A.sub.12 is a bond; and R.sub.9 and R.sub.11 are each independently C.sub.1-C.sub.24 hydrocarbyl, or C.sub.1-C.sub.24 heterohydrocarbyl containing O or S.

111. The amino lipid compound according to claim 110, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: ##STR00963## is ##STR00964## wherein c is 2, 3, or 4; and/or A.sub.5 and A.sub.6 are each independently C.sub.4, C.sub.5, or C.sub.6 alkenylene; or each independently C.sub.7, C.sub.8, or C.sub.9 alkenylene; and/or A.sub.7 and A.sub.8 are each independently C.sub.2, C.sub.3, or C.sub.4 hydrocarbylene; preferably, A.sub.7 and A.sub.8 are each independently C.sub.3 hydrocarbylene; and/or R.sub.9 and R.sub.11 are each independently a straight C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, or C.sub.10 alkyl.

112. An amino lipid compound having a structure of formula (II-VIa-c): ##STR00965## or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: Z.sub.1 and Z.sub.2 are C(O)O; A.sub.4 is a bond; A.sub.5 and A.sub.6 are each independently C.sub.3-C.sub.10 alkylene; A.sub.7 and A.sub.8 are each independently C.sub.2-C.sub.4 alkylene; each R.sub.9 is independently C.sub.3-C.sub.9 alkyl; each R is independently C.sub.3-C.sub.9 alkyl; and the dashed line connecting A.sub.1 and A.sub.2 and the dashed line connecting A.sub.1 and A.sub.3 are absent, A.sub.1 and A.sub.2 are each independently C.sub.1-C.sub.3 alkyl, and A.sub.3 is C.sub.2-C.sub.4 alkylene.

113. The amino lipid compound according to claim 112, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein A.sub.5 and A.sub.6 are each independently C.sub.4-C.sub.9 alkylene; preferably, A.sub.5 and A.sub.6 are each independently C.sub.5-C.sub.9 alkylene; more preferably, A.sub.5 and A.sub.6 are each independently C.sub.6, C.sub.7, or C.sub.8 alkylene; and/or A.sub.7 and A.sub.8 are C.sub.3 alkylene; and/or each R.sub.9 is independently C.sub.5-C.sub.8 alkyl; preferably, each R.sub.9 is independently C.sub.6, C.sub.7, or C.sub.8 alkyl; and/or each R.sub.11 is independently C.sub.5-C.sub.8 alkyl; preferably, each R.sub.11 is independently C.sub.6, C.sub.7, or C.sub.8 alkyl; and/or A.sub.1 and A.sub.2 are each independently C.sub.1-C.sub.2 alkyl; and/or A.sub.3 is C.sub.3 alkylene.

114. The amino lipid compound according to claim 107, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the amino lipid compound has one of the structures shown below: TABLE-US-00005 Amino lipid compounds Structural formulae 382 383 387 388 389 390 391 490 849 851 856 859 861 862 863 864 865 867 868 869 2035 2036 2039 2040 2041 2042 2043 2044 2045

115. A lipid nanoparticle comprising the amino lipid compound of claim 107; preferably, wherein the lipid nanoparticle further contains one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid); more preferably, the lipid nanoparticle further contains the helper lipid, the structural lipid, and the PEG-lipid; and/or preferably, wherein the lipid nanoparticle further comprises a biologically active ingredient; preferably, the biologically active ingredient is a nucleic acid; preferably, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotide, and DNA; more preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), cas9 mRNA, or a mixture thereof; more preferably, the DNA is a plasmid.

116. A pharmaceutical composition comprising the amino lipid compound of claim 107 or a lipid nanoparticle comprising said amino lipid compound, and a pharmaceutically acceptable carrier, diluent, or excipient.

117. A method of delivering a biologically active ingredient into a cell, tissue or organ, comprising contacting the lipid nanoparticle of claim 115 comprising the biologically active ingredient with the cell, tissue or organ.

118. A method for the treatment and/or prevention of a disease; preferably, for gene therapy, protein replacement therapy, antisense therapy, therapy by interfering RNA, or gene vaccination, comprising administering the lipid nanoparticle of claim 115, or a pharmaceutical composition comprising said lipid nanoparticle and a pharmaceutically acceptable carrier, diluent, or excipient.

119. A lipid nanoparticle comprising the amino lipid compound of claim 110; preferably, wherein the lipid nanoparticle further contains one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid); more preferably, the lipid nanoparticle further contains the helper lipid, the structural lipid, and the PEG-lipid; and/or preferably, wherein the lipid nanoparticle further comprises a biologically active ingredient; preferably, the biologically active ingredient is a nucleic acid; preferably, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotide, and DNA; more preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), cas9 mRNA, or a mixture thereof; more preferably, the DNA is a plasmid.

120. A pharmaceutical composition comprising the amino lipid compound of claim 110 or a lipid nanoparticle comprising said amino lipid compound, and a pharmaceutically acceptable carrier, diluent, or excipient.

121. A method of delivering a biologically active ingredient into a cell, tissue or organ, comprising contacting the lipid nanoparticle of claim 119 comprising the biologically active ingredient with the cell, tissue or organ.

122. A method for the treatment and/or prevention of a disease; preferably, for gene therapy, protein replacement therapy, antisense therapy, therapy by interfering RNA, or gene vaccination, comprising administering the lipid nanoparticle of claim 119, or a pharmaceutical composition comprising said lipid nanoparticle and a pharmaceutically acceptable carrier, diluent, or excipient.

123. A lipid nanoparticle comprising the amino lipid compound of claim 112; preferably, wherein the lipid nanoparticle further contains one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid); more preferably, the lipid nanoparticle further contains the helper lipid, the structural lipid, and the PEG-lipid; and/or preferably, wherein the lipid nanoparticle further comprises a biologically active ingredient; preferably, the biologically active ingredient is a nucleic acid; preferably, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotide, and DNA; more preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), cas9 mRNA, or a mixture thereof; more preferably, the DNA is a plasmid.

124. A pharmaceutical composition comprising the amino lipid compound of claim 112 or a lipid nanoparticle comprising said amino lipid compound, and a pharmaceutically acceptable carrier, diluent, or excipient.

125. A method of delivering a biologically active ingredient into a cell, tissue or organ, comprising contacting the lipid nanoparticle of claim 123 comprising the biologically active ingredient with the cell, tissue or organ.

126. A method for the treatment and/or prevention of a disease; preferably, for gene therapy, protein replacement therapy, antisense therapy, therapy by interfering RNA, or gene vaccination, comprising administering the lipid nanoparticle of claim 123, or a pharmaceutical composition comprising said lipid nanoparticle and a pharmaceutically acceptable carrier, diluent, or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[1100] FIGS. 1 to 2 show the results of in vivo safety (ALT enzymatic activity) test of lipid nanoparticles comprising different amino lipid compounds;

[1101] FIGS. 3 to 4 show the results of in vivo safety (AST enzymatic activity) test of lipid nanoparticles comprising different amino lipid compounds;

[1102] FIG. 5 shows the results of in vivo delivery of lipid nanoparticles comprising different amino lipid compounds;

[1103] FIG. 6 shows the delivery efficiency of lipid nanoparticles comprising different amino lipid compounds in different immune cell populations in the spleen;

[1104] FIG. 7 shows the delivery efficiency of lipid nanoparticles comprising different amino lipid compounds in different immune cell populations in PBMC.

[1105] In order to make the purposes, technical solutions, and advantages of the present invention clearer, the present invention is described below with reference to specific examples. The follow examples are merely illustrative of the present invention and are not intended to be limiting.

Examples

[1106] The following examples are provided for purposes of illustration and not limitation. The experimental methods for which specific conditions are not specified in the examples are usually under conventional conditions or conditions as recommended by the manufacturer of the raw material or commodity; and the reagents of unspecified origin are generally conventional reagents commercially available.

[1107] The abbreviations used in the examples have the following meanings: [1108] Pd/C Palladium/carbon; [1109] EA Ethyl acetate; [1110] DCM Dichloromethane; [1111] TEA Triethylamine; [1112] MPa Megapascal; [1113] DMF N,N-dimethylformamide; [1114] EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; [1115] DMAP 4-Dimethylaminopyridine; [1116] TBAF Tetrabutylammonium fluoride [1117] TEMPO 2,2,6,6-Tetramethylpiperidinooxy; [1118] NaDCC Sodium dichloroisocyanurate; [1119] TBSCl Tert-butyldimethylsilyl chloride; [1120] h Hour; [1121] min Minute.

Example 1: Synthesis of Amino Lipid Compound 383

##STR00871##

Step 1: Synthesis of DFSGN-T

Reaction Scheme:

##STR00872##

Experimental Procedure:

[1122] EDCI (4.43 g, 23.13 mmol), DCM (26 mL), and TEA (2.34 g, 23.13 mmol) were sequentially added to a 100 mL round-bottom flask, shaken and stirred well for 5 min. Then DMAP (0.47 g, 3.86 mmol), BHB (4,4-bis(heptyloxy) butanol) (4.2 g, 13.88 mmol), and DTN (10-oxononadecanedioic acid) (2.64 g, 7.71 mmol) were added and stirred at room temperature overnight. After the reaction was completed, DCM (100 mL) and water (150 mL) were added and stirred for extraction, and partitioned. The organic phase was collected, washed with 50 ml saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 7.7 g crude DFSGN-T, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 5.13 g DFSGN-T with a yield of 81%.

Step 2: Synthesis of DFSGN-C

##STR00873##

Experimental Procedure:

[1123] DFSGN-T (5.13 g, 5.62 mmol) was added to a 250 mL single-necked flask, dissolved in methanol (50 mL), and cooled to 0 C. Sodium borohydride (0.23 g, 6.18 mmol) was added portionwise with stirring, and the reaction was maintained for 1 h after the addition was completed. After the reaction was completed, the solvent was evaporated under reduced pressure, water (100 mL) and DCM (100 mL) were added and stirred for extraction, and partitioned. The organic phase was collected, washed with water (50 mL), and concentrated under reduced pressure at 45 C. to obtain 5.2 g crude DFSGN-C, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 4.5 g DFSGN-C with a yield of 88%.

Step 3: Synthesis of 383-D

Reaction scheme:

##STR00874##

Experimental Procedure:

[1124] DFSGN-C (1.8 g, 1.97 mmol), DCM (18 mL), DMAP (72 mg, 0.60 mmol), and pyridine (0.39 g, 4.93 mmol) were added to a 50 mL single-necked round-bottom flask and stirred at room temperature. P-nitrophenyl chloroformate (0.79 g, 3.94 mmol) was added portionwise and stirred for another 1 h. After the reaction was completed, the mixture was diluted by adding water (150 mL) and extracted with DCM (100 mL). The organic phase was collected, washed with water (80 mL), and then concentrated under reduced pressure to obtain 3.0 g crude 383-D, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 1.74 g 383-D with a yield of 82%.

Step 4: Synthesis of Amino Lipid Compound 383

Reaction Scheme:

##STR00875##

Experimental Procedure:

[1125] 383-D (1.74 g, 1.61 mmol), DCM (18 mL), DMAP (59 mg, 0.48 mmol), triethylamine (0.49 mg, 4.84 mmol), and diethylaminopropanol (1.27 g, 9.68 mmol) were added to a 50 mL single-necked round-bottom flask and stirred at room temperature for 2 days. After the reaction was completed, the mixture was diluted by adding water (100 mL) and extracted with DCM (100 mL). The organic phase was collected, washed with water (80 mL), and then concentrated under reduced pressure to obtain 2.8 g crude amino lipid compound 383, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 945 mg amino lipid compound 383 with a yield of 55% and a purity of 96.55%.

Amino Lipid Compound 383

##STR00876##

[1126] .sup.1H NMR in CDCl.sub.3 4.69-4.65 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.1 Hz, 4H), 3.58-3.54 (m, 4H), 3.42-3.38 (m, 4H), 2.53-2.49 (m, 6H), 2.28 (t, J=7.5 Hz, 4H), 1.83-1.79 (m, 2H), 1.71-1.66 (m, 12H), 1.61-1.52 (m, 12H), 1.36-1.28 (m, 52H), 1.01 (t, J=7.2 Hz, 6H) 0.88 (t, J=7.1 Hz, 12H).

[1127] LC-MS (ESI): Calculated for (M+H) 1070.92, Found 1071.5.

Example 2: Synthesis of Amino Lipid Compound 382

[1128] According to the general synthetic process, amino lipid compound 382 was prepared according to the method of Example 1, with replacing BHB with compound BSB, to obtain 655 mg amino lipid compound 382 with a yield of 58% and a purity of 97.63%.

BSB

##STR00877##

Amino Lipid Compound 382

##STR00878##

[1129] .sup.1H NMR in CDCl.sub.3 4.69-4.65 (m, 1H), 4.48 (t, J=5.1 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.07 (t, J=6.0 Hz, 4H), 3.58-3.55 (m, 4H), 3.42-3.38 (m, 4H), 2.53-2.49 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.84-1.79 (m, 2H), 1.74-1.65 (m, 12H), 1.61-1.52 (m, 12H), 1.37-1.27 (m, 44H), 1.01 (t, J=7.2 Hz, 6H) 0.89 (t, J=6.9 Hz, 12H).

[1130] LC-MS (ESI): Calculated for (M+H) 1014.85, Found 1015.4.

Example 3: Synthesis of Amino Lipid Compound 1245

Step 1: Synthesis of 3-Bromopropanal

Reaction Scheme:

##STR00879##

Experimental Procedure:

[1131] 3-Bromopropanol (10.0 g, 72 mmol) and DCM (150 mL) were added to a 500 mL single-necked flask. TEMPO (225 mg, 1.44 mmol), potassium bicarbonate (5.05 g, 50.4 mmol), and sodium bromide (296 mg, 2.88 mmol) were added with stirring. The reaction solution was cooled to 5 C. and added with aqueous NaDCC solution (9.5 g, 43.2 mmol) dropwise. After the dropwise addition was completed, the mixture was reacted at 5 C. for 3 h and filtered. The filter cake was washed once with 20 mL water and once with 20 mL DCM. The organic phase was collected and concentrated under reduced pressure to obtain crude 3-bromopropanal, which was purified by silica gel column chromatography and eluted with DCM to obtain 9.0 g 3-bromopropanal with a yield of 91.2%.

Step 2: Synthesis of 3,3-Dioctyloxybromopropane

Reaction Scheme:

##STR00880##

Experimental Procedure:

[1132] 3-Bromopropanal (9.0 g, 65.7 mmol), cyclopentyl methyl ether (80 mL), 1-octanol (21.4 g, 164.3 mmol), and ammonium bromide (322 mg, 3.3 mmol) were added to a 250 mL single-necked flask. The reaction solution was heated to 130 C. with stirring, refluxed for water separation, reacted for 6 h, and filtered. The filtrate was collected and concentrated under reduced pressure to obtain crude 3,3-dioctyloxybromopropane, which was purified by silica gel column chromatography and eluted with n-hexane to obtain 9.63 g 3,3-dioctyloxybromopropane with a yield of 38.6%.

Step 3: Synthesis of 1245-A

Reaction Scheme:

##STR00881##

Experimental Procedure:

[1133] 2-Hydroxymethyl-1,3-propanediol (4.5 g, 42.7 mmol) and DMF (250 mL) were added to a 500 mL two-necked flask. The reaction solution was cooled to 0 C. with stirring, added with sodium hydride (1.5 g, 37.0 mmol) portionwise, reacted at 0 C. for 1 h, and slowly added with a solution of 3,3-dioctyloxy-1-bromopropane in DMF (11.2 g, 29.5 mmol, dissolved in 20 mL DMF) dropwise. After the dropwise addition was completed, the reaction solution was warmed to 10 C. for 6 h, added with 200 mL water for quenching, and then transferred to a 1 L separatory funnel, added with 300 mL water, and extracted twice with 200 mL ethyl acetate. The organic phases were combined, washed with 300 mL water, and then concentrated under reduced pressure to obtain crude 1245-A, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=3:1 to obtain 5.87 g 1245-A with a yield of 49.2%.

Step 4: Synthesis of 1245-B

Reaction Scheme:

##STR00882##

Experimental Procedure:

[1134] 1245-A (5.87 g, 14.0 mmol) and DCM (280 mL) were added to a 500 mL two-necked flask. The reaction solution was cooled to 10 C. with stirring and added with linoleic acid (4.32 g, 15.4 mmol), EDCI (4.03 g, 21.0 mmol), DMAP (171 mg, 1.4 mmol), and pyridine (1.66 g, 21.0 mmol). After the dropwise addition was completed, the mixture was reacted at 10 C. for 8 h. The reaction solution was warmed to room temperature, added with 200 mL water for quenching, and then extracted twice with 200 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain crude 1245-B, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=5:1 to obtain 5.6 g 1245-B with a yield of 58.7%.

Step 5: Synthesis of Amino Lipid Compound 1245

Reaction Scheme:

##STR00883##

Experimental Procedure:

[1135] 1245-B (4.85 g, 7.12 mmol), DCM (70 mL), DMAP (261 mg, 2.14 mmol), and pyridine (1.41 g, 17.8 mmol) were added to a 250 mL two-necked flask and stirred at room temperature. Then p-nitrophenyl chloroformate (2.87 g, 15.54 mmol) was slowly added portionwise and reacted at room temperature for 1 h. 3-Diethylamino-1-propanol (2.8 g, 21.4 mmol) was added and reacted at room temperature for 12 h. The solvent was evaporated under reduced pressure, and 100 mL water was added. The mixture was extracted twice with 100 mL n-hexane. The organic phases were combined, washed with 150 mL water, and then concentrated under reduced pressure to obtain crude amino lipid compound 1245, which is purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=1:1 to obtain 3.28 g amino lipid compound 1245 with a yield of 55.0% and a purity of 96.30%.

[1136] LC-MS (ESI): Calculated for (M+H) 838.7, Found 839.1.

Example 4: Synthesis of Amino Lipid Compound 1246

Step 1: Synthesis of 4-(tert-butyldimethylsilyl)oxo-1-butanol

Reaction Scheme:

##STR00884##

Experimental Procedure:

[1137] 1,4-butanediol (20.0 g, 222 mmol), DCM (660 mL), and imidazole (22.67 g, 333 mmol) were added to a 1 L single-necked flask. The reaction solution was cooled to 0 C. with stirring, added with TBSCl (36.7 g, 244 mmol), reacted at 0 C. for 6 h, added with 400 mL water, and extracted twice with 300 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain crude 4-(tert-butyldimethylsilyl)oxo-1-butanol, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=5:1 to obtain 22.5 g 4-(tert-butyldimethylsilyl)oxo-1-butanol with a yield of 49.5%.

Step 2: Synthesis of 4-(tert-butyldimethylsilyl)oxo-1-butanal

Reaction Scheme:

##STR00885##

Experimental Procedure:

[1138] 4-(tert-butyldimethylsilyl)oxo-1-butanol (22.5 g, 110 mmol) and DCM (300 mL) were added to a 1 L single-necked flask, and TEMPO (344 mg, 2.2 mmol), potassium bicarbonate (7.7 g, 77 mmol), and sodium bromide (453 mg, 4.4 mmol) were added with stirring. The reaction solution was cooled to 5 C., added with aqueous NaDCC solution (14.5 g, 66 mmol, dissolved in 150 mL water) dropwise, reacted at 5 C. for 3 h, and filtered. The filter cake was washed once with 50 mL water and once with 50 mL DCM. The filtrate was collected and separated into phases. The organic phase was collected, and the aqueous phase was extracted twice with 150 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain crude 4-(tert-butyldimethylsilyl)oxo-1-butanal, which was purified by silica gel column chromatography and eluted with DCM to obtain 11.4 g 4-(tert-butyldimethylsilyl)oxo-1-butanal with a yield of 51.2%.

Step 3: Synthesis of 4,4-dioctyloxy-1-TBS butanol

Reaction Scheme:

##STR00886##

Experimental Procedure:

[1139] 4-(tert-butyldimethylsilyl)oxo-1-butanal (10.0 g, 49.4 mmol), cyclopentyl methyl ether (80 mL), 1-octanol (16.1 g, 123.5 mmol), and ammonium bromide (242 mg, 2.47 mmol) were added to a 250 mL single-necked flask. The reaction solution was heated to 130 C. with stirring, refluxed for water separation, reacted for 6 h, and filtered. The filtrate was collected and concentrated under reduced pressure to obtain crude 4,4-dioctyloxy-1-TBS butanol, which was purified by silica gel column chromatography and eluted with n-hexane to obtain 10.2 g 4,4-dioctyloxy-1-TBS butanol with a yield of 46.4%.

Step 4: Synthesis of 4,4-dioctyloxy-1-butanol

Reaction Scheme:

##STR00887##

Experimental Procedure:

[1140] 4,4-dioctyloxy-1-TBS butanol (10.0 g, 22.5 mmol), tetrahydrofuran (110 mL), and TBAF (10.6 g, 33.7 mmol) were added to a 250 mL single-necked flask, reacted at room temperature with stirring for 6 h, added with 80 mL saturated sodium bicarbonate solution, and then extracted twice with 80 mL ethyl acetate. The organic phases were combined, washed with 100 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was collected and concentrated under reduced pressure to obtain crude 4,4-dioctyloxy-1-butanol, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=5:1 to obtain 6.5 g 4,4-dioctyloxy-1-butanol with a yield of 87.4%.

Step 5: Synthesis of 1246-A1

Reaction Scheme:

##STR00888##

Experimental Procedure:

[1141] 5-hydroxymethyl-2,2-dimethyl-1,3-dioxane (13.0 g, 88.9 mmol) and DCM (270 mL) were added to a 500 mL two-necked flask. The reaction solution was cooled to 10 C. with stirring, added with linoleic acid (25.0 g, 88.9 mmol), EDCI (25.6 g, 133.5 mmol), DMAP (2.18 g, 17.8 mmol), and pyridine (10.6 g, 134.0 mmol), warmed to room temperature for 8 h, added with 200 mL water for quenching, and extracted twice with 200 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain crude 1246-A1, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=20:1 to obtain 23.6 g 1246-A1 with a yield of 64.9%.

Step 6: Synthesis of 1246-A

Reaction Scheme:

##STR00889##

Experimental Procedure:

[1142] 1246-A1 (23.6 g, 57.8 mmol) and ethanol (360 mL) were added to a 1 L round-bottom flask and stirred at room temperature. Hydrochloric acid (120 mL) was added dropwise and reacted at room temperature for 1 h. The reaction solution was added with 600 mL saturated sodium bicarbonate solution for quenching and extracted twice with 500 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain crude 1246-A, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=3:1 to obtain 17.5 g 1246-A with a yield of 82.2%.

Step 7: Synthesis of 1246-A-TBS

Reaction Scheme:

##STR00890##

Experimental Procedure:

[1143] 1246-A (21.3 g, 57.8 mmol) and DCM (600 mL) were added to a 1 L round-bottom flask. The reaction solution was cooled to 0 C. with stirring, added with imidazole (5.9 g, 86.7 mmol) and TBSCl (9.6 g, 63.7 mmol), reacted at 0 C. for 8 h, added with 500 mL saturated sodium bicarbonate solution for quenching, and extracted twice with DCM (500 mL). The organic phases were combined, washed with 400 mL saturated sodium chloride solution, and then concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=10:1 to obtain 12.4 g 1246-A-TBS with a yield of 44.4%.

Step 8: Synthesis of 1246-B

Reaction Scheme:

##STR00891##

Experimental Procedure:

[1144] 1246-A-TBS (4.5 g, 9.32 mmol), DCM (45 mL), and DMAP (0.34 g, 2.79 mmol) were added to a 100 mL two-necked flask, and pyridine (1.62 g, 20.5 mmol) was added dropwise with stirring. P-nitrophenyl chloroformate (3.75 g, 18.6 mmol) was slowly added portionwise and reacted at room temperature for 1 h. A mixture solution of 3-diethylamino-1-propanol (3.66 g, 27.9 mmol) and triethylamine (0.93 g, 9.32 mmol) was added and reacted at room temperature for 16 h. The solvent was evaporated under reduced pressure, and 200 mL water was added. The mixture was extracted twice with 200 mL n-hexane. The organic phases were combined, washed with 200 mL water and 150 mL saturated sodium bicarbonate solution, and then concentrated under reduced pressure to obtain 5.72 g oil.

[1145] The above oil (5.72 g) and tetrahydrofuran (57 mL) were added to a 250 mL round-bottom flask and stirred at room temperature. Triethylamine trihydrofluoride (14.35 g) was added dropwise and reacted at room temperature for 16 h. The reaction solution was concentrated, diluted with 100 mL ethyl acetate, adjusted to pH=8 with saturated sodium bicarbonate solution, and washed with 200 mL water. The aqueous phase was extracted twice with 200 mL ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain crude 1246-B, which was purified by silica gel column chromatography and eluted with DCM:methanol=10:1 to obtain 960 mg 1246-B with a yield of 19.6%.

Step 9: Synthesis of 1246-C

Reaction Scheme:

##STR00892##

Experimental Procedure:

[1146] 4,4-dioctyloxy-1-butanol (1.0 g, 3.02 mmol), DCM (10 mL), DMAP (74 mg, 0.6 mmol), and pyridine (0.36 g, 4.5 mmol) were added to a 25 mL single-necked flask and stirred at room temperature. P-nitrophenyl chloroformate (0.91 g, 4.5 mmol) was slowly added portionwise and reacted at room temperature for 1 h. The reaction solution was concentrated, added with 100 mL water, and extracted twice with 100 mL n-hexane. The organic phases were combined, washed with 150 mL water and 150 mL saturated sodium bicarbonate solution, and then concentrated under reduced pressure to obtain crude 1246-C, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=1:1 to obtain 905 mg 1246-C with a yield of 60.3%.

Step 10: Synthesis of Amino Lipid Compound 1246

Reaction Scheme:

##STR00893##

Experimental Procedure:

[1147] 1246-B (0.96 g, 1.82 mmol), DCM (10 mL), DMAP (67 mg, 0.55 mmol), and triethylamine (0.45 mL, 3.65 mmol) were added to a 25 mL two-necked flask and stirred at room temperature. 1246-C (0.91 g, 1.82 mmol) was added and reacted at room temperature for 12 h. The mixture was added with 100 mL water and extracted with 50 mL DCM. The aqueous phase was extracted twice with 100 mL DCM. The organic phases were combined, washed with 150 mL saturated sodium bicarbonate solution, and then concentrated under reduced pressure to obtain crude amino lipid compound 1246, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=1:1 to obtain 1.25 g amino lipid compound 1246 with a yield of 77.6% and a purity of 94.13%.

[1148] .sup.1H NMR (600 MHZ, CDCl.sub.3) 5.41-5.29 (m, 4H), 4.47 (t, J=5.5 Hz, 1H), 4.22-4.12 (m, 10H), 3.55 (dt, J=9.2, 6.7 Hz, 2H), 3.40 (dt, J=9.2, 6.7 Hz, 2H), 2.76 (t, J=6.9 Hz, 2H), 2.50 (q, J=7.2 Hz, 6H), 2.45 (dt, J=11.9, 5.9 Hz, 1H), 2.30 (t, J=7.6 Hz, 2H), 2.04 (q, J=7.0 Hz, 4H), 1.84-1.77 (m, 2H), 1.77-1.64 (m, 4H), 1.63-1.58 (m, 2H), 1.58-1.51 (m, 4H), 1.39-1.20 (m, 34H), 1.00 (t, J=7.1 Hz, 6H), 0.88 (q, J=6.9 Hz, 9H).

[1149] .sup.13C NMR (151 MHZ, CDCl.sub.3) 173.60, 155.13, 155.11, 130.36, 130.18, 128.18, 128.05, 102.70, 68.30, 67.12, 66.00, 65.03, 65.00, 61.39, 49.21, 47.01, 37.75, 34.23, 31.98, 31.66, 30.02, 29.93, 29.75, 29.58, 29.49, 29.41, 29.32, 29.26, 27.34, 26.60, 26.40, 25.77, 24.98, 24.20, 22.80, 22.71, 14.24, 14.22, 11.87.

[1150] LC-MS (ESI): Calculated for (M+H) 882.7, Found 883.1.

Example 5: Synthesis of Amino Lipid Compound 1247

[1151] According to the general synthetic process and the method of Example 3, amino lipid compound 1247 was prepared according to the following reaction scheme to obtain 2.0 g amino lipid compound 1247 with a yield of 54.9% and a purity of 95.75%.

##STR00894## ##STR00895##

[1152] .sup.1H NMR (600 MHz, CDCl.sub.3) 5.41-5.29 (m, 4H), 4.58 (t, J=5.8 Hz, 1H), 4.22-4.14 (m, 5H), 4.11 (dd, J=11.1, 6.4 Hz, 1H), 3.56 (dt, J=9.2, 6.7 Hz, 2H), 3.46 (dd, J=13.7, 6.1 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 2H), 2.77 (t, J=6.9 Hz, 2H), 2.62-2.49 (m, 6H), 2.33-2.28 (m, 3H), 2.04 (q, J=7.0 Hz, 4H), 1.85 (q, J=6.2 Hz, 2H), 1.83-1.78 (m, 2H), 1.63-1.58 (m, 2H), 1.58-1.52 (m, 4H), 1.38-1.22 (m, 34H), 1.00 (t, J=7.1 Hz, 6H), 0.88 (q, J=7.0 Hz, 9H).

[1153] .sup.13C NMR (151 MHz, CDCl.sub.3) 173.74, 155.31, 130.36, 130.18, 128.19, 128.05, 100.79, 68.34, 67.59, 66.95, 66.28, 66.27, 65.88, 62.18, 49.23, 47.01, 38.65, 34.33, 34.09, 31.99, 31.67, 30.07, 29.76, 29.60, 29.49, 29.44, 29.34, 29.29, 29.28, 27.34, 26.62, 26.42, 25.77, 25.05, 22.81, 22.72, 14.24, 14.22, 11.88.

[1154] LC-MS (ESI): Calculated for (M+H) 824.7, Found 825.1.

Example 6: Synthesis of Amino Lipid Compound 1248

[1155] According to the general synthetic process and the method of Example 3, amino lipid compound 1248 was prepared according to the following reaction scheme to obtain 5.47 g amino lipid compound 1248 with a yield of 82.6% and a purity of 96.52%.

##STR00896##

[1156] .sup.1H NMR (600 MHz, CDCl.sub.3) 5.41-5.19 (m, 4H), 4.44 (t, J=5.7 Hz, 1H), 4.23-4.14 (m, 5H), 4.11 (dd, J=11.2, 6.4 Hz, 1H), 3.55 (dt, J=9.2, 6.7 Hz, 2H), 3.44 (d, J=5.9 Hz, 2H), 3.42-3.36 (m, 4H), 2.77 (t, J=6.8 Hz, 2H), 2.62-2.49 (m, 6H), 2.33-2.28 (m, 3H), 2.04 (q, J=7.0 Hz, 4H), 1.83-1.77 (m, 2H), 1.63-1.58 (m, 4H), 1.58-1.52 (m, 6H), 1.41-1.21 (m, 36H), 1.00 (t, J=7.1 Hz, 6H), 0.88 (q, J=6.9 Hz, 9H).

[1157] .sup.13C NMR (151 MHz, CDCl.sub.3) 173.75, 155.31, 130.36, 130.18, 128.18, 128.05, 103.14, 71.44, 68.26, 66.93, 65.93, 65.73, 62.23, 49.23, 47.02, 38.69, 34.34, 33.42, 31.99, 31.67, 30.06, 29.76, 29.59, 29.57, 29.49, 29.43, 29.34, 29.28, 27.34, 26.62, 26.42, 25.77, 25.05, 22.81, 22.72, 21.52, 14.25, 14.22, 11.89.

[1158] LC-MS (ESI): Calculated for (M+H) 852.7, Found 853.1.

Example 7: Synthesis of Amino Lipid Compound 387

##STR00897##

[1159] According to the general synthetic process, amino lipid compound 387 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 387-A5, where 0.37 g amino lipid compound 387 was obtained from 387-A5 (1.7 g, 1.76 mmol) and 3-diethylamino-1-propanol (1.39 g, 10.56 mmol) with a yield of 21.93% and a purity of 95.40%.

[1160] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.55-2.47 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.85-1.76 (m, 2H), 1.74-1.63 (m, 8H), 1.63-1.48 (m, 16H), 1.38-1.20 (m, 36H), 1.00 (t, J=7.1 Hz, 6H), 0.90 (m, 12H).

[1161] LC-MS (ESI): Calculated for (M+H) 958.79, Found 959.3.

Example 8: Synthesis of Amino Lipid Compound 388

##STR00898##

[1162] According to the general synthetic process, amino lipid compound 388 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 388-A5, where 0.37 g amino lipid compound 388 was obtained from 388-A5 (1.2 g, 1.17 mmol) and 3-dimethylamino-1-propanol (0.72 g, 7.02 mmol) with a yield of 31.95% and a purity of 94.45%.

[1163] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.63 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.39-2.34 (m, 2H), 2.28 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.88-1.80 (m, 2H), 1.74-1.63 (m, 10H), 1.63-1.47 (m, 16H), 1.39-1.22 (m, 42H), 0.95-0.81 (m, 12H).

[1164] LC-MS (ESI): Calculated for (M+H) 986.82, Found 987.4.

Example 9: Synthesis of Amino Lipid Compound 389

##STR00899##

[1165] According to the general synthetic process, amino lipid compound 389 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 389-A5, where 1.05 g amino lipid compound 389 was obtained from 389-A5 (2.4 g, 2.23 mmol) and 3-diethylamino-1-propanol (1.75 g, 13.35 mmol) with a yield of 43.97% and a purity of 92.22%.

[1166] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.72-4.62 (m, 1H), 4.45 (t, J=5.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.05 (t, J=6.7 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.61-2.43 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.87-1.76 (m, 2H), 1.57 (m, 26H), 1.43-1.16 (m, 50H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (t, J=6.9 Hz, 12H).

[1167] LC-MS (ESI): Calculated for (M+H) 1070.67, Found.

Example 10: Synthesis of Amino Lipid Compound 390

##STR00900##

[1168] According to the general synthetic process, amino lipid compound 390 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 390-A5, where 0.44 g amino lipid compound 390 was obtained from 390-A5 (1.2 g, 1.17 mmol) and 3-diethylamino-1-propanol (0.92 g, 7.02 mmol) with a yield of 37.06% and a purity of 91.06%.

[1169] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.61 (m, 1H), 4.45 (t, J=5.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.05 (t, J=6.7 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.51 (td, J=7.1, 3.4 Hz, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.85-1.76 (m, 2H), 1.76-1.47 (m, 26H), 1.43-1.17 (m, 42H), 1.01 (t, J=7.1 Hz, 6H), 0.90 (m, 12H).

[1170] LC-MS (ESI): Calculated for (M+H) 1014.85, Found 1015.3.

Example 11: Synthesis of Amino Lipid Compound 391

##STR00901##

[1171] According to the general synthetic process, amino lipid compound 391 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 391-A5, where 0.847 g amino lipid compound 391 was obtained from 391-A5 (2 g, 2.03 mmol) and 3-diethylamino-1-propanol (1.6 g, 12.18 mmol) with a yield of 43.53% and a purity of 92.68%.

[1172] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.71-4.61 (m, 1H), 4.45 (t, J=5.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.05 (t, J=6.7 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.51 (td, J=7.1, 3.4 Hz, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.85-1.76 (m, 2H), 1.76-1.47 (m, 26H), 1.43-1.17 (m, 34H), 1.01 (t, J=7.1 Hz, 6H), 0.90 (m, 12H).

[1173] LC-MS (ESI): Calculated for (M+H) 958.46, Found 959.1.

Example 12: Synthesis of Amino Lipid Compound 392

##STR00902##

[1174] According to the general synthetic process, amino lipid compound 392 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 392-A5, where 0.29 g amino lipid compound 392 was obtained from 392-A5 (0.86 g, 0.85 mmol) and 3-diethylamino-1-propanol (0.67 g, 5.1 mmol) with a yield of 33.9% and a purity of 97.93%.

[1175] .sup.1H NMR (600 MHZ, CDCl.sub.3) 5.62-5.48 (m, 8H), 4.69-4.65 (m, 1H), 4.62-4.53 (m, 2H), 4.20-4.11 (m, 6H), 4.09-4.06 (m, 8H), 2.54-2.45 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 2.06-2.03 (m, 8H), 1.84-1.76 (m, 2H), 1.75-1.66 (m, 8H), 1.64-1.46 (m, 8H), 1.43-1.35 (m, 8H), 1.35-1.22 (m, 20H), 1.00 (t, J=7.1 Hz, 6H), 0.90 (t, J=7.4 Hz, 12H).

[1176] LC-MS (ESI): Calculated for (M+H) 1006.50, Found 1007.1.

Exaple 13: Synthesis of Amino Lipid Compound 396

##STR00903##

[1177] According to the general synthetic process, amino lipid compound 396 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 396-A5, where 0.29 g amino lipid compound 396 was obtained from 396-A5 (1.3 g, 1.27 mmol) and 1-(3-hydroxypropyl) pyrrolidine (0.67 g, 5.18 mmol) with a yield of 22.55% and a purity of 95.18%.

[1178] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.62 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.18 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.1 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.55-2.49 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.92-1.97 (m, 2H), 1.80-1.63 (m, 14H), 1.63-1.45 (m, 16H), 1.43-1.16 (m, 42H), 0.89 (t, J=6.9 Hz, 12H).

[1179] LC-MS (ESI): Calculated for (M+H) 1012.55, Found 1013.2.

Example 14: Synthesis of Amino Lipid Compound 398

##STR00904##

[1180] According to the general synthetic process, amino lipid compound 398 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 398-A5, where 0.29 g amino lipid compound 398 was obtained from 398-A5 (1 g, 0.98 mmol) and N-methyl-2-(2-hydroxyethyl) pyrrolidine (0.76 g, 5.88 mmol) with a yield of 29.23% and a purity of 96.77%.

[1181] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.72-4.62 (m, 1H), 4.48 (d, J=5.3 Hz, 2H), 4.25-4.18 (m, 1H), 4.18-4.12 (m, 1H), 4.07 (t, J=6.0 Hz, 4H), 3.56 (dt, J=8.5, 6.7 Hz, 4H), 3.40 (dt, J=8.9, 6.8 Hz, 4H), 3.05 (t, J=8.6 Hz, 1H), 2.28 (dd, J=16.8, 9.3 Hz, 7H), 2.20-2.02 (m, 3H), 2.00-1.94 (m, 1H), 1.83-1.63 (m, 12H), 1.63-1.43 (m, 18H), 1.40-1.19 (m, 42H), 0.88 (t, J=6.9 Hz, 12H).

[1182] LC-MS (ESI): Calculated for (M+H) 1012.55, Found 1013.2.

Example 15: Synthesis of Amino Lipid Compound 399

##STR00905##

[1183] According to the general synthetic process, amino lipid compound 399 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 399-A5, where 0.7 g amino lipid compound 399 was obtained from 399-A5 (1 g, 0.98 mmol) and 3-(2-methylpiperidin-1-yl) propan-1-amine (0.92 g, 5.88 mmol) with a yield of 68.71% and a purity of 96.77%.

[1184] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.69 (s, 1H), 4.48-4.47 (m, 2H), 4.08-4.06 (t, J=5.9 Hz, 4H), 3.56 (dt, J=13.6, 6.7 Hz, 4H), 3.40 (dt, J=13.8, 6.8 Hz, 4H), 3.32-3.21 (m, 1H), 3.17-3.16 (m, 1H), 2.85-2.83 (m, 1H), 2.79-2.74 (m, 1H), 2.28 (t, J=7.5 Hz, 6H), 2.06 (t, J=10.7 Hz, 1H), 1.81-1.41 (m, 30H), 1.37-1.27 (m, 46H), 1.04 (d, J=6.2 Hz, 3H), 0.89 (t, J=6.8 Hz, 12H).

[1185] LC-MS (ESI): Calculated for (M+H) 1039.62, Found 1040.3.

Example 16: Synthesis of Amino Lipid Compound 416

##STR00906##

[1186] According to the general synthetic process, amino lipid compound 416 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 416-A5, where 0.6 g amino lipid compound 416 was obtained from 416-A5 (1 g, 0.98 mmol) and 1-methylpiperidine-3-methanol (0.76 g, 5.88 mmol) with a yield of 60.47% and a purity of 96.77%.

[1187] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08-4.03 (m, 5H), 3.94 (dd, J=10.6, 7.4 Hz, 1H), 3.56 (dt, J=9.2, 6.7 Hz, 4H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.85 (d, J=10.2 Hz, 1H), 2.74 (d, J=10.5 Hz, 1H), 2.29-2.25 (m, 7H), 2.06-1.94 (m, 1H), 1.90 (t, J=10.8 Hz, 1H), 1.80-1.44 (m, 29H), 1.42-1.08 (m, 44H), 0.89 (t, J=6.9 Hz, 12H).

[1188] LC-MS (ESI): Calculated for (M+H) 1012.55, Found 1013.2.

Example 17: Synthesis of Amino Lipid Compound 423

##STR00907##

[1189] According to the general synthetic process, amino lipid compound 423 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 423-A5, where 0.7 g amino lipid compound 423 was obtained from 423-A5 (1 g, 0.98 mmol) and 3-dimethylamino-2,2-dimethyl-1-propanol (0.77 g, 5.88 mmol) with a yield of 70.4% and a purity of 91.78%.

[1190] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.72-4.63 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08 (t, J=6.1 Hz, 4H), 3.94 (s, 2H), 3.57 (dt, J=9.1, 6.7 Hz, 4H), 3.41 (dt, J=9.2, 6.7 Hz, 4H), 2.30-2.23 (m, 10H), 2.17 (s, 2H), 1.75-1.47 (m, 26H), 1.42-1.17 (m, 42H), 0.92 (s, 6H), 0.89 (t, J=6.9 Hz, 12H)

[1191] LC-MS (ESI): Calculated for (M+H) 1014.57, Found 1015.2.

Example 18. Synthesis of Amino Lipid Compound 199

##STR00908##

[1192] According to the general synthetic process, amino lipid compound 429 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 429-A5, where 0.84 g amino lipid compound 429 was obtained from 429-A5 (2 g, 1.94 mmol) and 3-diethylamino-1-propanol (1.53 g, 11.64 mmol) with a yield of 42.35% and a purity of 97.22%.

[1193] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.62 (m, 1H), 4.48 (t, J=5.1 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.07 (t, J=6.0 Hz, 4H), 3.67 (dt, J=9.3, 6.3 Hz, 4H), 3.55-3.36 (m, 20H), 2.53 (td, J=7.1, 3.4 Hz, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.86-1.81 (m, 10H), 1.73-1.63 (m, 8H), 1.63-1.45 (m, 8H), 1.27 (m, 20H), 1.19 (t, J=7.0 Hz, 12H), 1.03-1.00 (m, 6H).

[1194] LC-MS (ESI): Calculated for (M+H) 1022.45, Found 1023.1.

Example 19: Synthesis of Amino Lipid Compound 430

##STR00909##

[1195] According to the general synthetic process, amino lipid compound 430 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 430-A5, where 0.84 g amino lipid compound 430 was obtained from 430-A5 (2.3 g, 1.7 mmol) and 3-diethylamino-1-propanol (1.34 g, 10.2 mmol) with a yield of 36.7% and a purity of 98.29%.

[1196] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70-4.63 (m, 1H), 4.43 (t, J=5.2 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.46 (dd, J=9.2, 5.8 Hz, 4H), 3.27 (dd, J=9.2, 5.8 Hz, 4H), 2.57-2.45 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.86-1.76 (m, 2H), 1.74-1.45 (m, 24H), 1.42-1.18 (m, 80H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (q, J=6.9 Hz, 24H).

[1197] LC-MS (ESI): Calculated for (M+H) 1351.21, Found 1351.8.

Example 20: Synthesis of Amino Lipid Compound 490

##STR00910##

[1198] According to the general synthetic process, amino lipid compound 490 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 490-A5, where 1.3 g amino lipid compound 490 was obtained from 490-A5 (2.9 g, 2.69 mmol) and 3-dimethylamino-1-propanol (1.67 g, 16.14 mmol) with a yield of 46.35% and a purity of 93.50%.

[1199] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (s, 1H), 4.47 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.37-2.34 (m, 2H), 2.28 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.87-1.81 (m, 2H), 1.68 (t, J=6.6 Hz, 12H), 1.56 (dt, J=14.5, 4.6 Hz, 12H), 1.34-1.24 (m, 52H), 0.88 (t, J=7.0 Hz, 12H).

[1200] LC-MS (ESI): Calculated for 1042.62, Found (M+H): 1043.4.

Example 21: Synthesis of Amino Lipid Compound 849

##STR00911##

[1201] According to the general synthetic process, amino lipid compound 849 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 849-A5, where 0.8 g amino lipid compound 849 was obtained from 849-A5 (2.0 g, 1.68 mmol) and 3-dimethylamino-1-propanol (1.04 g, 10.08 mmol) with a yield of 41.2% and a purity of 93.97%.

[1202] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.66 (m, 1H), 4.47 (t, J=5.2 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.1 Hz, 4H), 3.55 (dt, J=9.2, 6.7 Hz, 4H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.38-2.33 (m, 2H), 2.27 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.87-1.80 (m, 2H), 1.71-1.63 (m, 8H), 1.63-1.49 (m, 16H), 1.37-1.20 (m, 66H), 0.87 (t, J=7.0 Hz, 12H).

[1203] LC-MS (ESI): Calculated for (M+H) 1154.84, Found 1155.5.

Example 22: Synthesis of Amino Lipid Compound 859

##STR00912##

[1204] According to the general synthetic process, amino lipid compound 859 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 859-A5, where 270 mg amino lipid compound 859 was obtained from 859-A5 (1.1 g, 0.97 mmol) and 3-diethylamino-1-propanol (0.76 g, 5.82 mmol) with a yield of 24.72% and a purity of 95.67%.

[1205] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.72-4.59 (m, 1H), 4.48 (t, J=5.1 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.1 Hz, 4H), 3.56 (dt, J=9.1, 6.7 Hz, 4H), 3.40 (dt, J=9.1, 6.7 Hz, 4H), 2.51 (td, J=7.1, 3.4 Hz, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.84-1.77 (m, 2H), 1.73-1.50 (m, 28H), 1.31 (dd, J=30.6, 6.5 Hz, 56H), 1.01 (t, J=7.1 Hz, 6H), 0.88 (t, J=7.0 Hz, 12H).

[1206] LC-MS (ESI): Calculated for 1126.78, Found (M+H): 1127.4.

Example 23: Synthesis of Amino Lipid Compound 2021

##STR00913##

Step 1: Synthesis of 2021-A2

##STR00914##

[1207] DCM (120 ml) and TEA (4.18 g, 41.75 mmol) were added to EDCI (8.0 g, 41.75 mmol), and 2021-A1 (9.6 g, 33.4 mmol) was added with stirring at room temperature. After the reaction solution was clear, 2-hydroxymethyl-1,3-propanediol (1.8 g, 16.7 mmol) and DMAP (377 mg, 3.09 mmol) were added and reacted at room temperature for 16 h. The reaction solution was washed with saturated potassium bicarbonate (150 ml), and partitioned. The organic phase was collected, and the aqueous phase was extracted once with DCM (80 ml). The organic phases were combined, and the solvent was evaporated under reduced pressure to obtain crude 2021-A2, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=20:1 to obtain 4.5 g 2021-A2 with a yield of 41.65%.

Step 2: Synthesis of 2021-A3

##STR00915##

[1208] 2012-A2 (2.0 g, 3.09 mmol) was added to a 50 mL single-necked round-bottom flask and dissolved in DCM (20 mL). DMAP (113 mg, 0.93 mmol) and pyridine (0.61 g, 7.73 mmol) were added and stirred at room temperature. P-nitrophenyl chloroformate (1.25 g, 6.18 mmol) was added portionwise and stirred for another 1 h. The mixture was diluted with water (150 mL) and extracted with DCM (100 mL). The organic phase was collected, washed with water (80 mL), and concentrated to obtain 3.5 g crude 2021-A3, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 2.13 g 2021-A3 with a yield of 85%.

Step 3: Synthesis of 2021

##STR00916##

[1209] 2021-A3 (2.0 g, 2.46 mmol) was added to a 50 mL single-necked round-bottom flask and dissolved in DCM (20 mL). DMAP (92 mg, 0.75 mmol), triethylamine (0.75 mg, 7.38 mmol), and dimethylaminopropanol (1.52 g, 14.76 mmol) were added and stirred at room temperature for 2 days. The mixture was diluted with water (100 mL) and extracted with dichloromethane (100 mL). The organic phase was collected, washed with water (80 mL), and concentrated under reduced pressure to obtain 2.6 g crude 2021, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 1.16 g amino lipid compound 2021 with a yield of 61% and a purity of 95.32%.

[1210] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.47 (t, J=5.6 Hz, 2H), 4.18 (t, J=6.1 Hz, 4H), 4.13 (dd, J=6.0, 1.8 Hz, 4H), 3.55 (dt, J=9.3, 6.7 Hz, 4H), 3.39 (dt, J=9.3, 6.7 Hz, 4H), 2.43-2.37 (m, 5H), 2.36-2.32 (m, 2H), 2.21 (s, 6H), 1.91 (td, J=7.6, 5.7 Hz, 4H), 1.85-1.80 (m, 2H), 1.57-1.51 (m, 8H), 1.35-1.24 (m, 24H), 0.88 (t, J=7.0 Hz, 12H).

[1211] LC-MS (ESI): Calculated for 776.11, Found (M+H): 776.7.

Example 24: Synthesis of Amino Lipid Compound 2035

##STR00917##

[1212] According to the general synthetic process, amino lipid compound 2035 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2035-A5, where 3.0 g amino lipid compound 2035 was obtained from 2035-A5 (4.1 g, 4.12 mmol) and 3-dimethylamino-1-propanol (2.55 g, 24.6 mmol) with a yield of 75.97% and a purity of 92.02%.

[1213] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.71-4.63 (m, 1H), 4.60 (t, J=5.8 Hz, 2H), 4.23-4.10 (m, 6H), 3.58 (dt, J=9.2, 6.7 Hz, 4H), 3.42 (dt, J=9.2, 6.7 Hz, 4H), 2.47-2.38 (m, 2H), 2.32-2.24 (m, 10H), 1.94 (q, J=6.4 Hz, 4H), 1.90-1.81 (m, 2H), 1.63-1.49 (m, 16H), 1.38-1.22 (m, 44H), 0.89 (t, J=7.0 Hz, 12H).

[1214] LC-MS (ESI): Calculated for (M+H) 958.46, Found 959.2.

Example 25: Synthesis of Amino Lipid Compound 2036

##STR00918##

[1215] According to the general synthetic process, amino lipid compound 2036 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2036-A5, where 0.9 g amino lipid compound 2036 was obtained from 2036-A5 (4.85 g, 4.62 mmol) and 3-dimethylamino-1-propanol (2.86 g, 27.7 mmol) with a yield of 19.2% and a purity of 92.23%.

[1216] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.67 (m, 1H), 4.46 (t, J=5.7 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.06 (t, J=6.7 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.40 (dt, J=9.3, 6.7 Hz, 4H), 2.39-2.34 (m, 2H), 2.28 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.84 (dd, J=14.2, 7.1 Hz, 2H), 1.66-1.54 (m, 30H), 1.45-1.27 (m, 42H), 0.89 (t, J=7.0 Hz, 12H).

[1217] LC-MS (ESI): Calculated for (M+H) 1014.57, Found 1015.3.

Example 26: Synthesis of Amino Lipid Compound 2038

##STR00919##

[1218] According to the general synthetic process, amino lipid compound 2038 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2038-A5, where 425 mg amino lipid compound 2038 was obtained from 2038-A5 (0.91 g, 1.02 mmol) and 3-dimethylamino-1-propanol (0.63 g, 6.12 mmol) with a yield of 47.77% and a purity of 94.67%.

[1219] .sup.1H-NMR (600 MHZ, CDCl.sub.3) 4.70-4.63 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.22 (t, J=5.8 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.2, 6.7 Hz, 4H), 3.41 (dt, J=9.2, 6.7 Hz, 4H), 2.65-2.57 (m, 4H), 2.30 (ddd, J=15.3, 11.8, 6.9 Hz, 14H), 1.70-1.54 (m, 20H), 1.38-1.27 (m, 28H), 0.89 (t, J=6.9 Hz, 12H).

[1220] LC-MS (ESI): Calculated for 874.29, Found (M+H): 875.1.

Example 27: Synthesis of Amino Lipid Compound 2039

##STR00920##

[1221] According to the general synthetic process, amino lipid compound 2039 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2039-A5, where 1.05 g amino lipid compound 2039 was obtained from 2039-A5 (5.0 g, 5.33 mmol) and 3-dimethylamino-1-propanol (3.3 g, 31.97 mmol) with a yield of 21.83% and a purity of 94.51%.

[1222] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.38-2.35 (m, 2H), 2.31-2.26 (m, 4H), 2.23 (s, 6H), 1.85 (dt, J=13.8, 6.7 Hz, 4H), 1.73-1.65 (m, 8H), 1.58 (m, 16H), 1.57-1.39-1.25 (m, 30H), 0.89 (t, J=7.0 Hz, 12H).

[1223] LC-MS (ESI): Calculated for 902.35, Found (M+H): 903.30.

Example 28: Synthesis of Amino Lipid Compound 2040

##STR00921##

[1224] According to the general synthetic process, amino lipid compound 2040 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2040-A5, where 0.77 g amino lipid compound 2040 was obtained from 2040-A5 (2.0 g, 2.07 mmol) and 3-dimethylamino-1-propanol (1.28 g, 12.42 mmol) with a yield of 40.0% and a purity of 92.49%.

[1225] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.68 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.38-2.35 (m, 2H), 2.31-2.26 (m, 4H), 2.23 (s, 6H), 1.85 (dt, J=13.8, 6.7 Hz, 4H), 1.73-1.65 (m, 8H), 1.58 (m, 16H), 1.39-1.25 (m, 34H), 0.89 (t, J=7.0 Hz, 12H).

[1226] LC-MS (ESI): Calculated for 930.40, Found (M+H): 931.2.

Example 29: Synthesis of Amino Lipid Compound 2041

##STR00922##

[1227] According to the general synthetic process, amino lipid compound 2041 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2041-A5, where 0.66 g amino lipid compound 2041 was obtained from 2041-A5 (2.5 g, 2.51 mmol) and 3-dimethylamino-1-propanol (1.56 g, 15.08 mmol) with a yield of 27.43% and a purity of 91.97%.

[1228] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.68 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.56-2.46 (m, 6H), 2.32-2.24 (m, 4H), 1.71-1.66 (m, 12H), 1.62 (m, 4H), 1.59-1.54 (m, 10H), 1.40-1.25 (m, 40H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (t, J=7.0 Hz, 12H).

[1229] LC-MS (ESI): Calculated for (M+H) 958.46, Found 959.0.

Example 30: Synthesis of Amino Lipid Compound 2042

##STR00923##

[1230] According to the general synthetic process, amino lipid compound 2042 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2038-A5, where 0.35 g amino lipid compound 2042 was obtained from 2038-A5 (1.0 g, 1.10 mmol) and 3-diethylamino-1-propanol (0.86 g, 6.60 mmol) with a yield of 35.26% and a purity of 96.36%.

[1231] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.68 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.56-2.46 (m, 6H), 2.32-2.24 (m, 4H), 1.71-1.66 (m, 12H), 1.62 (m, 4H), 1.59-1.54 (m, 10H), 1.40-1.25 (m, 28H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (t, J=7.0 Hz, 12H).

[1232] LC-MS (ESI): Calculated for 902.35, Found (M+H): 903.2.

Example 31: Synthesis of Amino Lipid Compound 2043

##STR00924##

[1233] According to the general synthetic process, amino lipid compound 2043 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2039-A5, where 0.56 g amino lipid compound 2043 was obtained from 2039-A5 (1.2 g, 1.28 mmol) and 3-diethylamino-1-propanol (1.01 g, 7.67 mmol) with a yield of 47.02% and a purity of 90.21%.

[1234] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.4 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.56-2.47 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.86-1.78 (m, 4H), 1.72-1.65 (m, 8H), 1.62-1.52 (m, 16H), 1.37-1.24 (m, 30H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (t, J=7.0 Hz, 12H).

[1235] LC-MS (ESI): Calculated for 930.40, Found (M+H): 931.3.

Example 32: Synthesis of Amino Lipid Compound 2044

##STR00925##

[1236] According to the general synthetic process, amino lipid compound 2044 was synthesized according to the procedure in Step 3 of Example 1, with replacing 383-D with compound 2040-A5, where 0.4 g amino lipid compound 2044 was obtained from 2044-A5 (0.93 g, 0.96 mmol) and 3-diethylamino-1-propanol (0.76 g, 5.76 mmol) with a yield of 43.50% and a purity of 90.45%.

[1237] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.60 (m, 1H), 4.48 (t, J=5.1 Hz, 2H), 4.20-4.12 (m, 2H), 4.08 (t, J=6.0 Hz, 4H), 3.57 (dt, J=9.2, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.57-2.45 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.93-1.84 (m, 4H), 1.73-1.65 (m, 8H), 1.65-1.51 (m, 16H), 1.35-1.25 (m, 34H), 1.02 (t, J=7.1 Hz, 6H), 0.89 (t, J=6.8 Hz, 12H).

[1238] LC-MS (ESI): Calculated for 958.46, Found (M+H): 959.3.

Example 33: Synthesis of Amino Lipid Compound 2045

##STR00926##

[1239] According to the general synthetic process, amino lipid compound 2045 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2041-A5, where 1.2 g amino lipid compound 2045 was obtained from 2045-A5 (2.5 g, 2.51 mmol) and 3-diethylamino-1-propanol (1.98 g, 15.08 mmol) with a yield of 48.46% and a purity of 90.78%.

[1240] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.61 (m, 1H), 4.47 (t, J=5.1 Hz, 2H), 4.16 (t, J=6.5 Hz, 2H), 4.06 (t, J=6.1 Hz, 4H), 3.56 (dt, J=9.3, 6.7 Hz, 4H), 3.39 (dt, J=9.3, 6.7 Hz, 4H), 2.58-2.43 (m, 6H), 2.27 (t, J=7.6 Hz, 4H), 1.90-1.74 (m, 6H), 1.74-1.45 (m, 20H), 1.42-1.16 (m, 40H), 1.00 (t, J=7.1 Hz, 6H), 0.88 (t, J=6.8 Hz, 12H).

[1241] LC-MS (ESI): Calculated for (M+H) 986.51, Found 987.1.

Example 34: Synthesis of Amino Lipid Compound 2061

##STR00927##

[1242] According to the general synthetic process, amino lipid compound 2061 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 2061-A5, where 0.942 g amino lipid compound 2061 was obtained from 2061-A5 (1.87 g, 1.99 mmol) and 3-diethylamino-1-propanol (1.23 g, 11.94 mmol) with a yield of 53.29% and a purity of 94.25%.

[1243] .sup.1H-NMR (600 MHZ, CDCl.sub.3) 4.70-4.63 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.22 (t, J=5.8 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.57 (dt, J=9.2, 6.7 Hz, 4H), 3.41 (dt, J=9.2, 6.7 Hz, 4H), 2.65-2.57 (m, 4H), 2.30 (ddd, J=15.3, 11.8, 6.9 Hz, 14H), 1.70-1.54 (m, 22H), 1.38-1.27 (m, 28H), 0.89 (t, J=6.9 Hz, 12H).

[1244] LC-MS (ESI): Calculated for 888.32, Found (M+H): 888.7.

Example 35: Synthesis of Amino Lipid Compound 851

##STR00928##

Step 1: Synthesis of 851-A1

Reaction Scheme:

##STR00929##

Experimental Procedure:

[1245] EDCI (2.8 g, 14.6 mmol), DCM (50 mL), and TEA (1.47 g, 14.6 mmol) were sequentially added to a 100 mL round-bottom flask, shaken and stirred well for 5 min. Then DMAP (0.89 g, 7.3 mmol), BHB (4,4-bis(heptyloxy) butanol) (3.09 g, 10.22 mmol), DTN (10-oxononadecanedioic acid) (5.00 g, 14.6 mmol) were added and stirred at room temperature overnight. DCM (100 mL) and water (150 mL) were added and stirred for extraction, and partitioned. The organic phase was collected, washed with 50 mL saturated sodium chloride solution, and then concentrated under reduced pressure to obtain 8.2 g crude 851-A1, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=2:1 to obtain 3.36 g 851-A1 with a yield of 52.3%.

Step 2: Synthesis of 851-A2

Reaction Scheme:

##STR00930##

Experimental Procedure:

[1246] EDCI (1.1 g, 5.74 mmol), DCM (30 mL), and TEA (0.58 g, 5.74 mmol) were sequentially added to a 100 mL round-bottom flask, shaken and stirred well for 5 min. Then DMAP (0.29 g, 2.39 mmol), BPB (4,4-bis(pentyloxy) butanol) (1.41 g, 5.74 mmol), and 851-A1 (3.00 g, 4.78 mmol) were added and stirred at room temperature overnight. DCM (100 mL) and water (150 mL) were added and stirred for extraction, and partitioned. The organic phase was collected, washed with 50 mL saturated sodium chloride solution, and then concentrated under reduced pressure to obtain 4.8 g crude 851-A2, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 3.42 g 851-A2 with a yield of 83.6%.

Step 3: Synthesis of 851-A3

Reaction Scheme:

##STR00931##

Experimental Procedure:

[1247] 851-A2 (3.00 g, 3.51 mmol) was added to a 250 mL single-necked flask, dissolved in methanol (30 mL), and cooled to 0 C. Sodium borohydride (0.135 g, 3.51 mmol) was added portionwise with stirring, and the reaction was maintained for 1 h after the addition was completed. The solvent was evaporated under reduced pressure, and water (100 mL) and DCM (100 mL) were added and stirred for extraction. The organic phase was collected, washed with water (50 mL), and then concentrated under reduced pressure to obtain 3.1 g crude 851-A3, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 2.86 g 851-A3 with a yield of 95.1%.

Step 4: Synthesis of 851-A4

Reaction Scheme:

##STR00932##

Experimental Procedure:

[1248] 851-A3 (2.86 g, 3.34 mmol) was added to a 50 mL single-necked round-bottom flask and dissolved in DCM (30 mL). DMAP (122 mg, 1.00 mmol) and pyridine (0.66 g, 8.35 mmol) were added and stirred at room temperature. P-nitrophenyl chloroformate (1.35 g, 6.68 mmol) was added portionwise and stirred for another 1 h. Water (150 mL) and DCM (100 mL) were added and stirred for extraction. The organic phase was collected, washed with water (80 mL), and then concentrated under reduced pressure to obtain 5.0 g crude 851-A4, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 3.06 g 851-A4 with a yield of 89.6%.

Step 5: Synthesis of Amino Lipid Compound 851

Reaction Scheme.

##STR00933##

Experimental Procedure:

[1249] 851-A4 (3.00 g, 2.93 mmol) was added to a 50 mL single-necked round-bottom flask and dissolved in DCM (30 mL). DMAP (108 mg, 0.88 mmol), triethylamine (0.88 mg, 8.8 mmol), and dimethylaminopropanol (1.81 g, 17.58 mmol) were added and stirred at room temperature for 2 days. Water (100 mL) and DCM (100 mL) were added and stirred for extraction. The organic phase was collected, washed with water (80 mL), and then concentrated under reduced pressure to obtain 3.6 g crude amino lipid compound 851, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1.5:1 to obtain 1.69 g amino lipid compound 851 with a yield of 58.3% and a purity of 97.21%.

[1250] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.66 (m, 1H), 4.47 (dd, J=5.3, 4.5 Hz, 2H), 4.16 (t, J=6.6 Hz, 2H), 4.07 (t, J=5.9 Hz, 4H), 3.56 (dtd, J=8.8, 6.7, 2.0 Hz, 4H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.37-2.33 (m, 2H), 2.27 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.87-1.80 (m, 2H), 1.68 (dt, J=13.0, 7.4 Hz, 8H), 1.62-1.50 (m, 16H), 1.35-1.24 (m, 44H), 0.92-0.85 (m, 12H).

[1251] LC-MS (ESI): Calculated for 986.51, Found (M+H): 987.2.

Example 3. Synthesis of Amino Lipid Compound 851

##STR00934##

[1252] According to the general synthetic process, amino lipid compound 854 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 854-A4, where 1.2 g amino lipid compound 854 was obtained from 854-A4 (2.37 g, 2.26 mmol) and 3-dimethylamino-1-propanol (1.40 g, 13.53 mmol) with a yield of 52.33% and a purity of 91.41%.

[1253] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.66 (s, 1H), 4.47 (t, J=5.2 Hz, 2H), 4.16 (t, J=6.6 Hz, 2H), 4.06 (t, J=6.2 Hz, 4H), 3.55 (dtd, J=8.3, 6.7, 1.5 Hz, 4H), 3.39 (dtd, J=7.4, 6.7, 0.6 Hz, 4H), 2.39-2.34 (m, 2H), 2.27 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.87-1.80 (m, 2H), 1.71-1.63 (m, 8H), 1.62-1.48 (m, 16H), 1.34-1.22 (m, 48H), 0.87 (m, 12H).

[1254] LC-MS (ESI): Calculated for 1014.57, Found (M+H): 1015.2.

Example 37: Synthesis of Amino Lipid Compound 856

##STR00935##

[1255] According to the general synthetic process, amino lipid compound 856 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 856-A4, where 500 mg amino lipid compound 856 was obtained from 856-A4 (1.5 g, 1.36 mmol) and 3-dimethylamino-1-propanol (0.84 g, 8.13 mmol) with a yield of 34.45% and a purity of 94.85%.

[1256] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.47 (t, J=5.0 Hz, 2H), 4.17 (t, J=6.6 Hz, 2H), 4.07 (t, J=6.0 Hz, 4H), 3.59-3.53 (m, 4H), 3.43-3.36 (m, 4H), 2.38-2.32 (m, 2H), 2.28 (t, J=7.6 Hz, 4H), 2.22 (s, 6H), 1.86-1.80 (m, 2H), 1.68 (dd, J=18.0, 8.4 Hz, 8H), 1.61-1.49 (m, 16H), 1.36-1.24 (m, 56H), 0.88 (m, 12H).

[1257] LC-MS (ESI): Calculated for 1070.67, Found (M+H): 1071.4.

Example 38: Synthesis of Amino Lipid Compound 861

##STR00936##

[1258] According to the general synthetic process, amino lipid compound 861 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 861-A4, where 900 mg amino lipid compound 861 was obtained from 861-A4 (1.5 g, 1.51 mmol) and 3-diethylamino-1-propanol (1.19 g, 9.06 mmol) with a yield of 60.48% and a purity of 94.43%.

[1259] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.63 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.1 Hz, 4H), 3.57 (dt, J=9.2, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 2.54-2.46 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.85-1.77 (m, 2H), 1.74-1.49 (m, 24H), 1.40-1.23 (m, 40H), 1.01 (t, J=7.1 Hz, 6H), 0.95-0.83 (m, 12H).

[1260] LC-MS (ESI): Calculated for 986.51, Found (M+H): 987.2.

Example 39: Synthesis of Amino Lipid Compound 862

##STR00937##

[1261] According to the general synthetic process, amino lipid compound 862 was synthesized according to the procedure in Step 5 of Example 35, where 910 mg amino lipid compound 862 was obtained from 851-A4 (2.2 g, 2.15 mmol) and 3-diethylamino-1-propanol (1.69 g, 12.91 mmol) with a yield of 41.69% and a purity of 93.61%.

[1262] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.62 (m, 1H), 4.48 (t, J=4.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=5.9 Hz, 4H), 3.57 (m, 4H), 3.41 (dt, J=9.0, 6.8 Hz, 4H), 2.56-2.45 (m, 6H), 2.33-2.25 (m, 4H), 1.85-1.76 (m, 2H), 1.74-1.49 (m, 26H), 1.39-1.22 (m, 42H), 1.01 (t, J=7.1 Hz, 6H), 0.93-0.83 (m, 12H).

[1263] LC-MS (ESI): Calculated for 1014.57, Found (M+H): 1015.3.

Example 40: Synthesis of Amino Lipid Compound 863

##STR00938##

[1264] According to the general synthetic process, amino lipid compound 863 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 863-A4, where 950 mg amino lipid compound 863 was obtained from 863-A4 (1.81 g, 1.72 mmol) and 3-diethylamino-1-propanol (1.36 g, 10.34 mmol) with a yield of 52.88% and a purity of 93.08%.

[1265] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.62 (m, 1H), 4.48 (t, J=4.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=5.7 Hz, 4H), 3.65-3.51 (m, 4H), 3.41 (dt, J=9.0, 6.8 Hz, 4H), 2.60-2.44 (m, 6H), 2.28 (t, J=7.5 Hz, 4H), 1.83-1.76 (m, 2H), 1.74-1.50 (m, 26H), 1.42-1.19 (m, 46H), 1.01 (t, J=7.1 Hz, 6H), 0.89 (dt, J=13.7, 7.0 Hz, 12H).

[1266] LC-MS (ESI): Calculated for 1042.62, Found (M+H): 1043.2.

Example 11: Synthesis of Amino Lipid Compound 864

##STR00939##

[1267] According to the general synthetic process, amino lipid compound 864 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 864-A4, where 1.2 g amino lipid compound 864 was obtained from 864-A4 (2.81 g, 2.61 mmol) and 3-diethylamino-1-propanol (2.08 g, 15.63 mmol) with a yield of 42.94% and a purity of 92.67%.

[1268] .sup.1H NMR (400 MHZ, CDCl.sub.3) 4.73-4.60 (m, 1H), 4.48 (dd, J=5.1, 4.5 Hz, 2H), 4.12 (dt, J=11.4, 6.3 Hz, 6H), 3.57 (dtd, J=8.8, 6.7, 2.0 Hz, 4H), 3.41 (dt, J=9.2, 6.7 Hz, 4H), 2.60-2.43 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.86-1.77 (m, 2H), 1.74-1.50 (m, 24H), 1.41-1.20 (m, 52H), 1.01 (t, J=7.1 Hz, 6H), 0.90 (dt, J=13.7, 4.4 Hz, 12H).

[1269] LC-MS (ESI): Calculated for 1070.67, Found (M+H): 1071.3.

Example 42: Synthesis of Amino Lipid Compound 865

##STR00940##

[1270] According to the general synthetic process, amino lipid compound 865 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 854-A4, where 0.65 g amino lipid compound 865 was obtained from 854-A4 (1.5 g, 1.43 mmol) and 3-diethylamino-1-propanol (1.12 g, 8.57 mmol) with a yield of 43.60% and a purity of 96.53%.

[1271] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.71-4.63 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (m, 4H), 3.44-3.36 (m, 4H), 2.51 (td, J=7.1, 3.4 Hz, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.85-1.77 (m, 2H), 1.74-1.50 (m, 24H), 1.41-1.20 (m, 48H), 1.32 (m, 6H), 0.89 (td, J=7.0, 3.5 Hz, 12H).

[1272] LC-MS (ESI): Calculated for 1042.62, Found (M+H): 1043.3.

Example 43: Synthesis of Amino Lipid Compound 867

##STR00941##

[1273] According to the general synthetic process, amino lipid compound 867 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 856-A4, where 0.60 g amino lipid compound 867 was obtained from 856-A4 (2.0 g, 1.81 mmol) and 3-diethylamino-1-propanol (1.42 g, 10.84 mmol) with a yield of 30.17% and a purity of 91.94%.

[1274] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.48 (dd, J=5.3, 4.7 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.0 Hz, 4H), 3.57 (m, 4H), 3.41 (m, 4H), 2.55-2.48 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.86-1.50 (m, 28H), 1.38-1.22 (m, 54H), 1.01 (t, J=7.1 Hz, 6H), 0.91-0.85 (m, 12H).

[1275] LC-MS (ESI): Calculated for 1098.73, Found (M+H): 1099.3.

Example 44: Synthesis of Amino Lipid Compound 868

##STR00942##

[1276] According to the general synthetic process, amino lipid compound 868 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 868-A4, where 84 mg amino lipid compound 868 was obtained from 868-A4 (1.0 g, 0.90 mmol) and 3-diethylamino-1-propanol (0.71 g, 5.4 mmol) with a yield of 8.49% and a purity of 92.68%.

[1277] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.67 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.56 (dt, J=9.1, 6.7 Hz, 4H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.54-2.46 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.84-1.77 (m, 2H), 1.73-1.50 (m, 28H), 1.37-1.23 (m, 52H), 1.01 (t, J=7.1 Hz, 6H), 0.88 (td, J=6.9, 1.4 Hz, 12H).

[1278] LC-MS (ESI): Calculated for 1098.73, Found (M+H): 1099.4.

Example 45: Synthesis of Amino Lipid Compound 869

##STR00943##

[1279] According to the general synthetic process, amino lipid compound 869 was synthesized according to the procedure in Step 5 of Example 35, with replacing 851-A4 with compound 869-A4, where 50 mg amino lipid compound 869 was obtained from 869-A4 (1.25 g, 1.08 mmol) and 3-diethylamino-1-propanol (0.85 g, 6.48 mmol) with a yield of 4.00% and a purity of 93.28%.

[1280] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.67 (m, 1H), 4.48 (t, J=5.3 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.56 (dt, J=9.1, 6.7 Hz, 4H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.54-2.46 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.84-1.77 (m, 2H), 1.73-1.50 (m, 28H), 1.37-1.23 (m, 60H), 1.01 (t, J=7.1 Hz, 6H), 0.88 (td, J=6.9, 1.4 Hz, 12H).

[1281] LC-MS (ESI): Calculated for 1154.84, Found (M+H): 1155.3.

Example 46: Synthesis of Amino Lipid Compound 434

##STR00944##

[1282] According to the general synthetic process, amino lipid compound 434 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 434-A4, where 1.1 g amino lipid compound 434 was obtained from 434-A4 (2.0 g, 1.74 mmol) and 3-diethylamino-1-propanol (1.37 g, 10.46 mmol) with a yield of 55.51% and a purity of 95.69%.

[1283] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.17 (t, J=6.5 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (m, 4H), 3.44-3.36 (m, 6H), 2.49-2.44 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.73-1.48 (m, 22H), 1.32 (m, 62H), 0.95 (m, 6H), 0.89 (td, J=7.0, 3.5 Hz, 12H).

[1284] LC-MS (ESI): Calculated for 1038.98, Found (M+H): 1039.4.

Example 47: Synthesis of Amino Lipid Compound 436

##STR00945##

[1285] According to the general synthetic process, amino lipid compound 436 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 436-A4, where 0.79 g amino lipid compound 436 was obtained from 436-A4 (2.0 g, 2.14 mmol) and 3-diethylamino-1-propanol (1.68 g, 12.84 mmol) with a yield of 39.6% and a purity of 90.91%.

[1286] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.58-4.53 (t, J=7.0 Hz, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.95 (q, J=7.1 Hz, 2H), 3.57 (m, 4H), 3.44-3.36 (m, 6H), 2.49-2.44 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.73-1.48 (m, 18H), 1.32 (m, 41H), 0.95 (m, 6H), 0.89 (t, J=7.0, 6H).

[1287] LC-MS (ESI): Calculated for 928.39, Found (M+H): 929.1.

Example 48: Synthesis of Amino Lipid Compound 437

##STR00946##

[1288] According to the general synthetic process, amino lipid compound 437 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 437-A4, where 0.6 g amino lipid compound 437 was obtained from 437-A4 (1.3 g, 1.24 mmol) and 3-diethylamino-1-propanol (0.98 g, 7.44 mmol) with a yield of 46.7% and a purity of 94.75%.

[1289] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.58-4.53 (t, J=7.0 Hz, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.95 (q, J=7.1 Hz, 2H), 3.57 (m, 4H), 3.44-3.36 (m, 6H), 3.25-3.16 (t, J=6.7, 2H), 2.98-2.89 (t, J=7.5, 2H), 2.49-2.44 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.73-1.48 (m, 18H), 1.32 (m, 46H), 1.05 (m, 3H), 0.89 (t, J=7.0, 12H).

[1290] LC-MS (ESI): Calculated for 1036.57, Found (M+H): 1037.3.

Example 49: Synthesis of Amino Lipid Compound 438

##STR00947##

[1291] According to the general synthetic process, amino lipid compound 438 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 438-A4, where 0.82 g amino lipid compound 438 was obtained from 438-A4 (1.6 g, 1.72 mmol) and 3-diethylamino-1-propanol (1.35 g, 10.32 mmol) with a yield of 51.6% and a purity of 92.59%.

[1292] .sup.1H NMR (600 MHZ, CDCl.sub.3) 5.53-5.51 (dt, J=11.0, 7.8 Hz, 3H), 5.48-5.46 (dt, J=10.7, 7.4 Hz, 3H), 4.71-4.63 (m, 1H), 4.58-4.53 (t, J=7.0 Hz, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.15 (d, J=9.66 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.57 (d, J=7.8 Hz, 4H), 3.44-3.36 (m, 6H), 3.25-3.16 (m, 6H), 2.49-2.44 (q, J=7.6 Hz, 6H), 2.28 (m, 6H), 1.73-1.48 (m, 20H), 1.32 (m, 16H), 0.96 (m, 3H), 0.89 (t, J=7.0, 12H).

[1293] LC-MS (ESI): Calculated for 922.34, Found (M+H): 923.1.

Example 50: Synthesis of Amino Lipid Compound 439

##STR00948##

[1294] According to the general synthetic process, amino lipid compound 439 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 439-A4, where 0.37 g amino lipid compound 439 was obtained from 439-A4 (1.2 g, 1.15 mmol) and 3-diethylamino-1-propanol (0.91 g, 6.90 mmol) with a yield of 31.6% and a purity of 95.92%.

[1295] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.86 (t, J=4.9 Hz, 1H), 4.71 (m, 1H), 4.26 (s, 4H), 4.24-4.16 (m, 2H), 4.16-4.04 (m, 4H), 3.40 (t, J=6.6 Hz, 2H), 3.37 (s, 3H), 2.58-2.50 (m, 6H), 2.32 (td, J=7.6, 1.9 Hz, 4H), 2.25 (t, J=7.2 Hz, 4H), 1.76 (t, J=7.8 Hz, 4H), 1.70-1.51 (m, 20H), 1.44-1.26 (m, 46H), 1.05 (t, J=7.1 Hz, 6H), 0.92 (t, J=6.8 Hz, 6H).

[1296] LC-MS (ESI): Calculated for 1032.58, Found (M+H): 1033.3.

Example 51: Synthesis of Amino Lipid Compound 440

##STR00949##

[1297] According to the general synthetic process, amino lipid compound 440 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 440-A4, where 0.52 g amino lipid compound 440 was obtained from 440-A4 (1.0 g, 1.08 mmol) and 3-diethylamino-1-propanol (0.85 g, 6.48 mmol) with a yield of 52.5% and a purity of 92.61%.

[1298] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70 (m, 1H), 4.51 (t, J=5.3 Hz, 1H), 4.21 (t, J=6.6 Hz, 2H), 4.10 (dt, J=13.5, 6.5 Hz, 4H), 3.60 (dt, J=9.3, 6.7 Hz, 2H), 3.48-3.40 (m, 2H), 3.35 (t, J=6.6 Hz, 4H), 2.43 (t, J=7.8 Hz, 2H), 2.31 (t, J=7.8 Hz, 4H), 2.28 (m, 4H), 1.75-1.54 (m, 24H), 1.37-1.25 (m, 38H), 1.05 (t, J=7.1 Hz, 6H), 0.92 (t, J=6.8 Hz, 9H).

[1299] LC-MS (ESI): Calculated for 914.40, Found (M+H): 914.6.

Example 52: Synthesis of Amino Lipid Compound 441

##STR00950##

[1300] According to the general synthetic process, amino lipid compound 441 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 441-A4, where 0.6 g amino lipid compound 441 was obtained from 441-A4 (1.8 g, 1.74 mmol) and 3-diethylamino-1-propanol (1.37 g, 10.44 mmol) with a yield of 33.2% and a purity of 94.67%.

[1301] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.58-4.53 (t, J=7.0 Hz, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.95 (q, J=7.1 Hz, 2H), 3.57 (m, 4H), 3.44-3.36 (m, 6H), 2.49-2.44 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.73-1.48 (m, 20H), 1.32 (m, 50H), 0.95 (m, 6H), 0.89 (t, J=7.0, 9H).

[1302] LC-MS (ESI): Calculated for 1026.58, Found (M+H): 1027.3.

Example 53. Synthesis of Amino Lipid Compound 393

##STR00951##

[1303] According to the general synthetic process, amino lipid compound 442 was synthesized according to the procedure in Step 4 of Example 1, with replacing 383-D with compound 442-A4, where 0.29 g amino lipid compound 442 was obtained from 442-A4 (1.3 g, 1.19 mmol) and 3-diethylamino-1-propanol (0.94 g, 7.14 mmol) with a yield of 22.8% and a purity of 94.29%.

[1304] .sup.1H NMR (600 MHZ, CDCl.sub.3) 4.70-4.62 (m, 1H), 4.58-4.53 (t, J=7.0 Hz, 1H), 4.48 (t, J=5.2 Hz, 2H), 4.08 (t, J=6.2 Hz, 4H), 3.97-3.94 (m, 2H), 3.66-3.62 (t, J=6.75 Hz, 2H), 3.44-3.36 (m, 6H), 2.49-2.44 (m, 6H), 2.28 (t, J=7.6 Hz, 6H), 1.73-1.48 (m, 16H), 1.32 (m, 56H), 1.25 (d, J=6.17 Hz, 6H), 0.95 (m, 6H), 0.89 (t, J=7.0, 9H).

[1305] LC-MS (ESI): Calculated for 1081.70, Found (M+H): 1082.3.

Example 54. Synthesis of Amina Lipid Compound 303

##STR00952## ##STR00953##

Step 1: Synthesis of DLSGN-T

Reaction Scheme:

##STR00954##

Experimental Procedure:

[1306] EDCI (8.40 g, 43.80 mmol), DMAP (0.64 g, 5.26 mmol), and triethylamine (4.44 g, 43.80 mmol) were sequentially added to a 250 mL single-necked flask, dissolved in DCM (60 mL), and stirred at room temperature. Then DTN (6.00 g, 17.5 mmol) and BSB (8.66 g, 31.54 mmol) were added and reacted at room temperature overnight. The reaction was quenched by addition of saturated aqueous ammonium chloride solution (100 mL), and the phases were separated. The organic phase was collected, and the aqueous phase was extracted twice with DCM (100 mL). The organic phases were combined and washed with 100 mL saturated sodium chloride solution. Then, the solvent was evaporated to obtain 15.9 g crude DFSGN-T, which was purified by silica gel column chromatography and eluted with ethyl acetate:n-heptane=1:10 to obtain 13.6 g DSLGN-T.

Step 2: Synthesis of 393-A1

Reaction Scheme:

##STR00955##

Experimental Procedure:

[1307] DSLGN-T (4 g, 4.71 mmol), DCM (20 mL), methanol (20 mL), and ammonium acetate (3.63 g, 47.1 mmol) were sequentially added to a 100 mL single-necked flask and stirred at room temperature for 1 h. Sodium cyanoborohydride (0.89 g, 14.13 mmol) was added and reacted at room temperature overnight. The solvent was evaporated under reduced pressure, and water (50 mL) was added. The mixture was extracted with DCM (50 mL2). The organic phase was collected, and the solvent was evaporated under reduced pressure to obtain 4.1 g crude 393-A1, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=5:1 to obtain 2.46 g 393-A1 with a yield of 61%.

Step 3: Synthesis of 393-A2

Reaction Scheme:

##STR00956##

Experimental Procedure:

[1308] P-nitrophenyl chloroformate (5.53 g, 27.43 mmol) and DCM (15 mL) were added to a 250 mL single-necked flask. Then DMAP (0.28 g, 2.29 mmol), 3-diethylamino-1-propanol (3 g, 22.86 mmol), and pyridine (2.17 g, 27.43 mmol) were dissolved in DCM (15 mL) and slowly added dropwise to the reaction system, followed by reacting for 2 h. The reaction was quenched by addition of 50 mL water. The organic phase was collected, and the aqueous phase was extracted once with 50 mL DCM. The organic phases were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography using n-hexane:ethyl acetate=20:1 to obtain 6.48 g 393-A1 with a yield of 95.6%.

Step 4: Synthesis of Amino Lipid Compound 393

Reaction Scheme:

##STR00957##

Experimental Procedure:

[1309] 383-A1 (1 g, 1.17 mmol), DCM (10 mL), DMAP (72 mg, 0.59 mmol), pyridine (0.28 g, 3.51 mmol), and 393-A2 (1.04 g, 3.51 mmol) were added to a 100 mL single-necked flask and reacted at room temperature overnight. Water (20 mL) was added, and the mixture was extracted with DCM (20 mL2). The organic phase was collected and concentrated under reduced pressure to obtain crude amino lipid compound 393, which was purified by silica gel column chromatography and eluted with n-hexane:ethyl acetate=3:1 to obtain 700 mg amino lipid compound 393 with a purity of 94.11% and a yield of 59%.

[1310] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.48 (t, J=5.2 Hz, 2H), 4.07 (t, J=6.1 Hz, 6H), 3.56 (dt, J=9.2, 6.7 Hz, 5H), 3.40 (dt, J=9.2, 6.7 Hz, 4H), 2.56-2.47 (m, 6H), 2.28 (t, J=7.6 Hz, 4H), 1.76 (m, 2H), 1.72-1.63 (m, 8H), 1.57 (m, 12H), 1.45 (m, 2H), 1.38-1.21 (m, 46H), 1.02 (t, J=7.1 Hz, 6H), 0.89 (t, J=6.9 Hz, 12H).

[1311] LC-MS (ESI): Calculated for (M+H) 1013.87, Found 1014.3.

Example 55: Synthesis of Amino Lipid Compound 394

##STR00958##

Experimental Procedure:

[1312] Triphosgene (214 mg, 0.73 mmol) and DCM (10 mL) were added to a 100 mL single-necked flask. A solution of 3-diethylaminopropylamine (190 mg, 1.46 mmol) in DCM (5 mL) was added dropwise at 10 C. and stirred at room temperature for 1 h. Triethylamine (295 mg, 2.92 mmol) was added dropwise and stirred for 10 min. The solvent was evaporated under reduced pressure to obtain a white solid. DCM (10 mL) and 393-A1 (1.2 g, 1.4 mmol) were added and stirred overnight. Water (20 mL) was added and stirred, and partitioned. The organic phase was collected, and the aqueous phase was extracted once with DCM (20 mL). The organic phases were combined and concentrated under reduced pressure to obtain crude amino lipid compound 394, which was purified by silica gel column chromatography and eluted with ethyl acetate:methanol=30:1 to obtain 1.18 g amino lipid compound 394 with a yield of 83.16%.

[1313] .sup.1H NMR (600 MHz, CDCl.sub.3) 4.48 (t, J=5.3 Hz, 2H), 4.07 (t, J=6.2 Hz, 4H), 3.66-3.59 (m, 1H), 3.57 (dt, J=9.3, 6.7 Hz, 4H), 3.41 (dt, J=9.3, 6.7 Hz, 4H), 3.38-3.32 (m, 2H), 3.11-3.07 (m, 4H), 3.03 (t, J=6.4 Hz, 2H), 2.28 (t, J=7.6 Hz, 4H), 2.02-1.94 (m, 2H), 1.73-1.63 (m, 8H), 1.63-1.51 (m, 12H), 1.43 (m, 2H), 1.38-1.23 (m, 52H), 0.89 (t, J=6.8 Hz, 12H).

[1314] LC-MS (ESI): Calculated for (M+H) 1012.89, Found 1013.6.

Experimental Example 1: Preparation of Lipid Nanoparticle Encapsulating Luciferase mRNA (Fluc mRNA)

(1) Formulating:

[1315] A specified amount of Fluc mRNA stock solution, 0.2 M sodium acetate buffer, and DEPC water were added to a container, and mixed well to obtain a water phase:

[1316] The amino lipid compound of the present disclosure, a helper lipid (DSPC), a structural lipid (cholesterol), and a PEG-lipid were separately dissolved in absolute ethanol to prepare respective solutions at concentrations of 20 mg/mL, 10 mg/mL, 20 mg/mL, and 25 mg/mL, respectively. The above four solutions were pipetted at a molar ratio of Lipid:DSPC:CHO-HP:M-DMG-2000 of 48:10:40.5:1.5, and mixed well to prepare an alcohol phase.

[1317] (2) Encapsulation: The water phase and the alcohol phase were aspirate into water phase and alcoholic phase syringes at a flow rate of water phase: alcohol phase=9 mL/min: 3 mL/min by using a microfluidic preparation instrument (MPE-L2), and encapsulation was carried out at a flow rate of water phase: alcohol phase=12 mL/min: 4 mL/min to obtain mRNA-encapsulating lipid nanoparticle (mRNA-LNP).

[1318] (3) Dialysis: The product of step (2) was loaded in a dialysis bag and placed in a Tris Buffer-8% (m/V) sucrose solution for replacement to remove ingredients such as residual ethanol, unassembled lipids. Dialysis was conducted for 2 h with magnetic stirring at room temperature and under protection from light (dialysate was replaced every 1 hour).

[1319] (4) The product of step (3) was sterilized by passing through a 0.22 m microporous membrane, and then packaged.

[1320] A variety of lipid nanoparticle formulations (LNP formulations) encapsulating Fluc mRNA were prepared, wherein the amino lipid compounds contained in the variety of LNP formulations were different from each other, and each LNP formulation had a Fluc mRNA concentration of 0.2 g/L, a mass ratio of Fluc mRNA to Lipid of 1:10, a particle size of 80 nm-130 nm, and an encapsulation efficiency of 85% or higher.

Experimental Example 2: Performance Evaluation of In Vivo Delivery of Lipid Nanoparticle

[1321] Animal preparation: Female BALB/c mice of 6-8 weeks old were selected and raised in an SPF grade breeding room. Animal testing was conducted in strict accordance with the guidelines of national health institutions and animal ethics requirements.

[1322] In vivo Delivery: prior to injection of the test LNP formulation, the LNP formulation was gently and repeatedly inverted to thoroughly mix the sample of the formulation. A corresponding amount of the LNP formulation was aspirated with a 1 ml insulin syringe, and the LNP formulation was injected by tail vein injection (IV), with 3 mice in duplicate per formulation. Each mouse was injected with 70 L of the corresponding LNP formulation encapsulating the luciferase mRNA (Fluc mRNA) prepared in Experimental Example 1.

[1323] 6 hours after injection of LNP formulations, mice were injected with 200 L D-Luciferin luciferase developing substrate (Catalog No. 122799; Manufacturer: Perkin Elmer). After the substrate was injected, the mice were anesthetized with isoflurane inhalation, and the injection time of luciferase developing substrate was recorded. 10 minutes after the substrate injection, the animals were placed in supine position, and the signal distribution and expression intensity of luciferase in the body and various organs of the animals were observed with In Vivo Imaging System (IVIS).

[1324] The encapsulation efficiency of the lipid nanoparticle encapsulating luciferase mRNA (Fluc mRNA) with a representative amino lipid compound and the fluorescence expression intensity induced by the same are shown in Table 4, with the LNP formulation containing SM-102, 1001 or ALC-0315 as a control LNP formulation. The preparation method of the control LNP formulation was basically the same as that of the LNP formulations in Experimental Example 1, with the difference lying in that the amino lipid compounds of the present disclosure were replaced with SM-102, 1001 or ALC-0315 in the preparation of the control LNP formulation.

##STR00959##

TABLE-US-00004 TABLE 4 Average number of Average number Average number Amino lipid photons (Total of photons of photons compounds in-vivo flux) (Liver) (Spleen) 383 6.10E+07 8.57E+06 1.53E+06 381 2.46E+08 4.40E+07 1.96E+07 380 4.05E+08 7.25E+07 6.79E+07 1246 4.92E+07 6.85E+06 3.83E+06 1248 8.20E+07 1.79E+07 3.48E+06 434 2.50E+07 3.52E+06 1.40E+05 436 2.50E+07 6.68E+06 5.08E+05 389 2.99E+07 4.14E+06 8.58E+04 390 5.23E+07 7.04E+06 3.84E+05 382 5.28E+07 6.78E+06 1.88E+05 388 1.94E+08 3.66E+07 1.34E+06 457 1.43E+07 1.55E+06 2.70E+06 437 1.66E+07 2.66E+06 2.62E+06 433 2.56E+07 3.83E+06 2.44E+05 451 2.86E+07 4.63E+06 1.74E+07 441 3.09E+07 5.62E+06 5.52E+05 438 5.78E+07 8.47E+06 1.07E+06 387 6.66E+07 7.15E+06 6.77E+05 392 8.20E+07 1.50E+07 6.65E+05 393 2.80E+08 5.44E+07 1.00E+07 452 3.27E+08 4.94E+07 2.22E+07 450 4.38E+07 7.96E+05 3.18E+06 399 9.00E+07 1.96E+07 2.05E+06 458 1.29E+08 2.69E+07 2.60E+06 449 1.65E+08 2.94E+07 7.31E+06 398 1.79E+08 2.78E+07 6.36E+05 396 1.85E+08 3.24E+07 9.78E+05 416 2.16E+08 5.13E+07 3.99E+06 459 3.42E+07 5.09E+06 2.52E+06 439 5.87E+07 1.25E+07 1.96E+06 440 2.30E+08 3.22E+07 2.14E+06 2044 4.94E+07 6.90E+06 1.18E+06 2045 5.83E+07 8.90E+06 1.89E+06 2040 1.53E+08 2.97E+07 1.11E+07 2043 1.64E+08 2.59E+07 1.07E+07 2039 2.05E+08 4.26E+07 3.75E+07 2041 2.08E+08 4.16E+07 1.07E+07 2021 4.49E+06 1.19E+06 1.04E+06 869 1.22E+07 2.15E+06 5.40E+04 867 2.15E+07 4.12E+06 1.36E+05 859 2.31E+07 3.71E+06 7.21E+04 490 2.65E+07 3.68E+06 5.06E+05 849 2.67E+07 4.12E+06 1.82E+05 868 3.04E+07 5.35E+06 1.74E+05 856 3.40E+07 9.34E+06 3.58E+05 864 3.66E+07 6.58E+06 2.33E+05 865 4.34E+07 8.60E+06 3.25E+05 863 4.43E+07 7.13E+06 2.88E+05 2036 5.60E+07 1.04E+07 5.75E+05 861 6.67E+07 1.17E+07 3.52E+05 862 8.57E+07 1.53E+07 6.67E+05 851 1.28E+08 2.21E+07 1.93E+06 2035 1.49E+08 3.12E+07 7.61E+05 ALC-0315 6.39E+08 1.23E+08 3.45E+06 1001 9.68E+07 9.38E+06 1.65E+07 SM-102 3.00E+08 6.19E+07 6.42E+06

Experimental Example 3: Evaluation of In Vivo Safety of Lipid Nanoparticle

[1325] A variety of lipid nanoparticle formulations (LNP formulations) encapsulating human erythropoietin mRNA (hEPO mRNA) were prepared according to the method as described in Experimental Example 1, with replacing the luciferase mRNA (Fluc mRNA) with human erythropoietin mRNA (hEPO mRNA), with a concentration of human erythropoietin mRNA (hEPO mRNA) of 0.5 g/L, a mass ratio of hEPO mRNA to Lipid of 1:10, a particle size of 90 nm-130 nm, and an encapsulation efficiency of above 90% or higher for each LNP formulation. MC3 in the MC3-containing LNP formulation was

##STR00960##

[1326] Animal preparation: Female SD rats of 6-8 weeks old were selected and raised in an SPF grade breeding room. Animal testing was conducted in strict accordance with the guidelines of national health institutions and animal ethics requirements.

[1327] In vivo Delivery: Prior to injection of the test LNP formulations, the LNP formulations were gently and repeatedly inverted to thoroughly mix the formulation samples. A corresponding amount of the formulation samples were aspirated with a 2 ml syringe, and the LNP formulations were injected by tail vein injection (IV), with 4 rats per formulation. Each rat was injected with (1200 L-hEPO 3 mpk) of the human erythropoietin mRNA (hEPO mRNA)-encapsulating LNP formulation. Tris buffer was used as a blank control.

[1328] Serum acquisition: Blood samples of rats were collected 12 h after injection, placed in tubes without anticoagulants, and naturally coagulated at room temperature for 30 min-60 min, and then centrifuged at a speed of 3500 rpm for 10 min to obtain the supernatant, which was the serum.

[1329] The detection of alanine transaminase was carried out according to instructions of the kit (Nanjing Jiancheng Bioengineering Institute, Catalog. No. C009-2-1), and a standard curve was made using the standard provided in the kit. D-PBS was used in the experiment, which was purchased from Sangon Biotech (Shanghai) Co., Ltd., Catalog No. E607009-0600.

[1330] The method of detection of enzyme activity of alanine aminotransferase (ALT) in serum of the mice is as follows:

Preparation of ALT Standard Curve:

[1331] (1) Enzymatic reaction: 0, 2, 4, 6, 8 and 10 L of 2 mol/mL sodium pyruvate standard solution were sequentially added to 5 L of 0.1 mol/L phosphate buffer, and the volume was supplemented to 25 L with a ALT matrix solution (alanine aminotransferase matrix solution), and repeatedly aspirating and spitting with a pipette for mixing well; [1332] (2) Addition reaction: 20 L 2,4-dinitrophenylhydrazine solution was added to all reaction wells in (1), mixed by aspirating and spitting, and then placed in an incubator at 37 C. to react for 20 min; [1333] (3) Developing: 200 L of 0.4 mol/L NaOH solution was added to all reaction wells in (2) to stop the reaction, mixed by aspirating and spitting, and incubated at room temperature for 15 min. The OD value of each well was measured at 510 nm in a microplate reader; and [1334] (4) Data processing of standard curve: The corresponding absolute OD value for each well was obtained by subtracting the OD value for the well with 0 L sodium pyruvate from the measured OD value for each well, the corresponding ALT Karmen units being 0, 28, 57, 97, 150 and 200, respectively. The standard curve was obtain by taking the absolute OD value as the abscissa and the corresponding Karmen unit as the ordinate.

Detection of ALT Enzyme Activity in Serum Samples:

[1335] (1) Reagent preparation: an ALT matrix solution was placed in an incubator at 37 C. for preheating; [1336] (2) Enzymatic reaction: 5 L diluted serum was aspirated and added to a 96-well plate, then 20 L matrix solution was added to the corresponding sample well and mixed by repeatedly aspirating and spitting to avoid bubbling, and then placed in an incubator at 37 C. for 30 min; [1337] (3) Addition reaction: 20 L 2,4-dinitrophenylhydrazine was added to all reaction wells in (2), mixed by aspirating and spitting, and then reacted in an incubator at 37 C. for 20 min; [1338] (4) Developing: 200 L of 0.4 mol/L NaOH solution was added to all reaction wells in (3) to stop the reaction, mixed by aspirating and spitting, and then incubated at room temperature for 15 min. The OD value for each well was measured at a wavelength of 510 nm in a microplate reader; and [1339] (5) Calculation of ALT enzyme activity in serum: The absolute OD value of the corresponding sample well was obtained by subtracting the OD value for the control well sample well without biochemical reaction) from the obtained OD value of the sample well, and was substituted into the standard curve formula to calculate the ALT enzyme activity (Karmen unit) for the corresponding serum sample. The Karman unit was converted into an activity unit (1 Karmen unit=0.482 U/L).

[1340] The method of detection of enzyme activity of aspartate aminotransferase (AST) in serum of the mice is as follows:

Preparation of AST Standard Curve:

[1341] (1) Enzymatic reaction: 0, 2, 4, 6, and 8 L of 2 mol/mL sodium pyruvate standard solution were sequentially added to 5 L of 0.1 mol/L phosphate buffer, and the volume was supplemented to 25 L with a AST matrix solution (aspartate aminotransferase matrix solution), and repeatedly aspirating and spitting with a pipette for mixing well; [1342] (2) Addition reaction: 20 L 2,4-dinitrophenylhydrazine solution was added to all reaction wells in (1), mixed by aspirating and spitting, and then placed in an incubator at 37 C. to react for 20 min; [1343] (3) Developing: 200 L of 0.4 mol/L NaOH solution was added to all reaction wells in (2) to stop the reaction, mixed by aspirating and spitting, and incubated at room temperature for 15 min. The OD value of each well was measured at 510 nm in a microplate reader; and [1344] (4) Data processing of standard curve: The corresponding absolute OD value for each well was obtained by subtracting the OD value for the well with 0 L sodium pyruvate from the measured OD value for each well, the corresponding AST Karmen units being 0, 24, 61, 114 and 190, respectively. The standard curve was obtain by taking the absolute OD value as the abscissa and the corresponding Karmen unit as the ordinate.

Detection of AST Enzyme Activity in Serum Samples:

[1345] (1) Reagent preparation: an AST matrix solution was placed in an incubator at 37 C. for preheating; [1346] (2) Enzymatic reaction: 5 L diluted serum was aspirated and added to a 96-well plate, then 20 L matrix solution was added to the corresponding sample well and mixed by repeatedly aspirating and spitting to avoid bubbling, and then placed in an incubator at 37 C. for 30 min; [1347] (3) Addition reaction: 20 L 2,4-dinitrophenylhydrazine was added to all reaction wells in (2), mixed by aspirating and spitting, and then reacted in an incubator at 37 C. for 20 min; [1348] (4) Developing: 200 L of 0.4 mol/L NaOH solution was added to all reaction wells in (3) to stop the reaction, mixed by aspirating and spitting, and then incubated at room temperature for 15 min. The OD value for each well was measured at a wavelength of 510 nm in a microplate reader; and [1349] (5) Calculation of AST enzyme activity in serum: The absolute OD value of the corresponding sample well was obtained by subtracting the OD value for the control well sample well without biochemical reaction) from the obtained OD value of the sample well, and was substituted into the standard curve formula to calculate the AST enzyme activity (Karmen unit) for the corresponding serum sample. The Karman unit was converted into an activity unit (1 Karmen unit=0.482 U/L).

[1350] The in vivo delivery and safety evaluation results of the lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA with the representative amino lipid compounds are shown in FIG. 1 to FIG. 5, with Tris, ALC-0315 and MC3 as controls. Tris refers to the control group injected with Tris buffer solution only.

[1351] As can be seen from FIG. 1 to FIG. 4, as compared to the commercially available ALC-0315 and MC3, the representative amino lipid compounds exhibit lower ALT enzyme activity and lower AST enzyme activity.

Experimental Example 4: Evaluation of the Delivery Efficiency of Lipid Nanoparticles to Immune Cell Populations in Different Tissues and Organs

[1352] C57BL/6 mice (3 mice per group) were intravenously injected with eGFP mRNA encapsulating LNP. After 12 hours, the mice were dissected, and the spleen and peripheral blood, were collected to prepare single-cell suspensions of various tissues and organs. After blocking with Fc blocking antibodies and surface staining with fluorescent-labeled antibodies, flow cytometry was conducted to analyze the expression levels of eGFP in various immune cell populations to confirm the delivery efficiency of lipid nanoparticles comprising different amino lipid compounds to immune cell populations in different tissues and organs.

[1353] The test results are shown in FIG. 6 to FIG. 7. Blank in FIG. 6 and FIG. 7 refers to the control group injected with 100 L Tris buffer. As can be seen from FIG. 6 to FIG. 7, the lipid nanoparticles comprising the amino lipid compounds 382, 388, 2039, 2041, 2042, 2043, and 2045 have different delivery preferences to different immune cells.

[1354] In addition to those described in this disclosure, various modifications to the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.