Ionizable lipids and use thereof

Abstract

The present invention designs and synthesizes a class of ionizable lipids. A lipid nanoparticle delivery system composed thereof can be used to efficiently and safely deliver nucleic acid drugs such as DNA and RNA, or small-molecule drugs, and not only has good encapsulation efficiency and stability, but also exhibits good safety and clearance rate in animals. They provide good delivery performance for nucleic-acid therapeutics or prophylactics, and have broad application prospects in fields related to drug delivery.

Description

Ionizable lipids and uses thereof

[0001] This application claims priority to Chinese patent application 2024110311424 with a filing date of 2024 / 7 / 30. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the field of drug delivery, such as DNA and RNA, and more particularly, to ionizable lipid compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof, as well as compositions thereof and uses for making lipid nanoparticles. BACKGROUND

[0003] Lipid nanoparticles are currently the most commonly used delivery system for mRNA vaccines and related DNA, RNA therapies. Although the COVID-19 mRNA vaccine has achieved great success, we still need to pay attention to the side effects of the COVID-19 mRNA vaccine, and the development of a safe and efficient delivery system is the key to reducing side effects in nucleic acid therapies such as mRNA. In addition, in other DNA and RNA nucleic acid related therapy fields, most of the development of nucleic acid drug therapies is still in the preclinical or clinical stage, and the key to the continued progress of nucleic acid drug therapies lies in the development of safe and efficient lipid nanoparticle delivery systems. Lipid nanoparticles are mainly composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol lipids, among which ionizable lipids are the key to the composition of lipid nanoparticle delivery systems, and therefore the development of new ionizable lipids is the focus of research on safe and efficient lipid nanoparticle delivery systems. SUMMARY

[0004] The present application provides a class of compounds (including stereoisomers, tautomers, or pharmaceutically acceptable salts thereof) as ionizable lipids, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids to form lipid nanoparticles as a delivery carrier for drugs; can be used to deliver nucleic acids such as antisense and / or messenger RNA, thereby ensuring the function of nucleic acids.

[0005] The present application provides the following solutions:

[0006] A compound having the structure shown in formula (I-B) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

[0007] wherein X0 is H, F, OH, methyl, methoxy, or absent;

[0008] T1, L1 are each independently C 1-4 alkyl or absent;

[0009] T2 and L2 are each independently -OC(O)-, -C(O)-O-, -NH-C(O)-, or -C(O)-NH;

[0010] T3, T4, L3, and L4 are each independently H and C. 4-20 Straight-chain or branched alkyl groups, C 4-20 Straight-chain or branched alkenyl, C 4-20 Straight-chain or branched alkynyl group, C with at least one ester bond 8-20 Straight-chain alkyl groups or C groups containing at least one disulfide bond 8-20 Straight-chain alkyl; wherein T3 and T4 are not both H; L3 and L4 are both H;

[0011] X1 and X2 are each independently C 1-4 Alkyl, -OC(O)-, -C(O)-O-, -OC(O)-O-, or none;

[0012] R2 and R3 are each independently C 1-4 Alkyl, C 1-4 Fluoroalkyl, C 1-4 hydroxyalkyl, Where n3 is an integer between 0 and 2.

[0013] In one implementation, T3, T4, L3, and L4 are each independently H and C. 4-9 Alkyl, C 14-20 Alkyl, C 9-20 alkenyl, C 9- 20 Alkyne group, C with at least one ester bond 13-20 Straight-chain alkyl groups or C groups containing at least one disulfide bond 13-20 Straight-chain alkyl groups.

[0014] In one implementation, T3, T4, L3, and L4 are each independently H, -C4H9, and -C6H, respectively. 13 -C8H 17 -C 10 H 21 -C 13 H 27 -C 14 H 29 -C 15 H 31 -C 16 H 33 C 17 H 35 -C 18 H 37 -C9H 17 -C 15 H29 , -C 17 H 33 , -C 17 H 31 , -C 18 H 35 , -C 18 H 33 , -C 10 H 17 , -(CH2) m1 -C(O)-O-(CH2) m2 CH3, -(CH2) m1 -O-C(O)-(CH2) m2 CH3or -(CH2) m1 -S-S-(CH2) m2 CH3;

[0015] wherein m1 is an integer from 2 to 10, and m2 is an integer from 5 to 14.

[0016] In one embodiment, m1 is an integer from 2 to 8, and m2 is an integer from 8 to 14.

[0017] In one embodiment, m1 is an integer from 3 to 6, and m2 is an integer from 8 to 12.

[0018] In one embodiment, m1 is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0019] In one embodiment, m2 is 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0020] In one embodiment, each of X1and X2in the structure of formula (I) is independently any one of a5-a7:

[0021] a5:

[0022] a6:

[0023] a7:

[0024] In one embodiment, each of X1and X2is independently C 1-4 ​​​​alkyl, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, or nothing.

[0025] In one embodiment, each of X1, X2is independently C 1-4 alkyl, or nothing.

[0026] In one embodiment, X0is H.

[0027] In one embodiment, each of T2, L2is independently -O-C(O)- or -C(O)-O-.

[0028] In one embodiment, each of R2, R3is independently C 1-4 alkyl.

[0029] In one embodiment, each of T1, L1is independently ethyl, propyl, or butyl.

[0030] In one embodiment, each of T1, L1is independently ethyl or propyl.

[0031] In one embodiment, each of T1, L1is independently ethyl or butyl.

[0032] In one embodiment, each of T1, L1is independently propyl or butyl.

[0033] In one embodiment, the compound is of the structure:

[0034] wherein n is 2, 3, or 4;

[0035] m3is 2, 3, or 4;

[0036] m4is 2, 3, or 4;

[0037] each of R2, R3is independently C 1-4 alkyl;

[0038] each of T3, T4is independently C 6-12 straight chain alkyl; each of L3, L4is independently H, C 6-20 straight chain alkyl, or C 13-20 straight chain alkenyl; and L3and L4are not both H.

[0039] In one embodiment, each of R2, R3is independently methyl, ethyl, propyl, or butyl.

[0040] Preferably, each of R2, R3is independently methyl, ethyl, or propyl.

[0041] Preferably; R2, R3 are each independently methyl or ethyl.

[0042] In one embodiment, T3, T4 are each independently:

[0043] (t1) T3, T4 are each independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , -C 11 H 23 or -C 12 H 25 ;

[0044] (t2) T3, T4 are each independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 or -C 11 H 23 ;

[0045] (t3) T3, T4 are each independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 or -C 10 H 21 ;

[0046] (t4) T3, T4 are each independently -C6H 13 , -C7H 15 , -C8H 17 or -C9H 19 ;

[0047] (t5) T3, T4 are each independently -C6H 13 , -C7H 15 or -C8H 17 ;

[0048] (t6) T3, T4 are each independently -C7H 15 , -C8H 17 or -C9H 19 ;

[0049] (t7) T3, T4 are each independently -C6H 13 or -C8H 17 ;

[0050] (t8) T3, T4are each independently -C6H 13 , -C8H 17、 , -C9H 19 , -C 10 H 21 , or -C 12 H 25 ;

[0051] (t9) T3, T4are each independently -C6H 13 , -C8H 17、 , -C 10 H 21 , or -C 12 H 25 ;

[0052] (t10) T3, T4are each independently -C6H 13 , -C8H 17、 , -C9H 19 , or -C 12 H 25 ;

[0053] (t11) T3, T4are each independently -C6H 13 , -C8H 17 , or -C 10 H 21 ;

[0054] (t12) T3, T4are each independently -C7H 15 , -C8H 17 , or -C9H 19 ;

[0055] (t13) T3, T4are each independently -C8H 17 , -C9H 19 , or -C 10 H 21 ;

[0056] (t14) T3, T4are each independently -C8H 17 , or -C 10 H 21 .

[0057] In one embodiment,

[0058] L3, L4are any one of the following:

[0059] (g1) when neither L3, L4is H; each L3, L4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C10 H 21 , -C 11 H 23 or -C 12 H 25 ;

[0060] (g2) L3, L4 are not both H; each of said L3, L4 is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 or -C 11 H 23 ;

[0061] (g3) L3, L4 are not both H; each of said L3, L4 is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 or -C 10 H 21 ;

[0062] (g4) L3, L4 are not both H; each of said L3, L4 is independently -C6H 13 , -C7H 15 , -C8H 17 or -C9H 19 ;

[0063] (g5) L3, L4 are not both H; each of said L3, L4 is independently -C6H 13 , -C7H 15 or -C8H 17 ;

[0064] (g6) L3, L4 are not both H; each of said L3, L4 is independently -C7H 15 , -C8H 17 or -C9H 19 ;

[0065] (g7) L3, L4 are not both H; each of said L3, L4 is independently -C6H 13 or -C8H 17 ;

[0066] (g8) L3 is H; said L4 is C 13-20 straight-chain alkyl or C 13-20 straight-chain alkenyl, wherein the number of double bonds in the C 13-20 straight-chain alkenyl is 1 or 2;

[0067] (g9) L3 is H; and said L4 is C 15-20 straight-chain alkyl or C 15-20 straight-chain alkenyl, wherein the number of double bonds in the straight-chain alkenyl is 1 or 2; 15-20 straight-chain alkenyl, wherein the number of double bonds in the straight-chain alkenyl is 1 or 2;

[0068] (g10) L3 is H; and said L4 is C 15-18 straight-chain alkyl or C 15-18 straight-chain alkenyl, wherein the number of double bonds in the straight-chain alkenyl is 1 or 2; 15-18 straight-chain alkenyl, wherein the number of double bonds in the straight-chain alkenyl is 1 or 2;

[0069] (g11) L3 is H; and said L4 is -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 32 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 13 H 25 , -C 14 H 27 , -C 15 H 29 , -C 16 H 30 , -C 17 H 33 , -C 18 H 35 , -C 19 H 37 , -C 20 H 39 , -C 13 H 23 , -C 14 H 25 , -C 15 H 27 , -C 16 H 29 , -C 17 H 31 , -C 18 H 33 , -C 19 H 35 or -C 20 H 37 ;

[0070] (g12) L3 is H; and said L4 is -C15 H 31 , -C 16 H 32 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 20 H 41 , -C 15 H 29 , -C 16 H 30 , -C 17 H 33 , -C 18 H 35 , -C 19 H 37 , -C 20 H 39 , -C 15 H 27 , -C 16 H 29 , -C 17 H 31 , -C 18 H 33 , -C 19 H 35 or -C 20 H 37 ;

[0071] (g13) L3 is H; and said L4 is -C 15 H 31 , -C 17 H 35 , -C 18 H 37 , -C 19 H 39 , -C 15 H 29 , -C 17 H 33 , -C 18 H 35 , -C 19 H 37 , -C 15 H 27 , -C 17 H 31 , -C 18 H 33 or -C 19 H 35 ;

[0072] (g14) L3 is H; and said L4 is -C 15 H31 , -C 17 H 35 , -C 18 H 37 , -C 15 H 29 , -C 17 H 33 , -C 18 H 35 , -C 15 H 27 , -C 17 H 31 , or -C 18 H 33 ;

[0073] (g15) L3 is H; L4 is -C 17 H 35 , -C 18 H 37 , -C 17 H 33 , -C 18 H 35 , -C 17 H 31 , or -C 18 H 33 ;

[0074] (g16) L3 is H; L4 is -C 15 H 31 , -C 17 H 35 , or -C 18 H 37 ;

[0075] (g17) L3 is H; L4 is -C 15 H 29 , -C 17 H 33 , or -C 18 H 35 ;

[0076] (g18) L3 is H; L4 is -C 15 H 27 , -C 17 H 31 , or -C 18 H 33 ;

[0077] (g19) L3 is H; L4 is -C 17 H 35 , -C 17 H 33 , or -C 17 H 31;

[0078] (g20) L3 is H; and said L4 is -C 18 H 37 , -C 18 H 35 or -C 18 H 33 .

[0079] In one embodiment, the compound is selected from one of the following structures:

[0080] The present application also protects a lipid nanoparticle comprising any of the foregoing compounds or stereoisomers thereof, and a therapeutic or prophylactic agent.

[0081] In one embodiment, the lipid nanoparticle further comprises one or more of a neutral lipid, a sterol, and a polymer-conjugated lipid.

[0082] In one embodiment, the neutral lipid is one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0083] In one embodiment, the neutral lipid is DSPC or DOPC.

[0084] In one embodiment, the neutral lipid is DSPC or DOPE.

[0085] In one embodiment, the neutral lipid is DSPC.

[0086] In one embodiment, the sterol is cholesterol.

[0087] In one embodiment, the polymer-conjugated lipid is a PEGylated lipid.

[0088] In one embodiment, the PEGylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxylpropyl carbamate.

[0089] In one embodiment, the PEGylated lipid is DMG-PEG 2000.

[0090] In one embodiment, the molar percentages of ionizable lipid, sterol, neutral lipid, and polymer-conjugated lipid in the lipid nanoparticle are 30-65%: 18.5-48.5%: 0-30%: 0-10%.

[0091] In one embodiment, the mole percent of ionizable lipid in the lipid nanoparticle is 30-55%, 35-55%, 35-52%, 40-52%, 45-52%, or 45-50%.

[0092] In one embodiment, the mole percent of ionizable lipid in the lipid nanoparticle is 45.0%, 45.3%, 45.5%, 45.8%, 46.0%, 46.3%, 46.5%, 46.8%, 47.0%, 47.3%, 47.5%, 47.8%, 48.0%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49.0%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, or 50.0%.

[0093] In one embodiment, the mole percent of sterol in the lipid nanoparticle is 20-48%, 25-45%, 30-45%, 35-45%, 35-43%, 35-40%, or 37-40%.

[0094] In one embodiment, the mole percent of sterol in the lipid nanoparticle is 35.0%, 35.5%, 35.8%, 36.0%, 36.5%, 37.0%, 37.5%, 37.6%, 37.7%, 37.8%, 37.9%, 38.0%, 38.1%, 38.2%, 38.3%, 38.4%, 38.5%, 38.6%, 38.7%, 38.8%, 38.9%, 39.0%, 39.1%, 39.2%, 39.3%, 39.4%, 39.5%, 39.6%, 39.7%, 39.8%, 39.9%, or 40.0%.

[0095] In one embodiment, the mole percent of neutral lipid in the lipid nanoparticle is 5-30%, 5-25%, 8-25%, 8-20%, 8-18%, 8-15%, or 8-12%.

[0096] In one embodiment, the molar percentage of neutral lipid in the lipid nanoparticle is: 8.0%, 8.2%, 8.5%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, 10.0%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11.0%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, or 12.0%.

[0097] In one embodiment, the molar percentage of polymer-conjugated lipid in the lipid nanoparticle is: 0.2-10%, 0.2-9%, 0.2-8%, 0.2-7%, 0.2-6%, 0.2-5%, 0.5-5%, 0.5-4.5%, 0.5-4.0%, 0.5-3.5%, 0.5-3.0%, 0.5-2.5%, 0.6-2.2%, 0.7-2.0%, 0.8-2.0%, 0.9-1.8%, or 1.0-1.8%.

[0098] In one embodiment, the molar percentage of polymer-conjugated lipid in the lipid nanoparticle is: 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10%, 1.20%, 1.25%, 1.30%, 1.35%, 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.65%, 1.68%, or 1.70%.

[0099] In one embodiment, the molar percentage of ionizable lipid, sterol, neutral lipid, and polymer-conjugated lipid in the lipid nanoparticle is: 40-62%: 18.5-42.8%: 3-15%: 0.5-5%.

[0100] In one embodiment, the molar percentage of ionizable lipid, sterol, neutral lipid, and polymer-conjugated lipid in the lipid nanoparticle is: 45-50%: 25-42.8%: 3-15%: 0.5-5%.

[0101] In one embodiment, the molar percentage of ionizable lipid, sterol, neutral lipid, and polymer-conjugated lipid in the lipid nanoparticle is: 48-50%: 30-42.8%: 8-15%: 0.8-3%.

[0102] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 48-50%: 35-42.8%: 9-13%: 1.0-2.5%.

[0103] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 48.5%: 38.9%: 11.1%: 1.5%.

[0104] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 62%: 32.65%: 4.35%: 1.0%.

[0105] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 50%: 38.8%: 9.7%: 1.5%.

[0106] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 50%: 38%: 9.5%: 2.5%.

[0107] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 50%: 42.8%: 5.7%: 1.5%.

[0108] In one embodiment, the mole percentage of ionizable lipid, sterol, neutral lipid and polymer conjugated lipid in the lipid nanoparticle is 65%: 19.7%: 13.1%: 2.2%.

[0109] In one embodiment, the therapeutic or prophylactic agent comprises a nucleic acid or a nucleic acid based drug.

[0110] In one embodiment, the nucleic acid is an antisense RNA or a messenger RNA.

[0111] The present application also protects a pharmaceutical composition comprising any of the aforementioned lipid nanoparticles and a pharmaceutically acceptable excipient.

[0112] In one embodiment, the pharmaceutically acceptable excipient includes, but is not limited to, a pharmaceutically acceptable diluent or excipient.

[0113] The present application also protects the use of any of the aforementioned compounds or stereoisomers thereof, or any of the aforementioned lipid nanoparticles, or any of the aforementioned pharmaceutical compositions as a drug delivery vehicle.

[0114] Advantages of the present application:

[0115] The present application designs and synthesizes a class of ionizable lipids, and the lipid nanoparticle delivery system composed of the same can be used for efficient and safe delivery of nucleic acid drugs such as DNA, RNA, or small molecule drugs. The lipid nanoparticle delivery system not only has good encapsulation efficiency and stability, but also has comparable or better delivery efficiency and expression amount compared with positive controls. Moreover, the lipid nanoparticle delivery system has good safety and clearance rate in animals, and has good delivery effect on nucleic acid therapeutic or prophylactic agents, and has wide application prospect in the field of drug delivery.

[0116] Definitions and general terminology

[0117] Unless otherwise indicated, all technical and scientific terms used within the application have the same meanings as those commonly understood by one of ordinary skill in the art to which the application pertains. All patents and publications referred to in this application are incorporated herein by reference in their entirety.

[0118] Unless otherwise indicated, or unless the context clearly indicates otherwise, as used herein the articles "a", "an", and "the" are intended to include both "only one" or "one or more" of the enumerated items. Thus, as used herein, these articles are intended to be used in the inclusive, as well as the exclusive, sense. For example, "a component" means one or more components.

[0119] "Stereoisomers" refer to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), atropisomers, and the like.

[0120] The stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0121] Any asymmetric atom (e.g., carbon, etc.) of a compound disclosed herein can exist in racemic or enantiomeric excess, e.g., in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least a 50% enantiomeric excess in the (R)- or (S)-configuration, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess, or at least a 99% enantiomeric excess.

[0122] "Pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0123] In particular embodiments, the present application provides ionizable lipids containing cyclic structures that can be used to formulate improved compositions for the delivery of mRNA and / or other oligonucleotides in vitro and in vivo; these improved compositions are lipid nanoparticles that can be used to deliver nucleic acids such as RNA and nucleic acid drugs; where the nucleic acids include, e.g., mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors, complementary RNA that interferes with messenger RNA (micRNA), DNA, polyvalent RNA, complementary DNA (cDNA), etc.

[0124] In general, the term "substituted" means that one or more hydrogen atoms in the given structure are replaced by a particular substituent. Unless otherwise indicated, a substituted group can have a substituent at each substitutable position of the group. When more than one position in the given structure can be substituted with one or more substituents selected from a particular group, the substituents can be the same or different at each position.

[0125] The term "unsubstituted" means that the designated group bears no substituents.

[0126] The term "optionally substituted" can be used interchangeably with the term "unsubstituted or substituted," i.e., the structure is either unsubstituted or substituted with one or more substituents described herein.

[0127] Unless the context requires otherwise, throughout the present application, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0128] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the nanometer (nm) scale (e.g., 1 to 1000 nm) that contains one or more types of lipid molecules. The LNPs provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule that is partially or completely encapsulated within a lipid shell. In particular, in some embodiments, where the payload is a negatively charged molecule (e.g., an mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is contemplated that ionizable lipids can interact with the negatively charged payload molecule and facilitate payload incorporation and / or encapsulation into the LNP during LNP formation. Other lipids that can form part of the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as sterols, polymer conjugated lipids, and various zwitterionic lipids.

[0129] The term "ionizable lipid" refers to a lipid that is positively charged at any pH or hydrogen ion activity of its environment, or is capable of becoming positively charged in response to the pH or hydrogen ion activity of its environment, e.g., the environment of its intended use.

[0130] The term "preventive agent" refers to any agent that can completely or partially inhibit the development, recurrence, onset, or spread of a disease and / or its associated symptoms in a subject.

[0131] The term "therapeutic agent" refers to any agent that can be used to treat, prevent, or alleviate a disease, disorder, or condition, including for use in treating, preventing, or alleviating a disease, disorder, or condition and / or one or more symptoms associated therewith.

[0132] In addition, it should be noted that the description mode "each independently" and "… each independently" and "… independently" used in the present application can be interchangeable unless explicitly indicated otherwise, and should be interpreted in a broad sense, which can mean that the specific options expressed between the same symbols in different groups do not affect each other, or can mean that the specific options expressed between the same symbols in the same group do not affect each other. DETAILED DESCRIPTION

[0133] The present application will be further described with reference to the following specific examples, which are intended to be illustrative only and not limiting of the scope of the application. Unless otherwise indicated, the procedures used in the following examples are routine procedures, and the materials used are commercially available unless otherwise indicated.

[0134] In the following examples, the starting materials were prepared by methods available in the art or purchased from regular commercial channels.

[0135] Example 1 Preparation of A-143

[0136] Step 1: Preparation of A-143-a

[0137] Dissolve 1,3-propanedione dicarbonylate diethyl ester (40.3 g, 1.5 mmol) and ethoxycarbonylmethylidene triphenylphosphonium (CAS: 1099-45-2, 100.0 g, 299.1 mmol, 1.5 eq) in toluene (15 mL) under nitrogen protection, stir the reaction at 120 °C for 4 h. TLC monitor the reaction is complete (PE:EA = 12:1), spin dry silica gel column chromatography purification (PE:EA = 50:1 / 20:1), get 27.0 g of A-143-a.

[0138] Step 2: Preparation of A-143-b

[0139] Dissolve A-143-a (23.0 g, 84.6 mmol, 1.0 eq) in ethanol (200 mL), add Raney nickel (6.0 g), replace with hydrogen three times, react at room temperature overnight, TLC monitor (PE:EA = 20:1,) the reaction is complete, filter, wash with EA, spin dry to get 23.0 g of A-143-b, yield 78%.

[0140] Step 3: Preparation of A-143-c

[0141] Dissolve A-143-b (23.0 g, 83.9 mmol, 1.0 eq) in anhydrous ethanol (120 mL), slowly add sodium borohydride (17.0 g, 449.7 mmol, 5.4 eq), react at room temperature for 3 days, TLC monitor the reaction (EA / EA:MeOH = 10:1), spin dry, drag with methanol 3 times, add methanol (150 mL) and water (50 mL), adjust pH = 4-5 with dilute hydrochloric acid in ice bath, vacuum oil pump dry, silica gel, column chromatography separation (EA→EA:MeOH = 8:1), get 7.3 g of A-143-c, yield 58%.

[0142] Step 4: Preparation of A-143-d

[0143] A-143-c (3.3 g, 22.2 mmol, 1.0 eq) was dissolved in DMF (120 mL), 2-hexyldecanoic acid (11.5 g, 45 mmol, 2.0 eq), DIEA (5.8 g, 45 mmol, 2.0 eq), DMAP (0.5 g, 4.5 mmol, 0.2 eq) were added, protected by nitrogen, cooled to -15 °C, EDCI (23.0 g, 67.5 mmol, 3.0 eq) was added, and the mixture was slowly warmed to room temperature and reacted overnight. Water (40 mL) was added and the pH was adjusted to 4-5 with dilute hydrochloric acid. The mixture was extracted with EA three times, the organic phases were combined, washed with water, saturated sodium chloride solution, dried, and the silica gel was mixed. Column chromatography was performed (PE:EA = 50:1 / 30:1 / 15; 1 / 8:1 / 5:1 / 2:1) to obtain 2.3 g of oil A-143-d.

[0144] Step 5: Preparation of A-143-e

[0145] A-143-d (2.3 g, 2.2 mmol, 1.0 eq) was dissolved in DCM (20 mL) and water (5 mL), potassium bromide (53 mg, 0.45 mmol, 0.2 eq), TBAB (3.6 mg, 0.16 mmol, 0.05 eq), sodium bicarbonate (0.32 g, 3.8 mmol, 1.7 eq) were added, and the mixture was cooled to 0 °C. Sodium hypochlorite solution (4%, 2 mL) was added dropwise, and the reaction was monitored by TLC (PE:EA = 12:1). After the reaction was completed, the mixture was washed with water and sodium chloride solution once. The organic phase was dried over sodium sulfate and rotary evaporated. The residue was separated by column chromatography on silica gel (PE / EA 20:1 eluent) to obtain 1.2 g of A-143-e with a yield of 87%.

[0146] Step 6: Preparation of A-143

[0147] A-143-e (0.5 g, 0.8 mmol, 1.0 eq) and pyrrolidine (87 mg, 1.2 mmol, 1.5 eq) were dissolved in THF (20 mL), and Raney nickel (1.0 g) was added. The mixture was replaced with hydrogen three times and reacted at room temperature overnight. The reaction was monitored by TLC (2% TEA, PE:EA = 12:1). After the reaction was completed, the mixture was filtered, washed with EA, and rotary evaporated. The residue was separated by column chromatography on silica gel (PE / EA 10:1 and 2% TEA, PE:EA = 20:1) to obtain 380 mg of oil A-143.

[0148] 1H NMR (500 MHz, MeOD) δ 4.17 - 4.08 (m, 4H), 4.14 (s, 6H), 2.55 (s, 4H), 2.49 (dd, J = 9.4, 6.9 Hz, 2H), 2.32 (td, J = 9.0, 4.5 Hz, 2H), 1.84 - 1.77 (m, 4H), 1.71 - 1.63 (m, 4H), 1.62 - 1.51 (m, 6H), 1.49 - 1.39 (m, 4H), 1.32 - 1.21 (m, 41H), 0.87 (t, J = 6.8 Hz, 12H).

[0149] Example 2 Preparation of A-147

[0150] Step 1: Preparation of A-147-d

[0151] A-143-c (4.8 g, 32.2 mmol, 1.0 eq) was dissolved in DMF (120 mL), 2-hexyl decanoic acid (12.0 g, 46.7 mmol, 1.5 eq), DIEA (7.06 g, 54.7 mmol, 1.7 eq), DMAP (0.78 g, 6.3 mmol, 0.2 eq) were added, nitrogen protection, cooling to -15 °C, EDCI (10.5 g, 54.76 mmol, 1.7 eq) was added, slowly warmed to room temperature and reacted overnight, TLC monitoring (PE:EA = 40:1 / 7:1 / 1:1 and EA:MeOH = 15:1), water (40 mL) was added and the pH was adjusted to 4-5 with concentrated hydrochloric acid, EA was extracted 3 times (50 mL*3), EA was combined, washed with water, salt washed, dried, silica gel sample, column chromatography separation (PE:EA = 50:1 / 30:1 / 15;1 / 8:1 / 5:1 / 2:1), 2.0 g of A-147-d was obtained as an oil.

[0152] Step 2: Preparation of A-147-e

[0153] A-147-d (2.0 g, 5.2 mmol, 1.0 eq) was dissolved in DCM (20 mL), oleic acid (1.9 g, 6.8 mmol, 1.3 eq), DIEA (0.66 g, 5.2 mmol, 1.0 eq), DMAP (63 mg, 0.5 mmol, 0.1 eq) were added, and the mixture was protected by nitrogen. EDCI (2.0 g, 10.3 mmol, 2.0 eq) was added, and the mixture was stirred overnight. TLC monitoring (PE:EA = 40:1 / 7:1 / 1:1) showed that the reaction was completed. Water (40 mL) was added, and the mixture was stirred. The mixture was extracted with EA (20 mL*3) three times. The EA was combined, washed with water, saturated sodium chloride solution, dried, and column chromatography was performed on silica gel (PE:EA = 4:1 and 2% TAE / 30:1 / 20:1) to obtain 1.4 g of A-147-e in the form of an oil.

[0154] Step 3: Preparation of A-147-f

[0155] Oxalyl chloride (0.9 g, 4.2 mmol, 3.0 eq) and dry DCM (20 mL) were mixed and cooled to -78 °C. DMSO (0.76 g, 6.3 mmol, 3.0 eq) was slowly added dropwise. The temperature was controlled below -65 °C. A-147-e (1.4 g, 5.2 mmol, 1.0 eq) in dry DCM (10 mL) was slowly added dropwise. The mixture was stirred for 1 h after the addition was completed. Dry TEA (1.0 g, 9.5 mmol, 5.0 eq) was slowly added dropwise at about -70 °C. The mixture was slowly warmed to room temperature and stirred for 1 h. TLC monitoring (PE:EA = 10:1) showed that the reaction was completed. Water (30 mL) was added, and the mixture was separated. The aqueous phase was extracted with EA three times. The organic phase was combined, dried, and column chromatography was performed on silica gel (PE:EA = 20:1 / 15:1) to obtain 1.1 g of A-147-f in the form of an oil.

[0156] Step 4: Preparation of A-147

[0157] A-147-f (0.7 g, 1.1 mmol, 1.0 eq) and pyrrolidine (120 mg, 1.6 mmol, 1.5 eq) were dissolved in THF (20 mL). Raney nickel (1.0 g) was added, and the mixture was replaced with hydrogen three times. The mixture was stirred at room temperature overnight. TLC monitoring (2% TEA, PE:EA = 12:1) showed that the reaction was completed. The mixture was filtered, washed with EA, dried, and column chromatography was performed on silica gel (2% TEA, PE:EA = 35:1 / 32:1 / 30:1) to obtain 200 mg of A-147 in the form of an oil.

[0158] 1H NMR (500 MHz, MeOD) δ 4.15 - 4.07 (m, 4H), 2.55 (s, 4H), 2.51 - 2.45 (m, 2H), 2.37 - 2.23 (m, 3H), 2.01 (dd, J = 13.8, 10.7 Hz, 5H), 1.80 (t, J = 3.4 Hz, 4H), 1.66 (dd, J = 6.7, 4.3 Hz, 4H), 1.63 - 1.51 (m, 7H), 1.49 - 1.39 (m, 2H), 1.36 - 1.18 (m, 43H), 0.87 (t, J = 6.8 Hz, 9H).

[0159] A-150, A-151 were prepared according to the route of A-147 by replacing the corresponding materials or intermediates.

[0160] Example 5 Preparation of B-447

[0161] Step 1: Preparation of B-447-a

[0162] Into a reaction flask was added mono-tert-butyl succinate (15 g, 86.1 mmol), hydroxylamine hydrochloride (10.1 g, 103.3 mmol) and HOBT (2.33 g, 17.22 mmol), then THF (150 mL) was added, followed by triethylamine (13.1 g, 129.2 mmol) and EDCI (24.76 g, 129.16 mmol), the reaction was stirred at room temperature for 5 hours. The reaction solution was added to ice water (200 mL), extracted twice with ethyl acetate (200 mL), the organic phase was collected, concentrated, and purified by column chromatography to obtain 16.9 grams of B-447-a. Yield 90%. 1 H NMR (500 MHz, Chloroform-d) δ 3.70 (s, 3H), 3.17 (s, 3H), 2.68 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 6.9 Hz, 2H), 1.43 (s, 9H).

[0163] Step 2: Preparation of B-447-b

[0164] A reaction flask was charged with magnesium (6.47 g, 269.7 mmol) and anhydrous THF (150 mL) under argon protection. A catalytic amount of 1,2-dibromoethane and about 10% of 2-(2-bromoethyl)-1,3-dioxolane (24.4 g, 134.86 mmol) THF solution (50 mL) were added. After initiation by heating, the remaining 90% solution was added dropwise slowly. After 1 hour of stirring at room temperature, another reaction flask was charged with B-447-a (14.65 g, 67.43 mmol) and anhydrous THF (70 mL) under argon protection and cooling to 0 °C. The above prepared Grignard reagent solution was then added dropwise. After 1 hour of stirring at 5 °C, the reaction solution was added to saturated aqueous ammonium chloride solution (200 mL) and extracted with ethyl acetate (200 mL) twice. The organic phase was collected, washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give the product 14.5 g. Yield 83.3%. 1 H NMR (500 MHz, Chloroform-d) δ 4.90 (t, J = 4.3 Hz, 1H), 4.01 - 3.89 (m, 2H), 3.87 - 3.78 (m, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.57 (t, J = 7.4 Hz, 2H), 2.49 (t, J = 6.6 Hz, 2H), 1.98 (td, J = 7.4, 4.3 Hz, 2H), 1.42 (s, 9H).

[0165] Step 3: Preparation of B-447-c

[0166] A reaction flask was charged with B-447-b (14.51 g, 56.17 mmol) and anhydrous THF (150 mL) under argon protection. Lithium aluminum hydride (6.4 g, 168.51 mmol) was added slowly in three portions with ice bath cooling. After stirring at room temperature overnight, the reaction solution was cooled to 0 °C. Water (5.3 g), 15% aqueous sodium hydroxide solution (5.3 g), and water (15.9 mL) were added dropwise slowly. After stirring for 30 minutes, the reaction solution was filtered with celite. The filtrate was collected and concentrated. B-447-c was obtained by column chromatography purification. Yield 9.3 g, 87%.

[0167] Step 4: Preparation of B-447-d

[0168] A flask was charged with B-447-c (8.3 g, 43.63 mmol) and anhydrous dichloromethane (100 mL), argon protection, ice bath cooling, triethylamine (6.62 g, 65.45 mmol) and DMAP (415 mg) were added, followed by dropwise addition of a solution of triphenylmethyl chloride (12.16 g, 43.63 mmol) in dichloromethane (50 mL), and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate (200 mL), washed with water, the organic phase was collected and concentrated, and column chromatography was used for purification to obtain 18 g of B-447-d. Yield 95%.

[0169] Step 5: Preparation of B-447-e

[0170] A flask was charged with B-447-d (10 g, 23.12 mmol) and anhydrous ethylene glycol dimethyl ether (100 mL), argon protection, potassium tert-butoxide (14.27 g, 127.16 mmol) was added, and the mixture was stirred at room temperature for 30 minutes, followed by addition of dimethylamino bromoethane hydrochloride (21.5 g, 92.48 mmol), and the mixture was stirred at 50°C overnight. Ice water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate, the organic phase was collected and concentrated, and column chromatography was used for purification to obtain 1.9 g of B-447-e. Yield 17%.

[0171] Step 6: Preparation of B-447-f

[0172] A flask was charged with B-447-e (1.9 g, 3.77 mmol) and 50% aqueous acetic acid (20 mL), argon protection, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure at low temperature, neutralized with sodium carbonate solid, dissolved in dichloromethane, filtered, the organic phase was collected and concentrated, and column chromatography was used for purification to obtain 860 mg of B-447-f. Yield 87%.

[0173] Step 7: Preparation of B-447-g

[0174] A flask was charged with B-447-f (860 mg, 3.29 mmol), dichloromethane (15 mL), oleic acid (929 mg, 3.29 mmol), DMAP (1.2 g, 9.87 mmol), and EDCI (946 mg, 4.94 mmol) were added in sequence, and the mixture was stirred overnight under nitrogen protection. The dichloromethane was removed by rotary evaporation, ethyl acetate and water were added, the mixture was stirred and separated, the organic phase was collected, the aqueous phase was extracted once more with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, rotary evaporated, and column chromatography was used for purification to obtain 775 mg of B-447-g.

[0175] Step 8: Preparation of B-447-h

[0176] A reaction flask was charged with B-447-g (775 mg) and acetone (5 mL), and 1 M aqueous hydrogen chloride (2 mL) was added dropwise. The reaction was stirred at room temperature overnight. The reaction was neutralized with solid sodium bicarbonate and extracted with ethyl acetate twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give B-447-h (520 mg). The crude product was used directly in the next step.

[0177] Step 9: Preparation of B-447-i

[0178] A reaction flask was charged with B-447-h (420 mg, 0.87 mmol), t-butyl alcohol (7 mL), 2-methyl-2-butene (7 mL), and water (7 mL), and cooled to 0 °C under argon. Sodium phosphate monobasic (630 mg, 5.24 mmol) and sodium chlorite (496 mg, 4.37 mmol) were added with stirring. The reaction was maintained at 0 °C for 6 h. The pH was adjusted to about 5 by the dropwise addition of 1 N aqueous hydrogen chloride. The reaction was extracted with ethyl acetate twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give B-447-i (387 mg). The crude product was used directly in the next step.

[0179] Step 10: Preparation of B-447

[0180] A reaction flask was charged with B-447-i (387 mg, 0.78 mmol), and then dichloromethane (25 mL), 9-heptadecanol (199 mg, 0.78 mmol), DMAP (285 mg, 2.33 mmol), and EDCI (224 mg, 1.17 mmol) were added sequentially. The reaction was stirred at 40 °C overnight. The reaction was concentrated, and methyl t-butyl ether was added. The pH was adjusted to 5 with water and dilute hydrochloric acid. The reaction was stirred and allowed to separate. The organic phase was collected and washed with saturated aqueous sodium bicarbonate once. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by column chromatography to give B-447 (100 mg). LC-MS [M+H] + 736.7.

[0181] 1H NMR (500 MHz, Chloroform-d) δ 5.40 - 5.29 (m, 2H), 4.86 (t, J = 6.2 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.55 (q, J = 5.9 Hz, 2H), 3.31 (t, J = 6.0 Hz, 1H), 2.56 (t, J = 5.9 Hz, 2H), 2.37 (td, J = 7.6, 2.3 Hz, 2H), 2.30 (d, J = 15.1 Hz, 8H), 2.00 (q, J = 6.5 Hz, 4H), 1.60 (q, J = 6.1, 5.1 Hz, 2H), 1.50 (q, J = 6.6, 6.0 Hz, 4H), 1.34 - 1.21 (m, 50H), 0.87 (t, J = 6.8 Hz, 9H).

[0182] Example 6 Preparation of B-448

[0183] Step 1: Preparation of B-448-a

[0184] Into a reaction vial was placed B-276-b (500 mg, 1.02 mmol), THF (8 mL) and H20 (2 mL), stirred to dissolve, sodium borohydride (39 mg, 1.02 mmol) was added, stirred at room temperature for 4 hours. The reaction mixture was cooled in an ice bath, 1 N aqueous hydrogen chloride was added dropwise to adjust the pH to about 5, extracted with methyl tert-butyl ether, collected the organic phase, washed with saturated brine once, dried over anhydrous sodium sulfate, filtered, concentrated, and dried by oil pump to give B-448-a 509 mg, which was used directly in the next step.

[0185] Step 2: Preparation of B-448-b

[0186] Into a reaction vial was placed B-448-a (509 mg, 1.04 mmol), 4-dimethylamino butyric acid hydrochloride (348 mg, 2.08 mmol) and DMAP (381 mg, 3.12 mmol), then dichloromethane (15 mL), THF (15 mL) and triethylamine (210 mg, 2.08 mmol) were added, stirred to dissolve, EDCI (399 mg, 2.08 mmol) was added, warmed to 40 °C and stirred overnight. The dichloromethane was concentrated, 1 N dilute hydrochloric acid was added to adjust the pH to about 5, extracted with ethyl acetate twice, collected the organic phase, washed with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by column chromatography to give B-448-b 502 mg, yield: 80%.

[0187] Step 3: Preparation of B-448-c

[0188] To a reaction flask was added B-448-b (502 mg, 0.83 mmol), methanol (20 mL), concentrated hydrochloric acid (5 drops), and 10% palladium on carbon (100 mg) under a hydrogen atmosphere. The reaction was stirred overnight, filtered, concentrated, and twice taken up in dioxane. B-448-c was obtained as an oil (450 mg) and used directly in the next step.

[0189] Step 4: Preparation of B-448

[0190] To a reaction flask was added B-448-c (450 mg, 0.88 mmol), dichloromethane (20 mL), oleic acid (297 mg, 1.05 mmol), DMAP (321 mg, 2.63 mmol), and EDCI (252 mg, 1.32 mmol). The reaction was stirred at 40 °C overnight, concentrated, diluted with 1 N hydrochloric acid, extracted twice with ethyl acetate, the organic layers were combined, washed with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give B-448 (425 mg, 62% yield). LC-MS [M+H] + : 778.7. 1 H NMR (500 MHz, Chloroform-d) δ 5.37 - 5.29 (m, 2H), 4.93 (q, J = 5.1, 4.6 Hz, 1H), 4.89 - 4.81 (m, 1H), 4.04 (t, J = 5.8 Hz, 2H), 2.36 - 2.30 (m, 3H), 2.27 (t, J = 7.5 Hz, 5H), 2.20 (s, 6H), 2.00 (q, J = 6.4 Hz, 4H), 1.85 - 1.73 (m, 4H), 1.61 (m, 6H), 1.35 - 1.16 (m, 48H), 0.87 (m, 9H).

[0191] B-449, B450 were prepared according to B-448.

[0192] B-449 was prepared according to the procedure for B-448 using N,N- dimethylglycine instead of 4-dimethylaminobutyric acid hydrochloride. LC-MS [M+H] + : 750.7. 1H NMR (500 MHz, Chloroform-d) δ 5.41 - 5.25 (m, 2H), 5.01 (dd, J = 8.2, 4.2 Hz, 1H), 4.91 - 4.79 (m, 1H), 4.04 (d, J = 4.0 Hz, 2H), 3.16 (s, 2H), 2.35 (s, 6H), 2.33 - 2.24 (m, 4H), 2.00 (q, J = 6.5 Hz, 4H), 1.66 - 1.56 (m, 6H), 1.49 (t, J = 6.2 Hz, 4H), 1.34 - 1.19 (m, 46H), 0.87 (m, 9H).

[0193] B-450 was prepared by replacing 4-dimethylaminobutyric acid hydrochloride with 3-dimethylaminopropionic acid hydrochloride in the preparation of B-448. LC-MS [M+H] + : 764.7. 1 H NMR (500 MHz, Chloroform-d) δ 5.40 - 5.24 (m, 2H), 4.96 (m, 1H), 4.90 - 4.78 (m, 1H), 4.04 (t, J = 5.9 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 2.40 - 2.26 (m, 4H), 2.22 (s, 6H), 2.00 (q, J = 6.5 Hz, 4H), 1.67 - 1.56 (m, 6H), 1.49 (m, 4H), 1.32 - 1.22 (m, 46H), 0.87 (m, 9H).

[0194] Example 9 Preparation of B-3

[0195] Step 1: Preparation of B-3-a

[0196] 1,3-propanedione diethyl ester (14.0 g, 1.0 eq, 69.14 mmol) and 2- (triphenylphosphoranyliden)acetonitrile (25.0 g, 1.2 eq, 82.97 mmol) were dissolved in toluene (150 mL) and stirred at 110 °C overnight under nitrogen protection. TLC monitoring (PE:EA = 5:1) showed that the reaction was completed. After drying, anhydrous zinc chloride (28.0 g) was added in four batches under ice bath stirring. After stirring for 1 h, the mixture was filtered, washed twice with EA, and dried. The residue was mixed with silica gel and purified by column chromatography (PE:EA = 15:1→8:1) to give 13.0 g of B-3-a as an oil.

[0197] Step 2: Preparation of B-3-b

[0198] B-3-a (13.0 g, 1.0 eq, 57.70 mmol) and ethanol (100 mL) were mixed, Raney nickel (5.5 g) was added, and the reaction was replaced with hydrogen three times for 2 h, TLC monitoring showed that the product spot was formed (PE:EA = 10:1 and 3:1), Boc anhydride (16.35 g, 1.3 eq, 74.98 mmol) was added, and the reaction was carried out under hydrogen pressure at room temperature overnight, TLC monitoring (DCM:MeOH = 15:1 and PE:EA = 8:1), the reaction was completed, filtered, washed twice with EA, and rotary evaporated to dryness. The dry sample was purified by column chromatography (PE:EA = 10:1→6:1) to obtain 11.9 g of oil B-3-b.

[0199] Step 3: Preparation of B-3-c

[0200] B-3-b (11.9 g, 35.9 mmol, 1.0 eq) was added to a 250 mL bottle, stirred with anhydrous ethanol (100 mL), and NaBH4 (2.7 g, 71.8 mmol, 2.0 eq) was slowly added. The reaction was carried out at room temperature overnight, and TLC monitoring showed that the reaction was completed. The sample was purified by column chromatography (2% TEA, PE:EA = 5:1) to obtain 16.8 g of yellowish oil B-3-c.

[0201] Step 4: Preparation of B-3-d

[0202] B-3-c (5.0 g, 20.2 mmol, 1.0 eq) was dissolved in 100 mL of dichloromethane, 2-hexyl decanoic acid (10.0 g, 40.4 mmol, 2.0 eq), EDCI (7.7 g, 40.4 mmol, 2.0 eq), DMAP (4.9 g, 40.4 mmol, 2.0 eq) were added, and the reaction was carried out at room temperature overnight. TLC monitoring (DCM:MeOH = 15:1, and PE:EA = 30:1) showed that the reaction was completed. After rotary evaporation to dryness, 20 mL of water and 70 EA were added and stirred, the organic phase was washed with 1M dilute hydrochloric acid, then with brine, dried, and the sample was purified by column chromatography (PE:EA = 50:1→30:1) to obtain 4.9 g of colorless oil B-3-d.

[0203] Step 5: Preparation of B-3-e

[0204] B-3-d (4.9 g, 6.8 mmol, 1.0 eq) was dissolved in dichloromethane (20 mL) under nitrogen protection, hydrogen chloride dioxane solution (4M, 9 mL, 33.8 mmol, 5.0 eq) was added, and the reaction was carried out at room temperature for 1 h. TLC monitoring (2% TEA, PE:EA = 10:1 and PE:EA = 30:1) showed that the reaction was completed. After washing with water, the sample was purified by column chromatography (2% TEA, PE:EA = 2:1) to obtain 1.85 g of oil B-3-e.

[0205] Step 6: Preparation of B-3

[0206] Compound B-3-e (0.9 g, 1.44 mmol, 1.0 eq) was dissolved in methanol (5 mL), aqueous formaldehyde (5 mL) was added, Pd / C (0.3 g, 10%) and the reaction was stirred at room temperature overnight, TLC monitored the reaction was completed (2% TEA, PE:EA = 10:1), filtered, washed with EA, rotary evaporated, and purified by silica gel column chromatography (2% TEA, PE:EA = 5:1→4:1), to give 80 mg of B-3 as an oil.

[0207] 1 H NMR (500 MHz, MeOD) δ 4.15 (t, J = 6.3 Hz, 4H), 2.57 (q, J = 7.2 Hz, 4H), 2.52 - 2.47 (m, 2H), 2.37 - 2.30 (m, 2H), 1.69 (q, J = 6.5 Hz, 4H), 1.65 - 1.41 (m, 12H), 1.34 - 1.21 (m, 44H), 1.06 (t, J = 7.2 Hz, 6H), 0.88 (t, J = 6.8 Hz, 12H).

[0208] Preparation of B-7 was according to the route of B-3 except using aqueous acetaldehyde instead of aqueous formaldehyde. LC-MS [M+H] + : 680.6. 1 H NMR (500 MHz, MeOD) δ 4.15 (t, J = 6.3 Hz, 4H), 2.57 (q, J = 7.2 Hz, 4H), 2.52 - 2.47 (m, 2H), 2.37 - 2.30 (m, 2H), 1.69 (q, J = 6.5 Hz, 4H), 1.65 - 1.41 (m, 12H), 1.34 - 1.21 (m, 44H), 1.06 (t, J = 7.2 Hz, 6H), 0.88 (t, J = 6.8 Hz, 12H).

[0209] Example 11 Preparation of B-62

[0210] Step 1: Preparation of B-62-b

[0211] B-3-c (5.0 g, 20.0 mmol) and 2-hexyldecanoic acid (6.66 g, 26.0 mmol) were dissolved in dichloromethane (100 mL), DMAP (242 mg, 2.0 mmol) and DIPEA (5.16 g, 40.0 mmol) were added, cooled to -15 °C, EDCI (7.68 g, 40.0 mmol) was added, and after the addition was complete, the reaction was slowly warmed to room temperature and stirred overnight. Spun down, water (50 mL) and ethyl acetate (50 mL) were added to the residue, and the mixture was partitioned. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, spun down, and the residue was purified by silica gel column chromatography to give 4.7 g of B-62-b.

[0212] Step 2: Preparation of B-62-c

[0213] B-62-b (1.1 g, 2.3 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and the reaction was stirred at room temperature for 3 hours. Spun down, an aqueous sodium bicarbonate solution (50 mL) and ethyl acetate (50 mL) were added to the residue, and the mixture was partitioned. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, spun down, and 0.9 g of B-62-c was obtained.

[0214] Step 3: Preparation of B-62-d

[0215] B-62-c (0.9 g, 2.3 mmol) was dissolved in THF (5 mL), an aqueous formaldehyde solution (5 mL) and 5% palladium on carbon (1.0 g) were added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. Filtered, the filtrate was spun down, and the resulting residue was purified by silica gel column chromatography to give 450 mg of B-62-d.

[0216] Step 4: Preparation of B-62

[0217] B-62-d (450 mg, 1.09 mmol) and oleic acid (461 mg, 1.64 mmol) were dissolved in dichloromethane (10 mL), DIPEA (211 mg, 1.64 mmol) and EDCI (313 mg, 1.64 mmol) were added, and the reaction was stirred at room temperature overnight after the addition was complete. Spun down, water (30 mL) was added to the residue, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, spun down, and the resulting residue was purified by silica gel column chromatography to give 511 mg of B-62. LC-MS [M+H] + : 678.6.

[0218] 1H NMR (500 MHz, MeOD) δ 5.37 - 5.29 (m, 2H), 4.16 - 4.12 (m, 4H), 2.38 - 2.29 (m, 5H), 2.26 (s, 6H), 2.03 - 1.99 (m, 4H), 1.71 - 1.51 (m, 11H), 1.45 (m, 2H), 1.36 - 1.24 (m, 40H), 0.88 (m, 9H).

[0219] Referring to the preparation route of B-62, replacing the materials or corresponding intermediates, the following compounds were prepared:

[0220] B-52: LC-MS [M+H] + : 568.5. 1 H NMR (500 MHz, MeOD) δ 4.17 - 4.12 (m, 4H), 2.37 - 2.29 (m, 5H), 2.26 (s, 6H), 1.71 - 1.51 (m, 11H), 1.49 - 1.41 (m, 2H), 1.34 - 1.24 (m, 34H), 0.88 (m, 9H).

[0221] B-53: LC-MS [M+H] + : 596.6. 1 H NMR (500 MHz, MeOD) δ 4.17 - 4.09 (m, 4H), 2.30 (m, 5H), 2.23 (s, 6H), 1.71 - 1.55 (m, 9H), 1.51 (m, 2H), 1.44 (m, 2H), 1.27 (s, 38H), 0.89 (m, 9H).

[0222] B-54: LC-MS [M+H] + : 596.6. 1 H NMR (500 MHz, Chloroform-d) δ 4.18 - 4.09 (m, 4H), 2.29 (m, 5H), 2.23 (s, 6H), 1.70 - 1.56 (m, 9H), 1.51 (m, 2H), 1.44 (m, 2H), 1.36 - 1.21 (m, 42H), 0.89 (m, 9H).

[0223] B-55: LC-MS [M+H] + : 652.6. 1H NMR (500 MHz, Chloroform-d) δ 4.16 - 4.07 (m, 4H), 2.35 - 2.26 (m, 5H), 2.23 (s, 6H), 1.63 (m, 9H), 1.51 (m, 2H), 1.44 (m, 2H), 1.35 - 1.21 (m, 39H), 0.89 (m, 9H).

[0224] B-56: LC-MS [M+H] + : 680.6. 1 H NMR (500 MHz, MeOD) δ 4.14 (m, 4H), 2.37 - 2.29 (m, 5H), 2.26 (s, 6H), 1.71 - 1.66 (m, 4H), 1.65 - 1.51 (m, 7H), 1.49 - 1.41 (m, 3H), 1.33 - 1.23 (m, 52H), 0.88 (m, 9H).

[0225] B-111: LC-MS [M+H] + : 708.7. 1 H NMR (500 MHz, Chloroform-d) δ 4.12 (t, J = 6.3 Hz, 4H), 2.37 - 2.26 (m, 5H), 2.23 (s, 6H), 1.71 - 1.55 (m, 9H), 1.51 (m, 2H), 1.43 (m 2H), 1.38 (s, 1H), 1.27 (s, 55H), 0.89 (m, 9H).

[0226] B-114: LC-MS [M+H] + : 720.7. 1 H NMR (500 MHz, CDC13) δ 5.36 - 5.26 (m, 2H), 4.13 - 4.01 (m, 6H), 2.35 - 2.27 (m, 4H), 2.23 (s, 6H), 2.04 - 1.95 (m, 7H), 1.69 - 1.52 (m, 7H), 1.51 - 1.38 (m, 3H), 1.35 - 1.18 (m, 44H), 0.89 (t, J = 6.9 Hz, 9H).

[0227] B-180: LC-MS [M+H] + : 676.6. 1H NMR (500 MHz, MeOD) δ 5.40 - 5.28 (m, 4H), 4.17 - 4.12 (m, 4H), 2.78 (t, J = 6.6 Hz, 2H), 2.38 - 2.30 (m, 5H), 2.27 (s, 6H), 2.09 - 2.03 (m, 4H), 1.72 - 1.51 (m, 11H), 1.51 - 1.42 (m, 2H), 1.40 - 1.24 (m, 36H), 0.92 - 0.86 (m, 9H).

[0228] B-451: LC-MS [M+H] + : 710.4. 1 H NMR (500 MHz, MeOD) δ 4.07 (m, 6H), 2.29 (m, 7H), 2.24 (s, 6H), 1.63 (m, 12H), 1.45 (m, 5H), 1.37 - 1.25 (m, 38H), 0.93 - 0.86 (m, 9H).

[0229] Example 22 Preparation of B-63

[0230] Step 1: Preparation of B-63-g

[0231] B-63-f (2.1 g, 7.3 mmol, 1.0 eq) was dissolved in DMF (25 mL) and stirred, 2-hexyldecanoic acid (2.5 g, 10.1 mmol, 1.4 eq), DIEA (1.87 g, 14.5 mmol, 2.0 eq), DMAP (0.2 g, 1.45 mmol, 0.2 eq) were added, and the temperature was lowered to -15 °C under nitrogen protection, EDCI (2.8 g, 14.5 mmol, 2.0 eq) was added dropwise for 6 h, and the temperature was slowly raised to room temperature and reacted overnight, TLC monitoring (PE:EA = 30:1 / 7:1) showed that the reaction was complete, EA was added, diluted hydrochloric acid was washed, water was washed, saturated sodium chloride solution was washed, dried and rotary evaporated, silica gel was added to the column and purified (PE:EA = 20:1→15:1), and 2.6 g of oily B-63-g was obtained.

[0232] Step 2: Preparation of compound B-63-h

[0233] B-63-g (2.2 g, 1.0 eq) was dissolved in DCM (20 mL) under nitrogen protection, cooled to 0 °C, then TFA (8 mL) was added dropwise, reacted at room temperature for 2 h, TLC monitoring showed that the reaction was complete (PE:EA = 20:1, 2% TEA, PE:EA = 20:1), rotary evaporation, DCM was dragged for 3 times, sodium bicarbonate solution was added to adjust pH = 8-9, EA was added, the phases were separated, dried and rotary evaporated, THF was dragged once to obtain 5.0 g of crude B-63-h.

[0234] Step 3: Preparation of B-63-i

[0235] B-63-h (1.2 g, 1.0 eq) and formaldehyde aqueous solution (5 mL) were dissolved in THF (8 mL), Raney nickel (1.0 g) was added, hydrogen was replaced for three times, and the reaction was carried out at room temperature overnight. TLC monitoring (2% TEA, PE:EA=12:1) showed that the reaction was completed. Filtration, EA washing, and rotary evaporation were carried out. The residue was separated by silica gel column chromatography (2% TEA, PE:EA=20:1 / 15:1 elution), and 1.2 g of oil B-63-i was obtained.

[0236] Step 4: Preparation of B-63

[0237] B-63-i (1.2 g of crude product, 1.1 mmol, 1.0 eq) was dissolved in DMF (20 mL), and oleic acid (0.3 g, 1.1 mmol, 1.1 eq), DIEA (0.2 g, 1.3 mmol, 1.2 eq), DMAP (16 mg, 10.13 mmol, 0.1 eq), EDCI (0.5 g, 2.6 mmol, 2.4 eq) were added under nitrogen protection. The reaction was carried out overnight. TLC monitoring (PE:EA=40:1 / 7:1 / 1:1) showed that the reaction was completed. Water (40 ml) was added and stirred. EA extraction was carried out three times (20 mL*3). The combined organic phase was washed with water and saturated sodium chloride solution, dried, and separated by silica gel column chromatography (PE:EA=4:1 and 2% TAE / 30:1 / 20:1), and 140 mg of oil B-63 was obtained.

[0238] 1 H NMR (500 MHz, MeOD) δ 5.38-5.28 (m, 2H), 4.15-4.04 (m, 6H), 2.37-2.29 (m, 4H), 2.25 (s, 6H), 2.05-1.96 (m, 7H), 1.70-1.54 (m, 7H), 1.53-1.40 (m, 3H), 1.37-1.20 (m, 40H), 0.88 (t, J=6.9 Hz, 9H).

[0239] The preparation method of B-58 is the same as that of B-63, except that stearic acid is used instead of oleic acid.

[0240] LC-MS [M+H] + : 694.4. 1H NMR (500 MHz, MeOD) δ 4.16-4.02 (m, 6H), 2.41-2.32 (m, 4H), 2.24 (s, 6H), 2.08-1.98 (m, 7H), 1.73-1.56 (m, 7H), 1.55-1.42 (m, 3H), 1.39 - 1.22 (m, 42H),, 0.89 (m, 9H).

[0241] Example 24 Preparation of B-211

[0242] Step 1: Preparation of B-211-b

[0243] Into a hydrogenation flask, B-211-a (6.5 g, 25.5 mmol), EtOH (65 mL), Boc20 (11.13 g, 51 mmol), TEA (6.45 g, 63.7 mmol) and Raney nickel (6.5 g) were added successively, and the reaction was pressurized to 1.2 MPa under hydrogen atmosphere and reacted for 48 hours. Filtration was performed using celite, and the filtrate was rotary evaporated to dryness, and column chromatography purification was performed to obtain 5.23 g of colorless liquid, which was B-211-b, yield: 57%.

[0244] Step 2: Preparation of B-211-c

[0245] B-211-b (1.0 g, 2.79 mmol) was added to THF (10 mL), and water (3 mL) and lithium hydroxide monohydrate (292 mg, 6.96 mmol) were added. Stirring was performed at room temperature for two days. Most of the THF was removed by concentration, extraction was performed using ethyl acetate, the aqueous phase was taken, 1 N HC1 was added to about pH 3, extraction was performed twice using ethyl acetate, the organic layers were combined, dried using anhydrous sodium sulfate, and rotary evaporation was performed to dryness to obtain 0.79 g of colorless liquid, which was B-211-c, yield: 94%.

[0246] Step 3: Preparation of B-211-d

[0247] To a solution of B-211-c (0.87 g, 2.77 mmol) in dichloromethane (20 mL) were added 9-heptadecanol (1.77 g, 6.93 mmol), DMAP (0.85 g, 6.93 mmol) and EDCI (1.17 g, 6.09 mmol) successively, and stirring was performed at room temperature overnight under nitrogen protection. The reaction solution was washed successively using 0.5 M HC1 and saturated brine once, the organic phase was dried using anhydrous magnesium sulfate again, concentrated, and column chromatography purification was performed on the residue to obtain 870 mg of oil, which was B-211-d, yield: 40%.

[0248] Step 4: Preparation of B-211

[0249] To a solution of B-211-d (1.43 g, 1.83 mmol) in dichloromethane (15 mL) was added TFA (1.5 mL) and stirred at room temperature overnight. Saturated sodium bicarbonate (15 mL) was added and stirred well, the aqueous phase was extracted with dichloromethane once more, the combined organic phase was dried over anhydrous sodium sulfate and concentrated to give 1.09 g of yellow oil. The oil was dissolved in THF (5 mL), Raney nickel (1.65 g) and formaldehyde aqueous solution (5 mL) were added and the reaction was stirred at room temperature overnight under hydrogen atmosphere. The mixture was filtered, concentrated to remove most of the THF, water was added and the mixture was extracted with ethyl acetate three times, the combined organic phase was dried over anhydrous sodium sulfate, concentrated and the residue was purified by column chromatography to give 167 mg of yellow liquid, which was B-211. Yield: 32%. LC-MS [M+H] + : 708.7.

[0250] 1 H NMR (500 MHz, Methanol-d4) δ 4.86 (m, 2H), 3.33 (m, 2H), 2.34 (m, 6H), 2.26 (s, 6H), 1.65 - 1.59 (m, 4H), 1.52 (m, 8H), 1.49 - 1.44 (m, 2H), 1.43 - 1.38 (m, 1H), 1.27 (m, 52H), 0.87 (m, 12H).

[0251] B-200 was prepared according to the procedure for B-211 except using 3-(cyanomethylidene)pentanedioic acid diethyl ester instead of B-211-a.

[0252] LC-MS [M+H] + : 680.6. 1 H NMR (500 MHz, Methanol-d4) δ 4.89 (p, J = 6.2 Hz, 2H), 3.10 - 3.03 (m, 2H), 2.78 (s, 6H), 2.43 (d, J = 6.2 Hz, 4H), 2.35 (p, J = 6.4 Hz, 1H), 1.84 - 1.77 (m, 2H), 1.55 (d, J = 6.7 Hz, 8H), 1.28 (d, J = 6.5 Hz, 53H), 0.88 (t, J = 6.8 Hz, 12H).

[0253] Example 26 Preparation of B-202

[0254] Step 1: Preparation of B-202-b

[0255] To a solution of B-202-a (4.00 g, 12.07 mmol) in THF (20 mL) was added hydrogen chloride / dioxane solution (20 mL) at room temperature and the reaction was stirred for 3 hours. The reaction was concentrated under reduced pressure, methanol (40 mL), formaldehyde (10 mL) and Raney nickel (4.00 g) were added and the reaction was stirred under hydrogen atmosphere overnight. The reaction was filtered, the filtrate was concentrated under reduced pressure, water (40 mL) was added, the pH was adjusted to weak alkaline with aqueous sodium bicarbonate solution and extracted with dichloromethane / methanol (8 / 1, 40 mL). The organic phase was concentrated under reduced pressure to give B-202-b as a yellowish oil 2.40 g, yield: 76.7%.

[0256] Step 2: Preparation of B-202-c

[0257] To a solution of B-202-b (2.30 g, 8.87 mmol) in THF (11 mL) was added 10% aqueous sodium hydroxide solution (11 mL) at room temperature and the reaction was stirred for 3 hours at 60 °C. The reaction was cooled to room temperature, washed with ethyl acetate (10 mL), the aqueous phase was collected and the pH was adjusted to 2 with concentrated hydrochloric acid. The reaction was concentrated under reduced pressure. DMF (10 mL) was added, the reaction was filtered and the filtrate was concentrated under reduced pressure to give B-202-c as a yellowish oil 1.80 g.

[0258] Step 3: Preparation of B-202-d

[0259] To a solution of B-202-c (1.80 g, 8.85 mmol), octadecanol (2.39 g, 8.85 mmol), DIPEA (3 mL, 17.70 mmol), DMAP (108 mg, 0.885 mmol) in dry DMF (30 mL) was added EDCI (2.54 g, 13.28 mmol) at room temperature and the reaction was stirred under argon overnight. Water (120 mL) was added, the pH was adjusted to 3 with 1 M aqueous hydrochloric acid solution and extracted with dichloromethane (50 mL). The organic phase was collected, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and the residue was purified by column chromatography on silica gel to give B-202-d as a yellowish solid 592 mg, yield: 15%.

[0260] Step 4: Preparation of B-202

[0261] EDCI (252 mg, 1.317 mmol) was added to a solution of B-202-d (300 mg, 0.658 mmol), 9-heptadecanol (219 mg, 0.856 mmol), DMAP (161 mg, 1.317 mmol) in dry DMF (4.5 mL) at room temperature and the reaction was stirred at 60 °C under argon overnight. Water (30 mL) was added and the reaction was extracted with ethyl acetate (30 mL), washed with saturated aqueous sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give B-202 as a colorless oil 336 mg, yield: 74%. LC-MS [M+H] + : 694.3.

[0262] 1 H NMR (500 MHz, Chloroform-d) δ 4.86 (m 1H), 4.05 (t, J = 6.8 Hz, 2H), 2.38 (m, 5H), 2.29 (t, J = 7.6 Hz, 2H), 2.19 (s, 6H), 1.61 (m, 2H), 1.57 - 1.45 (m, 6H), 1.25 (m, 57H), 0.87 (m, 9H).

[0263] Example 27 Preparation of 213

[0264] Step 1: Preparation of B-213-a

[0265] To a solution of B-211-c (1.5 g, 4.95 mmol) in THF (15 mL) was added stearyl alcohol (0.8 g, 2.97 mmol), DMAP (0.5 g, 4.0 mmol) and EDCI (1.2 g, 6.1 mmol) and the reaction was stirred at room temperature overnight. The reaction was concentrated under reduced pressure and the residue was purified by column chromatography to give 0.9 g of B-213-a as an oil.

[0266] Step 2: Preparation of B-213-b

[0267] To a solution of B-213-b (0.9 g, 1.62 mmol) in THF (15 mL) was added 9- heptadecanol (0.62 g, 2.43 mmol), DMAP (0.59 g, 4.86 mmol) and EDCI (0.47 g, 2.43 mmol) and the reaction was stirred at room temperature overnight. The reaction was poured into water and extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography to give 0.9 g of B-213-b as an oil.

[0268] Step 3: Preparation of B-213-c

[0269] B-213-b (0.90 g, 1.2 mmol) and 4N HC1-dioxane solution (3 mL) were added into a reaction flask at room temperature and stirred for 1 hour at room temperature. LC monitoring showed the reaction was complete, the solvent was removed under reduced pressure, the residue was dissolved in THF and concentrated, and the procedure was repeated twice. The residue was dissolved in THF (10 mL), formaldehyde solution (3 mL) and Raney nickel (2.0 g) were added, the system was replaced with hydrogen, and stirred overnight at room temperature. TLC monitoring showed the reaction was complete, and the mixture was filtered through celite. The filtrate was concentrated, diluted with water, and extracted with ethyl acetate. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 510 mg of B-213-c as an oil. LC-MS [M+H] + : 722.6.

[0270] 1 H NMR (500 MHz, CDC13) δ 4.87 (m, 1H), 4.07 (t, J = 6.7 Hz, 2H), 2.31 (m, 6H), 2.24 (s, 6H), 1.74 (m, 3H), 1.63 (m, 6H), 1.52 (m, 4H), 1.48 - 1.42 (m, 3H), 1.29 (m, 51H), 0.90 (m, 9H).

[0271] B-214 was prepared according to the procedure for B-213, using oleyl alcohol instead of stearyl alcohol. LC-MS [M+H] + : 720.6. 1 H NMR (500 MHz, CDC13) δ 5.36 - 5.31 (m, 2H), 4.88 (m, 1H), 4.05 (m, 2H), 2.33 (m, 6H), 2.23 (s, 6H), 1.72 (m, 3H), 1.64 (m, 6H), 1.53 (m, 4H), 1.47 - 1.41 (m, 3H), 1.29 (m, 47H), 0.89 (m, 9H).

[0272] Example 29 Preparation of B-452

[0273] Step 1: Preparation of B-452-a

[0274] To a reaction vial was added B-260-c (1.4 g, 2.52 mmol), HC1 / 1,4-dioxane (14 mL), stirred for 10 min at room temperature, concentrated, added THF (10 mL), stirred to dissolve, then added formaldehyde (5 mL) and 10% Pd / C (170 mg), replaced with hydrogen gas, stirred under hydrogen atmosphere overnight. Upon completion, filtered, added sodium bicarbonate to the filtrate, extracted with ethyl acetate twice, collected the organic phase, dried over anhydrous sodium sulfate, concentrated, purified by column chromatography to give 810 mg of B-452-a as an oil.

[0275] Step 2: Preparation of B-452-b

[0276] To a solution of B-452-a (810 mg, 1.68 mmol) in ethanol (10 mL) was added potassium hydroxide (131 mg, 3.35 mmol) in water, stirred at room temperature for 6 h, adjusted the pH to about 7-8 with dilute hydrochloric acid, concentrated the ethanol, added water, extracted with dichloromethane, combined the organic phases, dried over anhydrous sodium sulfate, concentrated, purified by column chromatography to give 385 mg of B-452-b as an oil.

[0277] Step 3: Preparation of B-452

[0278] To a reaction vial was added B-452-b (385 mg, 0.84 mmol), dichloromethane (4 mL), stearyl alcohol (272 mg, 1.0 mmol), DMAP (309 mg, 2.53 mmol), and EDCI (243 mg, 1.27 mmol), stirred at 30 °C overnight. Concentrated, added water, extracted with ethyl acetate, collected the organic phase, dried over anhydrous sodium sulfate, concentrated, purified by column chromatography to give 350 mg of B-452 as a colorless oil. LC-MS [M+H]: 708.6. + : 708.6.

[0279] 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (m, 1H), 4.07 (m, 2H), 2.31 (m, 4H), 2.23 (s, 6H), 2.05 (m, 4H), 1.63 (m, 6H), 1.50 (m, 4H), 1.49 (m, 3H), 1.37 - 1.20 (m, 50H), 0.90 (m, 9H).

[0280] Example 30 Preparation of B-239

[0281] Step 1: Preparation of B-239-a

[0282] Into a single-neck flask was placed cyanomethylphosphonic acid diethyl ester (9.5 g, 53.5 mmol), anhydrous THF (100 mL), and NaH (1.61 g, 40.3 mmol) was added in portions under nitrogen protection with ice-water bath cooling. After addition, the mixture was allowed to warm to room temperature and stirred for 30 minutes. Then a solution of 1,5-dibenzyloxy-3-pentanone (10 g, 33.6 mmol) in THF (20 mL) was added and the mixture was allowed to warm to 55 °C and stirred for 60 minutes. TLC tracking showed that the starting material was consumed. Water (50 mL) was added and the mixture was extracted with ethyl acetate twice. The combined organic phase was washed with saturated sodium chloride solution once, dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography gave B-239-a (9.95 g, 92% yield).

[0283] Step 2: Preparation of B-239-b

[0284] Into a hydrogenation flask was placed B-239-a (9.95 g, 31 mmol), ethanol (80 mL), Boc20 (10.1 g, 46.5 mmol), triethylamine (6.29 g, 62 mmol), and Raney nickel (10 g, 100% wt). The mixture was pressurized to 1.2 MPa with hydrogen atmosphere and stirred at room temperature for 2 hours. The mixture was filtered and concentrated. Purification by column chromatography gave B-239-a (12.1 g, 91% yield).

[0285] Step 3: Preparation of B-239-c

[0286] Into a hydrogenation flask was placed B-239-b (12.1 g, 28.3 mmol), THF (160 mL), and 10% Pd / C (1.21 g). The mixture was stirred at room temperature overnight under hydrogen atmosphere. TLC tracking showed that the starting material was consumed completely and a small amount of intermediate remained. The mixture was filtered and concentrated. Purification by column chromatography gave B-239-c (6.35 g, 91% yield).

[0287] Step 4: Preparation of B-239-d

[0288] Into a reaction flask was placed B-239-c (6.9 g, 27.9 mmol), DCM (140 mL), stearic acid (9.54 g, 33.5 mmol), and DMAP (5.11 g, 41.9 mmol). The mixture was cooled with ice-water bath and stirred at room temperature overnight after addition of EDCI (6.96 g, 36.3 mmol). TLC tracking showed that a small amount of starting material remained and the product was obvious. The mixture was stirred with 1 N dilute hydrochloric acid (50 mL) and the organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give B-239-d (4.8 g, 34% yield) as an oil.

[0289] Step 5: Preparation of B-239-e

[0290] To a solution of B-239-d (4.7 g, 9.15 mmol) in MeCN (25 mL) and carbon tetrachloride (50 mL) was added ruthenium trichloride (94 mg, 2% wt) and sodium periodate (5.93 g, 27.5 mmol) dissolved in water (50 mL) was added to the system. The system was stirred at room temperature for 2 hours under nitrogen protection. The reaction mixture was allowed to stand and was separated. The organic layer was dried over anhydrous sodium sulfate and was concentrated by rotary evaporation. The crude product B-239-e was purified by column chromatography. Yield: 840 mg, 17%.

[0291] Step 6: Preparation of B-239-f

[0292] To a reaction flask was added B-239-e (840 mg, 1.59 mmol) dissolved in DCM (10 mL). 9-Heptadecanol (613 mg, 2.39 mmol), DMAP (292 mg, 2.39 mmol) and EDCI (458 mg, 2.39 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was mixed with an appropriate amount of petroleum ether and was purified by column chromatography to obtain 327 mg of oil B-239-f. Yield: 27%.

[0293] Step 7: Preparation of B-239-g

[0294] To a reaction flask was added B-239-f (327 mg, 0.43 mmol) and 4N HCl / 1,4-dioxane (3 mL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated by rotary evaporation under reduced pressure to obtain the crude product B-239-g which was used directly in the next step.

[0295] Step 8: Preparation of B-239

[0296] The crude product B-239-g obtained in the previous step was dissolved in THF (3 mL). 30% Formaldehyde (1 mL) and Pd / C (30 mg, 10% wt) were added in sequence. The reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. The reaction mixture was filtered, concentrated, dissolved in DCM, washed with saturated sodium bicarbonate and saturated sodium chloride in sequence, dried over anhydrous sodium sulfate, concentrated by rotary evaporation and purified by column chromatography to obtain 217 mg of yellowish oil B-239. Yield: 72%. LC-MS [M+H]: 694.7. +

[0297] 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (t, J = 6.4 Hz, 1H), 4.18 - 4.09 (m, 2H), 2.37 - 2.25 (m, 6H), 2.23 (s, 6H), 2.04 (m, 1H), 1.83 (m, 2H), 1.70 (m, 2H), 1.63 (m, 2H), 1.53 (m, 6H), 1.27 (m, 50H), 0.90 (m, 9H).​

[0298] Example 31 Preparation of B-461

[0299] Step 1: Preparation of B-461-a

[0300] Into a reaction flask was placed Tosmic (384 mg, 1.97 mmol) and ethylene glycol dimethyl ether (8 mL), and the flask was protected with nitrogen. The reaction mixture was cooled to 0 °C, and 1 N potassium tert-butoxide in THF (4.1 mL, 4.1 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred for 10 minutes, and intermediate B-276-b (800 mg, 1.64 mmol) was added. The reaction mixture was stirred for 30 minutes in an ice bath, and then the ice bath was removed. The reaction mixture was stirred for 20 minutes at room temperature, and then the reaction was complete. The reaction mixture was quenched with ice water, and the pH was adjusted to neutral with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The mixture was concentrated, and the residue was purified by column chromatography to give 498 mg of B-461-a as an oil.

[0301] Step 2: Preparation of B-461-b

[0302] Into an autoclave was placed B-461-a (498 mg, 1.0 mmol), methanol (5 mL), triethylamine (404 mg, 4.0 mmol), 10% Pd / C, and di-tert-butyl dicarbonate (436 mg, 2.0 mmol). The reaction mixture was hydrogenated at high pressure for 2 hours. After the reaction was complete, the mixture was filtered, concentrated, and purified by column chromatography to give 344 mg of B-461-b as an oil.

[0303] Step 3: Preparation of B-461-c

[0304] Into a reaction flask was placed B-461-b (344 mg, 0.57 mmol) and HCl / 1,4-dioxane (3 mL), and the mixture was stirred at room temperature for 10 minutes. The mixture was concentrated, and THF (3 mL), aqueous formaldehyde (1 mL), and 10% Pd / C (30 mg) were added. The reaction mixture was hydrogenated for 1 hour. After the reaction was complete, the mixture was filtered, and water was added. The mixture was extracted with ethyl acetate, and the organic layers were combined and concentrated. Methanol (3 mL), 1 drop of hydrochloric acid, and 10% Pd / C (30 mg) were added, and the mixture was hydrogenated. After the reaction was complete, the mixture was filtered, and the filtrate was poured into water. The mixture was extracted twice with dichloromethane, and the organic layers were combined and washed with saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated to give 180 mg of B-461-c as an oil.

[0305] Step 4: Preparation of B-461

[0306] To a reaction vial was added B-461-c (180 mg, 0.41 mmol), dichloromethane (2 mL), oleic acid (115 mg, 0.41 mmol), DMAP (150 mg, 1.22 mmol), and EDCI (117 mg, 0.61 mmol) sequentially, and warmed to 40 °C overnight. Spun down, added water, extracted with ethyl acetate, washed the organic layer with saturated brine, dried over sodium sulfate, spun down, and purified by column chromatography to give 216 mg of B-461 as a light yellow oil. LC-MS [M+H] + : 706.5.

[0307] 1 H NMR (500 MHz, Chloroform-d) δ 5.38 - 5.35 (m, 2H), 4.89 (m, 1H), 4.07 (t, J = 6.8 Hz, 2H), 2.35 - 2.29 (m, 4H), 2.21 (s, 6H), 2.13 (m, 2H), 2.03 (m, 4H), 1.68 - 1.58 (m, 9H), 1.53 (m, 4H), 1.36 - 1.28 (m, 44H), 0.90 (t, J = 6.9 Hz, 9H).

[0308] Example 32 Preparation of B-276

[0309] Step 1: Preparation of B-276-a

[0310] To a 1 L three-necked flask was added succinic anhydride (58.6 g, 586.0 mmol), MeCN (300 mL), DMAP (4.76 g, 39.1 mmol) and 9-heptadecanol (100 g, 390.6 mmol) sequentially under nitrogen atmosphere. After completion, the reaction was stirred at 80 °C. TLC detection showed that the reaction was complete. The reaction was cooled to 40 ± 5 °C, water (100 mL) was added dropwise, and the reaction was cooled to 0 ± 5 °C. The stirring was continued for 1 h. The filter cake was washed with acetonitrile / water (150 mL, v / v = 3:1), and the filter cake was transferred to a vacuum drying oven for drying to give B-276-a, 131.1 g, in 94% yield.

[0311] Step 2: Preparation of B-276-b

[0312] Into a 2L 3-necked flask, was placed Mg (37.6g, 1.57mmol) and THF (720mL) under nitrogen atmosphere, then iodine (100mg) was added, stirred at room temperature, then a portion of 3-benzyloxybromopropane (163.0g, 712mmol) was added dropwise, heated to 45°C, after initiation, the rest of 3-benzyloxybromopropane was added dropwise; after the addition was completed, the reaction flask was placed in an 80°C water bath and stirred for 1 hour, after completion, cooled to room temperature for use. Into a 3L 3-necked flask, was placed B-276-a (123.4g, 346.6mmol), DMF (0.1mL, 1.3mmol) and dichloromethane (615mL) under nitrogen atmosphere, stirred in an ice water bath, (COCl)2(57.2g, 450.5mmol) was added dropwise into the above system, after completion, transferred to room temperature and stirred for 2 hours; the solvent was concentrated, THF (100mL) was added to the residue, then concentrated again under reduced pressure; after completion, THF (375mL) was added, cuprous iodide (135.6g, 693.2mmol) was added, stirred in -10 to -15°C; then the previously prepared format reagent was slowly added dropwise into the above system until the system turned black, acetic acid (25g, 416.4mmol) was added, then water (745g) was added, warmed to room temperature, filtered, the filter cake was washed with ethyl acetate (700mL), the filtrate was separated, the aqueous phase was extracted with ethyl acetate (500mL), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain B-276-b, 130.4g, yield 78%.

[0313] Step 3: Preparation of B-276-c

[0314] Into a 2L 3-necked flask, was placed Mg (37.6g, 1.57mmol) and THF (720mL) under nitrogen atmosphere, then iodine (100mg) was added, stirred at room temperature, then a portion of 3-benzyloxybromopropane (163.0g, 712mmol) was added dropwise, heated to 45°C, after initiation, the rest of 3-benzyloxybromopropane was added dropwise; after the addition was completed, the reaction flask was placed in an 80°C water bath and stirred for 1 hour, after completion, cooled to room temperature for use. Into a 3L 3-necked flask, was placed B-276-a (123.4g, 346.6mmol), DMF (0.1mL, 1.3mmol) and dichloromethane (615mL) under nitrogen atmosphere, stirred in an ice water bath, (COCl)2(57.2g, 450.5mmol) was added dropwise into the above system, after completion, transferred to room temperature and stirred for 2 hours; the solvent was concentrated, THF (100mL) was added to the residue, then concentrated again under reduced pressure; after completion, THF (375mL) was added, cuprous iodide (135.6g, 693.2mmol) was added, stirred in -10 to -15°C; then the previously prepared format reagent was slowly added dropwise into the above system until the system turned black, acetic acid (25g, 416.4mmol) was added, then water (745g) was added, warmed to room temperature, filtered, the filter cake was washed with ethyl acetate (700mL), the filtrate was separated, the aqueous phase was extracted with ethyl acetate (500mL), the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain B-276-b, 130.4g, yield 78%.

[0315] Step 4: Preparation of B-276-d

[0316] Into a slant four-port round-bottom flask, B-176-c (86.0 g, 154.1 mmol) and 1,4-dioxane (860 mL) were added successively under nitrogen atmosphere, and then dry palladium carbon (9.1 g) was added. After the reaction system was replaced with hydrogen atmosphere, it was stirred at room temperature for 2 hours. After TLC detection showed that the starting material was completely converted, the system was cooled to 15-20 °C, 1,1,3,3-tetramethyldisiloxane (166 g, 1.23 mol) was added dropwise, and after the dropwise addition was completed, the temperature was increased to 40-45 °C, and the reaction was stirred for 2 days, and then decreased to room temperature. Ethanol (518 mL) was added, and the reaction was stirred for 2 hours. Then, an ethanolic hydrochloric acid solution (103 mL, v / v = 1:3) was added dropwise, and the reaction was stirred for 0.5 hours. After completion, dry palladium carbon (4.3 g) was added, the reaction system was replaced with hydrogen, and the reaction was continued for 4 hours. Saturated sodium bicarbonate solution was added dropwise until the pH value was 7-8, and then it was filtered through diatomite. The filter residue was rinsed with dichloromethane (800 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane (500 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography was performed to purify the product, and B-276-d was obtained in a yield of 49.2 g, 70%.

[0317] Step 5: Preparation of B-276

[0318] Into a slant four-port round-bottom flask, oleic acid (43.9 g, 155.4 mmol) and dichloromethane (100 mL) were added under nitrogen atmosphere, and then oxalyl chloride (30.0 g, 236.4 mmol) was added dropwise. After the dropwise addition was completed, the reaction was continued to stir for 1 hour, and then concentrated under reduced pressure. Dichloromethane (175 mL) was added again, and the system was cooled to -5±5 °C. Into a round-bottom flask, B-276-d (47.2 g, 103.5 mmol), pyridine (16.5 g, 208.9 mmol), and dichloromethane (350 mL) were added successively under nitrogen atmosphere. After stirring uniformly, the above system was added dropwise. After HPLC detection showed that the starting material was completely converted, it was concentrated under reduced pressure until no distillate was obtained. Ethyl acetate / water (294 mL, v / v = 2:1) was added, and the pH value was adjusted to weakly acidic with 1M hydrochloric acid solution. The mixture was separated, and the aqueous phase was extracted with ethyl acetate (200 mL). The organic phases were combined, washed successively with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain B-276 in a yield of 72.0 g, 97%.

[0319] LC-MS [M+H] + : 720.5. 1H NMR δ 5.40 - 5.32 (m, 2H), 4.88 (p, J = 6.3 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 2.33 - 2.25 (m, 6H), 2.23 (s, 6H), 2.08 - 1.99 (m, 4H), 1.68 - 1.60 (m, 6H), 1.57 - 1.49 (m, 4H), 1.48 - 1.41 (m, 3H), 1.40 - 1.21 (br, 46H), 0.90 (t, J = 6.3 Hz, 9H).

[0320] Synthesis of B-436 was the same as B-276 except 1-decanoic acid was used instead of oleic acid in step 5. LC-MS [M+H] + : 610.5. 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 2.33 - 2.26 (m, 6H), 2.24 (s, 6H), 1.66 - 1.64 (m, 6H), 1.52 (m, 4H), 1.46 (m, 3H), 1.37 - 1.22 (m, 38H), 0.90 (t, J = 6.9 Hz, 9H).

[0321] Synthesis of B-438 was the same as B-276 except 1-tetradecanoic acid was used instead of oleic acid in step 5. LC-MS [M+H] + : 667.2. 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (t, J = 6.2 Hz, 1H), 4.06 (t, J = 6.7 Hz, 2H), 2.33 - 2.25 (m, 6H), 2.23 (s, 6H), 1.67 - 1.60 (m, 6H), 1.52 (m, 4H), 1.48 - 1.43 (m, 3H), 1.37 - 1.21 (m, 46H), 0.90 (t, J = 6.9 Hz, 9H).

[0322] Synthesis of B-277 was the same as B-276 except linoleic acid was used instead of oleic acid in step 5. LC-MS [M+H] + : 718.6. 1H NMR (500 MHz, MeOD) δ 5.40 - 5.28 (m, 4H), 4.87 (p, J = 6.2 Hz, 1H), 4.06 (t, J = 6.5 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.36 - 2.29 (m, 6H), 2.25 (s, 6H), 2.08 - 2.01 (m, 4H), 1.68 - 1.59 (m, 6H), 1.56 - 1.40 (m, 7H), 1.38 - 1.22 (m, 44H), 0.88 (t, J = 6.6 Hz, 9H).

[0323] B-441 was prepared in the same manner as B-276 except using 4-benzyloxybromobutane instead of 3-benzyloxybromopropane in step 1. LC-MS [M+H] + : 734.8. 1 H NMR (500 MHz, Chloroform-d) δ 5.38 - 5.33 (m, 2H), 4.88 (m, 1H), 4.07 (t, J = 6.7 Hz, 2H), 2.32 - 2.24 (m, 6H), 2.23 (s, 6H), 2.03 (m, 4H), 1.63 (m, 6H), 1.52 (m, 4H), 1.46 - 1.41 (m, 3H), 1.39 - 1.21 (m, 48H), 0.90 (m, 9H).

[0324] B-442 was prepared in the same manner as B-276 except using glutaric anhydride instead of succinic anhydride in step 1. LC-MS [M+H] + : 734.7. 1 H NMR (500 MHz, Chloroform-d) δ 5.37 - 5.35 (m, 2H), 4.88 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 2.32 - 2.24 (m, 6H), 2.23 (s, 6H), 2.03 (m, 4H), 1.63 (m, 6H), 1.52 (m, 4H), 1.46 - 1.42 (m, 3H), 1.36 - 1.22 (m, 48H), 0.89 (m, 9H).

[0325] B-275 was prepared in the same manner as B-276 except using stearic acid instead of oleic acid in step 5. LC-MS [M+H] + : 722.6. 1H NMR (500 MHz, MeOD) δ 4.90 - 4.83 (m, 1H), 4.06 (t, J = 6.5 Hz, 2H), 2.35 - 2.29 (m, 6H), 2.25 (s, 6H), 1.67 - 1.58 (m, 6H), 1.56 - 1.34 (m, 8H), 1.33 - 1.20 (m, 58H), 0.88 (t, J = 6.9 Hz, 9H).

[0326] The synthesis of B-286 was the same as B-275 except pentanedioic anhydride was used instead of succinic anhydride and 4-benzyloxy-1-bromobutane was used instead of 3-benzyloxy-1-bromopropane. LC-MS [M+H] + : 750.2. 1 H NMR (500 MHz, Chloroform-d) δ 4.89 (p, J = 6.3 Hz, 1H), 4.07 (t, J = 6.7 Hz, 2H), 2.33 - 2.24 (m, 6H), 2.23 (s, 6H), 1.63 (dt, J = 16.0, 5.9 Hz, 6H), 1.57 - 1.48 (m, 4H), 1.28 (d, J = 8.0 Hz, 61H), 0.90 (t, J = 6.8 Hz, 9H).

[0327] The synthesis of B-456 was the same as B-286 except stearyl alcohol was used instead of 9-heptadecanol and 2-octyldecanoic acid was used instead of stearic acid. LC-MS [M+H] + : 764.6. 1 H NMR (500 MHz, CDCl3) δ 4.08 (m, 4H), 2.29 (m, 4H), 2.23 (s, 6H), 1.62 (m, 8H), 1.44 (m, 4H), 1.28 (m, 63H), 0.94 - 0.86 (m, 9H).

[0328] The synthesis of B-266 was the same as B-276 except 2-hexyldecanoic acid was used instead of oleic acid in step 5. LC-MS [M+H] + : 694.7. 1 H NMR (500 MHz, MeOD) δ 4.91 - 4.84 (m, 1H), 4.08 (t, J = 5.3 Hz, 2H), 2.37 - 2.31 (m, 5H), 2.26 (s, 6H), 1.70 - 1.35 (m, 17H), 1.33 - 1.26 (m, 42H), 0.89 (t, J = 6.9 Hz, 12H).

[0329] The synthesis of B-273 was the same as B-266 except oleyl alcohol was used instead of 9-heptadecanol. LC-MS [M+H] + : 706.7. 1H NMR (500 MHz, MeOD) δ 5.39 - 5.31 (m, 2H), 4.07 (m, 4H), 2.35 - 2.29 (m, 5H), 2.24 (s, 6H), 2.01 (m, 3H), 1.69 - 1.53 (m, 9H), 1.50 - 1.39 (m, 6H), 1.37 - 1.22 (m, 48H), 0.88 (m, 9H).

[0330] Preparation of B-274 was same as B-266 except using linoleyl alcohol instead of 9- heptadecanol. LC-MS [M+H] + : 704.7. 1 H NMR (500 MHz, MeOD) δ 5.39 - 5.28 (m, 4H), 4.07 (m, 4H), 2.77 (t, J = 6.6 Hz, 2H), 2.36 - 2.29 (m, 5H), 2.25 (s, 6H), 2.04 (dd, J = 9.0, 5.0 Hz, 4H), 1.68 - 1.54 (m, 9H), 1.49 - 1.39 (m, 6H), 1.39 - 1.24 (m, 42H), 0.88 (m, 9H).

[0331] Preparation of B-268 was same as B-266 except using stearyl alcohol instead of 9- heptadecanol. LC-MS [M+H] + : 708.7. 1 H NMR (500 MHz, MeOD) δ 4.07 (m, 4H), 2.32 (m, 5H), 2.25 (s, 6H), 1.68 - 1.53 (m, 8H), 1.50 - 1.39 (m, 5H), 1.38 - 1.20 (m, 58H), 0.87 (t, J = 6.9 Hz, 9H).

[0332] Preparation of B-446 was same as B-246 except using 2-octyldecanoic acid instead of stearic acid and stearyl alcohol instead of 9-heptadecanol. LC-MS [M+H] + : 708.7. 1 H NMR (500 MHz, Chloroform-d) δ 4.14 (t, J = 6.7 Hz, 2H), 4.08 (t, J = 6.7 Hz, 2H), 2.38 - 2.27 (m, 5H), 2.23 (s, 6H), 2.05 (m, 1H), 1.76 - 1.66 (m, 2H), 1.66 - 1.50 (m, 6H), 1.45 (m, 2H), 1.39 (s, 1H), 1.27 (m, 58H), 0.90 (m, 9H).

[0333] Example 46 Preparation of B-260

[0334] Step 1: Preparation of B-260-a

[0335] Into a reaction flask was placed B-276-a (10 g, 28.09 mmol), dichloromethane (50 mL), CDI (5.5 g, 33.71 mmol), and the reaction was stirred at room temperature for 1 hour, noted as reaction 1. Into another three-necked flask was placed MeCN (50 mL), anhydrous magnesium chloride (5.3 g, 56.18 mmol), cyanoacetic acid (3.6 g, 42.13 mmol), and triethylamine (11.3 g, 112.40 mmol) was added dropwise under water bath cooling, and the reaction was stirred at 40 °C for 1 hour, noted as reaction 2. The contents of reaction 1 were added dropwise into reaction 2 via a constant pressure dropping funnel, and the reaction was stirred at 40 °C for 1 hour. Methanol was added dropwise to quench the reaction, and concentrated hydrochloric acid was added dropwise until the pH of the system was about 3. The organic phase was collected after extraction with dichloromethane and washed with aqueous sodium bicarbonate solution, and concentrated under reduced pressure. Purification by column chromatography gave 5.3 g of oil B-260-a.

[0336] Step 2: Preparation of B-260-b

[0337] Into a reaction flask was placed B-260-a (5.3 g, 14.02 mmol), toluene, ethoxycarbonylmethylidene triphenylphosphonium (10.6 g, 30.46 mmol), and the reaction was heated to 120 °C for 1 hour. After the reaction was completed, the reaction mixture was concentrated and purified by column chromatography to give 5.8 g of colorless oil B-260-b.

[0338] Step 3: Preparation of B-260-c

[0339] Into a high-pressure reaction kettle was placed B-260-b (5.8 g, 12.92 mmol), methanol (60 mL), triethylamine (3.3 g), Boc anhydride (4.2 g), and Raney nickel (6 g), and the reaction was carried out under a hydrogen atmosphere overnight. After the reaction was completed, the reaction mixture was filtered, washed with methanol, and stirred with water (100 mL) and methyl tert-butyl ether (100 mL). The upper organic phase was collected. Concentration and purification by column chromatography gave 5.6 g of colorless oil B-260-c.

[0340] Step 4: Preparation of B-260-d

[0341] Into a reaction flask was placed B-260-c (5 g), ethanol (50 mL), and sodium borohydride (1.2 g), and the reaction was stirred at room temperature overnight. Acetic acid was added dropwise to quench the reaction, and the reaction mixture was concentrated, stirred with ethyl acetate and water, and separated into two phases. The organic phase was collected, concentrated, and purified by column chromatography to give 1.7 g of oil B-260-d.

[0342] Step 5: Preparation of B-260-e

[0343] To a reaction flask was added B-260-d (1.7 g), HCl / 1,4-dioxane (8 mL), and the reaction was stirred at room temperature for 10 min. The reaction was concentrated, THF (10 mL) was added, formaldehyde (5 mL) was added, and 10% Pd / C (170 mg) was added. The reaction was stirred under a hydrogen atmosphere overnight. The reaction was filtered, and NaHCO3aq was added. The reaction was extracted with EtOAc twice, and the organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give 750 mg of B-260-e as an oil.

[0344] Step 6: Preparation of B-260

[0345] To a reaction flask was added B-260-e (750 mg), CH2Cl2(7 mL), and oleic acid (450 mg), DMAP (622 mg), and EDCI (490 mg) were added sequentially. The reaction was stirred at 30 °C overnight. The reaction was concentrated, water was added, and the reaction was extracted with EtOAc. The organic layers were combined, dried over Na2SO4, and concentrated. The residue was purified by column chromatography to give 760 mg of B-260 as a colorless oil. LC-MS [M+H] + 706.3.

[0346] 1 H NMR (500 MHz, Chloroform-d) δ 5.38-5.35 (m, 2H), 4.88 (m, 1H), 4.12 (t, J = 6.9 Hz, 2H), 2.29 (m, 6H), 2.23 (s, 6H), 2.03 (m, 4H), 1.65 (m, 6H), 1.57-1.45 (m, 7H), 1.36-1.28 (m, 46H), 0.89 (m, 9H).

[0347] Example 47 Preparation of B-326

[0348] Step 1: Preparation of B-326-a

[0349] To a reaction flask was added 4-oxoheptanedioic acid diethyl ester (10 g, 49.2 mmol), and EtOH (50 mL) was added to dissolve. Water (2 mL) was added, and LiOH H2O (2.07 g, 49.2 mmol) was added. The reaction was stirred at room temperature for 20 min. A large amount of solid was precipitated, and dilute HCl (2N) was added to adjust the pH to about 3. The reaction was concentrated, water was added, and the reaction was extracted with EtOAc. The organic layers were washed with saturated brine once, dried over Na2SO4, and concentrated to give 9.3 g of crude product.

[0350] Step 2: Preparation of B-326-b

[0351] To a reaction flask was added B-326-a crude (9.3 g), 9-heptadecanol (6.2 g, 24.3 mmol), DCM (60 mL), DMAP (9 g, 73.8 mmol), EDCI (7 g, 36.9 mmol), and the reaction was stirred at 40 °C overnight. 1 N dilute hydrochloric acid was added and the reaction was stirred, the organic phase was collected, washed once with saturated brine, concentrated, and purified by column chromatography to give 4.5 g of oil B-326-b.

[0352] Step 3: Preparation of B-326-c

[0353] To a reaction flask was added B-326-b (4.5 g, 10.2 mmol) and ethanol (30 mL), and a solution of lithium hydroxide monohydrate (644 mg, 15.3 mmol) in water (3 mL) was added, and the reaction was stirred at room temperature for 2 hours. The reaction was complete, the water was removed, water (40 mL) was added, the pH was adjusted to 4-5 with dilute hydrochloric acid, and the reaction was extracted twice with ethyl acetate, concentrated, and purified by column chromatography to give 3.3 g of oil B-326-c.

[0354] Step 4: Preparation of B-326-d

[0355] To a reaction flask was added B-326-c (3.3 g, 8 mmol), DCM (30 mL), triethylamine (3.6 g, 36 mmol), EDCI (2.3 g, 12 mmol), and dimethylamine hydrochloride (1.3 g, 16 mmol), and the reaction was stirred at 40 °C for 5 hours. Water was added and the reaction was stirred, extracted twice with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give 1.6 g of oil B-326-d.

[0356] Step 5: Preparation of B-326-e

[0357] To a reaction flask was added triethyl phosphonoacetate (1.3 g, 6 mmol), THF (13 mL), and the reaction was protected with nitrogen. LiHMDS (6 mL, 6 mmol) was added dropwise with ice bath cooling, and the reaction was stirred at room temperature for 2 hours. B-326-d (1.3 g, 3 mmol) was added and the reaction was stirred at room temperature overnight. The reaction was quenched by dropwise addition of saturated aqueous ammonium chloride solution with ice bath cooling. Water was added, the reaction was extracted twice with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 600 mg of oil B-326-e.

[0358] Step 6: Preparation of B-326-f

[0359] Into a reaction flask was added Raney nickel (1 g), rinsed with 1,4-dioxane to remove water, added 1,4-dioxane (5 mL), nitrogen protection, added B-326-e (600 mg, 1.17 mmol) and TMDS (5 g, 23.5 mmol). Warmed to 40 °C for 2 hours. After the reaction was completed, filtered, concentrated, and purified by column chromatography to obtain 600 mg of oil B-326-f.

[0360] Step 7: Preparation of B-326-g

[0361] Into a reaction flask was added B-326-f (600 mg, 1.21 mmol), ethanol (16 mL), ice bath cooling, then added sodium borohydride (192 mg, 5 mmol), warmed to 40 °C for 2 hours. The reaction was quenched with dilute hydrochloric acid, concentrated, added water, extracted with ethyl acetate, combined organic layers, dried over anhydrous sodium sulfate, rotary evaporation, and purified by column chromatography to obtain 100 mg of oil B-326-g.

[0362] Step 8: Preparation of B-326

[0363] Into a reaction flask was added B-326-g (100 mg, 0.22 mmol), dichloromethane (2 ml), oleic acid (134 mg, 0.44 mmol), EDCI (84 mg, 0.44 mmol) and DMAP (107 mg, 0.88 mmol) in sequence, warmed to 40 °C for 3 hours. After the reaction was completed, concentrated, added dilute hydrochloric acid, extracted with ethyl acetate twice, combined organic phases, washed with saturated aqueous sodium bicarbonate solution, dried the organic layer over anhydrous sodium sulfate, rotary evaporation, and purified by column chromatography to obtain 93 mg of oil B-326. LC-MS [M+H] + : 720.4.

[0364] 1 H NMR (500 MHz, Chloroform-d) δ 5.43-5.30 (m, 2H), 4.88 (m, 1H), 4.11 (t, J=6.9 Hz, 2H), 2.33-2.23 (m, 11H), 2.03 (m, 4H), 1.69-1.59 (m, 10H), 1.56-1.44 (m, 6H), 1.36-1.23 (m, 44H), 0.94-0.87 (m, 9H).

[0365] Example 48 Preparation of B-439

[0366] Step 1: Preparation of B-439-a

[0367] Into a reaction flask was placed mono-tert-butyl succinate (15 g, 86.11 mmol), hydroxylamine hydrochloride (10.08 g, 103.33 mmol) and HOBT (2.33 g, 17.22 mmol), followed by THF (150 mL), then triethylamine (13.07 g, 129.16 mmol) and EDCI (24.76 g, 129.16 mmol), and the reaction was stirred at room temperature for 5 hours. To the reaction was added ice water (200 mL), and the mixture was extracted twice with ethyl acetate (200 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography to give 16.9 g of B-439-a as an oil. Yield 90%.

[0368] Step 2: Preparation of B-439-b

[0369] Into a reaction flask was placed magnesium ribbon (3.9 g, 128.88 mmol), and THF (100 mL) was added under nitrogen protection. Five drops of 1,2-dibromoethane were added, followed by dropwise addition of a THF (40 mL) solution of 3-benzyloxybromopropane (14.76 g, 64.44 mmol). The dropwise addition was completed, and the Grignard reagent solution was obtained by stirring at room temperature for 2 hours. B-439-a (7.0 g, 32.22 mmol) was dissolved in THF (70 mL), and the solution was cooled in an ice bath under nitrogen protection. The Grignard reagent solution was added dropwise, and the dropwise addition was completed. The reaction was maintained at 5°C for 30 minutes. The reaction was quenched by dropwise addition of saturated ammonium chloride aqueous solution (100 mL). The reaction was extracted with ethyl acetate, and the organic phase was collected by liquid separation. The organic phase was concentrated, and the residue was purified by column chromatography to give 8.95 g of B-439-b as an oil. Yield 91%.

[0370] Step 3: Preparation of B-439-c

[0371] Into a reaction flask was placed diethyl (2-(dimethylamino)-2-oxoethyl)phosphonate (728 mg, 3.26 mmol), and anhydrous THF (6 mL) was added under nitrogen protection. LiHMDS (3.3 mL, 3.3 mmol) was added dropwise at 0°C. The dropwise addition was completed, and the reaction was stirred at room temperature for 1 hour. B-439-b (500 mg, 1.63 mmol) was added, and the reaction was stirred at room temperature overnight. The reaction was quenched by dropwise addition of saturated ammonium chloride aqueous solution at 0°C. The reaction was extracted twice with ethyl acetate, and the organic phase was collected. The organic phase was washed once with saturated brine, concentrated, and purified by column chromatography to give 547 mg of B-439-c as an oil. Yield 77%.

[0372] Step 4: Preparation of B-439-d

[0373] To a flask was added B-439-c (547 mg), nitrogen protection, dichloromethane (15 mL) was added, hydrogen chloride dioxane solution (4N, 7 ml) was added dropwise in ice bath, dropwise addition was completed, room temperature reaction overnight. The reaction liquid was concentrated, and was dragged with dioxane to obtain 468 mg of crude B-439-d, which was directly used in the next step.

[0374] Step 5: Preparation of B-439-e

[0375] To a flask was added B-439-d (468 mg, 1.46 mmol), 10-nonyl alcohol (417 mg, 1.46 mmol) and DMAP (357 mg, 2.92 mmol), then dichloromethane (15 mL) was added, after stirring and dissolving, EDCI (420 mg, 2.19 mmol) was added, and the temperature was raised to 40°C for overnight reaction. Dichloromethane was concentrated, 1N dilute hydrochloric acid was added, and was extracted twice with ethyl acetate, the organic phase was collected, and was washed with saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and rotary evaporated, and was purified by column chromatography to obtain 570 mg of oil B-439-e, with a yield of 67%.

[0376] Step 6: Preparation of B-439-f

[0377] To a flask was added B-439-e (570 mg, 0.97 mmol), DME (10 ml) was added, and Raney nickel (570 mg) was added, nitrogen protection, TMDS (1.3 g, 9.73 mmol) was added, and the temperature was raised to 40°C for overnight reaction. Filtration, concentration, and column chromatography purification were performed to obtain 450 mg of oil B-439-f.

[0378] Step 7: Preparation of B-439-g

[0379] To a flask was added B-439-f (450 mg), methanol (10 mL), concentrated hydrochloric acid (3 drops) and 10% palladium-carbon (100 mg) was added, hydrogen replacement, hydrogen atmosphere reaction overnight, filtration, concentration, and dioxane drag twice to obtain 418 mg of crude B-439-g, which was directly used in the next step.

[0380] Step 8: Preparation of B-439

[0381] To a reaction flask was added B-439-g (418 mg, 0.86 mmol), dichloromethane (20 mL), oleic acid (366 mg, 1.29 mmol), DMAP (420 mg, 3.44 mmol), and EDCI (264 mg, 1.38 mmol) sequentially, warmed to 40 °C overnight, concentrated the dichloromethane, added 1 N dilute hydrochloric acid, extracted with ethyl acetate twice, collected the organic phase, washed with saturated sodium bicarbonate, concentrated, and purified by column chromatography to give 407 mg of B-439 as a colorless oil. LC-MS [M+H] + : 748.8. 1 H NMR (500 MHz, Chloroform-d) δ 5.40 - 5.28 (m, 2H), 4.92 - 4.79 (m, 1H), 4.04 (t, J = 6.7 Hz, 2H), 2.31 - 2.22 (m, 6H), 2.21 (s, 6H), 2.00 (q, J = 6.5 Hz, 4H), 1.61 (dt, J = 11.2, 6.5 Hz, 6H), 1.46 - 1.39 (m, 3H), 1.37 - 1.17 (m, 54H), 0.87 (t, J = 6.8 Hz, 9H).

[0382] Prepared as for B-439 except using 13-pentacosanol instead of 10-nonadecanol. LC-MS [M+H] + : 832.9. 1 H NMR (500 MHz, Chloroform-d) δ 5.38 - 5.29 (m, 2H), 4.85 (t, J = 6.2 Hz, 1H), 4.04 (t, J = 6.7 Hz, 2H), 2.28 (td, J = 8.3, 7.9, 3.2 Hz, 6H), 2.23 (s, 6H), 2.00 (q, J = 6.5 Hz, 4H), 1.66 - 1.56 (m, 6H), 1.44 (dd, J = 8.6, 5.6 Hz, 3H), 1.34 - 1.20 (m, 66H), 0.87 (t, J = 6.8 Hz, 9H).

[0383] Example 50 Preparation of B-457

[0384] Step 1: Preparation of B-457-a

[0385] Compound SM1 (552 mg, 2.0 mmol) and 2-hexyldecanoic acid (1.28 g, 5.0 mmol) were dissolved in dichloromethane (20 mL), DMAP (242 mg, 2 mmol) and EDCI (1.15 g, 6 mmol) were added, and the reaction was stirred at room temperature overnight. The residue was added with water (30 mL) and ethyl acetate (30 mL), and the mixture was separated. The organic phase was washed with saturated sodium chloride three times, dried over anhydrous sodium sulfate, and concentrated. The residue was separated by silica gel column chromatography to give 1.4 g of B-457-a.

[0386] Step 2: Preparation of B-457-b

[0387] B-457-a (1.4 g, 1.86 mmol) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (10 mL) was added. The reaction was stirred at room temperature for 3 hours. The residue was added with saturated sodium bicarbonate solution (50 mL) and ethyl acetate (50 mL), and the mixture was separated. The organic phase was washed with saturated sodium chloride three times, dried over anhydrous sodium sulfate, and concentrated to give 1.2 g of B-457-b.

[0388] Step 3: Preparation of B-457

[0389] B-457-b (1.2 g) was dissolved in THF (10 mL), and formaldehyde aqueous solution (5 mL) and 10% palladium-carbon (0.5 g) were added, followed by replacement of hydrogen gas. The reaction was stirred at room temperature overnight. The mixture was filtered, and the filtrate was concentrated. The residue was separated by silica gel column chromatography to give 620 mg of B-457.

[0390] LC-MS [M+H] + : 680.6. 1 H NMR (500 MHz, MeOD) δ 4.08 (t, J = 6.4 Hz, 4H), 2.37-2.31 (m, 4H), 2.25 (s, 6H), 1.69-1.55 (m, 9H), 1.52-1.41 (m, 8H), 1.38 (m, 4H), 1.34-1.23 (m, 50H), 0.89 (m, 12H).

[0391] Example 51 Preparation of B-437

[0392] Step 1: Preparation of B-437-a

[0393] To a reaction flask was added geraniol (1 g, 6.48 mmol), adipic acid (1.9 g, 13.0 mmol) and dichloromethane (15 mL), followed by DMAP (159 mg, 1.30 mmol), DIPEA (2.5, 19.4 mmol) and EDCI (1.9 g, 9.91 mmol) and allowed to react at room temperature over the weekend. Spinned down, added water and extracted with ethyl acetate, dried the organic layer over sodium sulfate, spun down and purified by column chromatography to yield 700 mg of a yellowish oil, B-437-a.

[0394] Step 2: Preparation of B-437

[0395] To a reaction flask was added B-276-d (300 mg, 0.66 mmol) and dichloromethane (10 mL), followed by B-437-a (242 mg, 0.86 mmol), DMAP (241 mg, 2.0 mmol) and EDCI (190 mg, 1.0 mmol). Allowed to react at 40 °C for 1 h, spun down, added water and extracted with ethyl acetate, dried the organic layer over sodium sulfate, spun down and purified by column chromatography to yield 300 mg of a colorless oil, B-437.

[0396] 1 H NMR (500 MHz, Chloroform-d) δ 5.35 (m, 1H), 5.10 (m, 1H), 4.88 (m, 1H), 4.61 (d, J = 7.1 Hz, 2H), 4.06 (t, J = 6.7 Hz, 2H), 2.36 - 2.25 (m, 8H), 2.23 (s, 6H), 2.14 - 2.04 (m, 4H), 1.72 - 1.66 (m, 10H), 1.65 - 1.62 (m, 7H), 1.52 (m, 4H), 1.45 (m, 3H), 1.35 - 1.27 (m, 26H), 0.89 (t, J = 6.9 Hz, 6H).

[0397] Prepared as B-437 except geraniol was replaced with undecanol. LC-MS [M+H] + 738.7. 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (m, 1H), 4.07 (m, 4H), 2.63 (s, 4H), 2.36 - 2.25 (m, 8H), 2.23 (s, 6H), 1.69 - 1.61 (m, 10H), 1.52 (m, 4H), 1.46 - 1.43 (m, 4H), 1.36 - 1.22 (m, 41H), 0.89 (m, 9H).

[0398] Example 53 Preparation of B-443

[0399] Step 1: Preparation of B-443-a

[0400] Into a reaction flask was added 1-decanol (1.2 g, 7.59 mmol), glutaric acid (2.0 g, 15.2 mmol) and dichloromethane (15 mL), followed by DMAP (185 mg, 1.52 mmol), DIPEA (4.4 g, 34.1 mmol) and EDCI (2.2 g, 11.5 mmol) and allowed to react at room temperature overnight. Spun down, added water and extracted with methyl tert-butyl ether, washed the organic layer with saturated brine, dried over anhydrous sodium sulfate, spun down and purified by column chromatography to give 1 g of B-443-a as a light yellow oil.

[0401] Step 2: Preparation of B-443

[0402] Into a reaction flask was added B-276-d (300 mg, 0.66 mmol) and dichloromethane (10 mL), followed by B-443-a (234 mg, 0.85 mmol), DMAP (241 mg, 2.0 mmol) and EDCI (190 mg, 1.0 mmol). Allowed to react at 40 °C for 1 hour, spun down and added water, extracted with ethyl acetate, washed the organic layer with saturated brine, dried over anhydrous sodium sulfate, spun down and purified by column chromatography to give 190 mg of B-443 as a colorless oil.

[0403] LC-MS [M+H] + : 710.7. 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (m, 1H), 4.09 (m, 4H), 2.63 (s, 4H), 2.31-2.25 (m, 4H), 2.23 (s, 6H), 1.63 (m, 6H), 1.52 (m, 4H), 1.46-1.43 (m, 3H), 1.36-1.22 (m, 42H), 0.89 (m, 9H).

[0404] Prepared as B-443 except using succinic acid instead of glutaric acid. LC-MS [M+H] + : 696.6. 1H NMR (500 MHz, Chloroform-d) δ 4.88 (m, 1H), 4.09 (m, 4H), 2.63 (s, 4H), 2.31-2.25 (m, 4H), 2.23 (s, 6H), 1.63 (m, 6H), 1.52 (m, 4H), 1.46-1.43 (m, 3H), 1.36-1.22 (m, 40H), 0.89 (m, 9H).

[0405] Example 55 Preparation of B-453

[0406] Preparation of B-453 was the same as B-276 except using diethyl (2- (dimethylamino)-2-oxoethyl)phosphonate instead of diethyl (2- (dimethylamino)-2-oxoethyl)phosphonate.

[0407] LC-MS [M+H] + : 748.7. 1 H NMR δ 5.37-5.30 (m, 2H), 4.88 (m, 1H), 4.02 (m, 2H), 2.61 (q, J = 7.2 Hz, 4H), 2.31-2.23 (m, 6H), 2.06-1.95 (m, 4H), 1.65-1.57 (m, 6H), 1.53-1.45 (m, 4H), 1.45-1.38 (m, 3H), 1.36-1.18 (br, 55H), 0.89 (m, 9H).

[0408] Example 56 Preparation of B-454

[0409] Preparation of B-454 was the same as B-453 except using stearic acid instead of oleic acid.

[0410] LC-MS [M+H] + : 750.6. 1 H NMR (500 MHz, MeOD) δ 4.88 (m, 1H), 4.03 (t, J = 6.5 Hz, 2H), 2.59 (q, J = 7.2 Hz, 4H), 2.33-2.27 (m, 6H), 1.66-1.55 (m, 6H), 1.53-1.31 (m, 8H), 1.30-1.18 (m, 67H), 0.88 (m, 9H).

[0411] Example 57 Preparation of B-455

[0412] Preparation of B-455 is the same as B-442 except that (2-(diethylamino)-2- oxoethyl)phosphonic acid diethyl ester is used instead of (2-(dimethylamino)-2- oxoethyl)phosphonic acid diethyl ester.

[0413] LC-MS [M+H] + : 762.7. 1 H NMR (500 MHz, Chloroform-d) δ 5.38 - 5.36 (m, 2H), 4.88 (m, 1H), 4.08 (t, J = 6.7 Hz, 2H), 2.60 (q, J = 7.2 Hz, 4H), 2.34 - 2.27 (m, 6H), 2.05 (m, 4H), 1.65 (m, 6H), 1.54 (m, 4H), 1.48 - 1.45 (m, 3H), 1.37 - 1.24 (m, 57H), 0.90 (m, 9H).

[0414] Preparation and characterization of lipid nanoparticles

[0415] The N / P (nitrogen / phosphorus ratio) in the mRNA-LNP is maintained at 4.8, and the molar ratio of each lipid component in the LNP is: ionizable lipids: cholesterol: DSPC: PEG lipid (PEG2000-DMG) = 48.5:38.9:11.1:1.5. The ionizable lipids are the compounds prepared in this application. The Firefly Luciferase mRNA is dissolved in acetate buffer (200 mM, pH 5.0) to prepare an mRNA-buffer phase with a mRNA concentration of 85.38 pg / mL; the lipids are dissolved in anhydrous ethanol to prepare a lipid-ethanol phase with a total lipid concentration of 8.0 mM; the mRNA-buffer phase and the lipid-ethanol phase are rapidly mixed at a flow rate ratio of 3:1 using a microfluidic device (Dolomite SPMinjection pump, Fluidic-lab LNP B0 mixing chip) to obtain a suspension. The prepared suspension is subjected to ultrafiltration or dialysis using Tris salt buffer (20.5 mM, pH 7.5, containing 8.95% sucrose) to obtain the mRNA-LNP, and the ethanol concentration in the mRNA-LNP is about 0.7% (v / v).

[0416] The particle size and polydispersity coefficient (Malvern DLS) of the mRNA-LNP are detected, and the encapsulation efficiency (RiboGreen) is detected.

[0417] The results show that the part of ionizable lipids provided by the application can be well encapsulated in mRNA after being prepared into lipid nanoparticles, and most of the ionizable lipids prepared into lipid nanoparticles have an mRNA encapsulation rate of more than 97% and a PDI of less than 0.2.

[0418] Example 59 In vivo delivery and expression in animals

[0419] The in vivo delivery and test method of mice is as follows:

[0420] The mRNA of Firely Luciferase is encapsulated in LNP containing different ionizable lipids by using a microfluidic device (the ionizable lipids are the compounds prepared in the application, and the encapsulation is carried out according to the method in Example 58). After dialysis and concentration detection, the mRNA-LNP sample is diluted with Tris buffer, and the mRNA concentration of all samples is adjusted to 5 ng / uL. Each mouse (6-8 week old female Balb / c mice, Guangdong Yaoke) is injected with 0.2 mL of LNP sample, i.e. 1 ug per mouse. 3 hours after injection of the LNP sample, 15 mg / mL D-luciferin 0.2 mL is injected intraperitoneally. The mice are fully anesthetized with isoflurane, and 10-15 min after injection of D-luciferin, the mice are placed into a living imager for chemiluminescence photographic detection. The picture results are analyzed by Living Image 4.4 or 4.5 software.

[0421] The related characteristics of the lipid nanoparticles and the related results of the animal research test are shown in Table 1.

[0422] Table 1 Test results of LNP composed of different cationic lipids encapsulating the same mRNA

[0423] Note: A≥1.2; 0.8≤B<1.2; C<0.8.

[0424] The LNP composed of the ionizable lipids of the application has a good encapsulation rate for mRNA, and shows good delivery effect in animals, and the expression amount of mRNA after delivery is slightly better than or comparable to the positive control.

[0425] Example 60 PK and tissue distribution test in mice

[0426] According to the method in Example 58, mRNA-LNP samples containing different ionizable lipids are prepared, and the ionizable lipids of the positive control group are SM102.

[0427] Experimental process:

[0428] The test SD mice (male) were randomly divided into groups, each group of 3, and were intravenously administered 0.1 mg / kg of the mRNA-LNP sample described above. The animals were anesthetized with isoflurane and bled at 0.5, 3, 6, 24, and 48 h after administration, and were then sacrificed. After the whole blood sample was collected, it was placed in a centrifuge tube containing EDTA-K2, and was centrifuged at 1500-1600 g for 10 min to separate the plasma.

[0429] Collection of liver, spleen, kidney, heart, and lung tissues: After the tissues were removed, the collected tissues were rinsed with 2-8 °C pre-cooled physiological saline to remove the blood clots and contents on the surface and in the cavities. After washing, the water was absorbed with filter paper, and the liver tissue sample was weighed and placed in a grinding tube. 2-8 °C pre-cooled 50% methanol water was added at a ratio of 1:5 (weight-volume), and a rapid grinder was used for homogenization.

[0430] The concentration of lipids in the plasma and tissue samples of the SD mice was detected by LC-MS / MS, and the clearance rate of lipids in each tissue was calculated, as shown in Table 2.

[0431] The residual amount of ionizable lipids in the liver in Table 2 is the percentage ratio of the ionizable lipid content detected in the liver at different time points to the total amount of ionizable lipids in the total dose. The weight of the mice was about 20 g, and the weight of the liver was about 1 g.

[0432] Table 2 Residual amount of LNP encapsulating the same mRNA composed of different cationic lipids in the liver of mice

[0433] The results show that the mRNA-LNP sample prepared using the part of the ionizable lipids in the present application has a good clearance rate in rats, which is comparable to or significantly better than the positive control.

[0434] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the protection scope of the present application. For those skilled in the art, based on the above description and ideas, other different forms of changes or modifications can also be made, which are not required or impossible to enumerate all the embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A compound having the structure of Formula (I-B) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof: wherein, X0is H, F, OH, methyl, methoxy or absent; T1, L1are each independently C 1-4 alkyl or is absent; T2, L2are each independently -O-C(O)-, -C(O)-O-, -NH-C(O)- or -C(O)-NH-; T3, T4, L3, L4 are each independently H, C 4-20 linear or branched alkyl, C 4-20 linear or branched alkenyl, C 4-20 linear or branched alkynyl, C 8-20 linear alkyl or C 8-20 linear alkyl; wherein T3and T4are not simultaneously H; L3and L4are not simultaneously H; X1, X2are each independently C 1-4 alkyl, -O-C(O)-, -C(O)-O-, -O-C(O)-O-, or nothing; R2, R3are each independently C 1-4 alkyl, C 1-4 fluoroalkyl, C 1-4 hydroxyalkyl, wherein n3is an integer from 0 to 2.

2. The compound of claim 1, wherein each T3, T4 independently is H, C 4-9 alkyl, C 14-20 alkyl, C 9-20 alkenyl, C 9-20 alkynyl, C 13-20 linear alkyl or C 13-20 linear alkyl; Preferably, each T3, T4 is independently H, -C4H9, -C6H 13 , -C8H 17 , -C 10 H 21 , -C 13 H 27 , -C 14 H 29 , -C 15 H 31 , -C 16 H 33 , C 17 H 35 , -C 18 H 37 , -C9H 17 , -C 15 H 29 , -C 17 H 33 , -C 17 H 31 , -C 18 H 35 , -C 18 H 33 , -C 10 H 17 , -(CH2) m1 -C(O)-O-(CH2) m2 CH3, -(CH2) m1 -O-C(O)-(CH2) m2 CH3or -(CH2) m1 -S-S-(CH2) m2 CH3; wherein m1is an integer from 2 to 10, and m2is an integer from 5 to 14; Preferably, said For Preferably, said For Preferably, said For 3. The compound of claim 1 or 2, wherein said X0is H.

4. The compound of any one of claims 1 to 3, wherein said T1, L1are each independently ethyl, propyl or butyl.

5. The compound of any one of claims 1 to 4, wherein said T2, L2are each independently -O-C(O)- or -C(O)-O-.

6. The compound of any one of claims 1 to 5, wherein each T3, T4, L3, L4is independently H, C 4-20 linear or branched alkyl or C 4-20 linear or branched alkenyl.

7. The compound of any one of claims 1 to 6, wherein The structure of the compound or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof is shown as formula (I-C): wherein n is 2, 3 or 4; m3is 2, 3 or 4; m4is 2, 3 or 4; R2, R3are each independently C 1-4 alkyl; T3, T4are each independently C 6-12 linear alkyl; L3, L4are each independently H, C 6-20 linear alkyl or C 13-20 linear alkenyl; and L3and L4are not simultaneously H.

8. The compound of any one of claims 1 to 7, wherein said R2, R3are each independently methyl, ethyl, propyl or butyl; preferably; said R2, R3are each independently methyl, ethyl or propyl; preferably; said R2, R3are each independently methyl or ethyl.

9. The compound of any one of claims 1 to 8, wherein said T3, T4are any one of the following: (t1) each T3, T4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , -C 11 H 23 , or -C 12 H 25 ; (t2) each T3, T4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , or -C 11 H 23 ; (t3) each T3, T4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 or -C 10 H 21 ; (t4) each T3, T4is independently -C6H 13 , -C7H 15 , -C8H 17 , or -C9H 19 ; (t5) each T3, T4is independently -C6H 13 , -C7H 15 , or -C8H 17 ; (t6) each T3, T4is independently -C7H 15 , -C8H 17 , or -C9H 19 ; (t7) each T3, T4is independently -C6H 13 or -C8H 17 .

10. The compound of any one of claims 1 to 9, wherein said L3, L4are any one of the following: (g1) when neither L3, L4is H; each L3, L4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , -C 11 H 23 , or -C 12 H 25 ; (g2) L3, L4are not both H; said L3, L4are each independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , or -C 11 H 23 ; (g3) when neither L3, L4is H; each L3, L4is independently -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , or -C 10 H 21 ; (g4) when neither L3, L4is H; each L3, L4is independently -C6H 13 , -C7H 15 , -C8H 17 , or -C9H 19 ; (g5) L3, L4are not both H; said L3, L4are each independently -C6H 13 , -C7H 15 , or -C8H 17 ; (g6) L3, L4are not both H; said L3, L4are each independently -C7H 15 , -C8H 17 , or -C9H 19 ; (g7) when neither L3, L4is H; each of said L3, L4is independently -C6H 13 or -C8H 17 ; (g8) When L3 is H; L4 is C 13-20 Straight-chain alkyl or C 13-20 Straight-chain alkenyl, wherein C 13-20 The number of double bonds in the straight-chain alkenyl group is 1 or 2; (g9) L3is H; and said L4is C 15-20 straight-chain alkyl or C 15-20 straight-chain alkenyl, wherein the number of double bonds in C 15-20 the number of double bonds in straight-chain alkenyl is 1 or 2; (g10) When L3 is H; L4 is C 15-18 Straight-chain alkyl or C 15-18 Straight-chain alkenyl, wherein C 15-18 The number of double bonds in a straight-chain alkenyl group is 1 or 2.

11. A compound, characterized in that, The compound is selected from one of the following structures:

12. A lipid nanoparticle comprising the compound of any one of claims 1 to 11 or a stereoisomer thereof, and a therapeutic or prophylactic agent.

13. The lipid nanoparticle of claim 12, wherein, Also included are one or more of a neutral lipid, a sterol and a polymer-conjugated lipid.

14. The lipid nanoparticle of claim 13, wherein, said neutral lipid is one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM; said sterol is cholesterol; said polymer-conjugated lipid is a PEGylated lipid; said PEGylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkyloxypropyl carbamate; preferably, said PEGylated lipid is DMG-PEG 2000.

15. The lipid nanoparticle of claim 13 or 14, wherein, The molar ratio of the compound of any one of claims 1 to 11 or a stereoisomer thereof, the neutral lipid, the sterol and the polymer-conjugated lipid is: 30-65%: 18.5-48.5%: 0-30%: 0-10%; preferably, the molar ratio of the compound of any one of claims 1 to 11 or a stereoisomer thereof, the neutral lipid, the sterol and the polymer-conjugated lipid is: 40-62%: 18.5-42.8%: 3-15%: 0.5-5%; preferably, the molar ratio of the compound of any one of claims 1 to 11 or a stereoisomer thereof, the neutral lipid, the sterol and the polymer-conjugated lipid is: 45-50%: 25-42.8%: 3-15%: 0.5-5% preferably, the molar ratio of the compound of any one of claims 1 to 11 or a stereoisomer thereof, the neutral lipid, the sterol and the polymer-conjugated lipid is: 48-50%: 30-42.8%: 8-15%: 0.8-3%; preferably, the molar ratio of the compound of any one of claims 1 to 11 or a stereoisomer thereof, the neutral lipid, the sterol and the polymer-conjugated lipid is: 48-50%: 35-42.8%: 9-13%: 1.0-2.5%.

16. The lipid nanoparticle of any one of claims 12-15, wherein, said therapeutic or prophylactic agent comprises a nucleic acid or a nucleic acid-based drug.

17. The lipid nanoparticle of claim 16, wherein, said nucleic acid is an antisense RNA or a messenger RNA.

18. A pharmaceutical composition, characterized by, A lipid nanoparticle of any one of claims 12 to 17, and a pharmaceutically acceptable excipient.

19. Use of a compound according to any one of claims 1 to 11 or a stereoisomer thereof, or a lipid nanoparticle according to any one of claims 12 to 17, or a pharmaceutical composition according to claim 18 as a drug delivery vehicle.

20. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 11 or a stereoisomer thereof, or a lipid nanoparticle according to any one of claims 12 to 17, or a pharmaceutical composition according to

Images