Cationic lipids, liposomes, lipid nanoparticles, and uses

Abstract

Disclosed is a cationic lipid having the general formula (I) wherein A, B, C and D are each as defined in the specification. Also disclosed are liposomes and lipid nanoparticles comprising the cationic lipid and the use of the cationic lipid for the preparation of liposomes and lipid nanoparticles.

Description

[0001] This disclosure claims priority to Chinese Patent Application No. 202210349570.6, filed on April 2, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure generally relates to the field of gene therapy drug formulations, and more specifically, to a cationic lipid for forming liposomes or lipid nanoparticles. This disclosure also relates to liposomes comprising the cationic lipid and the use of the cationic lipid. Background Technology

[0003] In recent years, with the deepening understanding of disease pathogenesis at the cellular level, gene therapy has become a hot research topic. Various gene-engineered drugs are constantly being developed and commercialized.

[0004] In gene therapy, gene transfer vectors are crucial for the entry of exogenous genes into recipient cells. Among them, cationic liposomes and lipid nanoparticles are attracting increasing attention due to their good targeting, minimal side effects, good stability, and high transfection efficiency. Cationic liposomes and lipid nanoparticles are typically composed of cationic lipids, neutral assist lipids, and other materials under appropriate conditions, and their transfection efficiency is related to the structure of the cationic lipids used. Summary of the Invention

[0005] At least one embodiment of this disclosure provides a cationic lipid having the following general formula (I):

[0006]

[0007] Wherein, A is selected from methine and nitrazine, wherein the methine is optionally unsubstituted or substituted by R. a replace;

[0008] Where A is methine, B is R. 1 -L 1 -Y 1 -L 2 -,in

[0009] R 1 For R 2 R 3 N-, where R 2 and R 3 Each is independent as C 1-6 alkyl,

[0010] Or R 1 It is a 5- or 6-membered heterocyclic group containing 1 or 2 cyclic nitrogen atoms.

[0011] Wherein C1-6 alkyl or said 5- or 6-membered heterocyclyl is each independently unsubstituted or substituted with 1-3 R b substituents;

[0012] L 1 and L 2 are each independently selected from the group consisting of a bond and straight- chained or branched C 1-12 alkylene, said C 1-12 alkylene is optionally unsubstituted or substituted with 1-3 R c substituents; and

[0013] Y 1 is selected from the group consisting of -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-; and

[0014] B is C 1-6 alkyl, wherein said C 1-6 alkyl is optionally unsubstituted or substituted with 1-3 R d substituents;

[0015] C is -L 3 -Y 2 -L 4 -CH=CR 4 R 5 wherein

[0016] L 3 and L 4 are each independently selected from the group consisting of a bond and straight- chained or branched C 1-12 alkylene, said C 1-12 alkylene is optionally unsubstituted or substituted with 1-3 R e substituents,

[0017] Y 2 is selected from the group consisting of -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-, and

[0018] R 4 and R 5 are each independently selected from the group consisting of straight- chained or branched C 2-12 alkyl, said C 2-12 alkyl is optionally unsubstituted or substituted with 1-3 R f substituents;

[0019] D is -L 5 -Y 3 -L 6 -R 6 wherein

[0020] L 5 and L 6 are each independently selected from a bond and a straight-chained or branched C 1-12 alkylene group, said C 1-12 alkylene group being optionally unsubstituted in each occurrence by 1 to 3 R g substituents,

[0021] Y 3 is selected from -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-, and

[0022] R 6 is independently selected from a saturated or unsaturated straight-chained or branched C 6-18 hydrocarbon group or a steroid group, said C 6-18 hydrocarbon group or steroid group being optionally unsubstituted or substituted by 1 to 3 R h substituents;

[0023] or, D is -L 3 -Y 2 -L 4 -CH=CR 4 R 5 ;

[0024] X is independently in each occurrence O or S, and

[0025] R a , R b , R c , R d , R e , R f , R g and R h are each independently in each occurrence selected from C 1-3 alkyl, C 1-3 alkoxy, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkoxycarbonyl, C 1-3 alkylcarbonyloxy, C 1-3 alkylcarbonylamino, C 1-3 alkylaminocarbonyl, hydroxy, halogen, and cyano.

[0026] In one aspect, in the cationic lipid of formula (I), X is O.

[0027] In another aspect, in the cationic lipid of formula (I), B is R 1 -L 1 -Y 1 -L 2 -, R 1 is R 2 R 3 N-, and R 2 and R 3 are each independently C 1-6 alkyl, said C 1-6 alkyl is optionally unsubstituted or substituted with 1-3 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy, hydroxyl, and halo.

[0028] In another aspect, in the cationic lipid of formula (I), R 2 and R 3 are the same or different and are each independently selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-propyl, and t-butyl.

[0029] In another aspect, in the cationic lipid of formula (I), B is R 1 -L 1 -Y 1 -L 2 -, R 1 is a 5- or 6-membered heterocyclyl containing 1 or 2 ring nitrogen atoms, and said 5- or 6-membered heterocyclyl is a saturated 5- or 6-membered heterocyclyl unsubstituted or optionally substituted with 1-3 C 1-3 alkyl.

[0030] In another aspect, in the cationic lipid of formula (I), said 5- or 6-membered heterocyclyl is selected from the group consisting of N-methyl-piperidin-4-yl, N-methyl-piperidin-2-yl, N-methyl-pyrrolidin-2-yl, N-ethyl-piperidin-4-yl, N-ethyl-piperidin-2-yl, N-ethyl-pyrrolidin-2-yl, and piperidin-1-yl.

[0031] In another aspect, in the cationic lipid of formula (I), B is C 2-4 alkyl unsubstituted or substituted with 1-3 substituents selected from the group consisting of hydroxyl, halo, and cyano.

[0032] In another aspect, in the cationic lipid of formula (I), B is selected from the group consisting of primary hydroxyl-substituted methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.

[0033] In another aspect, in the cationic lipid of formula (I), C is -L3 -Y 2 -L 4 -CH=CR 4 R 5 , where R 4 and R 5 They are either the same or different, and each is independently selected from the C of the straight chain. 2-12 alkyl.

[0034] On the other hand, in the cationic lipids shown in formula (I), D is -L 5 -Y 3 -L 6 -R 6 And R 6 A straight-chain or branched C-type carbon containing one or two carbon-carbon double bonds. 6-18 Hydrocarbon group.

[0035] On the other hand, in the cationic lipids shown in formula (I), R 6 A straight-chain or branched C-type carbon containing two carbon-carbon double bonds. 6-18 Hydrocarbon group.

[0036] On the other hand, in the cationic lipids shown in formula (I), D is -L 5 -Y 3 -L 6 -R 6 And R 6 For being C 1-6 alkoxycarbonyl-substituted C 6-18 alkyl.

[0037] On the other hand, in the cationic lipids shown in formula (I), D is -L 3 -Y 2 -L 4 -CH=CR 4 R 5 , where R 4 and R 5 They are either the same or different, and each is independently selected from the C of the straight chain. 2-12 alkyl.

[0038] On the other hand, in the cationic lipids shown in formula (I), L 1 L 2 L 3 L 4 L 5 and L 2 Each is independently selected from chemical bonds and C 1-6 Alkylene, the C 1-6 The alkylene group is optionally unsubstituted or composed of 1-3 independently selected C14 groups. 1-3 Alkyl, C 1-3substituted with substituents selected from the group consisting of alkyl, alkoxy, hydroxyl, and halogen.

[0039] In another aspect, in the cationic lipid of formula (I), L 1 , L 2 , L 3 , L 4 , L 5 , and L 2 are each independently selected from the group consisting of a bond and an unsubstituted C 1-6 alkylene.

[0040] In another aspect, in the cationic lipid of formula (I), Y 1 , Y 2 , and Y 3 are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, and -O-C(=O)-.

[0041] In another aspect, the cationic lipid of formula (I) has a structure selected from the group consisting of:

[0042]

[0043]

[0044]

[0045] The present disclosure also discloses, in at least one embodiment, a liposome comprising the cationic lipid as described in any embodiment of the present disclosure.

[0046] The present disclosure also discloses, in at least one embodiment, a lipid nanoparticle comprising the cationic lipid as described in any embodiment of the present disclosure.

[0047] The present disclosure also discloses, in at least one embodiment, the use of the cationic lipid as described in at least one embodiment of the present disclosure for the manufacture of a liposome.

[0048] The present disclosure also discloses, in at least one embodiment, the use of the cationic lipid as described in at least one embodiment of the present disclosure for the manufacture of a lipid nanoparticle. DETAILED DESCRIPTION

[0049] For the purposes of the detailed description below, it is to be understood that the disclosure can assume various alternative arrangements and sequences of steps described herein without departing from the spirit of the disclosure. Also, any examples in the description are not presented to limit the scope of the application but are presented for the purpose of illustration. Furthermore, where a range of values is provided, it is understood that whenever a maximum and minimum values are stated to be included in the range, it is intended to disclose all values including the maximum value, minimum value, and any and all values between the maximum and minimum values. Also, the terms in the examples have their plain and ordinary meaning.

[0050] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0051] Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges of the same entire range. For example, a range of "1 to 10" is intended to include all sub-ranges, for example, 1 to 6.1, 5.1 to 10, 1.1 to 1.4, 8.7 to 9.8, etc.

[0052] In this disclosure, the use of the singular includes the plural, and vice-c versa, unless explicitly stated otherwise. Furthermore, in this disclosure, the use of "or" means "and / or" unless explicitly stated otherwise. Moreover, in this disclosure, the use of "a" or "an" means "at least one" unless explicitly stated otherwise. For example, "a" compound, "an" composition, and the like, means one or more of these items.

[0053] The compounds of the present disclosure can be synthesized by synthetic routes including processes analogous to those well-known in the chemical arts, particularly in light of the description contained herein. The starting materials are generally available from commercial sources, or are readily prepared using methods well known to persons of ordinary skill in the art. For the purposes of illustration, the reaction schemes depicted below show possible routes for synthesizing the compounds of the present disclosure, as well as key intermediates. For a more detailed description of the individual reaction steps, see the Example section below. Those skilled in the art will appreciate that other suitable starting materials, reagents and synthetic routes can be used to synthesize the compounds of the present disclosure and various derivatives thereof. In addition, using the methods described below, various compounds prepared can be further modified using routine chemistry well known to persons of ordinary skill in the art.

[0054] The compounds of the present disclosure can include stereoisomers (e.g., cis and trans isomers), optical isomers (e.g., R and S enantiomers), tautomers (e.g., keto-enol and imine-enamine tautomers), atropisomers (i.e., restricted rotation isomers), and the like, of the compounds of the present disclosure.

[0055] For the purposes of the present application as described and claimed herein, the following terms are defined as follows:

[0056] As used herein, the term “cationic lipid” refers to a class of charged lipid materials under physiological conditions, which can be used to prepare liposomes for delivering nucleic acid drugs. As used herein, the term “nucleic acid drug”, also known as “nucleotide drug”, refers to various ribonucleic acid (RNA) drugs and / or deoxyribonucleic acid (DNA) drugs with different functions, which mainly act at the genetic level. Examples of nucleic acid drugs can include, but are not limited to, RNA-based nucleic acid drugs such as transfer RNA (tRNA), small interfering RNA (siRNA), antisense oligonucleotide, microRNA (miRNA), long non-coding RNA (lncRNA), aptamer, messenger RNA (mRNA), circular RNA (circRNA), self-replicating RNA, and the like; and DNA-based nucleic acid drugs such as plasmid, linearized plasmid, nanoplasmid, doggybone DNA, closed-ended DNA, oligo DNA, and the like. Currently, nucleic acid drugs can be used as drugs for treating or preventing diseases including, but not limited to, tumors, autoimmune diseases, infectious diseases, and the like, and the like, for example, can be used as tumor-targeting therapeutic agents, new crown vaccines, and the like.

[0057] As used herein, the term "liposome" generally refers to a closed vesicle with a bilayer structure formed when lipids (lipidoids) and additional agents are dispersed in an aqueous phase, which can generally fuse with cell membranes to deliver bioactive substances (such as nucleic acid drugs or other drugs) into the interior of cells. In general, the basic structure of a cationic lipid consists of a positively charged polar head and one or more hydrophobic tails. The positively charged polar head group usually includes amines (primary, secondary, tertiary, quaternary ammonium salts), guanidines, amidines, etc., which can form complexes with negatively charged nucleic acids through electrostatic interactions, enter cells through endocytosis of cells. The hydrophobic tail can usually be a saturated / unsaturated aliphatic chain or a cholesteryl group, which can affect the pKa, lipophilicity, fluidity and fusogenicity of the cationic lipid, thereby affecting the formation and efficacy of the cationic liposome. There can also be a linker between the positively charged polar head group and the hydrophobic tail, which can connect the head and tail, or can be hidden in the tail. The linker can be divided into non-biodegradable linkers (such as ethers, etc.) and biodegradable linkers (such as esters, amides and thiols, etc.), which can affect the stability, biodegradability, cytotoxicity and transfection efficiency of the cationic liposome. The cationic lipid can be considered as a multi-component molecule, and each component needs to be precisely designed in order to safely and effectively package and deliver nucleic acids.

[0058] As used herein, the term "Lipid Nano Particles (LNP)" refers to a class of nanocarriers developed on the basis of liposomes for delivering nucleic acid substances to cells, usually composed of cationic lipids, neutral auxiliary phospholipids, cholesterol, PEG lipids. Lipid nanoparticles are one of the important technologies in the lipid carrier drug delivery system, and have become an important progress in nucleic acid-based therapeutic drugs. Oligonucleotides encapsulated in lipid nanoparticles are protected during delivery, not subject to enzymatic degradation, and effectively delivered to cells, where the contents in the carrier particles are released and translated into therapeutic proteins.

[0059] Unless otherwise indicated, the term "hydrocarbyl" as used herein refers to a monovalent linear chain group consisting of carbon and hydrogen atoms. The hydrocarbyl group can be linear or branched, saturated or unsaturated, and can be substituted or unsubstituted. Non-limiting examples of hydrocarbyl groups include, but are not limited to, alkyl, alkenyl, or alkynyl groups, and the like. For example, the term "C 2-18 hydrocarbyl" refers to a monovalent linear or branched alkyl, alkenyl, or alkynyl group having 2-18 carbon atoms. C 2-18Non-limiting examples of hydrocarbyl groups can include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, lauryl, butenyl, hexenyl, octenyl, decenyl, laurenlyl, 1,3-butadienyl, 1,3-pentadienyl, octynyl, and the like.

[0060] The term "alkyl," as used herein, unless otherwise indicated, means a saturated, monovalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n+1, where n is an integer between 1 and 18. The alkyl group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkyl" means an alkyl group having from 1 to 6 carbon atoms. The alkyl group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkyl group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkyl moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. n H2 n+1 The term "alkyl," as used herein, unless otherwise indicated, means a saturated, monovalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n+1, where n is an integer between 1 and 18. The alkyl group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkyl" means an alkyl group having from 1 to 6 carbon atoms. The alkyl group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkyl group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkyl moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. 1-6 The term "alkyl," as used herein, unless otherwise indicated, means a saturated, monovalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n+1, where n is an integer between 1 and 18. The alkyl group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkyl" means an alkyl group having from 1 to 6 carbon atoms. The alkyl group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkyl group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkyl moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. 1-6 The term "alkyl," as used herein, unless otherwise indicated, means a saturated, monovalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n+1, where n is an integer between 1 and 18. The alkyl group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkyl" means an alkyl group having from 1 to 6 carbon atoms. The alkyl group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkyl group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkyl moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain.

[0061] The term "alkenyl," as used herein, unless otherwise indicated, means a straight or branched chain aliphatic hydrocarbon chain having from 2 to 18 carbon atoms and containing at least one carbon-carbon double bond (e.g., -CH=CH-, -CH=CH2). Non-limiting examples of alkenyl groups include: ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-pentenyl, hexenyl, octenyl, decenyl, laurenlyl, 1,3-butadienyl, 1,3-pentadienyl, and the like.

[0062] The term "alkynyl," as used herein, unless otherwise indicated, means a straight or branched chain aliphatic hydrocarbon chain having from 2 to 18 carbon atoms and containing at least one carbon-carbon triple bond (e.g., -C≡C- or -C≡CH). Non-limiting examples of alkynyl groups include: ethynyl, 2-propynyl, 1-methyl-2-propynyl, 2-butynyl, 3-butynyl, 2-methyl-3-butynyl, and the like.

[0063] The term "alkylene," as used herein, unless otherwise indicated, means a saturated, divalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n, where n is an integer between 1 and 18. The alkylene group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkylene" means an alkylene group having from 1 to 6 carbon atoms. The alkylene group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkylene group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkylene moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. n H 2n The term "alkylene," as used herein, unless otherwise indicated, means a saturated, divalent straight or branched chain hydrocarbon radical of from 1 to 18 carbon atoms having the formula -CnH2n, where n is an integer between 1 and 18. The alkylene group can be straight or branched, and can be substituted or unsubstituted. For example, the term "C1-C6alkylene" means an alkylene group having from 1 to 6 carbon atoms. The alkylene group can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkylene group can be optionally substituted. Further, when used in a composite term such as alkoxy (alkoxy), the alkylene moiety has the same meaning as in the present disclosure and can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. 1-12"Alkylene" refers to a saturated divalent straight or branched chain hydrocarbon radical containing 1-12 carbon atoms. C 1-12 Non-limiting examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, hexylene, octylene, decylene, 2- ethylhexylene, and the like. The alkylene moiety can be attached to another chemical moiety through any one of the carbon atoms in the carbon chain. As described herein, the alkylene group can be optionally substituted.

[0064] The term "methine," as used herein, unless otherwise indicated, refers to a -CH< group, wherein the -CH< group can be optionally unsubstituted or substituted; the term "nitrido" refers to a -N< group.

[0065] The term "halogen," as used herein, unless otherwise indicated, refers to fluorine, chlorine, bromine, or iodine.

[0066] The term "hydroxyl," as used herein, unless otherwise indicated, refers to an -OH group.

[0067] The term "alkoxy," as used herein, unless otherwise indicated, refers to an alkyl-O- group, wherein alkyl is as defined in the present disclosure.

[0068] The term "amino," as used herein, unless otherwise indicated, refers to an -NH2 group.

[0069] The term "alkylamino," as used herein, unless otherwise indicated, refers to an alkyl-NH- group; the term "dialkylamino" refers to a (alkyl)2-N- group, wherein alkyl is as defined in the present disclosure.

[0070] The term "cyano," as used herein, unless otherwise indicated, refers to a -C≡N group.

[0071] The term "carbonyl," as used herein, unless otherwise indicated, refers to a -(C=O)- group.

[0072] The term "carbonyloxy," as used herein, unless otherwise indicated, refers to a -C(=O)-O- or -O-C(=O)- group.

[0073] The term "alkoxycarbonyl," as used herein, unless otherwise indicated, refers to an alkyl-O-C(=O)- group, wherein alkyl is as defined in the present disclosure.

[0074] The term "steroid group," as used herein, unless otherwise indicated, refers to a group comprising or derived from a cyclopenta-fused perhydrophenanthrene structure.

[0075] In one embodiment of the present disclosure, a cationic lipid is provided having the following general formula (I):

[0076]

[0077] wherein A is selected from the group consisting of methine and nitroso, wherein the methine can optionally be unsubstituted or substituted with R a substituted;

[0078] wherein when A is methine, B is R 1 -L 1 -Y 1 -L 2 -, wherein

[0079] R 1 is R 2 R 3 N-, wherein R 2 and R 3 are each independently C 1-6 alkyl,

[0080] or R 1 is a 5- or 6-membered heterocyclyl group containing 1 or 2 ring nitrogen atoms,

[0081] wherein the C 1-6 alkyl or the 5- or 6-membered heterocyclyl group is each independently unsubstituted or substituted with 1-3 R b substituted;

[0082] L 1 and L 2 are each independently selected from the group consisting of a bond and straight- or branched-chain C 1-12 alkylene, said C 1-12 alkylene being optionally unsubstituted or substituted with 1-3 R c substituted; and

[0083] Y 1 is selected from the group consisting of -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-; and

[0084] when A is nitroso, B is C 1-6 alkyl, wherein the C 1-6 alkyl is optionally unsubstituted or substituted with 1-3 R d substituted;

[0085] C is -L 3 -Y 2 -L 4 -CH=CR 4 R 5 , wherein

[0086] L 3 and L 4each independently selected from a chemical bond and straight- chained or branched C 1-12 alkylene, said C 1-12 alkylene is optionally, at each occurrence, unsubstituted or substituted with 1-3 R e substituted,

[0087] Y 2 is selected from -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-, and

[0088] R 4 and R 5 each independently selected from straight- chained or branched C 2-12 alkyl, said C 2-12 alkyl is optionally unsubstituted or substituted with 1-3 R f substituted;

[0089] D is -L 5 -Y 3 -L 6 -R 6 wherein

[0090] L 5 and L 6 each independently selected from a chemical bond and straight- chained or branched C 1-12 alkylene, said C 1-12 alkylene is optionally, at each occurrence, unsubstituted or substituted with 1-3 R g substituted,

[0091] Y 3 is selected from -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-, and

[0092] R 6 independently selected from saturated or unsaturated straight- chained or branched C 2-12 alkyl or a steroid group, said C 2-12 alkyl or a steroid group is optionally unsubstituted or substituted with 1-3 R h substituted;

[0093] or, D is -L 3 -Y 2 -L 4 -CH=CR 4 R 5 ;

[0094] X is independently, at each occurrence, O or S, and

[0095] R a , R b , R c , R d , R e , R f , R g and R h are each, independently at each occurrence, selected from the group consisting of C 1-3 alkyl, C 1-3 alkoxy, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkoxycarbonyl, C 1-3 alkylcarbonyloxy, C 1-3 alkylcarbonylamino, C 1-3 alkylaminocarbonyl, hydroxyl, halo, and cyano.

[0096] In some aspects of the disclosure, in the cationic lipid of formula (I), A can be a methine group. Alternatively, in another aspect of the disclosure, A can be a nitroso group.

[0097] In another aspect of the disclosure, in the cationic lipid of formula (I), X can be O or S. For example, X can be O.

[0098] In another aspect of the disclosure, in the cationic lipid of formula (I), A is a methine group, and B can be R 1 -L 1 -Y 1 -L 2 -, wherein R 1 can be R 2 R 3 N-, and R 2 and R 3 can each independently be C 1-3 alkyl optionally unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of C 1-3 alkyl, C 1-6 alkoxy, hydroxyl, and halo. For example, R 2 and R 3 can each independently be C 1-3 alkyl optionally unsubstituted or substituted with 1-3 substituents independently selected from the group consisting of C 1-3 alkyl, C 2-4 alkoxy, hydroxyl, and halo, e.g., methyl, ethyl, n-propyl, i-propyl, chloromethyl, chloroethyl, chloropropyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, methoxymethyl, methoxyethyl, and the like. In some aspects of the disclosure, R 2 and R 3may be the same and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isopropyl, or t-butyl. In some aspects of the disclosure, R 2 may be the same and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isopropyl, or t-butyl. In some aspects of the disclosure, R 3 may be the same and can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isopropyl, or t-butyl. In some aspects of the disclosure, R

[0099] Alternatively, in another aspect of the disclosure, in the cationic lipid represented by formula (I), A is a methine group, B can also be R 1 -L 1 -Y 1 -L 2 -, wherein R 1 may be a saturated 5- or 6-membered heterocyclic group containing 1 or 2 ring nitrogen atoms, unsubstituted or optionally substituted with 1-3 C 1-3 alkyl groups. For example, R 1 may be selected from the group consisting of N-methyl-piperidin-4-yl, N-methyl-piperidin-2-yl, N-methyl-pyrrolidin-2-yl, N-ethyl-piperidin-4-yl, N-ethyl-piperidin-2-yl, N-ethyl-pyrrolidin-2-yl, and piperidin-1-yl.

[0100] In another aspect of the disclosure, in the cationic lipid represented by formula (I), A is a nitrilo group, B can be a C 2-4 alkyl group, unsubstituted or substituted with 1-3 substituents selected from the group consisting of hydroxyl, halogen, and cyano. For example, B can be selected from the group consisting of primary hydroxyl-substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl.

[0101] In another aspect of the disclosure, in the cationic lipid represented by formula (I), C can be -L 3 -Y 2 -L 4 -CH=CR 4 R 5 , wherein R 4 and R 5 may be the same or different and each is independently selected from the group consisting of linear C 2-12 alkyl groups.

[0102] In another aspect of the disclosure, in the cationic lipid represented by formula (I), D can be -L 5 -Y 3 -L 6 -R 6 , wherein R 6 is a linear or branched C 6-18 hydrocarbon group containing one or more carbon-carbon double bonds; or, R 6 may be a linear or branched C 6-18 hydrocarbon group containing two carbon-carbon double bonds; or, R 6C can be -L 1-6 alkyl. 6-18 alkyl.

[0103] In alternative embodiments, in the cationic lipid of formula (I), D can be -L 3 -Y 2 -L 4 -CH=CR 4 R 5 wherein R 4 and R 5 are the same or different and each is independently selected from linear C 2-12 alkyl. For example, in some cases, C and D can be the same.

[0104] In another aspect of the disclosure, in the cationic lipid of formula (I), L 1 , L 2 , L 3 , L 4 , L 5 and L 6 may each be independently selected from a bond and C 1-8 alkylene, wherein the C 1-8 alkylene is optionally unsubstituted or substituted with 1-3 substituents independently selected from C 1-3 alkyl, C 1-3 alkoxy, hydroxyl, and halogen. In some cases, L 1 , L 2 , L 3 , L 4 , L 5 and L 6 may each be independently selected from a bond and unsubstituted C 1-8 alkylene, e.g., a bond, methylene, ethylene, propylene, butylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, and 2,2-dimethyl- propandiyl, etc.

[0105] In another aspect of the disclosure, in the cationic lipid of formula (I), Y 1 , Y 2 and Y 3 may each be independently selected from -C(=X)-X-, -X-C(=X)-, -C(=X)-NH-, -NH-C(=X)-, -X-C(=X)-NH-, -NH-C(=X)-X-, -X-C(=X)-X-, -NH-C(=X)-NH-, and -X-X-, wherein X can be independently O or S at each occurrence. In some cases, X can be O, and Y 1 , Y 2 and Y 3may each independently be selected from the group consisting of -C(=0)-0-, -0-C(=0)-, -C(=0)-NH-, -NH-C(=0)-, -0-C(=0)-NH-, -NH-C(=0)-0-, -0-C(=0)-0-, -NH-C(=0)-NH-, and -0-0-. For example, Y 1 , Y 2 , and Y 3 may each independently be selected from the group consisting of -C(=0)-0-, -0-C(=0)-, and -0-C(=0)-0-.

[0106] In some aspects of the present disclosure, the cationic lipid represented by Formula (I) can have a structure selected from the group consisting of:

[0107]

[0108]

[0109]

[0110] At least one embodiment of the present disclosure also discloses a liposome comprising any of the above cationic lipids.

[0111] At least one embodiment of the present disclosure also discloses a lipid nanoparticle comprising any of the above cationic lipids.

[0112] At least one embodiment of the present disclosure also discloses the use of any of the above cationic lipids for the preparation of a liposome.

[0113] At least one embodiment of the present disclosure also discloses the use of any of the above cationic lipids for the preparation of a lipid nanoparticle.

[0114] The liposome or the lipid nanoparticle according to at least one embodiment of the present disclosure can be used for encapsulating and delivering various nucleic acid drugs, including but not limited to RNA-based nucleic acid drugs such as tRNA, siRNA, antisense oligonucleotide, microRNA, IncRNA, aptamer, mRNA, circular RNA, self-replicating RNA, etc., and DNA-based nucleic acid drugs such as plasmid, linearized plasmid, nanoplasmid, “dog bone” DNA, blunt-ended RNA, oligomeric DNA, etc. In some examples, the liposome or the lipid nanoparticle according to the present disclosure can be used for delivering nucleic acid drugs for treating or preventing diseases including but not limited to tumors, autoimmune diseases, infectious diseases, etc., such as for delivering tumor-targeting therapeutic agents, new crown vaccines, etc.

[0115] Examples

[0116] The following examples provide more detailed descriptions of the preparation, characterization, and properties of the compounds of the application. It is to be understood, however, that the application as fully described herein and as set forth in the claims is not intended to be limited by the details of the following protocols or modes of preparation.

[0117] Abbreviations:

[0118] In the following examples the following abbreviations for terms are used:

[0119] r.t.: room temperature;

[0120] h / hr: hours

[0121] EA: ethyl acetate;

[0122] PE: petroleum ether;

[0123] DCM: dichloromethane;

[0124] DIEA: diisopropylethylamine;

[0125] EDCI: l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0126] DMAP: 4-dimethylaminopyridine;

[0127] THF: tetrahydrofuran;

[0128] tBuONa: sodium tert-butoxide;

[0129] tBuOK: potassium tert-butoxide;

[0130] MeOH: methanol;

[0131] TsOH: p-toluenesulfonic acid;

[0132] TfOH: trifluoroethanesulfonic acid;

[0133] ACN: acetonitrile;

[0134] DMF: N,N-dimethylformamide;

[0135] DMSO: dimethyl sulfoxide;

[0136] EtOH: ethanol;

[0137] NaH: sodium hydride;

[0138] DCC: dicyclohexylcarbodiimide;

[0139] TEA: triethylamine;

[0140] TsOH: p-methylbenzenesulfonic acid;

[0141] NaOH: sodium hydroxide;

[0142] LiOH: Lithium hydroxide;

[0143] NaI: Sodium iodide;

[0144] K2CO3: Potassium carbonate;

[0145] DSPC = Distearoylphosphatidylcholine;

[0146] DMG-PEG2000 = 1,2-Dimyristoyl-raC-glycerol-3-methoxypolyethylene glycol-2000;

[0147] FFluc = Firefly luciferase;

[0148] LNP = Liposome nanoparticle;

[0149] PDI = Polydispersity index;

[0150] pKa = Dissociation constant.

[0151] Preparation Example

[0152] Example 1: Preparation of 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2- (((3-nonyldodec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-001-01)

[0153]

[0154] Preparation of 3-nonyldodec-2-enoic acid ethyl ester (mixture 2)

[0155] To a mixture of ethyl 2-(diethoxyphosphoryl)acetate (33.6 g, 150 mmol, 2.5 eq) in THF (200 mL) was added tBuONa (14.4 g, 150 mmol, 2.5 eq) followed by compound 1 (16.92 g, 60 mmol, 1.0 eq) in THF (136 mL) at 0 °C under N2 atmosphere. The reaction was then stirred at reflux for 16 h. 100 mL of water was added to the compound and extracted with EA (50 mL x 3) and washed with NaCl aqueous solution (50 mL x 3). The organic layer was dried with Na2SO4, filtered, concentrated and purified by column chromatography on silica gel (PE:EA = 300 / 1) to give compound 2 (12.8 g, 60.6% yield) as a colorless oil.

[0156] Preparation of 3-nonyldodec-2-enoic acid (compound 3)

[0157] To a mixture of ethyl 3-nonyldodec-2-enoate (18 g, 33.21 mmol, 1.0 eq) in ethanol / water (180 mL / 180 mL) was added NaOH (5.31 g, 132.8 mmol, 4.0 eq) at 0 °C under N2 atmosphere. The reaction mixture was stirred at reflux for 16 h. The solvent was removed by evaporation, and the pH was adjusted to 4-5 with 1 N HC1, extracted with EA (120 mL x 3), washed with aqueous NaCl solution (120 mL). The organic layer was dried over Na2S04, filtered, concentrated, and purified by column chromatography on silica gel (PE:EA = 150 / 1) to give compound 3 (5.16 g, 30.2% yield) as a colorless oil.

[0158] 1 H NMR (400 MHz, CDC13) δ 5.64 (s, 1H), 2.65-2.55 (m, 2H), 2.20-2.11 (m, 2H), 1.46-1.26 (m, 28H), 0.91-0.85 (m, 6H).

[0159] Preparation of 3-hydroxy-2-(((3-nonyldodec-2-enoyl)oxy)methyl)propyl (9Z, 12Z)- octadeca-9, 12-dienoate (compound 5)

[0160] To a solution of compound 3 (1.32 g, 4.08 mmol, 1.0 eq) in DCM (10 mL) was added DIEA (2.11 g, 16.32 mmol, 4.0 eq), EDCI (1.18 g, 6.12 mmol, 1.5 eq) and DMAP (100 mg, 0.82 mmol, 0.2 eq) at 0 °C under N2 atmosphere. Then a solution of compound 4 (1.5 g, 4.08 mmol, 1.0 eq) in DCM (5 mL) was added. The reaction mixture was stirred at 30 °C for 16 h. 50 mL water was added to the mixture, and extracted with DCM (50 mL x 3), washed with aqueous NaCl solution (100 mL). The organic layer was dried over Na2S04, filtered, concentrated, and purified by column chromatography on silica gel (PE:EA = 6 / 1) to give compound 5 (1.53 g, 55.6% yield) as a colorless oil.

[0161] 1H NMR (400 MHz, CDC13) δ 5.62 (s, 1H), 5.42-5.26 (m, 4H), 4.30-4.08 (m, 4H), 3.68-3.54 (m, 2H), 2.82-2.72 (m, 2H), 2.64-2.52 (m, 2H), 2.36-2.31 (m, 2H), 2.17-2.10 (m, 2H), 2.09-2.01 (m, 4H), 1.66-1.57 (m, 2H), 1.46-1.23 (m, 43H), 0.91-0.85 (m, 9H).

[0162] Preparation of 3-nonyldodec-2-enoic acid ethyl ester (CPL-001-01)

[0163] To a solution of compound 5 (700 mg, 1.038 mmol, 1.0 eq) in DCM (10 mL) was added pyridine (820 mg, 10.38 mmol, 10.0 eq) and triphosgene (122 mg, 0.416 mmol, 0.4 eq) at 0 °C under N2atmosphere. The reaction mixture was stirred at 0 °C for 0.5 h. Then a solution of 3-(diethylamino)propan-1-ol (408 mg, 3.114 mmol, 3.0 eq) in DCM (1 mL) was added at 0 °C. The reaction mixture was stirred at 30 °C for 16 h. 20 mL water was added to the mixture and extracted with DCM (20 mL x 3), washed with NaCl aqueous solution (50 mL). The organic layer was dried over Na2S04, filtered, concentrated and purified by reverse phase chromatography to give the target CPL-001-01 (101 mg, 11.6% yield) as a colorless oil.

[0164] LCMS: LC / MS (ESI) m / z: 832.7 (M+H) + .

[0165] 1 H NMR (400 MHz, CDC13) δ 5.62 (s, 1H), 5.42-5.26 (m, 4H), 4.30-4.08 (m, 4H), 3.68-3.54 (m, 2H), 2.82-2.72 (m, 2H), 2.64-2.52 (m, 2H), 2.36-2.31 (m, 2H), 2.17-2.10 (m, 2H), 2.09-2.01 (m, 4H), 1.66-1.57 (m, 2H), 1.46-1.23 (m, 43H), 0.91-0.85 (m, 9H).

[0166] Example 2: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)- bis(3-propyltridec-2-enoate) (CPL-062-01)

[0167]

[0168] Preparation of (E)-3-propyltridec-2-enoic acid 6-bromohexyl ester (Compound 2)

[0169] A mixture of Compound 1 (514 mg, 2.02 mmol, 1.0 equiv), 6-bromohexan-1-ol (438 mg, 2.42 mmol, 1.2 equiv), TsOH-H2O (38 mg, 0.202 mmol, 0.1 equiv) in toluene (8 mL) was stirred at 120 °C for 16 h. The mixture was concentrated and purified by chromatography on silica gel (PE:EA = 150:1) to give Compound 2 (674 mg, 80% yield) as a yellow oil.

[0170] 1 H NMR (400 MHz, CDCL3) δ 5.62 (d, J = 5.6 Hz, 1H), 4.09 (t, J = 6.4 Hz, 2H), 3.42 (t, J = 6.8 Hz, 2H), 2.59-2.55 (m, 2H), 2.14-2.09 (m, 2H), 1.90-1.83 (m, 2H), 1.69-1.62 (m, 2H), 1.50-1.37 (m, 8H), 1.29-1.26 (m, 14H), 0.97-0.86 (m, 6H).

[0171] Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-propyltridec-2-enoate) (CPL-062-01)

[0172] A mixture of Compound 2 (674 mg, 1.62 mmol, 2.2 equiv), 3-aminopropan-1-ol (55 mg, 0.736 mmol, 1.0 equiv), K2CO3 (406 mg, 2.94 mmol, 4.0 equiv) and NaI (44 mg, 0.294 mmol, 0.1 equiv) in ACN (10 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by preparative HPLC to give the target CPL-062-01 (257 mg, 46.2% yield) as a colorless oil.

[0173] LCMS: LC / MS (ESI) m / z: 748.23 (M+H) + .

[0174] LCMS: LC / MS (ESI) m / z: 748.23 (M+H)1 H NMR (400 MHz, CDC13) δ 5.62 (d, J = 5.2 Hz, 1H 2H), 4.08 (t, J = 6.4 Hz, 4H), 3.83 (t, J = 5.2 Hz, 2H), 3.20-3.05 (m, 2H), 3.02-2.80 (m, 2H), 2.59-2.55 (m, 4H), 2.14-2.10 (m, 4H), 2.00-1.90 (m, 2H), 1.69-1.62 (m, 6H), 1.52-1.26 (m, 48H), 0.96-0.86 (m, 12H).

[0175] Example 3: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)- bis(3-pentylundec-2-enoate) (CPL-069-01)

[0176]

[0177] Preparation of (E)-3-pentylundec-2-enoic acid 6-bromohexyl ester (Compound 2)

[0178] To a solution of (E)-3-pentylundec-2-enoic acid (600 mg, 2.4 mmol, 1.0 eq) and 6-bromohexan-1-ol (514 mg, 2.12 mmol, 1.2 eq) in toluene (5 mL) was added TfOH.H2O (50 mg, 0.264 mmol, 0.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 h. The solvent was removed under vacuum. The residue was purified by silica gel column (PE / EA = 50 / 1) to give Compound 2 (600 mg, 60% yield) as a colorless oil. LCMS: LC / MS (ESI) m / z: 417.14 (M+H) + .

[0179] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 1H), 4.10-4.06 (m, 2H), 3.46-3.40 (m, 2H), 2.60-2.56 (m, 2H), 2.40-2.37 (m, 2H), 2.18-2.10 (m, 2H), 1.89-1.85 (m, 2H), 1.68-1.55 (m, 2H), 1.51-1.23 (m, 21H), 0.86-0.92 (m, 6H).

[0180] Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-pentylundec-2-enoate

[0181] To a solution of (Z)-3-propylhept-2-enoic acid 7-bromoheptyl ester (600 mg, 1.43 mmol, 2.1 eq) and 3-aminopropan-1-ol (49 mg, 0.65 mmol, 1.0 eq) in ACN (5 mL) was added K2CO3 (360 mg, 2.535 mmol, 4.0 eq) and Nal (49 mg, 0.33 mmol, 0.5 eq) at 25 °C, the reaction mixture was stirred at 80 °C for 20 h. 30 mL water was added to the mixture, and extracted with EA (20 mL x 3). The organic layer was washed with aqueous NaCl (30 mL x 3), and dried over Na2SO4, filtered, concentrated and purified by prep-HPLC to give the target CPL-069-01 (127.1 mg, 7% yield) as colorless oil. LCMS: LCMS (ESI) m / z: 748.23 (M+H) + .

[0182] 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 2H), 4.08-4.05 (m, 4H), 3.81-3.72 (m, 2H), 2.65-2.56 (m, 6H), 2.42-2.39 (m, 4H), 2.16-2.11 (m, 4H), 1.62-1.27 (m, 54H), 0.93-0.86 (m, 12H).

[0183] Example 4: Preparation of ((3-hydroxypropyl)azanediyl)bis(heptane-7,1-diyl) (2E,2'E)- bis(3-pentyltridec-2-enoate) (CPL-092-01)

[0184]

[0185] Preparation of (E)-3-propyltridec-2-enoic acid 7-bromohexyl ester (Compound 2)

[0186] To a solution of (E)-3-pentyltridec-2-enoic acid (600 mg, 2.126 mmol, 1.0 eq) and 7-bromoheptan-1-ol (412.5 mg, 2.126 mmol, 1.0 eq) in toluene (10 mL) was added TfOH.H2O (48.48 mg, 0.213 mmol, 0.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 h. The solvent was removed under vacuum. The residue was purified by silica gel column (PE / EA = 50 / 1) to give compound 2 (700 mg, 71.8% yield) as colorless oil. LCMS: LC / MS (ESI) m / z: 459.28 (M+H) + .

[0187] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 1H), 4.10-4.06 (m, 2H), 3.42-3.40 (m, 2H), 2.63-2.53 (m, 2H), 2.15-2.11 (m, 2H), 1.90-1.82 (m, 2H), 1.69-1.61 (m, 2H), 1.48-1.41 (m, 4H), 1.38-1.23 (m, 24H), 0.90-0.86 (m, 6H).

[0188] Preparation of ((3-hydroxypropyl)azanediyl)bis(octane-8, 1-diyl) (2E, 2'E)-bis(3- pentylundec-2-enoate) (CPL-113-01)

[0189] To a solution of (E)-3-pentylundec-2-enoic acid 8-bromooctyl ester (700 mg, 1.527 mmol, 2.1 eq) and 3-aminopropan-1-ol (54.57 mg, 0.727 mmol, 1.0 eq) in ACN (10 mL) was added K2CO3 (401.30 mg, 2.908 mmol, 4.0 eq) and NaI (54.41 mg, 0.363 mmol, 0.5 eq) at 25 °C, the reaction mixture was stirred at 80 °C for 20 h. 30 mL water was added to the mixture, and extracted with EA (20 mL x 3). The organic layer was washed with aqueous NaCl (30 mL x 3) and dried over Na2SO4, filtered, concentrated and purified by prep-HPLC to give the target CPL-113-01 (265.94 mg, 44.0% yield) as colorless oil. LCMS: LCMS (ESI) m / z: 832.77 (M+H) + .

[0190] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.08-4.05 (m, 4H), 3.81-3.78 (m, 2H), 2.64-2.41 (m, 10H), 2.14-2.10 (m, 4H), 1.68-1.62 (m, 6H), 1.50-1.26 (m, 60H), 0.91-0.86 (m, 12H).

[0191] Example 5: Preparation of ((3-hydroxypropyl)azanediyl)bis(octane-8, 1-diyl) (2E, 2'E)-bis(3-pentylundec-2-enoate) (CPL-113-01)

[0192]

[0193] Preparation of (E)-3-pentylundec-2-enoic acid 8-bromooctyl ester

[0194] To a solution of (E)-3-pentylundec-2-enoic acid (800 mg, 3.1 mmol, 1.0 eq) and 6-bromoheptan-1-ol (774 mg, 3.7 mmol, 1.2 eq) in toluene (5 mL) was added TfOH.H2O (59 mg, 0.31 mmol, 0.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 h. The solvent was removed under vacuum. The residue was purified by silica gel column (PE / EA = 50 / 1) to give compound 2 (880 mg, 60% yield) as a colorless oil.

[0195] 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 1H), 4.09-4.05 (m, 2H), 3.42-3.39 (m, 2H), 2.60-2.56 (m, 2H), 2.14-2.11 (m, 2H), 1.89-1.60 (m, 2H), 1.44-1.27 (m, 28H), 0.91-0.86 (m, 6H).

[0196] Preparation of ((3-hydroxypropyl)azanediyl)bis(octane-8,1-diyl) (2E,2'E)-di(3- propyltridec-2-enoate

[0197] To a solution of (Z)-3-propylhept-2-enoic acid 7-bromoheptyl ester (880 mg, 1.97 mmol, 2.1 eq) and 3-aminopropan-1-ol (68 mg, 0.9 mmol, 1.0 eq) in ACN (5 mL) was added K2CO3 (497 mg, 3.6 mmol, 4.0 eq) and Nal (67 mg, 0.45 mmol, 0.5 eq) at 25 °C, and the reaction mixture was stirred at 80 °C for 20 h. 30 mL of water was added to the mixture, and extracted with EA (20 mL x 3). The organic layer was washed with aqueous NaCl (30 mL x 3), and dried over Na2SO4, filtered, concentrated and purified by prep-HPLC to give the target CPL-113-01 (101.27 mg, 5% yield) as a colorless oil. LCMS: LCMS (ESI) m / z: 804.34 (M+H) + .

[0198] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.05 (m, 4H), 3.81-3.78 (m, 2H), 2.64-2.56 (m, 6H), 2.42-2.38 (m, 4H), 2.14-2.11 (m, 4H), 1.69-1.60 (m, 6H), 1.46-1.21 (m, 56H), 0.91-0.83 (m, 12H)

[0199] Example 6: Preparation of ((3-hydroxypropyl)azanediyl)bis(nona-9, 1-diyl) (2E, 2'E)-bis(3-butyl oct-2-enoate) (CPL-133-01)

[0200]

[0201] Preparation of (E)-3-butyl oct-2-enoic acid 9-bromononyl ester

[0202] A mixture of compound 1 (500 mg, 2.53 mmol, 1.0 equiv), 9-bromononan-1-ol (840 mg, 3.78 mmol, 1.5 equiv), TsOH-H20 (96 mg, 0.51 mmol, 0.2 equiv) in toluene (10 mL) was stirred at 120 °C for 16 h. The mixture was concentrated and purified by chromatography on silica gel (PE:EA = 300:1) to give compound 2 (720 mg, 70.8% yield) as a yellow oil.

[0203] 1 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.05 (m, 2H), 3.42-3.38 (m, 2H), 2.60-2.56 (m, 2H), 2.15-2.11 (m, 2H), 1.87-1.81 (m, 2H), 1.65-1.62 (m, 2H), 1.48-1.25 (m, 20H), 0.93-0.87 (m, 6H).

[0204] Preparation of ((3-hydroxypropyl)azanediyl)bis(nona-9, 1-diyl) (2E, 2'E)-bis(3-butyl oct-2-enoate) (CPL-133-01)

[0205] A mixture of compound 2 (720 mg, 1.79 mmol, 2.4 eq), 3-aminopropan-1-ol (56 mg, 0.75 mmol, 1.0 eq), K2CO3(412 mg, 2.98 mmol, 4.0 eq) and Nal (23 mg, 0.15 mmol, 0.2 eq) in ACN (10 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-133-01 (129 mg, 24% yield) as a colorless oil.

[0206] LCMS: LC / MS (ESI) m / z: 720.6 (M+H) + .

[0207] 1 H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.03 (m, 4H), 3.83-3.81 (m, 2H), 3.03-2.93 (m, 2H), 2.73-2.60 (m, 4H), 2.59-2.56 (m, 4H), 2.15-2.11 (m, 4H), 2.01-1.86 (m, 2H), 1.65-1.27 (m, 48H), 0.96-0.82 (m, 12H).

[0208] Example 7: Preparation of ((3-hydroxypropyl)azanediyl)bis(undecan-11,1-diyl) (2E,2'E)-di(3-butylnon-2-enoate) (CPL-174-01)

[0209]

[0210] Preparation of (E)-3-butyl non-2-enoic acid 11-bromoundecyl ester (compound 2)

[0211] To a solution of (Z)-3-butyl non-2-enoic acid (500 mg, 2.356 mmol, 1.0 eq) and 11-bromoundecan-1-ol (589.21 mg, 2.356 mmol, 1.0 eq) in toluene (10 mL) was added TfOH (44.85 mg, 0.236 mmol, 0.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 h. The solvent was removed under vacuum. The residue was purified by silica gel column (PE / EA = 50 / 1) to give compound 2 (730 mg, 69.8% yield) as a colorless oil. LCMS: LC / MS (ESI) m / z: 445.26 (M+H) + .

[0212] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 1H), 4.09-4.05 (m, 2H), 3.42-3.39 (m, 2H), 2.61-2.56 (m, 2H), 2.17-2.11 (m, 2H), 1.89-1.82 (m, 2H), 1.67-1.62 (m, 2H), 1.48-1.28 (m, 26H), 0.94-0.86 (m, 6H).

[0213] Preparation of ((3-hydroxypropyl)azanediyl)di(undecan-11,1-diyl)(2E,2'E)-di(3- butyloct-2-enoate) (CPL-174-01)

[0214] To a solution of (E)-3-butylnon-2-enoic acid 11-bromoundecyl ester (730 mg, 1.643 mmol, 2.1 eq) and 3-aminopropan-1-ol (58.70 mg, 0.782 mmol, 1.0 eq) in ACN (10 mL) was added K2CO3 (431.66 mg, 3.128 mmol, 4.0 eq) and NaI (58.61 mg, 0.391 mmol, 0.5 eq) at 25 °C, the reaction mixture was stirred at 80 °C for 20 h. 40 mL water was added to the mixture, and extracted with EA (30 mL x 3). The organic layer was washed with aqueous NaCl (40 mL x 3), and dried over Na2SO4, filtered, concentrated and purified by prep-HPLC to give the target CPL-174-01 (181.92 mg, 28.9% yield) as a colorless oil. LCMS: LCMS (ESI) m / z: 804.74 (M+H) + .

[0215] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 1H), 4.09-4.05 (m, 2H), 3.42-3.39 (m, 2H), 2.61-2.56 (m, 2H), 2.17-2.11 (m, 2H), 1.89-1.82 (m, 2H), 1.67-1.62 (m, 2H), 1.48-1.28 (m, 26H), 0.94-0.86 (m, 6H).

[0216] Example 8: Preparation of ((3-hydroxypropyl)azanediyl)di(undecan-11,1-diyl)(2E,2'E)- di(3-pentyltridecan-2-enoate) (CPL-180-01)

[0217]

[0218] Preparation of (Z)-3-propyltridecan-2-enoic acid 11-bromoundecyl ester (compound 2)

[0219] To a solution of (Z)-3-pentyltridec-2-enoic acid (600 mg, 1.569 mmol, 1.0 eq) and 11-bromoundecan-1-ol (392.39 mg, 1.569 mmol, 1.0 eq) in toluene (5 mL) was added TfOH.H2O (29.64 mg, 0.156 mmol, 0.1 eq) under nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 h. The solvent was removed under vacuum. The residue was purified by silica gel column (PE / EA = 50 / 1) to give compound 2 (440 mg, 54.6% yield) as colorless oil. LCMS: LC / MS (ESI) m / z: 515.34 (M+H) + .

[0220] 1H NMR (400 MHz, CDC13-d) δ 5.61 (s, 1H), 4.07 (m, 2H), 3.40 (m, 2H), 2.61-2.53 (m, 2H), 2.12 (m, 2H), 1.85 (m, 2H), 1.63 (m, 2H), 1.45 (d, J = 7.4 Hz, 4H), 1.29 (m, 32H), 0.89 (m, 6H).

[0221] Preparation of ((3-hydroxypropyl)azanediyl)bis(undecan-11,1-diyl) (2E,2'E)-bis(3-pentyltridec-2-enoate) (CPL-180-01)

[0222] To a solution of (Z)-3-pentyltridec-2-enoic acid 11-bromoundecyl ester (440 mg, 0.855 mmol, 2.1 eq) and 3-aminopropan-1-ol (30.55 mg, 0.407 mmol, 1.0 eq) in ACN (5 mL) was added K2CO3 (224.66 mg, 1.628 mmol, 4.0 eq) and Nal (30.58 mg, 0.204 mmol, 0.5 eq) at 25 °C, the reaction mixture was stirred at 80 °C for 20 h. 30 mL water was added to the mixture, and extracted with EA (20 mL x 3). The organic layer was washed with aqueous NaCl (30 mL x 3), and dried over Na2SO4, filtered, concentrated and purified by prep-HPLC to give the target CPL-180-01 (194.57 mg, 50.6% yield) as colorless oil. LCMS: LCMS (ESI) m / z: 944.89 (M+H) + .

[0223] 1H NMR (400 MHz, CDC13-d) δ 5.61 (s, 2H), 4.08-4.05 (m, 4H), 3.82-3.81 (m, 2H), 2.84-2.56 (m, 10H), 2.14-2.10 (m, 4H), 1.80-1.61 (m, 6H), 1.48-1.41 (m, 8H), 1.38-1.23 (m, 68H), 0.91-0.86 (m, 12H).

[0224] Example 9: Preparation of ((2-hydroxyethyl)azanediyl)bis(octan-8, 1-diyl) (2E, 2'E)-di(3-butyl non-2-enoate) (CPL-455-01)

[0225]

[0226] Preparation of 8-bromooctyl 3-butylhept-2-enoate (Compound 2)

[0227] A mixture of Compound 1 (1.5 g, 7.07 mmol, 1.0 eq), 8-bromooctan-1-ol (1.77 g, 8.48 mmol, 1.2 eq), and TsOH-H20 (135 mg, 0.707 mmol, 0.1 eq) in toluene (30 mL) was stirred at 120 °C under nitrogen atmosphere for 16 h. The mixture was concentrated and purified by silica gel column to give Compound 2 (2.69 g, 94.7% yield) as a colorless oil.

[0228] 1 1H NMR (400 MHz, CDC13-d) δ 5.61 (s, 2H), 4.08-4.05 (m, 4H), 3.82-3.81 (m, 2H), 2.84-2.56 (m, 10H), 2.14-2.10 (m, 4H), 1.80-1.61 (m, 6H), 1.48-1.41 (m, 8H), 1.38-1.23 (m, 68H), 0.91-0.86 (m, 12H).

[0229] Example 9: Preparation of ((2-hydroxyethyl)azanediyl)bis(octan-8, 1-diyl) (2E, 2'E)-di(3-butyl non-2-enoate) (CPL-455-01)

[0230] A mixture of compound 2 (906 mg, 2.248 mmol, 2.2 eq), 2-aminoethan-1-ol (62 mg, 1.022 mmol, 1.0 eq), K2CO3(564 mg, 4.088 mmol, 4.0 eq) and Nal (15 mg, 0.1022 mmol, 0.1 eq) in ACN (15 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-455-01 (322 mg, 44.6% yield) as a colorless oil.

[0231] LCMS: LC / MS (ESI) m / z: 706.7 (M+H) + .

[0232] 1 H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.03 (m, 4H), 3.90-3.80 (m, 2H), 3.05-2.87 (m, 6H), 2.60-2.56 (m, 4H), 2.15-2.11 (m, 4H), 1.70-1.60 (m, 4H), 1.46-1.27 (m, 44H), 0.93-0.86 (m, 12H).

[0233] Example 10: Preparation of ((3-hydroxypropyl)azanediyl)bis(octane-8, 1-diyl) (2E, 2'E)-di(3-butylnon-2-enoate) (CPL-456-01)

[0234]

[0235] ((3-hydroxypropyl)azanediyl)bis(octane-8, 1-diyl) (2E, 2'E)-di(3-butylnon-2-enoate) (CPL-456-01)

[0236] A mixture of compound 1 (903 mg, 2.24 mmol, 2.2 eq), 3-aminopropan-1-ol (76 mg, 1.02 mmol, 1.0 eq), K2CO3(562 mg, 4.07 mmol, 4.0 eq) and Nal (15 mg, 0.10 mmol, 0.1 eq) in ACN (15 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-456-01 (351 mg, 47.8% yield) as a colorless oil.

[0237] LCMS: LC / MS (ESI) m / z: 720.0 (M+H) + .

[0238] 1 H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.03 (m, 4H), 3.83-3.80 (m, 2H), 3.03-2.85 (m, 2H), 2.88-2.65 (m, 2H), 2.60-2.56 (m, 4H), 2.15-2.11 (m, 4H), 1.95-1.80 (m, 2H), 1.72-1.55 (m, 6H), 1.46-1.27 (m, 44H), 0.93-0.86 (m, 12H).

[0239] Example 11: Preparation of ((4-hydroxybutyl)azanediyl)bis(octan-8,1-diyl) (2E,2'E)- di(3-butylnon-2-enoate) (CPL-457-01)

[0240]

[0241] ((4-hydroxybutyl)azanediyl)bis(octan-8,1-diyl) (2E,2'E)-di(3-butylnon-2-enoate) (CPL-457)

[0242] A mixture of compound 1 (881 mg, 2.186 mmol, 2.2 eq), 4-aminobutan-1-ol (88 mg, 0.994 mmol, 1.0 eq), K2CO3(548 mg, 3.976 mmol, 4.0 eq), and Nal (15 mg, 0.0994 mmol, 0.1 eq) in ACN (15 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-457-01 (334 mg, 45.8% yield) as a colorless oil.

[0243] LCMS: LC / MS (ESI) m / z: 734.7 (M+H) + .

[0244] 1 H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.08-4.03 (m, 4H), 3.73-3.70 (m, 2H), 3.03-2.87 (m, 4H), 2.60-2.56 (m, 4H), 2.15-2.12 (m, 4H), 2.00-1.93 (m, 2H), 1.72-1.62 (m, 6H), 1.46-1.28 (m, 46H), 0.93-0.86 (m, 12H).

[0245] Example 12: Preparation of 5-((2-hydroxyethyl)(6-((3-octylundec-2-enoyl)oxy)hexyl)amino)pentyl dodecanoate (CPL-002)

[0246]

[0247] A mixture of compound dodecanoic acid-5-bromopentyl ester (131 mg, 0376 mmol, 1.1 eq), 6-(2-hydroxyethylamino)-hexan-1-yl 3-octylundec-2-enoate (150 mg, 0.342 mmol, 1.0 eq), K2CO3(47 mg, 0.342 mmol, 1 eq), Cs2CO3(33 mg, 0.1026 mmol, 0.3 eq) and Nal (15 mg, 0.1026 mmol, 0.3 eq) in ACN (6 mL) was stirred at 85 °C for 16 hours. 40 mL of water was added and extracted with 20 mL of ethyl acetate three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-002 (60 mg, 25% yield) as a colorless oil.

[0248] LCMS: LC / MS (ESI) m / z: 708.7 (M+H) + .

[0249] 1H NMR (400 MHz, CDC13) δ 5.54 (s, 1H), 3.97-4.01 (m, 4H), 3.50-3.53 (m, 2H), 2.56-2.59 (m, 2H), 2.54 - 2.39 (m, 6H), 2.22 (t, J = 7.5 Hz, 2H), 2.09 - 2.00 (m, 2H), 1.53 - 1.61 (m, 6H), 1.48 - 1.42 (m, 3H), 1.40 - 1.34 (m, 4H), 1.33 - 1.28 (m, 4H), 1.20 (d, J = 10.3 Hz, 40H), 0.79 - 0.82 (m, 9H).

[0250] Example 13: Preparation of ((4-hydroxybutyl)azanediyl)bis(pentan-5,1-diyl) (2E,2'E)-bis(3-hexylundec-2-enoate) (CPL-003)

[0251]

[0252] A mixture of (E)-5-bromopentyl-3-hexylundec-2-enoate (50 mg, 0.12 mmol, 1.0 equiv), 4-amino-1-butanol (5.34 mg, 0.06 mmol, 0.3 equiv), K2CO3(49.68 mg, 0.36 mmol, 3.0 equiv) and Nal (5.4 mg, 0.036 mmol, 0.3 equiv) in ACN (1 mL) was stirred at 80 °C for 24 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-003 (28.81 mg, 30.7% yield) as a colorless oil.

[0253] LCMS: LCMS (ESI) m / z: 762.69 (M+H) + .

[0254] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 1H), 4.07 (q, J = 6.2 Hz, 4H), 3.73 - 3.65 (m, 2H), 3.00 (d, J = 23.7 Hz, 2H), 2.90 (d, J = 13.6 Hz, 4H), 2.58 (m, 3H), 2.13 (t, J = 7.7 Hz, 3H), 2.06 - 2.00 (m, 2H), 1.92 - 1.85 (m, 2H), 1.83 - 1.74 (m, 4H), 1.69 (d, J = 6.9 Hz, 4H), 1.43 (m, 11H), 1.29 (m, 36H), 0.89 (m, 12H).

[0255] Example 14: Preparation of ((3-hydroxypropyl)azanediyl)bis(butan-4, 1-diyl) (2E, 2'E)-bis(3-butylundec-2-enoate) (CPL-021)

[0256]

[0257] A mixture of (E)-4-bromobutyl-3-butylandec-2-enoate (286 mg, 0.762 mmol, 2.4 equiv), 3-amino-1-propanol (24 mg, 0.317 mmol, 1.0 equiv), K2CO3(175 mg, 1.268 mmol, 4.0 equiv) and Nal (24 mg, 0.159 mmol, 0.5 equiv) in ACN (15 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-021 (49 mg, 23.1% yield) as a colorless oil.

[0258] LCMS: LC / MS (ESI) m / z: 663.58 (M+H) +

[0259] 1H NMR (400 MHz, CDCL3) δ 5.62 (s, 1H), 4.10-4.06 (m, 4H), 3.81-3.78 (m, 2H), 2.65-2.56 (m, 6H), 2.48-2.44 (m, 4H), 2.15-2.10 (m, 4H), 1.70-1.54 (m, 6H), 1.50-1.19 (m, 37H), 0.93-0.87 (m, 12H).

[0260] Example 15: Preparation of ((3-hydroxypropyl)azanediyl)bis(butan-4, 1-diyl) (2E, 2'E)-bis(2- ylidenehexyl decanoate) (CPL-025)

[0261]

[0262] A mixture of (E)-4-bromobutyl-3-pentylundec-2-enoate (300 mg, 0.77 mmol, 2.2 eq), 3- amino-1-propanol (24.19 mg, 0.32 mmol, 1.0 eq), K2CO3(89.02 mg, 0.64 mmol, 2.0 eq) and Nal (9.65 mg, 0.06 mmol, 0.2 eq) in DMF (5 mL) was stirred at 80 °C for 48 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-025 (66.75 mg, 29.93% yield) as a colorless oil.

[0263] LCMS: LC / MS (ESI) m / z: 892.16 (M+H) + .

[0264] 1H NMR (400 MHz, CDCL3) δ 5.62 (s, 2H), 4.08 (s, 4H), 3.79 (s, 2H), 2.64 (d, J = 3.7 Hz, 2H), 2.58 (s, 4H), 2.45 (s, 4H), 2.13 (s, 4H), 1.25 (s, 46H), 0.88 (s, 12H).

[0265] Example 16: Preparation of ((3-hydroxypropyl)azanediyl)bis(butan-4, 1-diyl) (2Z, 2'Z)-bis(3- pentyltridecyl-2-enoate) (CPL-026)

[0266]

[0267] A mixture of (Z)-4-bromobutyl-3-pentyltridec-2-enoate (410 mg, 0.985 mmol, 2.1 eq), 3-amino-1-propanol (35.78 mg, 0.47 mmol, 1.0 eq), K2CO3(259.44 mg, 1.88 mmol, 4.0 eq) and Nal (35.22 mg, 0.235 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-026 (111.03 mg, 31.6% yield) as a colorless oil.

[0268] LCMS: LCMS (ESI) m / z: 748.67 (M+H) + .

[0269] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (d, J = 1.3 Hz, 2H), 4.08 (t, J = 6.3 Hz, 4H), 3.79 (t, J = 5.2 Hz, 2H), 2.65 (s, 2H), 2.61 - 2.55 (m, 4H), 2.46 (s, 4H), 2.13 (m, 4H), 1.66 (m, 4H), 1.57 (d, J = 7.0 Hz, 4H), 1.44 (t, J = 7.1 Hz, 6H), 1.35 - 1.23 (m, 40H), 0.90 - 0.86 (m, 12H).

[0270] Example 17: Preparation of ((3-hydroxypropyl)azanediyl)bis(butan-4,1-diyl) (2E,2'E)-bis(3-hexyldecyl-2-enoate) (CPL-031)

[0271]

[0272] A mixture of (Z)-4-bromobutyl-3-hexyldec-2-enoate (180 mg, 0.4 mmol, 2.1 eq), 3-amino-1-propanol (15 mg, 0.23 mmol, 1.0 eq), K2CO3(110.5 mg, 0.8 mmol, 4.0 eq) and Nal (15 mg, 0.1 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-031 (49.42 mg, 9% yield) as a colorless oil.

[0273] LCMS (ESI) m / z: 692.12 (M+H) + .

[0274] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.09 (t, J = 6.4 Hz, 4H), 3.84 - 3.73 (m, 2H), 2.72 - 2.54 (m, 6H), 2.46 (s, 4H), 2.15 - 2.08 (m, 4H), 1.65 (s, 4H), 1.31 - 1.24 (m, 42H), 0.94-0.87 (m, 12H).

[0275] Example 18: Preparation of ((3-hydroxypropyl)azanediyl)bis(butan-4, 1-diyl) (2Z, 2’Z)-bis(3-heptylundec-2-enoate) (CPL-032)

[0276]

[0277] A mixture of (Z)-4-bromobutyl-3-heptylundec-2-enoate (230 mg, 0.553 mmol, 2.1 eq), 3-amino-1-propanol (19.74 mg, 0.236 mmol, 1.0 eq), K2CO3(145.59 mg, 1.052 mmol, 4.0 eq) and Nal (19.59 mg, 0.131 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-032 (79.86 mg, 40.6% yield) as a colorless oil.

[0278] LCMS (ESI) m / z: 748.67 (M+H) + .

[0279] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.13 - 4.06 (m, 4H), 3.81 (t, J = 5.3 Hz, 2H), 3.18 - 2.41 (m, 10H), 2.18 - 2.09 (m, 4H), 1.58 - 1.42 (m, 10H), 1.29 (t, J = 6.7 Hz, 44H), 0.88 (m, 12H).

[0280] Example 19: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5, 1-diyl) (2E, 2'E)-bis(3-propylundec-2-enoate) (CPL-039)

[0281]

[0282] A mixture of (E)-5-bromopentyl-3-propylundec-2-enoate (313 mg, 0.834 mmol, 2.4 eq), 3-amino-1-propanol (26 mg, 0.347 mmol, 1.0 eq), K2CO3(191 mg, 1.388 mmol, 4.0 eq), and Nal (5.2 mg, 0.0347 mmol, 0.1 eq) in ACN (8 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-039 (72 mg, 31.3% yield) as a colorless oil.

[0283] LCMS: LC / MS (ESI) m / z: 664.5 (M+H) + .

[0284] 1H NMR (400 MHz, CDCL3) δ 5.62 (d, J = 5.2 Hz, 2H), 4.09 (t, J = 6.8 Hz, 4H), 3.80 (t, J = 5.2 Hz, 2H), 2.64 (t, J = 5.2 Hz, 2H), 2.59-2.55 (m, 4H), 2.44-2.40 (m, 4H), 2.14-2.09 (m, 4H), 1.70-1.62 (m, 6H), 1.54-1.41 (m, 10H), 1.37-1.26 (m, 26H), 0.96-0.86 (m, 12H).

[0285] Example 20: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5, 1-diyl) (2E, 2'E)-bis(3-butylundec-2-enoate) (CPL-043)

[0286]

[0287] A mixture of (E)-5-bromopentyl-3-butyloctadeca-2-enate (379 mg, 0.973 mmol, 2.4 equiv), 3-amino-1-propanol (30 mg, 0.406 mmol, 1.0 equiv), K2CO3(224 mg, 1.624 mmol, 4.0 equiv) and Nal (30 mg, 0.203 mmol, 0.5 equiv) in ACN (4 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-043 (99 mg, 35.9% yield) as a colorless oil.

[0288] LCMS: LC / MS (ESI) m / z: 691.61 (M+H) + .

[0289] 1H NMR (400 MHz, CDCL3) δ 5.62 (s, 1H), 4.09-4.06 (m, 4H), 3.80-3.78 (m, 2H), 2.66-2.56 (m, 6H), 2.44-2.41 (m, 4H), 2.15-2.12 (m, 4H), 1.79-1.63 (m, 6H), 1.56-1.19 (m, 41H), 0.96-0.83 (m, 12H).

[0290] Example 21: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5,1-diyl) (2E,2'E)-bis(3-butyltridec-2-enoate) (CPL-044)

[0291]

[0292] A mixture of (E)-5-bromopentyl-3-butyloctadeca-2-enate (379 mg, 0.973 mmol, 2.4 equiv), 3-amino-1-propanol (30 mg, 0.406 mmol, 1.0 equiv), K2CO3(224 mg, 1.624 mmol, 4.0 equiv) and Nal (30 mg, 0.203 mmol, 0.5 equiv) in ACN (4 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-043 (99 mg, 35.9% yield) as a colorless oil.

[0293] LCMS: LC / MS (ESI) m / z: 691.61 (M+H) + .

[0294] 1H NMR (400 MHz, CDCL3) δ 5.61 (s, 2H), 4.09-4.05 (m, 4H), 3.80-3.77 (m, 2H), 2.64-2.63 (m, 2H), 2.58-2.53 (m, 4H), 2.52-2.43 (m, 4H), 2.13-2.04 (m, 4H), 1.68-1.65 (m, 6H), 1.35-1.26 (m, 49H), 0.93-0.79 (m, 12H).

[0295] Example 22: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-pentylundec-2-enoate) (CPL-047)

[0296]

[0297] A mixture of (E)-5-bromopentyl-3-pentylundec-2-enoate (570 mg, 1.4 mmol, 2.2 eq), 3-amino-1-propanol (45 mg, 0.6 mmol, 1.0 eq), K2CO3(333 mg, 1.31 mmol, 2 eq) and Nal (44 mg, 0.02 mmol, 0.2 eq) in DMF (5 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-047 (23.12 mg, 2% yield) as a colorless oil.

[0298] LCMS: LC / MS (ESI) m / z: 720.1 (M+H) + .

[0299] 1H NMR (400 MHz, CDCL3) δ 5.61 (s, 2H), 4.09-4.05 (m, 4H), 3.80-3.77 (m, 2H), 2.64-2.63 (m, 2H), 2.58-2.53 (m, 4H), 2.52-2.43 (m, 4H), 2.13-2.04 (m, 4H), 1.68-1.65 (m, 6H), 1.35-1.26 (m, 49H), 0.93-0.79 (m, 12H).

[0300] Example 23: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5,1-diyl) (2E,2'E)-bis(3-pentyltridecyl-2-enoate) (CPL-048)

[0301]

[0302] A mixture of (E)-5-bromopentyl-3-pentyltridec-2-enoate (210 mg, 0.488 mmol, 2.1 eq), 3-amino-1-propanol (17.44 mg, 0.232 mmol, 1.0 eq), K2CO3(325.82 mg, 0.928 mmol, 4.0 eq) and Nal (17.38 mg, 0.116 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-048 (67.42 mg, 37.4% yield) as a colorless oil.

[0303] LCMS (ESI) m / z: 776.71 (M+H) + .

[0304] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.07 (t, J = 6.7 Hz, 4H), 3.79 (t, J = 5.1 Hz, 2H), 2.64 (s, 2H), 2.60 - 2.53 (m, 4H), 2.46 - 2.38 (m, 4H), 2.16 - 2.09 (m, 4H), 1.66 (s, 4H), 1.49 - 1.39 (m, 10H), 1.35 - 1.23 (m, 44H), 0.90 - 0.86 (m, 12H).

[0305] Example 24: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5,1-diyl) (2E,2'E)-bis(3-hexylnon-2-enoate) (CPL-049)

[0306]

[0307] A mixture of (E)-6-bromopentyl-3-hexylnon-2-enoate (480 mg, 1.24 mmol, 2.4 eq), 3-amino-1-propanol (39 mg, 0.51 mmol, 1.0 eq), K2CO3(285 mg, 2.06 mmol, 4.0 eq) and Nal (9 mg, 0.1 mmol, 0.2 eq) in ACN (10 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-049 (78 mg, 21% yield) as a colorless oil.

[0308] LC / MS (ESI) m / z: 692.7 (M+H) + .

[0309] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.17 - 4.00 (m, 4H), 3.94 - 3.60 (m, 2H), 3.11 - 2.85 (m, 2H), 2.86 - 2.62 (m, 4H), 2.62 - 2.51 (m, 4H), 2.20 - 2.09 (m, 4H), 1.94 - 1.79 (m, 2H), 1.73 - 1.33 (m, 26H), 0.97 - 0.81 (m, 12H).

[0310] Example 25: Preparation of ((3-hydroxypropyl)azanediyl)bis(pentan-5, 1-diyl) (2E, 2'E)-bis(3-butyldec-2-enoate) (CPL-052)

[0311]

[0312] A mixture of (E)-5-bromopentyl-3-butyldec-2-enoate (420 mg, 1.12 mmol, 2.1 eq), 3-amino-1-propanol (38.2 mg, 0.48 mmol, 1.0 eq), K2CO3(280.6 mg, 1.9 mmol, 4.0 eq) and Nal (45 mg, 0.24 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-052 (93.01 mg, 12% yield) as a colorless oil.

[0313] LCMS (ESI) m / z: 664 (M+H) + .

[0314] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.07 (t, J = 6.8 Hz, 4H), 3.86 - 3.74 (m, 2H), 2.69 - 2.52 (m, 6H), 2.45 - 2.36 (m, 4H), 2.12 (d, J = 6.8 Hz, 4H), 1.52 - 1.24 (m, 22H), 0.94 - 0.87 (m, 12H).

[0315] Example 26: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6, 1-diyl) (2E, 2'E)-bis(3-propylundec-2-enoate) (CPL-061)

[0316]

[0317] A mixture of (E)-6-bromohexyl-3-propylundec-2-enoate (440 mg, 1.13 mmol, 2.4 equiv), 3-amino-1-propanol (35 mg, 0.47 mmol, 1.0 equiv), K2CO3(259 mg, 1.88 mmol, 4.0 equiv), and Nal (7 mg, 0.047 mmol, 0.1 equiv) in ACN (6 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-061 (105 mg, 32.3% yield) as a colorless oil.

[0318] LCMS: LC / MS (ESI) m / z: 692.6 (M+H) + .

[0319] 1H NMR (400 MHz, CDCL3) δ 5.62 (d, J = 5.2 Hz, 2H), 4.08 (t, J = 6.8 Hz, 4H), 3.80 (t, J = 5.2 Hz, 2H), 2.64 (t, J = 5.2 Hz, 2H), 2.59-2.55 (m, 4H), 2.41-2.38 (m, 4H), 2.14-2.09 (m, 4H), 1.69-1.61 (m, 6H), 1.52-1.27 (m, 40H), 0.96-0.86 (m, 12H).

[0320] Example 27: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-butylundec-2-enoate) (CPL-065)

[0321]

[0322] A mixture of (E)-6-bromohexyl-3-propylundec-2-enoate (440 mg, 1.13 mmol, 2.4 equiv), 3-amino-1-propanol (35 mg, 0.47 mmol, 1.0 equiv), K2CO3(259 mg, 1.88 mmol, 4.0 equiv), and Nal (7 mg, 0.047 mmol, 0.1 equiv) in ACN (6 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-061 (105 mg, 32.3% yield) as a colorless oil.

[0323] LCMS: LC / MS (ESI) m / z: 692.6 (M+H) + .

[0324] 1H NMR (400 MHz, CDCL3) δ 5.61 (s, 1H), 4.08-4.05 (m, 4H), 3.81-3.78 (m, 2H), 2.64-2.56 (m, 6H), 2.43-2.39 (m, 4H), 2.15-2.11 (m, 4H), 1.69-1.61 (m, 6H), 1.50-1.19 (m, 45H), 0.94-0.87 (m, 12H).

[0325] Example 28: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-butyl-2-tridecenoate) (CPL-066)

[0326]

[0327] A mixture of (E)-6-bromohexyl-3-butyltridec-2-enoate (408 mg, 0.946 mmol, 2.4 eq), 3-amino-1-propanol (30 mg, 0.394 mmol, 1.0 eq), K2CO3(218 mg, 1.576 mmol, 4.0 eq) and Nal (30 mg, 0.197 mmol, 0.5 eq) in ACN (4 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-066 (126.09 mg, 41.22% yield) as a colorless oil.

[0328] LCMS: LC / MS (ESI) m / z: 776.29 (M+H) + .

[0329] 1H NMR (400 MHz, CDCL3) δ 5.61 (s, 1H), 4.08-4.05 (m, 4H), 3.81-3.78 (m, 2H), 2.64-2.56 (m, 6H), 2.43-2.39 (m, 4H), 2.15-2.11 (m, 4H), 1.69-1.61 (m, 6H), 1.50-1.19 (m, 45H), 0.94-0.87 (m, 12H).

[0330] Example 29: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2Z,2'Z)-bis(3-pentyltridecyl-2-enoate) (CPL-070)

[0331]

[0332] A mixture of (E)-6-bromohexyl-3-pentyltridec-2-enoate (430 mg, 0.968 mmol, 2.1 eq), 3-amino-1-propanol (34.57 mg, 0.461 mmol, 1.0 eq), K2CO3(254.47 mg, 1.84 mmol, 4.0 eq), and Nal (34.5 mg, 0.230 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate three times. The organic phase was washed with 30 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-070 (90.91 mg, 24.32% yield) as a colorless oil.

[0333] LCMS (ESI) m / z: 804.74 (M+H) + .

[0334] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.07 (t, J = 6.7 Hz, 4H), 3.79 (t, J = 5.1 Hz, 2H), 2.64 (s, 2H), 2.61 - 2.54 (m, 4H), 2.41 (d, J = 8.6 Hz, 4H), 2.12 (t, J = 7.7 Hz, 4H), 1.64 (d, J = 7.7 Hz, 4H), 1.46 - 1.37 (m, 10H), 1.34 - 1.22 (m, 48H), 0.89 (m, 12H).

[0335] Example 30: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl) (2E,2'E)-bis(3-hexylnon-2-enoate) (CPL-071)

[0336]

[0337] A mixture of (E)-6-bromohexyl-3-hexyl non-2-enoate (400 mg, 0.99 mmol, 2.4 eq), 3-amino-1-propanol (31 mg, 0.41 mmol, 1.0 eq), K2CO3(229 mg, 1.576 mmol, 4.0 eq), and Nal (13 mg, 0.08 mmol, 0.2 eq) in ACN (10 mL) was stirred at 80 °C for 16 h. The mixture was concentrated and purified by preparative HPLC to give the target CPL-071 (101 mg, 34% yield) as a colorless oil.

[0338] LC / MS (ESI) m / z: 720.7 (M+H) + .

[0339] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.19 - 3.97 (m, 4H), 3.93 - 3.71 (m, 2H), 3.04 - 2.87 (m, 2H), 2.85 - 2.64 (m, 4H), 2.64 - 2.49 (m, 4H), 2.20 - 2.08 (m, 4H), 1.92 - 1.79 (m, 2H), 1.77 - 1.35 (m, 48H), 0.97 - 0.78 (m, 12H).

[0340] Example 31: Preparation of ((3-hydroxypropyl)azanediylbis(hexan-6,1-diyl)(2E,2'E)-bis(3-butyldec-2-enoate) (CPL-074)

[0341]

[0342] A mixture of (E)-6-bromohexyl-3-butanoyldec-2-enoate (387 mg, 0.95 mmol, 2.1 eq), 3-amino-1-propanol (36 mg, 0.48 mmol, 1.0 eq), K2CO3(263 mg, 1.9 mmol, 4.0 eq) and Nal (36 mg, 0.24 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-074 (86.55 mg, 13% yield) as a colorless oil.

[0343] LCMS: LCMS (ESI) m / z: 692.12 (M+H) + .

[0344] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 2H), 4.19 - 3.97 (m, 4H), 3.93 - 3.71 (m, 2H), 3.04 - 2.87 (m, 2H), 2.85 - 2.64 (m, 4H), 2.64 - 2.49 (m, 4H), 2.20 - 2.08 (m, 4H), 1.92 - 1.79 (m, 2H), 1.77 - 1.35 (m, 48H), 0.97 - 0.78 (m, 12H).

[0345] Example 32: Preparation of ((3-hydroxypropyl)azanediyl)bis(hexan-6,1-diyl)(2E,2'E)-bis(3-octyltridecyl-2-enoate) (CPL-079)

[0346]

[0347] A mixture of (E)-6-bromohexyl-3-octyltridec-2-enoate (420 mg, 0.8642 mmol, 2.3 eq), 3-amino-1-propanol (28 mg, 0.3757 mmol, 1.0 eq), K2CO3(156 mg, 1.1271 mmol, 3.0 eq), and Cs2CO3(61 mg, 0.1879 mmol, 0.5 eq) in ACN (8 mL) was stirred at 90 °C for 24 h. 40 mL of water was added and extracted with 20 mL of ethyl acetate for three times. The organic phase was washed with 40 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-079 (76.07 mg, 10% yield) as a colorless oil.

[0348] LCMS: LC / MS (ESI) m / z: 888.9 (M+H) + .

[0349] 1H NMR (400 MHz, CDCl3) δ 5.54 (s, 2H), 4.00 (t, J = 6.7 Hz, 4H), 3.71 (dd, J = 10.5, 5.4 Hz, 2H), 2.52 (dd, J = 19.0, 11.4 Hz, 6H), 2.34 (s, 2H), 2.10 - 2.02 (m, 4H), 1.98 - 1.93 (m, 1H), 1.59 (dd, J = 13.9, 7.0 Hz, 6H), 1.40 - 1.36 (m, 6H), 1.19 (s, 64H), 0.81 (dd, J = 6.9, 5.6 Hz, 12H).

[0350] Example 33: Preparation of ((3-hydroxypropyl)azanediyl)bis(heptane-7,1-diyl) (2E,2'E)-bis(3-pentylnon-2-enoate) (CPL-090)

[0351]

[0352] A mixture of (E)-6-bromoheptyl-3-pentanylnon-2-enoate (565 mg, 1.4055 mmol, 2.3 eq), 3-amino-1-propanol (45 mg, 0.6111 mmol, 1.0 eq), K2CO3(253 mg, 1.833 mmol, 3.0 eq), Cs2CO3(99 mg, 0.3056 mmol, 0.5 eq), and Nal (45 mg, 0.3056 mmol, 0.5 eq) in ACN (8 mL) was stirred at 80 °C for 48 h. 40 mL of water was added and extracted with 20 mL of ethyl acetate three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-090 (148.8 mg, 15% yield) as a colorless oil.

[0353] LCMS: LC / MS (ESI) m / z: 720.7 (M+H) + .

[0354] 1H NMR (400 MHz, CDCl3) δ 5.54 (s, 2H), 4.00 (t, J = 6.7 Hz, 4H), 3.75 - 3.70 (m, 2H), 2.52 (dd, J = 20.2, 12.6 Hz, 6H), 2.41 - 2.27 (m, 4H), 2.10 - 2.02 (m, 4H), 1.58 (dd, J = 13.9, 7.2 Hz, 6H), 1.38 (d, J = 7.0 Hz, 8H), 1.23 (ddd, J = 18.4, 10.2, 5.8 Hz, 36H), 0.84 - 0.80 (m, 12H).

[0355] Example 34: Preparation of ((3-hydroxypropyl)azanediyl)bis(heptane-7,1-diyl) (2E,2'E)-bis(3-butyldecan-2-enoate) (CPL-096)

[0356]

[0357] A mixture of (E)-6-bromoheptyl-3-butyloct-2-enoate (421 mg, 0.95 mmol, 2.1 eq), 3-amino-1-propanol (33 mg, 0.44 mmol, 1.0 eq), K2CO3(235 mg, 1.7 mmol, 4.0 eq) and Nal (32 mg, 0.22 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-096 (101.27 mg, 5% yield) as a colorless oil.

[0358] LCMS: LCMS (ESI) m / z: 720.18 (M+H) + .

[0359] 1H NMR (400 MHz, CDC13) δ 5.63 (d, J = 9.3 Hz, 2H), 4.07 (s, 4H), 3.81 (s, 2H), 2.59 (s, 8H), 2.41 (s, 5H), 2.13 (s, 6H), 1.66 (s, 10H), 1.28 (s, 50H), 0.90 (s, 12H)

[0360] Example 35: Preparation of ((3-hydroxypropyl)azanediyl)bis(heptan-7,1-diyl) (2Z,2’Z)-bis(3-octyltridecan-2-enoate) (CPL-101)

[0361]

[0362] A mixture of (Z)-6-bromoheptyl-3-octyltridec-2-enoate (400 mg, 0.80 mmol, 2.3 eq), 3-amino-1-propanol (26 mg, 0.3478 mmol, 1.0 eq), K2CO3(144 mg, 1.0434 mmol, 3.0 eq), Cs2CO3(57 mg, 0.1739 mmol, 0.5 eq) and Nal (26 mg, 0.1739 mmol, 0.5 eq) in ACN (6 mL) was stirred at 85 °C for 48 h. 40 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 40 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-101 (145.15 mg, 20% yield) as a colorless oil.

[0363] LCMS: LC / MS (ESI) m / z: 916.7 (M+H)+ .

[0364] 1H NMR (400 MHz, CDC13) δ 5.54 (s, 2H), 4.01 - 3.97 (m, 4H), 3.77 - 3.74 (m, 2H), 2.96 (s, 2H), 2.90 (s, 1H), 2.53 - 2.49 (m, 2H), 1.99 (ddd, J = 31.2, 16.6, 9.0 Hz, 10H), 1.37 (dd, J = 3.4, 1.3 Hz, 2H), 1.29 (s, 6H), 1.21 (d, J = 14.6 Hz, 70H), 0.82 (d, J = 6.5 Hz, 12H).

[0365] Example 36: Preparation of ((3-hydroxypropyl)azanediyl)bis(octane-8, 1-diyl) (2E, 2'E)-bis(3-pentylnon-2-enoate) (CPL-112)

[0366]

[0367] A mixture of (E)-8-bromooctan-3-pentyl non-2-enoate (400 mg, 0.9615 mmol, 2.3 eq), 3-amino-1-propanol (31 mg, 0.4181 mmol, 1.0 eq), K2CO3(173 mg, 1.2543 mmol, 3.0 eq), Cs2CO3(68 mg, 0.2091 mmol, 0.5 eq) and NaI (31 mg, 0.2091 mmol, 0.5 eq) in ACN (8 mL) was stirred at 85 °C for 48 h. 40 mL of water was added and extracted with 20 mL of ethyl acetate for three times. The organic phase was washed with 40 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-112 (118.53 mg, 17% yield) as a colorless oil.

[0368] LC / MS (ESI) m / z: 748.7 (M+H) + .

[0369] 1H NMR (400 MHz, CDC13) δ 5.54 (s, 2H), 4.00 (t, J = 6.7 Hz, 4H), 3.77 - 3.66 (m, 2H), 2.52 (dd, J = 20.6, 13.0 Hz, 6H), 2.34 (s, 2H), 2.10 - 2.02 (m, 4H), 1.61 - 1.53 (m, 8H), 1.38 (d, J = 5.1 Hz, 8H), 1.30 - 1.17 (m, 40H), 0.85 - 0.80 (m, 12H).

[0370] Example 37: Preparation of ((3-hydroxypropyl)azanediyl)bis(nonane-9,1-diyl) (2E,2'E)-bis(3-propylundec-2-enoate) (CPL-127)

[0371]

[0372] A mixture of (E)-9-bromononanyl-3-propylundec-2-enoate (514 mg, 1.193 mmol, 2.4 equiv), 3-amino-1-propanol (37 mg, 0.497 mmol, 1.0 equiv), K2CO3(275 mg, 1.988 mmol, 4.0 equiv) and Nal (37 mg, 0.249 mmol, 0.5 equiv) in ACN (4 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by prep-HPLC to give the target CPL-127 (200.08 mg, 51.89% yield) as a colorless oil.

[0373] LCMS: LC / MS (ESI) m / z: 775.71 (M+H) + .

[0374] 1H NMR (400 MHz, CDCL3) δ 5.62-5.61 (m, 2H), 4.09-4.05 (m, 4H), 3.80 (t, J = 5.2 Hz, 2H), 2.70-2.66 (m, 2H), 2.59-2.54 (m, 4H), 2.55-2.40 (m, 4H), 2.14-2.09 (m, 4H), 1.68-1.65 (m, 6H), 1.35-1.26 (m, 53H), 0.96-0.86 (m, 12H).

[0375] Example 38: Preparation of (Z)-3-pentyltridec-2-enoic acid 9-((3- hydroxypropyl)(9-(E)-3-pentyltridec-2-enoyl)oxy)nonyl)amino)nonyl ester (CPL-136)

[0376]

[0377] A mixture of (E)-9-bromononanyl-3-pentanoyltridec-2-enoate (600 mg, 1.23 mmol, 2.1 eq), 3-amino-1-propanol (44 mg, 0.587 mmol, 1.0 eq), K2CO3(324.02 mg, 2.348 mmol, 4.0 eq), and Nal (43.92 mg, 0.293 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate three times. The organic phase was washed with 30 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-136 (269.34 mg, 51.63% yield) as a colorless oil.

[0378] LCMS (ESI) m / z: 888.83 (M+H) + .

[0379] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.07 (t, J = 6.8 Hz, 4H), 3.79 (t, J = 5.1 Hz, 2H), 2.70 - 2.61 (m, 2H), 2.61 - 2.52 (m, 4H), 2.46 - 2.36 (m, 4H), 2.12 (t, J = 7.7 Hz, 4H), 2.09 - 1.99 (m, 2H), 1.65 - 1.60 (m, 4H), 1.47 - 1.43 (m, 6H), 1.34 - 1.24 (m, 62H), 0.89 (d, J = 6.2 Hz, 12H).

[0380] Example 39: Preparation of ((3-hydroxypropyl)azanediyl)bis(decan-10,1-diyl) (2E,2'E)-bis(3-propylnon-2-enoate) (CPL-148)

[0381]

[0382] A mixture of (E)-10-bromodecanyl-3-propyl non-2-enoate (611 mg, 1.469 mmol, 2.4 eq), 3-amino-1-propanol (46 mg, 0.306 mmol, 0.5 eq), K2CO3(338 mg, 2.448 mmol, 4.0 eq), and Nal (46 mg, 0.306 mmol, 0.5 eq) in ACN (4 mL) was stirred at 80 °C for 40 h. The mixture was concentrated and purified by preparative HPLC to give the target CPL-148 (151.61 mg, 33.18% yield) as a colorless oil.

[0383] LCMS: LC / MS (ESI) m / z: 747.67 (M+H) + .

[0384] 1H NMR (400 MHz, CDCL3) δ 5.62-5.61 (m, 2H), 4.09-4.05 (m, 4H), 3.80 (t, J = 5.2 Hz, 2H), 2.70-2.66 (m, 2H), 2.59-2.54 (m, 4H), 2.55-2.40 (m, 4H), 2.14-2.09 (m, 4H), 1.68-1.65 (m, 6H), 1.35-1.26 (m, 49H), 0.96-0.86 (m, 12H).

[0385] Example 40: Preparation of ((3-hydroxypropyl)azanediylbis(decan-10,1-diyl) (2Z,2'Z)-bis(3-butylnon-2-enate) (CPL-152)

[0386]

[0387] A mixture of (E)-10-bromodecanyl-3-butanyl non-2-enate (215 mg, 0.5 mmol, 2.1 eq), 3-amino-1-propanol (17.86 mg, 0.238 mmol, 1.0 eq), K2CO3(131.38 mg, 0.952 mmol, 4.0 eq) and Nal (17.84 mg, 0.119 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL water was added and extracted with 20 mL ethyl acetate for three times. The organic phase was washed with 30 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-152 (103.64 mg, 56.13% yield) as a colorless oil.

[0388] LCMS (ESI) m / z: 776.71 (M+H) + .

[0389] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 2H), 4.07 (t, J = 6.8 Hz, 4H), 3.83 (t, J = 5.3 Hz, 2H), 2.95 (d, J = 63.2 Hz, 4H), 2.58 (m, 4H), 2.17 - 2.09 (m, 4H), 1.91 (s, 2H), 1.62 (d, J = 7.2 Hz, 6H), 1.45 - 1.25 (m, 52H), 0.89 (m, 12H).

[0390] Example 41: Preparation of ((3-hydroxypropyl)azanediyl)bis(undecan-11,1-diyl) (2E,2'E)-bis(3-butyldecan-2-enoate) (CPL-184)

[0391]

[0392] A mixture of (E)-11-bromoundecyl-3-butyldodec-2-enoate (154 mg, 0.95 mmol, 2.1 eq), 3-amino-1-propanol (11.25 mg, 0.15 mmol, 1.0 eq), K2CO3(82.8 mg, 0.6 mmol, 4.0 eq), and Nal (11 mg, 0.075 mmol, 0.5 eq) in ACN (5 mL) was stirred at 80 °C for 20 h. 30 mL of water was added and extracted with 20 mL of ethyl acetate three times. The organic phase was washed with 30 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-184 (26.73 mg, 9% yield) as a colorless oil.

[0393] LCMS (ESI) m / z: 832.39 (M+H) + .

[0394] 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 2H), 4.07 (t, J = 6.7 Hz, 4H), 3.86 - 3.74 (m, 2H), 2.69 - 2.52 (m, 6H), 2.45 - 2.36 (m, 4H), 2.12 (d, J = 6.8 Hz, 4H), 1.52 - 1.24 (m, 69H), 0.94 - 0.87 (m, 12H).

[0395] Example 42: Preparation of 3-(((E)-3-butyloct-2-enoyl)oxy)-2-((((2- (dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-199)

[0396]

[0397] A mixture of 3-((E)-3-butyloct-2-enate)-2-(hydroxymethyl)propane-1,2- diyl-(9Z,12Z)-octadeca-9,12-dienoate (250 mg, 0.27 mmol, 1.0 equiv), pyridine (216 mg, 2.73 mmol, 10.0 equiv) and triphosgene (33 mg, 0.10 mmol, 0.4 equiv) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. N,N-dimethylethanolamine (74 mg, 0.82 mmol, 3.0 equiv) was added and stirred at 30 °C for 16 h. 10 mL of water was added and extracted with 10 mL of dichloromethane for three times. The organic phase was washed with 20 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-199 (62 mg, 34% yield) as a colorless oil.

[0398] LC / MS (ESI) m / z: 664.6 (M+H) + .

[0399] 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 1H), 5.46 - 5.19 (m, 4H), 4.33 - 3.98 (m, 8H), 2.83 - 2.69 (m, 2H), 2.68 - 2.53 (m, 4H), 2.50 - 2.38 (m, 1H), 2.36 - 2.29 (m, 4H), 2.28 (s, 6H), 2.17 - 2.10 (m, 2H), 2.09 - 1.99 (m, 4H), 1.50 - 1.23 (m, 26H), 0.99 - 0.84 (m, 9H).

[0400] Example 43: Preparation of 3-((((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-((((E)-3- hexylundec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-203)

[0401]

[0402] A mixture of 3-((E)-3-hexylundec-2-enoyloxy)-2-(hydroxymethyl)propanoyl- (9Z,12Z)-octadeca-9,12-dienoate (200 mg, 0.339 mmol, 1.0 equiv), pyridine (268 mg, 3.39 mmol, 10.0 equiv) and triphosgene (41 mg, 0.136 mmol, 0.4 equiv) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. N,N-dimethylethanolamine (61 mg, 0.678 mmol, 2.0 equiv) was added and stirred at 30 °C for 16 h. 10 mL of water was added and extracted with 10 mL of dichloromethane for three times. The organic phase was washed with 20 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-203 (40 mg, 16.8% yield) as a colorless oil.

[0403] LC / MS (ESI) m / z: 706.6 (M+H) + .

[0404] 1H NMR (400 MHz, CDCl3) δ 5.60 (s, 1H), 5.46 - 5.30 (m, 4H), 4.34 - 4.07 (m, 8H), 2.85 - 2.71 (m, 2H), 2.71 - 2.62 (m, 2H), 2.62 - 2.54 (m, 2H), 2.48 - 2.23 (m, 9H), 2.18 - 2.09 (m, 2H), 2.09 - 1.94 (m, 5H), 1.69 - 1.20 (m, 30H), 0.91 - 0.82 (m, 9H).

[0405] Example 44: Preparation of 3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-((((E)-3- heptylundec-2-enoyl)oxy)methyl)propanoyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-208)

[0406]

[0407] A mixture of 3-((E)-3-heptanoyloxyundec-2-enoyl)-2-(hydroxymethyl)propanoyl- (9Z,12Z)-octadeca-9,12-dienoate (200 mg, 0.316 mmol, 1.0 equiv), pyridine (249.9 mg, 3.16 mmol, 10.0 equiv) and triphosgene (38.57 mg, 0.13 mmol, 0.4 equiv) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. N,N-dimethylpropan-1-amine (56.29 mg, 0.632 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-208 (28.01 mg, 11.84% yield) as a colorless oil.

[0408] LCMS (ESI) m / z: 748.6 (M+H) + .

[0409] 1H NMR (400 MHz, Chloroform-d) δ 5.60 (s, 1H), 5.40 - 5.29 (m, 4H), 4.32 - 4.19 (m, 4H), 4.15 (m, 4H), 2.81 - 2.61 (m, 4H), 2.61 - 2.54 (m, 2H), 2.44 (m, 1H), 2.42 - 2.25 (m, 8H), 2.13 (t, J = 7.7 Hz, 2H), 2.04 (t, J = 6.9 Hz, 4H), 1.44 (d, J = 7.3 Hz, 2H), 1.38 - 1.25 (m, 36H), 0.91 - 0.86 (m, 9H).

[0410] Example 45: Preparation of 3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2- (((3-octyloxyundec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-210)

[0411]

[0412] A mixture of 3-((E)-3-octylundec-2-enoyloxy)-2-(hydroxymethyl)propanyl- (9Z,12Z)-octadeca-9,12-dienoate (200 mg, 0.3096 mmol, 1.0 equiv), N,N- dimethylpropan-1 -amine (55 mg, 0.6192 mmol, 2.0 equiv), pyridine (245 mg, 3.096 mmol, 10.0 equiv) and triphosgene (37 mg, 0.1238 mmol, 0.4 equiv) in dichloromethane (7 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-210 (25.97 mg, 11% yield) as a colorless oil.

[0413] LC / MS (ESI) m / z: 761.62 (M+H) + .

[0414] 1H NMR (400 MHz, CDC13) δ 5.60 (s, 1H), 5.43 - 5.29 (m, 4H), 4.25 - 4.18 (m, 4H), 4.17 - 4.10 (m, 4H), 2.77 (t, J = 6.4 Hz, 2H), 2.58 (dd, J = 15.0, 7.3 Hz, 3H), 2.48 - 2.31 (m, 2H), 2.29 (s, 6H), 2.12 (dd, J = 18.8, 10.9 Hz, 2H), 2.07 - 2.01 (m, 4H), 1.44 (dd, J = 14.6, 7.0 Hz, 4H), 1.36 - 1.23 (m, 38H), 0.90 - 0.86 (m, 9H).

[0415] Example 46: Preparation of 3-(((Z)-3-butylnon-2-enoyl)oxy)-2-((((3- (dimethylamino)propoxy)carbonyl)oxy)methyl)propanyl (9Z,12Z)-octadeca-9,12- dienoate (CPL-218)

[0416]

[0417] A mixture of 3-((E)-3-butylnon-2-enoyloxy)-2-(hydroxymethyl)propanoyl-(9Z, 12Z)- octadeca-9, 12-dienoate (150 mg, 0.267 mmol, 1.0 equiv), pyridine (212.2 mg, 2.67 mmol, 10.0 equiv) and triphosgene (31.69 mg, 0.107 mmol, 0.4 equiv) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. N,N-dimethylpropan-1 -amine (54.95 mg, 0.533 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-218 (27.16 mg, 14.71 % yield) as a colorless oil.

[0418] LCMS (ESI) m / z: 692.54 (M+H) + .

[0419] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (s, 1H), 5.42 - 5.29 (m, 4H), 4.25 - 4.12 (m, 8H), 2.77 (t, J = 6.5 Hz, 2H), 2.63 - 2.53 (m, 2H), 2.45 (m, 1H), 2.39 (t, J = 7.3 Hz, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.25 (s, 6H), 2.18 - 2.11 (m, 2H), 2.05 (m, 4H), 1.43 (m, 4H), 1.40 - 1.21 (m, 26H), 0.90 (m, 9H).

[0420] Example 47: Preparation of 3-(((E)-3-butyl oct-2-enoyl)oxy)-2-((((3- (dimethylamino)propoxy)carbonyl)oxy)methyl)propanoyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-221)

[0421]

[0422] A mixture of 3-((E)-3-pentylnon-2-enoyloxy)-2-(hydroxymethyl)propanoyl- (9Z,12Z)-octadeca-9,12-dienoate (150 mg, 0.27 mmol, 1.0 equiv), pyridine (216 mg, 2.73 mmol, 10.0 equiv) and triphosgene (33 mg, 0.10 mmol, 0.4 equiv) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. N,N-dimethylpropan-1 -amine (83 mg, 0.82 mmol, 3.0 equiv) was added and stirred at room temperature for 16 h. 10 mL of water was added and extracted with 10 mL of dichloromethane for three times. The organic phase was washed with 20 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-221 (60 mg, 32% yield) as a colorless oil.

[0423] LC / MS (ESI) m / z: 678.6 (M+H) + .

[0424] 1H NMR (400 MHz, CDC13) δ 5.61 (s, 1H), 5.48 - 5.20 (m, 4H), 4.31 - 4.01 (m, 8H), 2.82 - 2.69 (m, 2H), 2.64 - 2.50 (m, 2H), 2.50 - 2.39 (m, 1H), 2.38 - 2.28 (m, 4H), 2.23 (s, 6H), 2.17 - 2.10 (m, 2H), 2.09 - 2.00 (m, 4H), 1.90 - 1.78 (m, 2H), 1.50 - 1.24 (m, 26H), 0.97 - 0.85 (m, 9H).

[0425] Example 48: Preparation of 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-2- ( (((E)-3-pentylnon-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-222)

[0426]

[0427] A mixture of 3-((E)-3-pentyl non-2-enate)-2-(hydroxymethyl)propane-(9Z,12Z)- octadeca-9,12-dienoate (200 mg, 0.3472 mmol, 1.0 equiv), N,N-dimethylpropan-1- amine (72 mg, 0.6944 mmol, 2.0 equiv), pyridine (274 mg, 3.472 mmol, 10.0 equiv), and triphosgene (41 mg, 0.1389 mmol, 0.4 equiv) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-222 (71.12 mg, 29% yield) as a colorless oil.

[0428] LC / MS (ESI) m / z: 705.9 (M+H) + .

[0429] 1H NMR (400 MHz, CDCl3) δ 5.54 (s, 1H), 5.33 - 5.21 (m, 4H), 4.16 - 4.07 (m, 8H), 2.70 (t, J = 6.5 Hz, 2H), 2.55 - 2.47 (m, 2H), 2.39 - 2.29 (m, 3H), 2.24 (t, J = 7.6 Hz, 2H), 2.18 (s, 6H), 2.09 - 2.04 (m, 2H), 1.98 (q, J = 6.9 Hz, 4H), 1.82 - 1.75 (m, 2H), 1.38 (dd, J = 10.0, 6.4 Hz, 4H), 1.22 (dd, J = 13.9, 11.3 Hz, 26H), 0.82 (dd, J = 7.0, 4.4 Hz, 9H).

[0430] Example 49: Preparation of 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-2- ( (((E)-3-pentyl undec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-223)

[0431]

[0432] A mixture of 3-((E)-3-pentylundec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z,12Z)- octadeca-9,12-dienoate (150 mg, 0.237 mmol, 1.0 equiv), pyridine (188 mg, 2.37 mmol, 10.0 equiv) and triphosgene (29 mg, 0.09 mmol, 0.4 equiv) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. A solution of N,N-dimethylpropan-1-amine (73 mg, 0.71 mmol, 3.0 equiv) in 1 mL of dichloromethane was added and stirred at 30 °C for 16 h. 10 mL of water was added and extracted with 10 mL of dichloromethane for three times. The organic phase was washed with 20 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-223 (36 mg, 21% yield) as a colorless oil.

[0433] LC / MS (ESI) m / z: 734.6 (M+H) + .

[0434] 1H NMR (400 MHz, CDCl3) δ 5.61 (s, 1H), 5.44 - 5.26 (m, 4H), 4.30 - 4.00 (m, 8H), 2.84 - 2.69 (m, 2H), 2.62 - 2.53 (m, 2H), 2.49 - 2.39 (m, 1H), 2.39 - 2.27 (m, 4H), 2.23 (s, 6H), 2.17 - 2.10 (m, 2H), 2.10 - 1.99 (m, 4H), 1.92 - 1.78 (m, 2H), 1.47 - 1.27 (m, 34H), 0.96 - 0.85 (m, 9H).

[0435] Example 50: Preparation of 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-2-((((E)-3- pentyltridec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-224)

[0436]

[0437] A mixture of 3-((E)-3-pentyltridec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z,12Z)- octadeca-9,12-dienoate (200 mg, 0.327 mmol, 1.0 equiv), pyridine (252 mg, 3.2 mmol, 10.0 equiv) and triphosgene (37 mg, 0.128 mmol, 0.4 equiv) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. N,N-dimethylpropan-1 -amine (98.9 mg, 0.96 mmol, 3.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-224 (45.26 mg, 18% yield) as a colorless oil.

[0438] Example 51: Preparation of 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-2- ( (((E)-3-heptyltridec-2-enoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (CPL-231)

[0439]

[0440] A mixture of 3-((E)-3-pentyltridec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z,12Z)- octadeca-9,12-dienoate (200 mg, 0.327 mmol, 1.0 equiv), pyridine (252 mg, 3.2 mmol, 10.0 equiv) and triphosgene (37 mg, 0.128 mmol, 0.4 equiv) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. N,N-dimethylpropan-1 -amine (98.9 mg, 0.96 mmol, 3.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-224 (45.26 mg, 18% yield) as a colorless oil.

[0441] LCMS (ESI) m / z: 790.65 (M+H) + .

[0442] 1H NMR (400 MHz, Chloroform-d) δ 5.60 (s, 1H), 5.35 (m, 4H), 4.23 - 4.13 (m, 8H), 2.77 (t, J = 6.5 Hz, 2H), 2.61 - 2.53 (m, 2H), 2.49 - 2.42 (m, 1H), 2.39 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.6 Hz, 2H), 2.25 (s, 6H), 2.13 (t, J = 7.7 Hz, 2H), 2.05 (m, 4H), 1.86 (m, 2H), 1.44 (m, 6H), 1.28 (m, 36H), 0.88 (m, 9H).

[0443] Example 52: Preparation of 3-((3-decyltridecyl-2-enoyl)oxy)-2-((((3- (dimethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-234)

[0444]

[0445] A mixture of 3-((E)-3-decyltridec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z, 12Z)- octadeca-9, 12-dienoate (190 mg, 0.2707 mmol, 1.0 equiv), N,N-dimethylpropan-1- amine (56 mg, 0.5413 mmol, 2.0 equiv), pyridine (214 mg, 2.707 mmol, 10.0 equiv), and triphosgene (32 mg, 0.1083 mmol, 0.4 equiv) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-234 (40.04 mg, 18% yield) as a colorless oil.

[0446] LC / MS (ESI) m / z: 832.7 (M+H) + .

[0447] 1H NMR (400 MHz, CDC13) δ 5.53 (s, 1H), 5.31 - 5.22 (m, 4H), 4.17 - 4.05 (m, 8H), 2.70 (t, J = 6.2 Hz, 2H), 2.55 - 2.47 (m, 2H), 2.33 (dd, J = 14.9, 7.4 Hz, 2H), 2.27 - 2.15 (m, 9H), 2.09 - 2.02 (m, 2H), 1.98 (q, J = 6.7 Hz, 5H), 1.84 - 1.73 (m, 3H), 1.37 (d, J = 4.5 Hz, 4H), 1.24 - 1.17 (m, 42H), 0.83 - 0.80 (m, 9H).

[0448] Example 53: Preparation of 3-(((4-(dimethylamino)butyloxy)carbonyl)oxy)-2- (((E)-3-heptylundec-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-252)

[0449]

[0450] A mixture of 3-((E)-3-heptylundec-2-enate)-2-(hydroxymethyl)propanoyl-(9Z, 12Z)- octadeca-9, 12-dienoate (200 mg, 0.316 mmol, 1.0 equiv), pyridine (249.9 mg, 3.16 mmol, 10.0 equiv) and triphosgene (38.57 mg, 0.130 mmol, 0.4 equiv) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. N,N-dimethylbutanolamine (74.02 mg, 0.632 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-252 (30.28 mg, 12.3% yield) as a colorless oil.

[0451] LCMS (ESI) m / z: 776.63 (M+H) + .

[0452] 1H NMR (400 MHz, Chloroform-d) δ 5.61 (d, J = 6.2 Hz, 1H), 5.42 - 5.29 (m, 4H), 4.26 - 4.09 (m, 8H), 2.77 (t, J = 6.5 Hz, 2H), 2.58 (t, J = 7.8 Hz, 2H), 2.44 (m, 1H), 2.35 - 2.18 (m, 10H), 2.13 (t, J = 7.7 Hz, 2H), 2.06 (t, J = 6.6 Hz, 4H), 1.70 (m, 2H), 1.44 (d, J = 7.6 Hz, 4H), 1.37 - 1.24 (m, 36H), 0.89 (m, 9H).

[0453] Example 54: Preparation of 3-((3-decyltridecyl-2-enoyl)oxy)-2-((((4- (dimethylamino)butyloxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-256)

[0454]

[0455] A mixture of 3-((E)-3-decyltridec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z, 12Z)- octadeca-9, 12-dienoate (190 mg, 0.2707 mmol, 1.0 equiv), N,N-dimethylbutan-1-amine (63 mg, 0.5413 mmol, 2.0 equiv), pyridine (214 mg, 2.707 mmol, 10.0 equiv), and triphosgene (32 mg, 0.1083 mmol, 0.4 equiv) in dichloromethane (6 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-256 (36.08 mg, 16% yield) as a colorless oil.

[0456] LC / MS (ESI) m / z: 846.7 (M+H) + .

[0457] 1H NMR (400 MHz, CDC13) δ 5.53 (s, 1H), 5.35 - 5.21 (m, 4H), 4.18 - 4.03 (m, 8H), 2.70 (t, J = 6.5 Hz, 2H), 2.55 - 2.45 (m, 2H), 2.37 (dt, J = 9.2, 4.7 Hz, 1H), 2.27 - 2.18 (m, 4H), 2.14 (s, 6H), 2.09 - 2.03 (m, 2H), 1.98 (q, J = 6.6 Hz, 5H), 1.63 (dd, J = 13.0, 6.3 Hz, 3H), 1.35 (d, J = 4.7 Hz, 2H), 1.24 - 1.18 (m, 46H), 0.82 - 0.79 (m, 9H).

[0458] Example 55: Preparation of 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((E)-3- pentylnon-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-266)

[0459]

[0460] A mixture of 3-((E)-3-pentyl non-2-enate)-2-(hydroxymethyl)propanyl-(9Z, 12Z)- octadeca-9, 12-dienoate (200 mg, 0.3472 mmol, 1.0 equiv), N,N-diethylpropan-1- amine (91 mg, 0.6944 mmol, 2.0 equiv), pyridine (274 mg, 3.407 mmol, 10.0 equiv), and triphosgene (41 mg, 0.1389 mmol, 0.4 equiv) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-266 (35.80 mg, 14% yield) as a colorless oil.

[0461] LC / MS (ESI) m / z: 735.1 (M+H) + .

[0462] 1H NMR (400 MHz, CDC13) δ 5.54 (s, 1H), 5.33 - 5.22 (m, 4H), 4.16 - 4.07 (m, 8H), 2.70 (t, J = 6.5 Hz, 2H), 2.53 - 2.34 (m, 9H), 2.24 (t, J = 7.6 Hz, 2H), 2.10 - 2.03 (m, 2H), 1.98 (q, J = 6.8 Hz, 4H), 1.77 (s, 2H), 1.38 (dd, J = 8.5, 5.5 Hz, 4H), 1.22 (dd, J = 13.9, 11.3 Hz, 26H), 0.97 (t, J = 7.0 Hz, 6H), 0.82 (dd, J = 7.0, 4.3 Hz, 9H).

[0463] Example 56: Preparation of 3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-((((E)-3- heptylundec-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-274)

[0464]

[0465] A mixture of 3-((E)-3-heptylundec-2-enate)-2-(hydroxymethyl)propanyl-(9Z, 12Z)- octadeca-9, 12-dienoate (200 mg, 0.316 mmol, 1.0 equiv), pyridine (249.9 mg, 3.16 mmol, 10.0 equiv) and triphosgene (38.57 mg, 0.130 mmol, 0.4 equiv) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. N,N-diethylpropanolamine (82.93 mg, 0.632 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-274 (34.53 mg, 13.8% yield) as a colorless oil.

[0466] LCMS (ESI) m / z: 790.65 (M+H) + .

[0467] 1H NMR (400 MHz, Chloroform-d) δ 5.60 (s, 1H), 5.41 - 5.30 (m, 4H), 4.23 - 4.14 (m, 8H), 2.77 (t, J = 6.5 Hz, 2H), 2.67 - 2.38 (m, 9H), 2.30 (t, J = 7.6 Hz, 2H), 2.13 (t, J = 7.7 Hz, 2H), 2.04 (t, J = 6.9 Hz, 4H), 1.84 (s, 2H), 1.48 - 1.40 (m, 4H), 1.35 - 1.25 (m, 34H), 1.04 (s, 6H), 0.89 (m, 9H).

[0468] Example 57: Preparation of 3-(((2-(l-methylpiperidin-4-yl)ethoxy)carbonyl)oxy)- 2-((((E)-3-propylnon-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-280)

[0469]

[0470] To a mixture of 3-((E)-3-propylnon-2-enoyl)-2-(hydroxymethyl)propanyl-(9Z, 12Z)- octadeca-9, 12-dienoate (183 mg, 0.334 mmol, 1.0 equiv) and triphosgene (39 mg, 0.133 mmol, 0.4 equiv) in dichloromethane (6 mL) at 0 °C under nitrogen protection, 4-dimethylaminopyridine (81 mg, 0.668 mmol, 2.0 equiv) was added dropwise and stirred for 20 min. N-methyl-4-(2-hydroxyethyl)piperidine (95 mg, 0.668 mmol, 2.0 equiv) was added dropwise and stirred at room temperature overnight. 15 mL ice water was added and extracted with 20 mL dichloromethane for three times. The organic phase was washed with 20 mL sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-280 (72 mg, 30.1% yield) as a colorless oil.

[0471] LC / MS (ESI) m / z: 718.7 (M+H) + .

[0472] 1H NMR (400 MHz, CDCL3) δ 5.62 (d, J = 5.2 Hz, 1H), 5.40-5.31 (m, 4H), 4.22-4.12 (m, 8H), 2.78-2.72 (m, 4H), 2.65-2.54 (m, 3H), 2.45-2.42 (m, 1H), 2.32 (t, J = 7.6 Hz, 2H), 2.15-2.10 (m, 2H), 2.07-1.99 (m, 5H), 1.95-1.80 (m, 3H), 1.72-1.57 (m, 4H), 1.52-1.25 (m, 29H), 0.97-0.83 (m, 9H).

[0473] Example 58: Preparation of 3-(((2-(l-methylpiperidin-4-yl)ethoxy)carbonyl)oxy)-2- ( ((E)-3-pentylundec-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-289)

[0474]

[0475] A mixture of 3-((E)-3-pentylundec-2-enoyl)-2-(hydroxymethyl)propanyl-(9Z, 12Z)- octadeca-9, 12-dienoate (100 mg, 0.1653 mmol, 1.0 equiv), N-methyl-4-(2- hydroxyethyl)piperidine (71.03 mg, 0.4959 mmol, 3.0 equiv), pyridine (130.76 mg, 1.65 mmol, 10.0 equiv) and triphosgene (19.62 mg, 0.0601 mmol, 0.4 equiv) in dichloromethane (5 mL) was stirred at room temperature for 16 hours. 40 mL of water was added and extracted with 20 mL of dichloromethane three times. The organic phase was washed with 40 mL of sodium chloride solution three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-289 (89.14 mg, 64.66% yield) as a colorless oil.

[0476] LC / MS (ESI) m / z: 774.8 (M+H) + .

[0477] 1H NMR (400 MHz, CDC13) δ 5.60 (s, 1H), 5.37 - 5.31 (m, 4H), 4.20 (d, J = 4.8 Hz, 2H), 4.18 - 4.14 (m, 6H), 2.82 (s, 2H), 2.76 (d, J = 6.4 Hz, 2H), 2.61 - 2.55 (m, 2H), 2.46 - 2.42 (m, 1H), 2.30 (d, J = 6.8 Hz, 2H), 2.26 (s, 2H), 2.12 (d, J = 7.4 Hz, 2H), 2.03 (d, J = 5.8 Hz, 3H), 1.91 (d, J = 12.4 Hz, 2H), 1.61 (s, 6H), 1.25 (s, 36H), 0.89 (d, J = 2.8 Hz, 10H).

[0478] Example 59: Preparation of 3-(((2-(l-methylpiperidin-4-yl)ethoxy)carbonyl)oxy)- 2-(((E)-3-pentyltridec-2-enoyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-290)

[0479]

[0480] A mixture of 3-((E)-3-pentyltridec-2-enoyl)-2-(hydroxymethyl)propanoyl-(9Z, 12Z)- octadeca-9, 12-dienoate (300 mg, 0.45 mmol, 1.0 equiv), pyridine (308.50 mg, 4.5 mmol, 10.0 equiv) and triphosgene (46.29 mg, 0.18 mmol, 0.4 equiv) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. N-methyl-4-(2-hydroxyethyl)piperidine (113.13 mg, 0.90 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-290 (32.51 mg, 8.99% yield) as a colorless oil.

[0481] LCMS (ESI) m / z: 803.1 (M+H) + .

[0482] 1H NMR (400 MHz, Chloroform-d) δ 5.60 (s, 1H), 5.43 - 5.28 (m, 4H), 4.23 - 4.14 (m, 8H), 2.87 (d, J = 11.3 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.62 - 2.54 (m, 2H), 2.49 - 2.40 (m, 1H), 2.31 (d, J = 7.5 Hz, 2H), 2.28 (d, J = 1.6 Hz, 3H), 2.16 - 2.10 (m, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.95 (t, J = 11.4 Hz, 2H), 1.76 - 1.67 (m, 3H), 0.89 (tt, J = 4.2, 2.4 Hz, 9H).

[0483] Example 60: Preparation of 3-(((E)-3-hexylundec-2-enoyl)oxy)-2-((((2-(1- methylpiperidin-4-yl)ethoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate (CPL-292)

[0484]

[0485] A mixture of 3-((E)-3-hexylundec-2-enoyl)-2-(hydroxymethyl)propanyl-(9Z, 12Z)- octadeca-9, 12-dienoate (150 mg, 0.24 mmol, 1.0 equiv), pyridine (0.2 mL, 2.5 mmol, 10.0 equiv) and triphosgene (30 mg, 0.101 mmol, 0.4 equiv) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. N-methyl-4-(2-hydroxyethyl)piperidine (75 mg, 0.52 mmol, 2.0 equiv) was added and stirred at room temperature for 16 h. 30 mL of water was added and extracted with 20 mL of dichloromethane for three times. The organic phase was washed with 30 mL of sodium chloride solution for three times, filtered and dried over sodium sulfate. The mixture was concentrated and purified by prep-HPLC to give the target CPL-292 (17.10 mg, 8.95% yield) as a colorless oil.

[0486] LCMS (ESI) m / z: 788.6 (M+H) + .

[0487] 1H NMR(400MHz,Chloroform-d)δ5.60(s,1H),5.35(s,4H),4.24–4.10(m,6H),3.99(d,J=4.6Hz,2H),2.98(s,2H),2.77(s,2H),2.57(s,2H),2. 42(d,J=11.8Hz,2H),2.36–2.24(m,3H),2.12(d,J=8.0Hz,2H),2.04(d,J=6.8Hz,8H),1.74(s,4H),1.47–1.05(m,63H),0.94–0.74(m,18H).

[0488] Example 61: Preparation of 3-(((3-(dimethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)-2-((((E)-3-propyl-2-enoyl)oxy)methyl)propyl(9E,12E)-octadec-9,12-dienoate (CPL-304)

[0489]

[0490] 3-((E)-3-propanetridecyl-2-enoate)-2-(hydroxymethyl)propane-(9Z,12Z)-octadecyl-9,12-dienoate (237 mg, 0.392 mmol, 1.0 equivalent) and triphosgene (46 mg, 0.157 mmol, 0.4 equivalent) were mixed in dichloromethane (4 mL). Under nitrogen protection at 0 °C, 4-dimethylaminopyridine (95 mg, 0.784 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred for 20 minutes. 3-Dimethylamino-2,2-dimethyl-1-propanol (103 mg, 0.784 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred overnight at room temperature. 15 mL of ice water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-304 (34 mg, 11.4% yield), which was a colorless oil.

[0491] LC / MS (ESI) m / z: 762.6 (M+H) + .

[0492] 1H NMR (400MHz, CDCL3) δ5.55-5.53(m,1H),5.34-5.23(m,4H),4.15-4.08(m,6H),3.87(s,2H),2.71(t,J=6.4Hz,2H),2.51-2.47(m,2H),2.41-2 .35(m,1H),2.25-2.20(m,8H),2.09-2.03(m,4H),2.00-1.95(m,4H),1 .53(s,6H),1.47-1.35(m,4H),1.32-1.19(m,24H),0.89-0.76(m,15H).

[0493] Example 62: Preparation of 3-(((E)-3-butyloctyl-2-enoyl)oxy)-2-((((3-(dimethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-309)

[0494]

[0495] A mixture of 3-((E)-3-butyloctyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (150 mg, 0.27 mmol, 1.0 equivalent), pyridine (216 mg, 2.73 mmol, 10.0 equivalent), and triphosgene (33 mg, 0.10 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. 3-Dimethylamino-2,2-dimethyl-1-propanol (130 mg, 0.82 mmol, 3.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 10 mL of water was added, and the mixture was extracted three times with 10 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-309 (95 mg, 49% yield), which was a colorless oil.

[0496] LC / MS (ESI) m / z: 706.6 (M+H) + .

[0497] 1H NMR(400MHz, CDCl3)δ5.61(s,1H),5.46–5.26(m,4H),4.28–4.09(m,6H),3.95(s,2H),2.82–2.70(m,2H),2.63–2.54(m,2H),2.52–2.37( m,1H),2.32–2.28(m,4H),2.28(s,3H),2.19–2.10(m,4H),2.10–1.97(m,4H),1.66–1.60(m,2H),1.47–1.20(m,25H),1.00–0.75(m,15H).

[0498] Example 63: Preparation of 3-(((E)-3-butyldec-2-enoyl)oxy)-2-((((3-(dimethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-316)

[0499]

[0500] A mixture of 3-((E)-3-butyldecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.33 mmol, 1.0 equivalent), pyridine (260.7 mg, 3.3 mmol, 10.0 equivalent), and triphosgene (40 mg, 0.13 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. 3-Dimethylamino-2,2-dimethyl-1-propanol (131 mg, 1.04 mmol, 3.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-316 (98.2 mg, 76% yield), which was a colorless oil.

[0501] LCMS(ESI) m / z: 734.12 (M+H) + .

[0502] 1H NMR(400MHz, CDCl3)δ5.61(s,1H),5.43–5.24(m,4H),4.22-4.12(m,6H),3.94(s,2H),2.77(s,2H),2.62–2.51(m,1H),2. 47–2.41(m,1H),2.36–2.23(m,8H),2.16(s,3H),2.08–1.98(m,6H),1.61(s,2H),1.33–1.27(m,28H),0.93-0.80(m,15H).

[0503] Example 64: Preparation of 3-((3-decyltridecyl-2-enoyl)oxy)-2-((((3-(dimethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-322)

[0504]

[0505] A mixture of 3-((E)-3-decyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (190 mg, 0.2707 mmol, 1.0 equivalent), 3-dimethylamino-2,2-dimethyl-1-propanol (71 mg, 0.5413 mmol, 2.0 equivalent), pyridine (214 mg, 2.707 mmol, 10.0 equivalent), and triphosgene (32 mg, 0.1083 mmol, 0.4 equivalent) in dichloromethane (6 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-322 (39.69 mg, 17% yield), which was a colorless oil.

[0506] LC / MS (ESI) m / z: 860.8 (M+H) + .

[0507] 1HNMR (400MHz, CDCl3) δ5.53 (s, 1H), 5.35–5.22 (m, 4H), 4.10 (ddd, J = 14.5 ,11.9,7.4Hz,6H),3.88(s,2H),2.70(t,J=6.5Hz,2H),2.58–2.45(m,2H), 2.38(dt,J=11.8,5.9Hz,1H),2.29–2.16(m,8H),2.02(ddd,J=20.3,14.5, 7.3Hz, 8H), 1.37 (d, J = 7.4Hz, 2H), 1.26–1.17 (m, 46H), 0.85–0.79 (m, 15H).

[0508] Example 65: Preparation of 3-((((1-methylpyrrolidone-3-yl)methoxy)carbonyl)oxy)-2-((((E)-3-propylnon-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-324)

[0509]

[0510] 3-((E)-3-propylnon-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (300 mg, 0.547 mmol, 1.0 equivalent) and triphosgene (65 mg, 0.218 mmol, 0.4 equivalent) were mixed in dichloromethane (6 mL). Under nitrogen protection at 0 °C, 4-dimethylaminopyridine (133 mg, 1.094 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred for 20 minutes. 1-Methyl-3-pyrrolidinemethanol (126 mg, 1.094 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred overnight at room temperature. 15 mL of ice water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-324 (112 mg, 29.7% yield), which was a colorless oil.

[0511] LC / MS (ESI) m / z: 690.6 (M+H) + .

[0512] 1H NMR (400MHz, CDCL3) δ5.61 (d, J=5.2Hz, 1H), 5.39-5.31 (m, 4H), 4.22-4.01 (m, 8H),2.78(t,J=6.8Hz,2H),2.66-2.64(m,1H),2.59-2.51(m,5H),2.45-2.42(m ,1H),2.35(s,3H),2.32(t,J=7.6Hz,2H),2.15-2.10(m,2H),2.07-2.00(m,5H) ,1.62-1.60(m,2H),1.52-1.42(m,4H),1.37-1.25(m,22H),0.96-0.85(m,9H).

[0513] Example 66: Preparation of 3-(((E)-3-butylundec-2-enoyl)oxy)-2-(((((1-methylpyrrolidone-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-329)

[0514]

[0515] 3-((E)-3-butyl undecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.339 mmol, 1.0 equivalent) and triphosgene (40 mg, 0.135 mmol, 0.4 equivalent) were mixed in dichloromethane (5 mL). Under nitrogen protection at 0 °C, 4-dimethylaminopyridine (82 mg, 0.678 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred for 20 minutes. 1-Methyl-3-pyrrolidinemethanol (78 mg, 0.678 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred overnight at room temperature. 15 mL of ice water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-329 (57 mg, 22.9% yield), which was a colorless oil.

[0516] LC / MS (ESI) m / z: 732.1 (M+H) + .

[0517] 1H NMR (400MHz, CDCL3) δ5.60(s,1H),5.40-5.31(m,4H),4.22-4.12(m,8H),2.90-2.65(m,7H),2.59-2.55(m,2H),2.48-2.40(m,1H),2. 32-2.20(m,3H),2.16-2.11(m,2H),2.07-2.02(m,6H),1.90-1.78(m,2H),1.62-1.59(m,2H),1.46-1.26(m,30H),0.93-0.83(m,9H).

[0518] Example 67: Preparation of 3-(((E)-3-butyldec-2-enoyl)oxy)-2-(((((1-methylpyrrolidone-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-338)

[0519]

[0520] A mixture of 3-((E)-3-butyldecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.35 mmol, 1.0 equivalent), pyridine (276.5 mg, 3.5 mmol, 10.0 equivalent), and triphosgene (41.16 mg, 0.14 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. 1-Methyl-3-pyrrolidinemethanol (115.17 mg, 1.04 mmol, 3.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-338 (36.08 mg, 14% yield), which was a colorless oil.

[0521] LCMS(ESI) m / z: 718.07 (M+H) + .

[0522] 1H NMR(400MHz, CDCl3)δ5.61(s,1H),5.36(dt,J=12.4,6.8Hz,4H),4.26–4.01(m,8H),2.77(t,J=6.5Hz,2H),2.59(dd,J=29.1,20.1Hz ,6H),2.47–2.40(m,1H),2.34(d,J=11.0Hz,4H),2.18–2.10(m,2H),2.07–2.01(m,4H),1.28(d,J=18.0Hz,34H),0.96–0.89(m,9H).

[0523] Example 68: Preparation of 3-((((1-methylpyrrolidone-3-yl)methoxy)carbonyl)oxy)-2-((((Z)-3-octyltridecane-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-343)

[0524]

[0525] A mixture of 3-((E)-3-octyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (190 mg, 0.2819 mmol, 1.0 equivalent), 1-methyl-3-pyrrolidinemethanol (65 mg, 0.5638 mmol, 2.0 equivalent), pyridine (223 mg, 2.819 mmol, 10.0 equivalent), and triphosgene (33 mg, 0.1128 mmol, 0.4 equivalent) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-343 (31.78 mg, 14% yield) as a colorless oil.

[0526] LC / MS (ESI) m / z: 816.6 (M+H) + .

[0527] 1HNMR (400MHz, CDCl3) δ5.53 (s, 1H), 5.35–5.22 (m, 4H), 4.17–3.93 (m, 8H), 2.70 (t, J = 6.1Hz, 2H), 2.50 (dd, J = 14.6, 6.5Hz, 5H), 2.41–2.33 (m, 1H),2.25(dd,J=17.3,9.8Hz,6H),2.10–2.02(m,2H),1.98(dd,J=13.5,6.5Hz,5H),1.40–1.34(m,4H),1.25–1.17(m,42H),0.82–0.79(m,9H).

[0528] Example 69: Preparation of 3-(((E)-3-butylundec-2-enoyl)oxy)-2-(((((1-methylpyrrolidone-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-348)

[0529]

[0530] 3-((E)-3-propanetridecyl-2-enoate)-2-(hydroxymethyl)propane-(9Z,12Z)-octadecyl-9,12-dienoate (250 mg, 0.414 mmol, 1.0 equivalent) and triphosgene (49 mg, 0.165 mmol, 0.4 equivalent) were mixed in dichloromethane (6 mL). Under nitrogen protection, a 0.5 mL solution of 4-dimethylaminopyridine (101 mg, 0.828 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred for 20 minutes. 3-Dimethylamino-2,2-dimethyl-1-propanol (131 mg, 0.828 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred overnight at room temperature. 15 mL of ice water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-348 (151 mg, 46.1% yield), which was a colorless oil.

[0531] LC / MS (ESI) m / z: 791.2 (M+H) + .

[0532] 1H NMR(400MHz,CDCL3)δ5.62(d,J=5.6Hz,1H),5.41-5.29(m,4H),4.21-4.12(m ,6H),3.91(s,2H),2.78(t,J=6.8Hz,2H),2.58-2.54(m,2H),2.52-2.41(m,5H ),2.32(t,J=7.6Hz,2H),2.23(s,2H),2.14-2.10(m,2H),2.07-2.00(m,4H),1 .62-1.60(m,2H),1.54-1.42(m,4H),1.39-1.26(m,28H),0.97-0.85(m,21H).

[0533] Example 70: Preparation of 3-(((E)-3-butylundec-2-enoyl)oxy)-2-(((((diethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-351)

[0534]

[0535] 3-((E)-3-butyl undecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.339 mmol, 1.0 equivalent) and triphosgene (40 mg, 0.135 mmol, 0.4 equivalent) were mixed in dichloromethane (5 mL). Under nitrogen protection at 0 °C, 4-dimethylaminopyridine (82 mg, 0.678 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred for 20 minutes. 3-Dimethylamino-2,2-dimethyl-1-propanol (107 mg, 0.678 mmol, 2.0 equivalent) was added dropwise, and the mixture was stirred overnight at room temperature. 15 mL of ice water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-351 (60 mg, 22.8% yield), which was a colorless oil.

[0536] LC / MS (ESI) m / z: 776.7 (M+H) + .

[0537] 1H NMR (400MHz, CDCL3) δ5.60(s,1H),5.41-5.29(m,4H),4.21-4.12(m,6H),3.91(s,2H),2.78(t,J=6.4Hz,2H),2.59-2.41(m,6H),2.32( t,J=7.6Hz,2H),2.26-2.20(m,1H),2.15-2.12(m,2H),2.07-2.00(m,6H),1.62-1.60(m,2H),1.46-1.26(m,35H),0.96-0.87(m,15H).

[0538] Example 71: Preparation of 3-(((3-(diethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)-2-((((E)-3-pentylundec-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-355)

[0539]

[0540] A mixture of 3-((E)-3-pentyl undecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.3306 mmol, 1.0 equivalent), 3-dimethylamino-2,2-dimethyl-1-propanol (157.97 mg, 0.9918 mmol, 3.0 equivalent), pyridine (261.51 mg, 3.31 mmol, 10.0 equivalent), and triphosgene (39.12 mg, 0.1322 mmol, 0.4 equivalent) in dichloromethane (5 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-355 (114.7 mg, 43.9% yield), which was a colorless oil.

[0541] LC / MS (ESI) m / z: 790.8 (M+H) + .

[0542] 1HNMR (400MHz, CDCl3) δ5.60 (s, 1H), 5.35 (d, J = 6.4Hz, 4H), 4.22–4.12 (m, 6 H),3.92(s,2H),2.77(t,J=5.8Hz,2H),2.62–2.54(m,2H),2.48(d,J=6.8Hz ,2H),2.30(t,J=7.8Hz,2H),2.24(s,2H),2.16–2.11(m,2H),2.04(s,4H),1 .56(s,6H),1.44(s,6H),1.25(s,32H),0.94(d,J=7.8Hz,6H),0.88(s,8H).

[0543] Example 72: Preparation of 3-(((3-(diethylamino)-2,2-dimethylpropoxy)carbonyl)oxy)-2-((((E)-3-heptyltridecane-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-363)

[0544]

[0545] A mixture of 3-((E)-3-heptyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.303 mmol, 1.0 equivalent), pyridine (239.67 mg, 3.03 mmol, 10.0 equivalent), and triphosgene (35.9 mg, 0.121 mmol, 0.4 equivalent) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. 3-Dimethylamino-2,2-dimethyl-1-propanol (96.45 mg, 0.606 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-363 (42.5 mg, 16.57% yield), which was a colorless oil.

[0546] LCMS(ESI) m / z: 846.71 (M+H) + .

[0547] 1H NMR(400MHz,Chloroform-d)δ5.60(s,1H),5.42–5.29(m,4H),4.23–4.14(m ,6H),3.92(s,2H),2.77(t,J=6.5Hz,2H),2.61–2.54(m,2H),2.53–2.41(m,5 H),2.30(t,J=7.6Hz,2H),2.24(s,2H),2.17–2.10(m,2H),2.05(m,4H),1.4 4(d,J=7.4Hz,2H),1.39–1.25(m,40H),0.95(t,J=7.1Hz,6H),0.88(m,15H).

[0548] Example 73: Preparation of 3-(((Z)-3-butylnon-2-enoyl)oxy)-2-(((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-372)

[0549]

[0550] A mixture of 3-((E)-3-butylnon-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (150 mg, 0.267 mmol, 1.0 equivalent), pyridine (212.2 mg, 2.67 mmol, 10.0 equivalent), and triphosgene (31.69 mg, 0.107 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. 1-Methylpiperidine-3-methanol (68.82 mg, 0.533 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-372 (40.26 mg, 21.01% yield), which was a colorless oil.

[0551] LCMS(ESI) m / z: 718.55 (M+H) + .

[0552] 1H NMR(400MHz,Chloroform-d)δ5.61(s,1H),5.43–5.28(m,4H),4.24–4.11(m,6H),4.06 (m,1H),3.96(m,1H),2.87(d,J=11.1Hz,1H),2.77(t,J=6.6Hz,2H),2.58(t,J=6.5Hz, 1H),2.44(m,1H),2.30(d,J=6.9Hz,4H),2.17(s,7H),2.13(d,J=8.2Hz,2H),2.05(m,5 H),1.72(d,J=11.6Hz,3H),1.46–1.40(m,3H),1.38–1.25(m,24H),0.93–0.86(m,9H).

[0553] Example 74: Preparation of 3-((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-(((E)-3-pentylundecoxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-377)

[0554]

[0555] A mixture of 3-((E)-3-pentyl undecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.33 mmol, 1.0 equivalent), pyridine (261 mg, 3.3 mmol, 10.0 equivalent), and triphosgene (40 mg, 0.13 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. A solution of 1-methylpiperidine-3-methanol (185 mg, 0.99 mmol, 3.0 equivalent) in dichloromethane (1 mL) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-377 (22 mg, 8% yield), which was a colorless oil.

[0556] LC / MS (ESI) m / z: 760.6 (M+H) + .

[0557] 1H NMR(400MHz, CDCl3)δ5.61(s,1H),5.44–5.27(m,4H),4.25–4.11(m,6H),4.11– 4.03(m,1H),3.99–3.89(m,1H),2.88–2.71(m,4H),2.61–2.55(m,2H),2.48–2. 40(m,1H),2.35–2.30(m,2H),2.26(s,3H),2.18–2.10(m,2H),2.09–1.97(m,4H ),1.77–1.66(m,4H),1.45–1.28(m,35H),1.05–0.99(m,2H),0.94–0.85(m,9H).

[0558] Example 75: Preparation of 3-((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-((((E)-3-pentyltridecane-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-378)

[0559]

[0560] A mixture of 3-((E)-3-pentyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.31 mmol, 1.0 equivalent), pyridine (250 mg, 3.1 mmol, 10.0 equivalent), and triphosgene (38 mg, 0.12 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. A solution of 1-methylpiperidine-3-methanol (125 mg, 0.95 mmol, 3.0 equivalent) in dichloromethane (1 mL) was added, and the mixture was stirred at room temperature for 16 h. 10 mL of water was added, and the mixture was extracted three times with 10 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-378 (81 mg, 32% yield), which was a colorless oil.

[0561] LC / MS (ESI) m / z: 788.6 (M+H) + .

[0562] 1H NMR(400MHz, CDCl3)δ5.60(s,1H),5.44–5.28(m,4H),4.28–4.11(m,6H),4.10– 3.89(m,2H),2.88–2.69(m,4H),2.64–2.54(m,2H),2.48–2.42(m,1H),2.36–2. 29(m,2H),2.27(s,3H),2.18–2.10(m,2H),2.09–1.96(m,5H),1.96–1.85(m,1H ),1.77–1.66(m,4H),1.45–1.22(m,37H),1.08–0.93(m,2H),0.92–0.83(m,9H).

[0563] Example 76: Preparation of 3-(((E)-3-hexyltridecane-2-enoyl)oxy)-2-(((((1-methylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadecane-9,12-dienoate (CPL-381)

[0564]

[0565] A mixture of 3-((E)-3-hexyltridecyl-2-enoate)-2-(hydroxymethyl)propane-(9Z,12Z)-octadecyl-9,12-dienoate (150 mg, 0.23 mmol, 1.0 equivalent), pyridine (184 mg, 2.3 mmol, 10.0 equivalent), and triphosgene (28 mg, 0.09 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. A solution of 1-methylpiperidine-3-methanol (92 mg, 0.69 mmol, 3.0 equivalent) in dichloromethane (1 mL) was added, and the mixture was stirred at room temperature for 16 h. 10 mL of water was added, and the mixture was extracted three times with 10 mL of dichloromethane. The organic phase was washed three times with 20 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-381 (52 mg, 28% yield), which was a colorless oil.

[0566] LC / MS (ESI) m / z: 802.6 (M+H) + .

[0567] 1H NMR(400MHz, CDCl3)δ5.60(s,1H),5.44–5.28(m,4H),4.27–4.19(m,2H),4.19– 4.11(m,4H),4.09–4.01(m,1H),4.01–3.92(m,1H),2.88–2.70(m,4H),2.62–2.5 3(m,2H),2.51–2.39(m,1H),2.38–2.28(m,2H),2.28(s,3H),2.16–2.11(m,2H) ,2.08–1.86(m,6H),1.80–1.66(m,4H),1.46–1.26(m,38H),1.07–0.85(m,12H).

[0568] Example 77: Preparation of 3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)-2-((((E)-3-hexyldecyl-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-383)

[0569]

[0570] A mixture of 3-((E)-3-hexyldecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.33 mmol, 1.0 equivalent), pyridine (260.7 mg, 3.3 mmol, 10.0 equivalent), and triphosgene (40 mg, 0.13 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. 1-Methylpiperidine-3-methanol (129 mg, 1.04 mmol, 3.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-383 (25.83 mg, 6% yield) as a colorless oil.

[0571] LCMS(ESI) m / z: 760.59 (M+H) + .

[0572] 1H NMR(400MHz, CDCl3)δ5.60(s,1H),5.44–5.25(m,4H),4.17-3.93(m,8H),2.89–2.67(m,4H),2.61–2.54(m,1H),2. 49–2.39(m,1H),2.33–2.24(m,5H),2.19–1.80(m,10H),1.78–1.66(m,4H),1.32–1.25(m,32H),0.91-0.85(m,9H)

[0573] Example 78: Preparation of 3-(((Z)-3-butylnon-2-enoyl)oxy)-2-((((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-394)

[0574]

[0575] A mixture of 3-((E)-3-butylnon-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (150 mg, 0.267 mmol, 1.0 equivalent), pyridine (212.2 mg, 2.67 mmol, 10.0 equivalent), and triphosgene (31.69 mg, 0.107 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. A dichloromethane solution (1 mL) of 1-(3-hydroxypropyl)-4-methylpiperazine (69.89 mg, 0.533 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-394 (30.22 mg, 15.2% yield), which was a colorless oil.

[0576] LCMS(ESI) m / z: 747.58 (M+H) + .

[0577] 1H NMR(400MHz,Chloroform-d)δ5.61(s,1H),5.42–5.28(m,4H),4.25–4.11(m,8H),2.77(t,J=6.3Hz,2H),2.63–2.39(m,11H ),2.35–2.27(m,5H),2.14(t,J=7.6Hz,2H),2.05(m,4H),1.87(m,2H),1.48–1.40(m,4H),1.40–1.22(m,26H),0.89(m,9H).

[0578] Example 79: Preparation of 3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)-2-((((E)-3-pentylnon-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-398)

[0579]

[0580] A mixture of 3-((E)-3-pentylnon-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.3472 mmol, 1.0 equivalent), 1-(3-hydroxypropyl)-4-methylpiperazine (110 mg, 0.6944 mmol, 2.0 equivalent), pyridine (274 mg, 3.472 mmol, 10.0 equivalent), and triphosgene (41 mg, 0.1389 mmol, 0.4 equivalent) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-398 (25.79 mg, 9.8% yield) as a colorless oil.

[0581] LC / MS (ESI) m / z: 761.6 (M+H) + .

[0582] 1HNMR(400MHz, CDCl3)δ5.54(s,1H),5.33–5.22(m,4H),4.15–4.07(m,8H ),2.70(t,J=6.6Hz,2H),2.53–2.48(m,2H),2.43–2.31(m,8H),2.27–2.20 (m,6H),2.09–2.03(m,2H),1.98(q,J=6.4Hz,4H),1.79(dd,J=14.2,6.8Hz ,2H),1.39–1.34(m,4H),1.24–1.18(m,28H),0.82(dd,J=6.9,4.4Hz,9H).

[0583] Example 80: Preparation of 3-(((E)-3-heptyltridecane-2-enoyl)oxy)-2-((((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-407)

[0584]

[0585] A mixture of 3-((E)-3-heptyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.303 mmol, 1.0 equivalent), pyridine (239.67 mg, 3.03 mmol, 10.0 equivalent), and triphosgene (35.9 mg, 0.121 mmol, 0.4 equivalent) in dichloromethane (5 mL) was stirred at 0 °C for 30 min. 1-(3-hydroxypropyl)-4-methylpiperazine (95.83 mg, 0.606 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-407 (21.72 mg, 8.48% yield), which was a colorless oil.

[0586] LCMS(ESI) m / z: 845.69 (M+H) + .

[0587] 1H NMR(400MHz,Chloroform-d)δ5.60(s,1H),5.41–5.30(m,4H),4.22–4.14(m,8H),2.77(t,J=6.5Hz,2H),2.57(t,J=7.8Hz,7H),2.45(m,4H),2.34(d ,J=10.7Hz,3H),2.29(d,J=7.6Hz,2H),2.16–2.10(m,2H),2.05(m,4H),1 .87(m,2H),1.44(d,J=7.3Hz,4H),1.36–1.24(m,40H),0.91–0.86(m,9H).

[0588] Example 81: Preparation of 3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)-2-((((Z)-3-octyltridecane-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadecane-9,12-dienoate (CPL-409)

[0589]

[0590] A mixture of 3-((E)-3-octyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (190 mg, 0.2819 mmol, 1.0 equivalent), 1-(3-hydroxypropyl)-4-methylpiperazine (89 mg, 0.5638 mmol, 2.0 equivalent), pyridine (223 mg, 2.819 mmol, 10.0 equivalent), and triphosgene (33 mg, 0.1128 mmol, 0.4 equivalent) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-409 (23.57 mg, 10% yield) as a colorless oil.

[0591] LC / MS (ESI) m / z: 859.7 (M+H) + .

[0592] 1H NMR(400MHz, CDCl3)δ5.53(s,1H),5.34–5.22(m,4H),4.18–4.03(m,8H),2.7 0(t,J=6.5Hz,2H),2.53–2.48(m,4H),2.38(dd,J=14.3,6.7Hz,4H),2.28–2. 21(m,5H),2.10–2.03(m,2H),1.98(q,J=6.8Hz,4H),1.83–1.74(m,2H),1.58 –1.49(m,3H),1.37(d,J=7.9Hz,4H),1.25–1.17(m,42H),0.83–0.79(m,9H).

[0593] Example 82: Preparation of 3-(((Z)-3-butylnon-2-enoyl)oxy)-2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-416)

[0594]

[0595] A mixture of 3-((E)-3-butylnon-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (150 mg, 0.267 mmol, 1.0 equivalent), pyridine (212.12 mg, 2.67 mmol, 10.0 equivalent), and triphosgene (31.69 mg, 0.107 mmol, 0.4 equivalent) in dichloromethane (2 mL) was stirred at 0 °C for 30 min. 1-piperidinpropanol (76.28 mg, 0.533 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-416 (29.03 mg, 14.9% yield), which was a colorless oil.

[0596] LCMS(ESI) m / z: 732.57 (M+H) + .

[0597] 1H NMR (400MHz, Chloroform-d) δ5.61 (s, 1H), 5.35 (m, 4H), 4.24–4.11 (m, 8H), 2.77 (t, J = 6.5Hz, 2H), 2.60–2.56 (m, 1H), 2.51–2. 35(m,6H),2.30(t,J=7.6Hz,2H),2.14(m,2H),2.05(q,J=6.9Hz,4H),1.90(t,J=7.2Hz,2H),1.58–1.19(m,36H),0.89(m,9H).

[0598] Example 83: Preparation of 3-(((E)-3-pentylundec-2-enoyl)oxy)-2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-421)

[0599]

[0600] A mixture of 3-((E)-3-pentyl undecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.3306 mmol, 1.0 equivalent), 1-piperidinpropanol (142.06 mg, 0.9918 mmol, 3.0 equivalent), pyridine (261.51 mg, 3.31 mmol, 10.0 equivalent), and triphosgene (39.24 mg, 0.1322 mmol, 0.4 equivalent) in dichloromethane (5 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-421 (86.45 mg, 33.78% yield) as a colorless oil.

[0601] LC / MS (ESI) m / z: 774.6 (M+H) + .

[0602] 1H NMR(400MHz, CDCl3)δ5.60(s,1H),5.35(qd,J=8.4,2.7Hz,4H),4.21–4.14(m,8H),2.76(d,J=6.8Hz,2H),2.60 –2.53(m,2H),2.34(t,J=15.8Hz,6H),2.13–2.03(m,6H),1.88(s,2H),1.54–1.27(m,42H),0.91–0.87(m,10H).

[0603] Example 84: Preparation of 3-(((E)-3-heptylundec-2-enoyl)oxy)-2-((((3-(piperidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-428)

[0604]

[0605] A mixture of 3-((E)-3-heptylundecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.32 mmol, 1.0 equivalent), pyridine (253.12 mg, 3.2 mmol, 10.0 equivalent), and triphosgene (35.60 mg, 0.12 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. 1-Piperidinpropanol (90.23 mg, 0.63 mmol, 2.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to obtain the target CPL-428 (33.21 mg, 12.93% yield), which was a colorless oil.

[0606] LCMS(ESI) m / z: 803.7(M+H) + .

[0607] 1H NMR(400MHz, CDCl3)δ5.60(d,J=5.8Hz,1H),5.42–5.28(m,4H),4.28–4.03(m,8H),2.77(t,J=6.4Hz,2H),2.63–2.54(m,2H),2.54–2.34(m,6H), 2.34–2.19(m,3H),2.17–2.09(m,2H),2.07–2.01(m,4H),1.84(ddd,J=6 2.8, 46.7, 40.0Hz, 4H), 1.60–1.20 (m, 42H), 0.88 (dd, J=6.9, 4.2Hz, 9H).

[0608] Example 85: Preparation of 3-((((1-ethylpiperidin-4-yl)methoxy)carbonyl)oxy)-2-((((E)-3-hexyldecyl-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-449)

[0609]

[0610] A mixture of 3-((E)-3-hexyldec-2-enoate)-2-(hydroxymethyl)propane-(9Z,12Z)-octadec-9,12-dienoate (200 mg, 0.33 mmol, 1.0 equivalent), pyridine (260.7 mg, 3.3 mmol, 10.0 equivalent), and triphosgene (40 mg, 0.13 mmol, 0.4 equivalent) in dichloromethane (3 mL) was stirred at 0 °C for 30 min. 1-piperidinol (143.23 mg, 1.04 mmol, 3.0 equivalent) was added, and the mixture was stirred at room temperature for 16 h. 30 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 30 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-449 (50.72 mg, 39% yield) as a colorless oil.

[0611] LCMS(ESI) m / z: 774.18 (M+H) + .

[0612] 1H NMR (400MHz, CDCl3) δ5.60 (s, 1H), 5.41–5.26 (m, 4H), 4.22–4.14 (m, 6H), 3.98 (d, J = 6. 4Hz,2H),2.96(d,J=14.0Hz,2H),2.77(t,J=6.4Hz,2H),2.61–2.54(m,1H),2.47–2.37 (m,3H),2.29(d,J=7.7Hz,2H),2.16–1.98(m,6H),1.89(t,J=10.8Hz,2H),1.75(d,J=1 3.2Hz, 2H), 1.61 (s, 6H), 1.38–1.23 (m, 38H), 1.08 (t, J = 7.2Hz, 4H), 0.91-0.83 (m, 9H).

[0613] Example 86: Preparation of 3-(((1-ethylpiperidin-4-methoxy)carbonyl)oxy)-2-((((Z)-3-octyltridecane-2-enoyl)oxy)methyl)propyl(9Z,12Z)-octadecane-9,12-dienoate (CPL-453)

[0614]

[0615] A mixture of 3-((E)-3-octyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.2967 mmol, 1.0 equivalent), N-ethylpiperidine-1-methanol (85 mg, 0.5935 mmol, 3.0 equivalent), pyridine (234 mg, 2.967 mmol, 10.0 equivalent), and triphosgene (35 mg, 0.1187 mmol, 0.4 equivalent) in dichloromethane (8 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-453 (44.16 mg, 18% yield) as a colorless oil.

[0616] LC / MS (ESI) m / z: 844.7 (M+H) + .

[0617] 1HNMR(400MHz, CDCl3)δ5.53(s,1H),5.35–5.22(m,4H),4.17–4.04(m,6H),3.92(d,J=6 .4Hz,2H),2.70(t,J=6.5Hz,2H),2.57–2.46(m,2H),2.37(dt,J=11.9,5.9Hz,2H),2.22 (dd,J=17.7,10.1Hz,2H),2.09–2.02(m,2H),1.98(dd,J=13.6,6.7Hz,4H),1.65(d,J=3 0.6Hz, 4H), 1.37 (d, J = 6.7Hz, 4H), 1.32–1.17 (m, 46H), 1.05 (s, 3H), 0.84–0.80 (m, 9H).

[0618] Example 87: Preparation of 3-((3-decyltridecyl-2-enoyl)oxy)-2-(((((1-ethylpiperidin-4-yl)methoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadec-9,12-dienoate (CPL-454)

[0619]

[0620] A mixture of 3-((E)-3-decyltridecyl-2-enoate)-2-(hydroxymethyl)propyl-(9Z,12Z)-octadecyl-9,12-dienoate (200 mg, 0.2849 mmol, 1.0 equivalent), N-ethylpiperidine-1-methanol (81 mg, 0.5698 mmol, 2.0 equivalent), pyridine (225 mg, 2.849 mmol, 10.0 equivalent), and triphosgene (34 mg, 0.1114 mmol, 0.4 equivalent) in dichloromethane (6 mL) was stirred at room temperature for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-454 (35.65 mg, 14% yield) as a colorless oil.

[0621] LC / MS (ESI) m / z: 873.7 (M+H) + .

[0622] 1HNMR (400MHz, CDCl3) δ5.53 (s, 1H), 5.35–5.21 (m, 4H), 4.19–4.04 (m, 6H), 3.92 (d, J= 6.5Hz,2H),2.70(t,J=6.5Hz,2H),2.56–2.46(m,2H),2.41–2.33(m,2H),2.22(dd,J=17 .5,9.9Hz,2H),2.09–2.03(m,2H),1.98(q,J=6.6Hz,4H),1.66(t,J=19.1Hz,4H),1.37 (dd,J=12.1,4.5Hz,4H),1.31–1.17(m,50H),1.03(t,J=6.9Hz,3H),0.83–0.79(m,9H).

[0623] Example 88: Preparation of 5-((5-(E)-3-butyltridecane-2-enoyl)oxy)pentyl((3-hydroxypropyl)amino)pentyl(E)-3-propyl-2-tridecenoate (CPL-1092)

[0624]

[0625] A mixture of compound (E)-5-bromopentyl-3-propyl tridecyl-2-enoate (108 mg, 0.27 mmol, 1.1 equivalents), 5-(3-hydroxypropylamino)-pentyl-3-butyl tridecyl-2-enoate (100 mg, 0.24 mmol, 1.0 equivalents), K₂CO₃ (67 mg, 0.49 mmol, 2.0 equivalents), and NaI (1.8 mg, 0.01 mmol, 0.05 equivalents) in 10 mL of ACN was stirred at 85 °C for 16 hours. 40 mL of water was added, and the mixture was extracted three times with 20 mL of ethyl acetate. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-1092 (75.02 mg, 42.06% yield) as a colorless oil.

[0626] LC / MS(ESI) m / z: 734.7(M+H)+.

[0627] 1H NMR (400MHz, CDCl3) δ5.55(s,2H),4.02-3.99(m,4H),3.74-3.71(m,2H),2.52-2.39(m ,10H),2.10-2.05(m,4H),1.63-1.56(m,6H),1.45-1.23(m,46H),0.87-0.81(m,12H).

[0628] Example 89: Preparation of 10-(((5-(E)-3-butyltridecane-2-enoyl)oxy)pentyl)((3-hydroxypropyl)amino)decyl(E)-3-propyl-2-tridecenoate (CPL-1093)

[0629]

[0630] A mixture of compound (E)-10-bromodecyl-3-propyl tridecyl-2-enoate (126 mg, 0.27 mmol, 1.1 equivalents), 5-(3-hydroxypropylamino)-pentyl-3-butyl tridecyl-2-enoate (100 mg, 0.24 mmol, 1.0 equivalents), K₂CO₃ (67 mg, 0.49 mmol, 2.0 equivalents), and NaI (1.8 mg, 0.01 mmol, 0.05 equivalents) in 10 mL of ACN was stirred overnight at 80 °C. 40 mL of water was added, and the mixture was extracted three times with 20 mL of ethyl acetate. The organic phase was washed three times with 40 mL of sodium chloride solution, filtered, and dried over sodium sulfate. The mixture was concentrated and purified by preparative HPLC to give the target CPL-1093 (80.54 mg, 41.30% yield) as a colorless oil.

[0631] LC / MS(ESI) m / z: 805.7(M+H)+.

[0632] 1H NMR (400MHz, CDCl3) δ5.55 (d, J=4.8Hz, 2H), 4.02-3.98 (m, 4H), 3.74-3.72 (m, 2H) ,2.64-2.41(m,10H),2.10-1.97(m,4H),1.65–1.25(m,62H),0.85–0.80(m,12H).

[0633] Test case

[0634] The properties of the cationic lipids prepared in the above examples were tested according to the following methods.

[0635] Preparation and characterization of lipid nanoparticles

[0636] Following the steps described below, taking mRNA as an example of drug encapsulation, lipid nanoparticles (LNPs) are prepared using the cationic lipids disclosed herein.

[0637] 1. The cationic lipid (50 mg / mL) prepared in the above embodiments of this disclosure is dissolved in ethanol with cholesterol (20 mg / mL), DSPC (20 mg / mL) and DMG-PEG2000 (20 mg / mL), and the solution is placed in a 55°C metal constant temperature bath to aid dissolution as needed, to obtain a lipid mixture with the following molar composition: 1.5% DMG-PEG2000, 50% cationic lipid, 38.5% cholesterol and 10% DSPC.

[0638] 2. Dilute the mRNA encoding FFluc with 10 mM pH 4.0 citrate buffer to prepare a 0.2 mg / mL mRNA solution.

[0639] 3. Using a syringe pump with a three-way valve, mix the lipid mixture obtained in step 1 with the mRNA solution obtained in step 2 at a total flow rate of 40 mL / min.

[0640] 4. Collect the LNP mixture after the three-way valve and place it in a dialysis bag with a molecular weight cutoff of 100 kDa.

[0641] 5. Dialyze the LNP mixture overnight relative to 50 mM Tris buffered saline at pH 7.4.

[0642] 6. The LNPs obtained from sieving were then measured to determine their PDI using dynamic laser scattering; mRNA encapsulation efficiency and concentration were measured using the Ribogreen assay; and pKa was measured using the TNS-α assay.

[0643] The results are shown in Table 1 below.

[0644] Table 1 Performance parameters of lipid nanoparticles (LNPs) made from cationic lipids of this disclosure

[0645]

[0646]

[0647]

[0648]

[0649] As can be seen from the results in the table above, LNPs made from the cationic lipids of this disclosure have superior drug encapsulation efficiency, enabling effective drug encapsulation for targeted in vivo delivery.

[0650] Animal research

[0651] The prepared encapsulation solution was administered to mice using the following method, and its delivery efficiency in mice was assessed. DLin-MC3-DMA (MC3) is a cationic liposome for delivering small nucleic acids, approved by the FDA in 2018. It exhibits high nucleic acid drug delivery efficiency and biocompatibility, and is typically used as the industry gold standard in cationic liposome screening. In mouse experiments, a ratio of nucleic acid delivery efficiency normalized to MC3 greater than 0.1 was considered a cationic liposome with high delivery efficiency.

[0652] intramuscular administration

[0653] 1. Dilute the LNP solution with 50 mM TBS, pH 7.4 to a concentration of 0.1 mg / mL mRNA.

[0654] 2. The mice used in this study were female Balb / C mice, weighing 18-22g, 6-8 weeks old, with 5 mice in each group.

[0655] 3. Keep the administration solution at 4°C before injection.

[0656] 4. Inject 50 μL of LNP solution into the right thigh of the mouse.

[0657] 5. Six hours after injection, mice were injected intraperitoneally with 200 μL of 1.5 mg / mL D-fluorescein solution.

[0658] 6. Acquire in vivo luminescence images of mice using the IVIS system (Aniview).

[0659] 7. Measure the luminescence intensity of each mouse using Aniview software.

[0660] 8. MC3 lipid nanoparticles were used as a positive control. The in vivo potency of each cationic lipid tested was recorded as a fold change in luminescence intensity relative to MC3 (ratio normalized to MC3).

[0661] intravenous administration

[0662] 1. Dilute the LNP solution with 50 mM TBS, pH 7.4 to a concentration of 0.1 mg / mL mRNA.

[0663] 2. The mice used in this study were female Balb / C mice, weighing 18-22g, 6-8 weeks old, with 5 mice in each group.

[0664] 3. Keep the administration solution at 4°C before injection.

[0665] 4. Each mouse was administered 0.06 mg / kg via tail vein injection.

[0666] 5. Six hours after injection, mice were injected intraperitoneally with 200 μL of 1.5 mg / mL D-fluorescein solution.

[0667] 6. Acquire in vivo luminescence images of mice using the IVIS system (Aniview).

[0668] 7. Measure the luminescence intensity of each mouse using Aniview software.

[0669] 8. MC3 lipid nanoparticles were used as a positive control. The in vivo potency of each cationic lipid tested was recorded as a fold change in luminescence intensity relative to MC3 (ratio normalized to MC3).

[0670] The results are shown in Table 2 below.

[0671] Table 2. In vivo delivery efficiency of lipid nanoparticles prepared from cationic lipids of this disclosure

[0672]

[0673]

[0674]

[0675] As shown in the table above, the nucleic acid delivery efficiency of LNPs prepared from the cationic lipids of this disclosure in mice, after normalization to MC3, is consistently above 0.10, with some reaching as high as 9.31. Therefore, the cationic lipids of this disclosure achieve good, even excellent, in vivo targeted delivery of nucleic acid drugs.

[0676] Although the nucleic acid delivery efficiency of cationic lipids was tested using mRNA as an example in this disclosure, the nucleic acid drugs adapted to cationic lipids in this disclosure are not limited to this, but can be used to effectively deliver various nucleic acid drugs including but not limited to DNA drugs, RNA drugs, or both.

[0677] It should be understood that the present invention is not limited to the specific embodiments described above, as changes can be made to the specific embodiments, and these changes still fall within the scope of the appended claims.

Claims

1. A cationic lipid having the general formula (I): ###0001### Formula (I) wherein, A is a nitrilo group; B is selected from the group consisting of primary hydroxyl substituted methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl.

3. The cationic lipid according to claim 1 or 2, having a structure selected from the group consisting of: ###0003### ###0004### ###0005### 4. A liposome comprising the cationic lipid according to any one of claims 1 to 3.

5. A lipid nanoparticle comprising the cationic lipid according to any one of claims 1 to 3. B is C substituted by one hydroxy group 1-6 alkyl; C and D are each independently -L 3 -Y 2 -L 4 -CH=CR 4 R 5 wherein L 3 selected from linear C 1-12 alkylene, said C 1-12 alkylene is unsubstituted, Y 2 is -X-C(=X)-, wherein X is O, L 4 is a chemical bond, and R 4 and R 5 are each independently selected from the group consisting of linear C 2-12 alkyl, said C 2-12 alkyl being unsubstituted, wherein said R 4 and R 5 are different.

2. The cationic lipid of claim 1, wherein, 6. Use of the cationic lipid according to any one of claims 1 to 3 for the preparation of a liposome.

7. Use of the cationic lipid according to any one of claims 1 to 3 for the preparation of a lipid nanoparticle. ， ， ， ， ， , and 。 ​ ​ ​ ​

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