Ionizable lipids and lipid nanoparticle compositions for delivery of nucleic acids

Abstract

The present invention provides novel ionizable lipids. Also provided are novel lipid nanoparticle compositions for delivering nucleic acid materials to cells in vitro and in vivo, which have different and improved pharmacokinetic characteristics compared to those typically observed in the art. Methods of using the compositions for research and as therapeutic agents are also provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 425,969, filed Nov. 16, 2022, and U.S. Provisional Application No. 63 / 455,243, filed Mar. 28, 2023, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] There are many situations in which nucleic acids need to be delivered, including research, diagnostic, and therapeutic applications. An example of such a therapeutic application is gene therapy, which can be used to treat genetic disorders and other conditions. Although individual genetic disorders are rare, collectively they represent a significant disease burden, especially for children, resulting in severe disability and death.

[0004] In the field of gene therapy, viral vectors, such as AAV-based vectors, are commonly used to deliver genes into cells. However, AAV vectors are limited in the size of the genetic payload that they can package. Thus, any genetic payload greater than 4.7 kB is not suitable for delivery by AAV vectors, which limits the utility of such vectors for many indications. In addition, viral vectors (such as AAV) induce an antibody response, which limits readministration, which is not applicable in some indications. In addition, in indications where target cells, such as in the liver, are dividing, expression from successfully transduced cells may decrease or be lost with cell division and turnover, requiring readministration, which may be impossible or ineffective due to immune memory. In addition, many individuals have pre-existing immunity to commonly used viral vectors such as AAV, which may even limit the initial treatment with AAV gene therapy. In addition, viral vectors such as AAV may be toxic at the doses required to achieve therapeutic benefit in some indications.

[0005] Lipid nanoparticles (LNPs) provide an alternative to viral gene therapy. Although lipid nanoparticles have been developed and used to deliver many RNA therapeutics, the RNA delivered by lipid nanoparticles has a limited therapeutic lifespan. DNA delivered by lipid nanoparticles designed for RNA delivery has poor efficiency and significant activation of the innate immune response in the treated individual. New delivery vectors for delivering nucleic acids, such as DNA, into cells have significantly advanced many scientific studies, especially in vivo studies for patients in need of gene therapy and in vitro studies for research applications. Summary of the Invention

[0006] The present disclosure provides novel ionizable lipids having an ionizable head group attached to a lipid tail via a straight-chain alkyl core. The straight-chain alkyl core may have n carbon atoms, where n-1 carbon atoms in the straight-chain alkyl core are attached to the lipid tail. The present disclosure also provides novel lipid nanoparticle (LNP) compositions for delivering nucleic acid materials to cells in vitro and in vivo, which have different and improved pharmacokinetic characteristics compared to those commonly observed in the art. Methods of using the compositions of the present invention for research and as therapeutic agents are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A -C describes a study conducted to evaluate how the structure of ionizable lipids affects the efficacy and toxicity of DNA-LNPs. ( Figure 1A ) Formulation details of the test articles. The ionizable lipid varies in all formulations. The ionizable cationic lipids tested were ALC-0315 [bis(2-hexyldecanoic acid)(4-hydroxybutyl)azanediyl] bis(hexane-6,1-diyl) ester, MC3 4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester, and two ionizable cationic lipids L-2 and L-3 of the present disclosure. The phospholipid in all formulations was DSPC. The nucleic acid carrier used in all formulations was a nanoparticle plasmid DNA (npDNA) containing the hAAT promoter driving the expression of the EPO transgene. All test articles were observed to have good encapsulation efficiency and small size. ( Figure 1B ) Four hours after intravenous (i.v.) administration of a 1 mg / kg dose of the test article to wild-type BALB / c mice, the EPO serum level was measured. ( Figure 1C ) Four hours after intravenous (i.v.) administration of a 1 mg / kg dose of the test article to wild-type BALB / c mice, the IL-6 cytokine level in the serum was measured.

[0008] Figure 2A -O describes a further study conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs. ( Figure 2A - 2B)Formulation details of the test articles. The ionizable lipids vary in these formulations. Two benchmark ionizable lipids, ALC-0315 and MC3, were described above. Other benchmark ionizable lipids were tested, including LP01 (see Finn et al., Cell Reports, 2018, 22:2227), SM102 (see Sabnis et al., Molecular Therapy, 2018, 26:1509), and ARCT (see Rajappan et al., Organic Process R&D, 2021, 25:1383). Three ionizable lipids of the present disclosure, L-5, L-15, and L-9, were also tested. The phospholipid in these formulations is DSPC ( Figure 2B ) or DOPE ( Figure 2C ). The nucleic acid carrier used in all formulations is a nanoparticle plasmid DNA (npDNA) containing the hAAT promoter that drives the expression of the EPO transgene. All test articles were observed to have good encapsulation efficiency and small size.( Figure 2B )( Figure 2C ) Seven days after i.v. administration of a 1 or 0.3 mg / kg dose of the test articles in Figure 2A and 2B to wild-type BALB / c mice, the serum levels of EPO protein in the serum of wild-type BALB / c mice were measured.( Figure 2D -I) Four hours after i.v. administration of a 1 or 0.3 mg / kg dose of the test articles in Figure 2A (formulated with DSPC phospholipid) to wild-type BALB / c mice, the levels of IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC in their serum were measured.( Figure 2J -O) Four hours after i.v. administration of the test articles in Figure 2B (formulated with DOPE phospholipid) to wild-type BALB / c mice, the levels of IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC in their serum were measured.

[0009] Figure 3A -F describes further studies conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs.( Figure 3A - 3B)Formulation details of the test articles. The ionizable lipids vary in these formulations. The reference ionizable lipid ALC-0315 is as described above. Other reference ionizable lipids were tested, including A9 (see Han et al., Nature Communications, 2021, 12:7233) and ssOP (see Tanaka et al., Pharmaceutics, 2021, 13:544). Three ionizable lipids of the present disclosure, L-12, L-13, and L-14, were also tested. The phospholipid in these formulations is DSPC( Figure 3A ) or DOPE( Figure 3B ). The nucleic acid carrier used in all formulations is nanoparticle plasmid DNA (npDNA) containing the hAAT promoter that drives the expression of the EPO transgene. All test articles were observed to have good encapsulation efficiency and small size.( Figure 3C ) Seven days after i.v. administration of 1 or 0.3 mg / kg dose of the test articles to wild-type BALB / c mice, the EPO protein level in their sera was measured.( Figure 3D -I) Four hours after i.v. administration of 1 or 0.3 mg / kg dose of the Figure 3A test articles (formulated with DSPC phospholipid) to wild-type BALB / c mice, the levels of IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC in their sera were measured.( Figure 3J -O) Four hours after i.v. administration of Figure 3B test articles (formulated with DOPE phospholipid) to wild-type BALB / c mice, the levels of IL-6, IFNγ, TNFα, IL-1β, IL-12, and KC in their sera were measured.

[0010] Figure 4A -C describes further studies conducted to evaluate how the structures of additional ionizable lipids affect the efficacy and toxicity of DNA-LNPs.( Figure 4A ) Formulation details of the test articles. The ionizable lipids vary in these formulations. The reference ionizable lipid ALC-0315 is as described above. Five ionizable lipids of the present disclosure, L-9, L-10, L-11, L-15, and L-16, were also tested. The phospholipid in these formulations is DSPC. The nucleic acid carrier used in all formulations is nanoparticle plasmid DNA (npDNA) containing the hAAT promoter that drives the expression of the EPO transgene. All test articles were observed to have good encapsulation efficiency and small size.( Figure 4B ) Three days after i.v. administration of 1 or 0.3 mg / kg dose of the test articles to wild-type BALB / c mice, the EPO protein level in their sera was measured.( Figure 4C) The IL-6 cytokine levels in the serum were measured 4 hours after i.v. administration of 1 or 0.3 mg / kg dose of the test article to wild-type BALB / c mice.

[0011] Figure 5A -C describes further studies conducted to evaluate how the structure of additional ionizable lipids affects the efficacy and toxicity of DNA-LNPs. ( Figure 5A ) Details of the formulations of the test articles. The ionizable lipids vary in these formulations. Two benchmark ionizable lipids, ALC-0315 and ARCT, are described above. An additional benchmark ionizable lipid, CL1 (see Lam et al., Advanced Materials, 2023, 35: 2209624), was also tested. Four ionizable lipids of the present disclosure, L-17, L-21, L-19, and L-20, were also tested. The phospholipid in these formulations is DSPC. The nucleic acid carrier used in all formulations is a nanoplasmid DNA (npDNA) containing the hAAT promoter that drives the expression of the EPO transgene. All test articles were observed to have good encapsulation efficiency and small size. ( Figure 5B ) The EPO protein levels in the serum of wild-type BALB / c mice were measured 3 days after i.v. administration of 1 or 0.3 mg / kg dose of the test article to wild-type balb / c mice. ( Figure 5C ) The IL-6 cytokine levels in the serum were measured 4 hours after i.v. administration of 1 or 0.3 mg / kg dose of the test article to wild-type BALB / c mice.

[0012] Figure 6A -K describes studies conducted to evaluate how the structure of ionizable lipids affects the efficacy and toxicity of LNPs co-formulated with DNA and mRNA. ( Figure 6A ) Details of the formulations of the test articles. The ionizable lipids vary in these formulations. The benchmark ionizable lipid CL1 is as described above. Three ionizable lipids of the present disclosure, L-15, L-17, and L-18, were also tested. The phospholipid in these formulations is DOPE. The nucleic acid carrier used in all formulations contains nanoplasmid DNA (npDNA) and mRNA mixed at a DNA:mRNA ratio of 1:3 (w / w), where the npDNA contains the TTR promoter that drives the expression of the human factor IX (FIX) transgene. All test articles were observed to have good encapsulation efficiency and small size. ( Figure 6B ) The human FIX protein levels in the serum were measured 21 days after i.v. administration of 0.5 mg / kg DNA (1.5 mg / kg mRNA) dose of the test article to wild-type balb / c mice. ( Figure 6C-K) The cytokine levels in the serum were measured 4 hours after i.v. administration of a test article at a dose of 0.5 mg / kg DNA (1.5 mg / kg mRNA) to wild-type BALB / c mice. Detailed Description

[0013] 4.1 Lipid Nanoparticle Compositions

[0014] Provided are novel lipid nanoparticle compositions for delivering nucleic acids to cells in vitro and in vivo, which have distinct and improved pharmacokinetic profiles compared to those commonly observed in the art. Also provided are methods of using the lipid nanoparticle compositions of the present disclosure for research and as therapeutic agents.

[0015] "Lipid nanoparticle" refers to a lipid composition that can be used to deliver an active agent or therapeutic agent such as a nucleic acid (e.g., DNA and / or RNA), protein, small molecule, etc. to a target site of interest. In a lipid nanoparticle, the nucleic acid reagent can be encapsulated in the lipid, thereby protecting the reagent from enzymatic degradation.

[0016] Typically, lipid nanoparticles include several lipid components, including, for example, an ionizable lipid, one or more helper lipids (e.g., non-cationic lipids), and a lipid that prevents nanoparticle aggregation (also referred to as a coating lipid or conjugated lipid, e.g., PEG-lipid). In some aspects, the present disclosure provides a lipid nanoparticle (LNP) composition as described herein that comprises a nucleic acid, wherein the nucleic acid is substantially encapsulated by the lipid components of the LNP.

[0017] 4.2 Ionizable Lipids

[0018] The lipid nanoparticles (LNPs) of the present disclosure can include an ionizable lipid. As outlined above, novel ionizable lipids are provided herein. Ionizable lipids are commonly used in lipid nanoparticles (LNPs) to condense their nucleic acid cargo, e.g., DNA or RNA, at low pH and drive membrane association and fusion. The term "ionizable lipid" refers to a lipid that contains an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or lower), but can remain neutral at, for example, a higher pH (e.g., physiological pH). The pH sensitivity of such ionizable lipids may be desirable to provide intracellular delivery of the nucleic acid cargo. When neutral, the ionizable lipid can have less interaction with the cell membrane and then become charged when internalized into an endosome in a target cell, where the pH is lower than that in the extracellular environment. The protonated and thus positively charged ionizable lipid can promote membrane destabilization and facilitate endosomal escape of the nanoparticle.

[0019] In some embodiments, the ionizable lipid is a cationic lipid. The term "cationic lipid" refers to a lipid that carries a net positive charge at a selected pH (e.g., pH 6.5 or lower). In some embodiments, the ionizable lipid is a cationic lipid that includes at least one ionizable amino group that is positively charged or becomes protonated at a selected pH (e.g., pH 6.5 or lower). In some embodiments, the cationic lipid includes one or more tertiary amino groups, such as trialkylamino groups.

[0020] As disclosed herein, ionizable lipids include ionizable head groups (e.g., ionizable amino groups) connected to lipid tails via a straight chain alkyl core. The straight chain alkyl core may have n carbon atoms, wherein n-1 carbon atoms in the straight chain alkyl core are connected to lipid tails. In certain embodiments, the straight chain alkyl core has 3 carbon atoms and 2 lipid tails. In certain embodiments, the straight chain alkyl core has 4 carbon atoms and 3 lipid tails. In certain embodiments, the straight chain alkyl core has 5 carbon atoms and 4 lipid tails. In certain embodiments, the straight chain alkyl core has 6 carbon atoms and 5 lipid tails.

[0021] In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, a linear alkyl core, a hydrocarbon chain (e.g., C8-C 20 Carbon chains, such as C 18 In some embodiments, the cationic lipid comprises a hydrocarbon chain having the same number of ether bonds as the ether bonds. In some embodiments, the cationic lipid comprises a protonatable tertiary amine head group, a straight chain alkyl core, a hydrocarbon chain (such as described herein), and an ester bond between the straight chain alkyl core and the hydrocarbon chain. In some embodiments, the cationic lipid comprises a hydrocarbon chain having the same number of ester bonds as the ester bonds. In some embodiments, the cationic lipid comprises a protonatable tertiary amine head group, a straight chain alkyl core, a hydrocarbon chain (such as described herein), and a carbonate bond between the straight chain alkyl core and the hydrocarbon chain. In some embodiments, the cationic lipid comprises a hydrocarbon chain having the same number of carbonate bonds as the ester bonds. In some embodiments, the cationic lipid comprises 2 or more hydrocarbon chains, such as 3 or more hydrocarbon chains, or 4 or more hydrocarbon chains.

[0022] Aspects of the present disclosure include an ionizable lipid compound of formula (I):

[0023] (ZLY)-W n -(XR) (n-1)

[0024] (I)

[0025] in:

[0026] Z is an ionizable head group;

[0027] L is an optionally substituted (C 1- C 12 ) alkylene;

[0028] Y is a linking group;

[0029] W n is a straight-chain alkyl core having n carbon atoms, where n is from 3 to 6;

[0030] X is an optional linking group; and

[0031] each R is independently a lipid tail.

[0032] In some embodiments of formula (I), n is from 4 to 6, such that the straight-chain alkyl core has 4 to 6 carbon atoms. In some cases, n is 4, such that the straight-chain alkyl core has 4 carbon atoms. In some cases, n is 5, such that the straight-chain alkyl core has 5 carbon atoms. In some cases, n is 6, such that the straight-chain alkyl core has 6 carbon atoms. In some embodiments, n is 3, such that the straight-chain alkyl core has 3 carbon atoms.

[0033] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0034]

[0035] where * depicts the attachment point to Y and each ** depicts the attachment point to X.

[0036] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0037]

[0038] where * depicts the attachment point to Y and each ** depicts the attachment point to X; and each G 2 is independently H or -CH2OH. In certain cases, at least one G 2 is H. In certain cases, both G 2 groups are H. In certain cases, at least one G 2 is -CH2OH. In certain cases, both G 2 groups are -CH2OH. In certain cases, one G 2 is H and the other is -CH2OH.

[0039] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0040]

[0041] where * depicts the attachment point to Y and each ** depicts the attachment point to X; and G 2 is H or -CH2OH. In some cases, G 2 is H. In some cases, G 2 is -CH2OH.

[0042] In some embodiments of formula (I), W n is:

[0043]

[0044] where * depicts the attachment point to Y and each ** depicts the attachment point to X; and G 2 is H or -CH2OH. In some cases, G 2 is H. In some cases, G 2 is -CH2OH.

[0045] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0046]

[0047] where * depicts the attachment point to Y and each ** depicts the attachment point to X; and G 1 is H or a group that is cyclically linked to Y, and the group forms a heterocycle together with the carbon atom of the W n to which it is attached. In some cases, G 1 is cyclically linked to Y to form a 5-membered heterocycle. In some cases, G 1 is cyclically linked to Y to form a 6-membered heterocycle. In some cases, G 1 is H.

[0048] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0049]

[0050] where * depicts the attachment point to Y and each ** depicts the attachment point to X; G 1 is H or a group that is cyclically linked to Y, and the group forms a heterocycle together with the carbon atom of the W n to which it is attached; and G 2 is H or -CH2OH. In some cases, G 1 is cyclically linked to Y to form a 5-membered heterocycle. In some cases, G 1 is cyclically linked to Y to form a 6-membered heterocycle. In some cases, G1 is H. In some cases, G 2 is H. In some cases, G 2 is -CH2OH. In some cases, G 1 and G 2 at least one of them is H. In some cases, G 1 and G 2 are both H. In some cases, G 1 is H and G 2 is -CH2OH. In some cases, G 1 is cyclically connected to Y to form a heterocycle, and G 2 is H. In some cases, G 1 is cyclically connected to Y to form a heterocycle, and G 2 is -CH2OH.

[0051] In some embodiments of formula (I), the straight-chain alkyl core W n is:

[0052]

[0053] where * depicts the attachment point to Y and each ** depicts the attachment point to X; G 1 is H or a group cyclically connected to Y, and the group together with the carbon atom of W n to which it is attached forms a heterocycle; and G 2 is H or -CH2OH. In some cases, G 1 is cyclically connected to Y to form a 5-membered heterocycle. In some cases, G 1 is cyclically connected to Y to form a 6-membered heterocycle. In some cases, G 1 is H. In some cases, G 2 is H. In some cases, G 2 is -CH2OH. In some cases, G 1 and G 2 at least one of them is H. In some cases, G 1 and G 2 are both H. In some cases, G 1 is H and G 2 is -CH2OH. In some cases, G 1 is cyclically connected to Y to form a heterocycle, and G 2 is H. In some cases, G 1 is cyclically connected to Y to form a heterocycle, and G 2 is -CH2OH.

[0054] As described above, in formula (I), the alkyl straight-chain core W nIt is connected to the ionizable head group Z through a linking group Y. A "linking group" refers to a linking moiety that connects two groups via a covalent bond. The linking group Y can be a straight chain, branched chain, cyclic, single atom, or covalent bond. Examples of such linking groups include, but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, amido, urea, imide, ether, thioether, carbonate, alkyldioxy, oxyimino, amino, carbonyl, heterocycle (such as cyclic acetal), etc.

[0055] In some embodiments of formula (I), Y is selected from -O-, -C(R 10 )2-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, where R 10 is selected from H and C 1-6 alkyl. In some cases, Y is selected from -O-, -OC(O)-, -C(O)O- and -OC(O)NR 10 -. In some cases, Y is -O-. In some cases, Y is -OC(O)-. In some cases, Y is -OC(O)NR 10 ]-, where R 10 is H. In some cases, Y is -C(R 10 )2-, where each R 10 is H. In some cases, Y is -C(O)O-. In some cases, Y is -OC(O)O-. In some cases, Y is -SC(O)NR 10 -, where each R 10 is H. In some cases, Y is -C(O)NR 10 -, where each R 10 is H. In some cases, Y is -NR 10 C(O)-, where each R 10 is H. In some cases, Y is -S-. In some cases, Y is -NR 2 -. In some cases, Y is -NR 10 C(O)O-, where each R 10 is H. In some cases, Y is -NR 10 C(O)S-, where each R 10 is H.

[0056] In some embodiments, W n comprises a group G adjacent to the attachment point of the linking group Y 1 . In some embodiments, G 1 is cyclically linked to the linking group Y to form a heterocycle. In some embodiments, G 1 is cyclically linked to Y to form a 5-membered heterocycle. In some embodiments, the 5-membered heterocycle is a cyclic acetal. In some embodiments, G 1 is cyclically linked to Y to form a 6-membered heterocycle. In some embodiments, the 6-membered heterocycle is a cyclic acetal.

[0057] As described above, in formula (I), the linking group Y is linked to the ionizable head group Z through an optionally substituted (C 1- C 12 ) alkylene L. In some embodiments of formula (I), L is a (C2-C6) alkylene or a substituted (C2-C6) alkylene. In some embodiments, L is a (C2-C4) alkylene or a substituted (C2-C4) alkylene. In certain cases, L is a C2-alkylene or a substituted C2-alkylene. In certain cases, L is a C3-alkylene or a substituted C3-alkylene. In certain cases, L is a C4-alkylene or a substituted C4-alkylene. In certain cases, L is a C5-alkylene or a substituted C5-alkylene. In certain cases, L is a C6-alkylene or a substituted C6-alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3. In certain cases, L is -(CH2)4-. In certain cases, L is -(CH2)5-. In certain cases, L is -(CH2)6-.

[0058] In some embodiments, -Y-L-Z has the formula -O(CH2) r Z, where r is from 2 to 6. In some embodiments, -Y-L-Z has the formula -OC(O)(CH2) r Z, where r is from 2 to 6. In some embodiments, -Y-L-Z has the formula -OC(O)NH(CH2) r Z, where r is from 2 to 6. In some embodiments, -Y-L-Z has the formula -CH2(CH2) r Z, where r is from 2 to 6. In some cases, r is from 2 to 4. In some cases, r is 2. In some cases, r is 3. In some cases, r is 4.

[0059] As described herein, the ionizable lipids of formula (I) include an ionizable head group. In some embodiments, the ionizable head group includes a primary amine, secondary amine, or tertiary amine that can be protonated at physiological pH. In some embodiments, the ionizable head group includes a tertiary amino group. In certain embodiments, the ionizable head group has the formula -NR11 R 12 , wherein R 11 and R 12 are each independently an alkyl group or a substituted alkyl group. In some embodiments, R 11 and R 12 are each independently a C 1-6 alkyl group or a substituted C 1-6 alkyl group. In some embodiments, R 11 and R 12 are each independently a C 1-3 alkyl group or a substituted C 1-3 alkyl group. In some embodiments, R 11 and R 12 are each a C 1-3 alkyl group. In some embodiments, R 11 and R 12 are each a methyl group. In some embodiments, R 11 and R 12 are each an ethyl group. In certain cases, R 11 and R 12 are both propyl groups. In certain cases, R 11 and R 12 are both n-propyl groups. In certain cases, R 11 and R 12 are both isopropyl groups. In certain cases, R 11 and R 12 are both isopropyl groups. In certain cases, R 11 and R 12 are each independently an optionally substituted butyl group. In certain cases, R 11 and R 12 are each independently an optionally substituted n-butyl group. In certain cases, R 11 and R 12 are each independently an optionally substituted sec-butyl group. In certain cases, R 11 and R 12 are each independently an optionally substituted butyl group. In certain cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted C 1-4 alkyl group, a C 1-3 alkyl group, a C 1-4 heteroalkyl group, and a C 1-3 heteroalkyl group.

[0060] As described herein, formula (I) includes 2 to 5 lipid tails R, which are optionally linked to the core W via an additional linking group X (such as -(X-R) (n-1) ) n connected.

[0061] In some embodiments, the ionizable lipid of formula (I) includes a linking group X. The linking group X can be linear, branched, cyclic, or a single atom. Examples of such linking groups include, but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkyl, amido, urea, imide, ether, thioether, thiocarbamate, carbonate, alkyldioxy, oxyimino, amino, carbonyl, etc. In some embodiments of formula (I), each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O-, -(CH2) s OC(O)O-, -(CH2) s OC(O)NR 10 -, -(CH2) s O-, -(CH2) s SC(O)NR 10 -, -(CH2) s C(O)NR 10 -, -(CH2) s NR 10 C(O)-, -(CH2) s S-, -(CH2) s NR 10 -, -(CH2) s NR 10 C(O)O- and -(CH2) s NR 10 C(O)S-, where R 10 is selected from H and C 1-6 alkyl and s is from 0 to 6. In some embodiments, each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O- and -(CH2) s OC(O)O-. In some embodiments, each X is -(CH2) s OC(O)-, where s is 0, 1, or 2. In some embodiments, each X is -(CH2) s C(O)O-, where s is 0, 1, or 2. In some embodiments, each X is -(CH2) s OC(O)O-, where s is 0, 1, or 2. In some embodiments, at least one X group is -(CH2) sO⁻, where s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s OC(O)NR 10 ⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s SC(O)NR 10 ⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s C(O)NR 10 ⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s NR 10 C(O)⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s S⁻, where s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s NR 10 ⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s NR 10 C(O)O⁻, where R 10 is H and s is 0, 1, or 2. In some embodiments, at least one X group is -(CH₂) s NR 10 C(O)S⁻, where R 10 is H and s is 0, 1, or 2.

[0062] In some embodiments of formula (I), each X is independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -O-, -OC(O)NR 10 ⁻, -SC(O)NR 10 ⁻, -C(O)NR 10 ⁻, -NR 10 C(O)⁻, -S⁻, -NR 10 ⁻, -NR 10 C(O)O⁻ and -NR 10 C(O)S⁻, where R 10 is selected from H and C 1-6Alkyl. In some embodiments, each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In some embodiments, each X is -OC(O)-. In some embodiments, each X is -C(O)O-. In some embodiments, each X is -OC(O)O-. In some embodiments, at least one X group is -O-. In some embodiments, at least one X group is -OC(O)NR 10 -, where R 10 is H. In some embodiments, at least one X group is -SC(O)NR 10 -, where R 10 is H. In some embodiments, at least one X group is -C(O)NR 10 -, where R 10 is H. In some embodiments, at least one X group is -NR 10 C(O)-, where R 10 is H. In some embodiments, at least one X group is -S-. In some embodiments, at least one X group is -NR 10 -, where R 10 is H. In some embodiments, at least one X group is -NR 10 C(O)O-, where R 10 is H. In some embodiments, at least one X group is -NR 10 C(O)S-, where R 10 is H.

[0063] In some embodiments of formula (I), each -X-R is independently selected from -(CH2) s OC(O)R, -(CH2) s C(O)OR, -(CH2) s OC(O)OR, -(CH2) s OR, -(CH2) s OC(O)NR 10 R, -(CH2) s SC(O)NR 10 R, -(CH2) s C(O)NR 10 R, -(CH2) s NR 10 C(O)R, -(CH2) s SR, -(CH2) s NR 10 R, -(CH2) s NR 10 C(O)OR and -(CH2) s NR 10 C(O)SR, where R10 Selected from H and C 1-6 an alkyl group, s is from 0 to 6, and each R is independently a lipid tail. In some embodiments, each -X-R is -(CH2) s OC(O)R. In some embodiments, each -X-R is -(CH2) s C(O)OR. In some embodiments, each -X-R is -(CH2) s OC(O)OR. In some embodiments, each -X-R is -(CH2) s OR. In some embodiments, each -X-R is -(CH2) s OC(O)NR 10 R. In some embodiments, each -X-R is -(CH2) s SC(O)NR 10 R. In some embodiments, each -X-R is -(CH2) s C(O)NR 10 R. In some embodiments, each -X-R is -(CH2) s NR 10 C(O)R. In some embodiments, each -X-R is -(CH2) s SR. In some embodiments, each -X-R is -(CH2) s NR 10 R. In some embodiments, each -X-R is -(CH2) s NR 10 C(O)OR. In some embodiments, each -X-R is -(CH2) s NR 10 C(O)SR.

[0064] In some embodiments of formula (I), each -X-R is independently selected from -OC(O)R, C(O)OR, OC(O)OR, OR, OC(O)NR 10 R, SC(O)NR 10 R, C(O)NR 10 R, NR 10 C(O)R, SR, NR 10 R, NR 10 C(O)OR and NR 10 C(O)SR, wherein R 10 is selected from H and C 1-6an alkyl group, and each R is independently a lipid tail. In some embodiments, each -X-R is OC(O)R. In some embodiments, each -X-R is C(O)OR. In some embodiments, each -X-R is OC(O)OR. In some embodiments, each -X-R is OR. In some embodiments, each -X-R is OC(O)NR 10 R. In some embodiments, each -X-R is SC(O)NR 10 R. In some embodiments, each -X-R is -C(O)NR 10 R. In some embodiments, each -X-R is -NR 10 C(O)R. In some embodiments, each -X-R is -SR. In some embodiments, each -X-R is -NR 10 R. In some embodiments, each -X-R is -NR 10 C(O)OR. In some embodiments, each -X-R is -NR 10 C(O)SR. In some embodiments of formula (I), each lipid tail is independently a straight-chain or branched-chain, saturated or unsaturated, and / or optionally cyclic-group-containing aliphatic hydrocarbon group.

[0065] In some embodiments of formula (I), each R is a straight-chain hydrocarbon group optionally containing one or more cyclic groups. In some embodiments, each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl straight-chain hydrocarbon groups. In some embodiments, each R is independently selected from C6-C 12 alkyl and C6-C 12 alkenyl straight-chain hydrocarbon groups. In some embodiments, at least one R is a straight-chain hydrocarbon group containing a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any one of the monocyclic or bicyclic groups is optionally substituted.

[0066] In some embodiments of formula (I), at least one R is a branched hydrocarbon group optionally containing one or more cyclic groups. In some embodiments, each R is a branched hydrocarbon group optionally containing one or more cyclic groups. In some embodiments, the branched hydrocarbon group contains 8 to 20 carbon atoms. In some embodiments, the branched hydrocarbon group contains 8 carbon atoms. In some embodiments, the branched hydrocarbon group contains 9 carbon atoms. In some embodiments, the branched hydrocarbon group contains 10 carbon atoms. In some embodiments, the branched hydrocarbon group contains 11 carbon atoms. In some embodiments, the branched hydrocarbon group contains 12 carbon atoms. In some embodiments, the branched hydrocarbon group contains 13 carbon atoms. In some embodiments, the branched hydrocarbon group contains 14 carbon atoms. In some embodiments, the branched hydrocarbon group contains 15 carbon atoms. In some embodiments, the branched hydrocarbon group contains 16 carbon atoms. In some embodiments, the branched hydrocarbon group contains 17 carbon atoms. In some embodiments, the branched hydrocarbon group contains 18 carbon atoms. In some embodiments, the branched hydrocarbon group contains 19 carbon atoms. In some embodiments, the branched hydrocarbon group contains 20 carbon atoms. In some embodiments, the branched hydrocarbon group is saturated. In some embodiments, the branched hydrocarbon group is unsaturated. In some embodiments, R has the formula -CH(R 7 )2, where each R 7 is independently a C5-C 12 alkyl or a C5-C 12 alkenyl. In some embodiments, each R 7 is a C5-alkyl or a C5-alkenyl. In some embodiments, each R 7 is a C6-alkyl or a C6-alkenyl. In some embodiments, each R 7 is a C7-alkyl or a C7-alkenyl. In some embodiments, each R 7 is a C8-alkyl or a C8-alkenyl. In some embodiments, each R 7 is a C9-alkyl or a C9-alkenyl. In some embodiments, each R 7 is a C 10 -alkyl or a C 10 -alkenyl. In some embodiments, each R 7 is a C 11 -alkyl or a C 11 -alkenyl. In some embodiments, each R 7 is a C 12 -alkyl or a C 12 -alkenyl. In some embodiments, at least one R is a branched hydrocarbon group containing a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, where any one of the monocyclic or bicyclic groups is optionally substituted.

[0067] In some embodiments, R is a straight-chain or branched-chain hydrocarbon group containing one or more cyclic groups. In some embodiments, the cyclic group is an optionally substituted monocyclic cycloalkyl group. In some embodiments, the cyclic group is an optionally substituted bicyclic cycloalkyl group. In some cases, the cyclic group is an optionally substituted monocyclic aryl group. In some cases, the cyclic group is an optionally substituted bicyclic aryl group. In some cases, the cyclic group is an optionally substituted monocyclic or bicyclic heterocyclic group. In some cases, the cyclic group is an optionally substituted monocyclic or bicyclic heteroaryl group.

[0068] In some embodiments of formula (I), R is wherein

[0069] Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5- to 12-membered monocyclic groups, bicyclic groups, bridged polycyclic groups and spirocyclic groups;

[0070] R x and R y are each independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 20 aliphatic group;

[0071] r, p and q are each independently an integer from 0 to 20.

[0072] In some embodiments, R is wherein

[0073] Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group selected from 5- to 12-membered monocyclic groups, bicyclic groups, bridged polycyclic groups and spirocyclic groups;

[0074] R x and R y are each independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 10 aliphatic group;

[0075] r, p and q are each independently an integer from 0 to 10.

[0076] In some embodiments, R is wherein

[0077] Cy A and Cy BEach is independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, and the cyclic group is selected from a 5- to 12-membered monocyclic group, bicyclic group, bridged polycyclic group and spirocyclic group;

[0078] R x and R y Each is independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C6 aliphatic group;

[0079] r, p and q are each independently an integer from 0 to 6.

[0080] In some embodiments, R comprises a group selected from the following:

[0081]

[0082]

[0083] where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is where each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R.

[0084] In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R. In some embodiments, R is Each # represents an attachment point to X, or an attachment point to a straight-chain or branched hydrocarbon chain of R.

[0085] In some embodiments, the compound of formula (I) has the formula (IIA):

[0086]

[0087] wherein X, R, Y, L and Z are as defined above.

[0088] In certain embodiments of formula (IIA), Y is selected from -O-, -OC(O)- and -OC(O)NR 10 -, where R 10 is H and C 1-6 alkyl. In certain embodiments of formula (IIA), Y is -O-. In certain embodiments of formula (IIA), Y is -OC(O)-. In certain embodiments of formula (IIA), Y is -OC(O)NR 10 -, where R 10 is H.

[0089] In certain embodiments of formula (IIA), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In certain cases, L is (C2-C6) alkylene. In certain cases, L is (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0090] In certain embodiments of formula (IIA), Z is a tertiary amine. In certain cases, Z is -NR 11 R 12 where R 11 and R 12 are each independently C 1-6 alkyl or substituted C 1-6 alkyl. In certain cases, R 11 and R 12 are each C 1-3 alkyl. In certain cases, R 11 and R 12 are each methyl. In certain cases, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 are both propyl. In certain cases, R 11 and R 12 are both n-propyl. In certain cases, R 11 and R 12 are both isopropyl. In certain cases, R 11 and R 12 are both isopropyl. In certain cases, R 11 and R 12Each is independently an optionally substituted butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted n-butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted sec-butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted butyl group. In some cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In some cases, each R 11 and R 12 is independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkyl, C 1-4 heteroalkyl, and C 1-3 heteroalkyl.

[0091] In certain embodiments of formula (IIA), each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In certain embodiments of formula (IIA), at least one X is -OC(O)-. In some cases, each X is -OC(O)-. In certain embodiments of formula (IIA), at least one X is -C(O)O-. In some cases, each X is -C(O)O-. In certain embodiments of formula (IIA), at least one X is -OC(O)O-. In some cases, each X is -OC(O)O-.

[0092] In certain embodiments of formula (IIA), each -X-R has the formula -OC(O)R. In certain embodiments, each -X-R has the formula -C(O)OR. In certain embodiments, each -X-R has the formula -OC(O)OR.

[0093] In certain embodiments of formula (IIA), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl. In some cases, each R is C5-C 12 alkyl, C5-C 12 alkenyl, and C5-C 12 alkynyl. In some cases, each R is C5-C 12Alkyl. In certain cases, each R is a C5 alkyl. In certain cases, each R is a C6 alkyl. In certain cases, each R is a C7 alkyl. In certain cases, each R is a C8 alkyl. In certain cases, each R is a C9 alkyl. In certain cases, each R is a C 10 Alkyl. In certain cases, each R is a C 11 Alkyl. In certain cases, each R is a C 12 Alkyl.

[0094] In certain embodiments of formula (IIA), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups (such as those described herein). In certain embodiments of formula (IIA), R is -CH(R 7 )2, where each R 7 is independently a C5-C 12 alkyl or a C5-C 12 alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkenyl.

[0095] In certain embodiments of formula (IIA), at least one R is a straight-chain or branched hydrocarbon group containing one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is a phenyl group. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0096] In certain embodiments of formula (IIA), the compound has formula (IIIA):

[0097]

[0098] Where:

[0099] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0100] q is 1 to 4;

[0101] Y is selected from -O-, -OC(O)-, and -OC(O)NR10 -; and

[0102] Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

[0103] In some embodiments of formula (IIA), the compound has formula (IIIA), wherein:

[0104] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0105] q is 1 to 4;

[0106] Y is selected from -O-, -OC(O)- and -OC(O)NR 10 -; and

[0107] Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl and -CH(R 7 )2, where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

[0108] In certain embodiments of formula (IIIA), Y is selected from -O-, -OC(O)- and -OC(O)NR 10 -, where R 10 is selected from H and C 1-6 alkyl. In certain embodiments of formula (IIIA), Y is -O-. In certain embodiments of formula (IIIA), Y is -OC(O)-. In certain embodiments of formula (IIA), Y is -OC(O)NR 10 -, where R 10 is H.

[0109] In certain embodiments of formula (IIIA), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.

[0110] In certain embodiments of formula (IIIA), R11 and R 12 are different. In some cases, at least one of R 11 and R 12 is methyl. In some cases, R 11 and R 12 are the same. In some cases, R 11 and R 12 are both methyl. In some cases, at least one of R 11 and R 12 is ethyl. In some cases, R 11 and R 12 are both ethyl. In some cases, R 11 and R 12 are both propyl. In some cases, R 11 and R 12 are both n-propyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are both independently optionally substituted butyl. In some cases, R 11 and R 12 are both independently optionally substituted n-butyl. In some cases, R 11 and R 12 are both independently optionally substituted sec-butyl. In some cases, R 11 and R 12 are both independently optionally substituted butyl. In some cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In some cases, each R 11 and R 12 is independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkyl, C 1-4 heteroalkyl, and C 1-3 heteroalkyl.

[0111] In certain embodiments of formula (IIIA), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl. In some cases, each R is selected from C5-C 12 alkyl, C5-C12 Alkenyl and C5-C 12 Alkynyl. In certain cases, each R is C5-C 12 Alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is C6 alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is C8 alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C 10 Alkyl. In certain cases, each R is C 11 Alkyl. In certain cases, each R is C 12 Alkyl.

[0112] In certain embodiments of formula (IIIA), at least one R is -CH(R 7 )2, where each R 7 is independently C5-C 12 Alkyl or C5-C 12 Alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 Alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 Alkenyl.

[0113] In certain embodiments of formula (IIIA), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is aryl. In some embodiments, J is phenyl. In certain embodiments, t and u are both 1 to 5. In some cases, t is 2 and u is 3.

[0114] In certain embodiments of the compound of formula (I), the compound has formula (IIB):

[0115]

[0116] where X, R, Y, L, and Z are as defined above.

[0117] In certain embodiments of formula (IIB), Y is selected from -O-, -OC(O)-, -OC(O)NR 10 -, -NR 10 C(O)-, -NR 10 C(O)O-, and -NR 10 C(O)S-, where R 10 is selected from H and C 1-6Alkyl. In certain embodiments of formula (IIB), Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-. In certain cases, Y is -NHC(O)-. In certain cases, Y is -NHC(O)O-. In certain cases, Y is -NHC(O)S-.

[0118] In certain embodiments of formula (IIB), L is a (C2-C6) alkylene or a substituted (C2-C6) alkylene. In certain cases, L is a (C2-C6) alkylene. In certain cases, L is a (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0119] In certain embodiments of formula (IIB), Z is a tertiary amine. In certain cases, Z is -NR 11 R 12 , where R 11 and R 12 are each independently a C 1-6 alkyl or a substituted C 1-6 alkyl. In certain cases, R 11 and R 12 are each a C 1-3 alkyl. In certain cases, R 11 and R 12 are each methyl. In certain cases, R 11 and R 12 are each ethyl. In certain cases, R 11 and R 12 are both propyl. In certain cases, R 11 and R 12 are both n-propyl. In certain cases, R 11 and R 12 are both isopropyl. In certain cases, R 11 and R 12 are both isopropyl. In certain cases, R 11 and R 12 are both independently optionally substituted butyl. In certain cases, R 11 and R 12 are both independently optionally substituted n-butyl. In certain cases, R 11 and R 12 are both independently optionally substituted sec-butyl. In certain cases, R 11 and R 12 are both independently optionally substituted butyl. In certain cases, each R 11 and R 12Independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In some cases, each R 11 and R 12 are independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkenyl, C 1-4 heteroalkyl, and C 1-3 heteroalkenyl.

[0120] In certain embodiments of formula (IIB), each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-. In certain embodiments of formula (IIB), at least one X is -OC(O)-. In some cases, each X is -OC(O)-. In certain embodiments of formula (IIB), at least one X is -C(O)O-. In certain cases, each X is -C(O)O-. In certain embodiments of formula (IIB), at least one X is -OC(O)O-. In certain cases, each X is -OC(O)O-.

[0121] In certain embodiments of formula (IIB), each -X-R has the formula -OC(O)R. In certain embodiments of formula (IIB), each -X-R has the formula -C(O)OR. In certain embodiments of formula (IIB), each -X-R has the formula -OC(O)OR.

[0122] In certain embodiments of formula (IIB), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl. In certain cases, each R is C5-C 12 alkyl, C5-C 12 alkenyl, and C5-C 12 alkynyl. In certain cases, each R is C5-C 12 alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is C6 alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is C8 alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C 10 alkyl. In certain cases, each R is C 11 alkyl. In certain cases, each R is C 12 alkyl.

[0123] In certain embodiments of formula (IIB), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups (such as those described herein). In certain embodiments of formula (IIB), R is -CH(R 7 )2, where each R 7 is independently a C5-C 12 alkyl or a C5-C 12 alkenyl. In some cases, each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl. In some cases, each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkenyl.

[0124] In certain embodiments of formula (IIB), at least one R is a straight-chain or branched hydrocarbon group containing one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is a phenyl group. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0125] In certain embodiments of formula (IIB), the compound has formula (IIIB):

[0126]

[0127] where:

[0128] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0129] q is 1 to 4;

[0130] Y is selected from -NHC(O)-, -NHC(O)O- and -NHC(O)S-; and

[0131] each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently a C5-C 12 alkyl or a C5-C12 An alkenyl group, J is a cyclic group and t and u are each independently from 1 to 10.

[0132] In some embodiments of formula (IIB), the compound has formula (IIIB), wherein:

[0133] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0134] q is from 1 to 4;

[0135] Y is selected from -NHC(O)-, -NHC(O)O- and -NHC(O)S-; and

[0136] each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl and -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

[0137] In certain embodiments of formula (IIIB), Y is -NHC(O)-. In some cases, Y is -NHC(O)O-. In some cases, Y is -NHC(O)S-.

[0138] In certain embodiments of formula (IIIB), q is 1. In some cases, q is 2. In some cases, q is 3. In some cases, q is 4.

[0139] In certain embodiments of formula (IIIB), R 11 and R 12 are different. In some cases, at least one of R 11 and R 12 is methyl. In some cases, R 11 and R 12 are the same. In some cases, R 11 and R 12 are both methyl. In some cases, at least one of R 11 and R 12 is ethyl. In some cases, R 11 and R 12 are both ethyl. In some cases, R 11 and R 12 are both propyl. In some cases, R 11 and R 12 are both n-propyl. In some cases, R 11and R 12 are both isopropyl groups. In some cases, R 11 and R 12 are both isopropyl groups. In some cases, R 11 and R 12 are each independently an optionally substituted butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted n-butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted sec-butyl group. In some cases, R 11 and R 12 are each independently an optionally substituted butyl group. In some cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In some cases, each R 11 and R 12 is independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkenyl, C 1-4 heteroalkyl, and C 1-3 heteroalkyl.

[0140] In certain embodiments of formula (IIIB), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl. In some cases, each R is selected from C5-C 12 alkyl, C5-C 12 alkenyl, and C5-C 12 alkynyl. In some cases, each R is C5-C 12 alkyl. In some cases, each R is C5 alkyl. In some cases, each R is C6 alkyl. In some cases, each R is C7 alkyl. In some cases, each R is C8 alkyl. In some cases, each R is C9 alkyl. In some cases, each R is C 10 alkyl. In some cases, each R is C 11 alkyl. In some cases, each R is C 12 alkyl.

[0141] In certain embodiments of formula (IIIB), at least one R is -CH(R 7 )2, where each R 7 is independently C5-C 12 alkyl or C5-C12 Alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 alkenyl.

[0142] In certain embodiments of formula (IIIB), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is aryl. In some embodiments, J is phenyl. In certain embodiments, both t and u are 1 to 3. In some cases, t is 2 and u is 3.

[0143] In some embodiments, the compound of formula (I) has formula (IIC):

[0144]

[0145] where X, R, Y, L, and Z are as defined above.

[0146] In certain embodiments of formula (IIC), Y is selected from -C(R 10 )2- and -O-, where R 10 is selected from H and C 1-6 alkyl. In certain embodiments of formula (IIC), Y is -O-. In certain embodiments of formula (IIC), Y is -C(R 10 )2-, where R 10 is H.

[0147] In certain embodiments of formula (IIC), L is (C2-C6) alkylene or substituted (C2-C6) alkylene. In certain cases, L is (C2-C6) alkylene. In certain cases, L is (C2-C4) alkylene. In certain cases, L is -(CH2)2-. In certain cases, L is -(CH2)3-. In certain cases, L is -(CH2)4-.

[0148] In certain embodiments of formula (IIC), Z is a tertiary amine. In certain cases, Z is -NR 11 R 12 , where R 11 and R 12 are each independently C 1-6 alkyl or substituted C 1-6 alkyl. In certain cases, R 11 and R 12Each is C 1-3 alkyl. In some cases, R 11 and R 12 each is methyl. In some cases, R 11 and R 12 each is ethyl. In some cases, R 11 and R 12 are both propyl. In some cases, R 11 and R 12 are both n - propyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are both independently optionally substituted butyl. In some cases, R 11 and R 12 are both independently optionally substituted n - butyl. In some cases, R 11 and R 12 are both independently optionally substituted sec - butyl. In some cases, R 11 and R 12 are both independently optionally substituted butyl. In some cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n - propyl, n - butyl, sec - butyl, tert - butyl, - CH2CH2OH, - CH(CH3)CH2OH, - CH2CH(OH)CH3 and - CH2CH2CH2OH. In some cases, each R 11 and R 12 is independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkyl, C 1-4 heteroalkyl and C 1-3 heteroalkyl.

[0149] In certain embodiments of formula (IIC), each X is independently selected from - (CH2) s OC(O)-, - (CH2) s C(O)O-, - (CH2) s OC(O)O-, where s is from 0 to 6. In some cases, at least one X is - (CH2) s OC(O)-, where s is 0, 1 or 2. In some cases, each X is - (CH2) s OC(O)-, where s is 0, 1 or 2. In certain embodiments of formula (IIC), at least one X is - (CH2) sC(O)O-, where s is 0, 1 or 2. In some cases, each X is -(CH2) s C(O)O-, where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 1 and each X is -CH2C(O)O-. In certain embodiments of formula (IIC), s is 2 and X is -(CH2)2C(O)O-. In certain embodiments of formula (IIC), at least one X is -(CH2) s OC(O)O-, where s is 0, 1 or 2. In some cases, each X is -(CH2) s OC(O)O-, where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 0 and X is -OC(O)O-.

[0150] In certain embodiments of formula (IIC), each -X-R has the formula -(CH2) s OC(O)R, where s is from 0 to 6. In certain embodiments, each -X-R has the formula -(CH2) s C(O)OR, where s is from 0 to 6. In certain embodiments, each -X-R has the formula -(CH2) s OC(O)OR, where s is from 0 to 6.

[0151] In certain embodiments of formula (IIC), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl. In some cases, each R is C5-C 12 alkyl, C5-C 12 alkenyl and C5-C 12 alkynyl. In some cases, each R is C5-C 12 alkyl. In some cases, each R is C5 alkyl. In some cases, each R is C6 alkyl. In some cases, each R is C7 alkyl. In some cases, each R is C8 alkyl. In some cases, each R is C9 alkyl. In some cases, each R is C 10 alkyl. In some cases, each R is C 11 alkyl. In some cases, each R is C 12 alkyl.

[0152] In certain embodiments of formula (IIC), at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups (such as those described herein). In certain embodiments of formula (IIC), R is -CH(R 7 )2, where each R 7 is independently C5-C 12 alkyl or C5-C12 Alkenyl. In some cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 alkyl. In some cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 alkenyl. In some cases, each R is -CH(R 7 )2 and each R 7 is C6-C9 alkenyl.

[0153] In certain embodiments of formula (IIC), at least one R is a straight-chain or branched-chain hydrocarbyl group containing one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is an aryl group. In some embodiments, J is a phenyl group. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.

[0154] In certain embodiments of formula (IIC), the compound has formula (IIIC):

[0155]

[0156] where:

[0157] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0158] q is 1 to 4;

[0159] Y is selected from -O- and -C(R 10 )2-;

[0160] each s is independently 0, 1, or 2;

[0161] W is -O- or -CH2-; and

[0162] each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12An alkenyl group, J is a cyclic group, and each of t and u is from 1 to 10.

[0163] In some embodiments of formula (IIC), the compound has formula (IIIC), wherein:

[0164] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0165] q is from 1 to 4;

[0166] Y is selected from -O- and -C(R 10 )2-;

[0167] s is from 0 to 2;

[0168] W is O or CH2; and

[0169] each R is -CH(R 7 )2, where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

[0170] In certain embodiments of formula (IIIC), Y is selected from -C(R 10 )2- and -O-, where R 10 is selected from H and C 1-6 alkyl. In certain embodiments of formula (IIIC), Y is -O-. In certain embodiments of formula (IIIC), Y is -C(R 10 )2-, where R 10 is H.

[0171] In certain embodiments of formula (IIIC), q is 1. In some cases, q is 2. In some cases, q is 3. In some cases, q is 4.

[0172] In certain embodiments of formula (IIIC), R 11 and R 12 are different. In some cases, at least one of R 11 and R 12 is methyl. In some cases, R 11 and R 12 are the same. In some cases, R 11 and R 12 are both methyl. In some cases, at least one of R 11 and R 12 is ethyl. In some cases, R 11 and R 12are both ethyl. In some cases, R 11 and R 12 are both propyl. In some cases, R 11 and R 12 are both n-propyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are both isopropyl. In some cases, R 11 and R 12 are each independently an optionally substituted butyl. In some cases, R 11 and R 12 are each independently an optionally substituted n-butyl. In some cases, R 11 and R 12 are each independently an optionally substituted sec-butyl. In some cases, R 11 and R 12 are each independently an optionally substituted butyl. In some cases, each R 11 and R 12 is independently selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, -CH2CH2OH, -CH(CH3)CH2OH, -CH2CH(OH)CH3, and -CH2CH2CH2OH. In some cases, each R 11 and R 12 is independently selected from optionally substituted C 1-4 alkyl, C 1-3 alkyl, C 1-4 heteroalkyl, and C 1-3 heteroalkyl.

[0173] In certain embodiments of formula (IIIC), W is -CH2-. In certain embodiments of formula (IIIC), W is -O-.

[0174] In certain embodiments of formula (IIIC), s is 0. In certain embodiments, s is 1. In certain embodiments, s is 2.

[0175] In certain embodiments of formula (IIIC), W is -CH2- and s is 0. In some cases, W is -CH2- and s is 1. In some cases, W is -O- and s is 0.

[0176] In certain embodiments of formula (IIIC), each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl, and C5-C 20 alkynyl. In some cases, each R is selected from C5-C 12 alkyl, C5-C12 Alkenyl and C5-C 12 Alkynyl. In certain cases, each R is C5-C 12 Alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is C6 alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is C8 alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C 10 Alkyl. In certain cases, each R is C 11 Alkyl. In certain cases, each R is C 12 Alkyl.

[0177] In certain embodiments of formula (IIIC), at least one R is -CH(R 7 )2, where each R 7 is independently C5-C 12 Alkyl or C5-C 12 Alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 Alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C5-C 12 Alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C6-C9 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is C6-C9 alkenyl.

[0178] In certain embodiments of formula (IIIC), R is -(CH2) t J(CH2) u , where J is a cyclic group and t and u are each independently 1 to 10. In some embodiments, J is aryl. In some embodiments, J is phenyl. In certain embodiments, both t and u are 1 to 3. In some cases, t is 2 and u is 3.

[0179] In some embodiments of formulas (I), (IIA), (IIIA), (IIB), (IIIB), (IIC) and (IIIC), each R is independently where

[0180] Cy A and Cy BEach is independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, and the cyclic group is selected from 5- to 12-membered monocyclic groups, bicyclic groups, bridged polycyclic groups and spirocyclic groups;

[0181] R x and R y Each is independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 20 aliphatic group;

[0182] r, p and q are each independently an integer from 0 to 20.

[0183] In some embodiments, R is wherein

[0184] Cy A and Cy B Each is independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, and the cyclic group is selected from 5- to 12-membered monocyclic groups, bicyclic groups, bridged polycyclic groups and spirocyclic groups;

[0185] R x and R y Each is independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 10 aliphatic group;

[0186] r, p and q are each independently an integer from 0 to 10.

[0187] In some embodiments, R is wherein

[0188] Cy A and Cy B Each is independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, and the cyclic group is selected from 5- to 12-membered monocyclic groups, bicyclic groups, bridged polycyclic groups and spirocyclic groups;

[0189] R x and R y Each is independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C6 aliphatic group;

[0190] r, p and q are each independently an integer from 0 to 6.

[0191] In certain embodiments, the lipid is selected from the compounds in Table 1:

[0192] Table 1: Exemplary ionizable lipids

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] 4.3 Additional Ionizable Lipids

[0202] In some embodiments, in addition to the ionizable lipid of formula (I) (e.g., as described above), the lipid nanoparticle composition may further comprise one or more additional ionizable lipid components. Any suitable lipid that carries a net positive charge at physiological pH or about physiological pH can be used as the additional ionizable lipid in the compositions described herein.

[0203] Non-limiting examples of cationic lipids are described in detail herein. Cationic lipids and related analogs for use in the lipid nanoparticles of the present disclosure include, but are not limited to, those lipids described in the following: U.S. Patent Publications Nos. 20060083780 and 20060240554; U.S. Patents Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are incorporated herein by reference for all purposes. Additional cationic lipids of interest include, but are not limited to: 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), and 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N,N-triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”); 3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (“DMRIE”). Additionally, many commercial formulations of cationic lipids can be used, such as LIPOFECTIN (comprising DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO / BRL). In a specific embodiment, the cationic lipid is an amino lipid comprising one or two fatty acyl or fatty alkyl chains.

[0204] Other exemplary ionizable lipids that can be applied to the lipid nanoparticles of the present disclosure are described below: International PCT patent publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 086373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346 and WO2013 / 086354;and U.S. Patent Publications US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 0128760, US2010 / 0324120, US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 0338210, US2012 / 0101148, US2012 / 0027796, US2012 / 0058144, US2013 / 0323269, US2011 / 0117125, US2011 / 0256175, US2012 / 0202871, US2011 / 0076335, US2006 / 0083780, US2013 / 0123338, US2015 / 0064242, US2006 / 0051405, US2013 / 0065939, US2006 / 0008910, US2003 / 0022649, US2010 / 0130588, U52013 / 0116307, US2010 / 0062967, US2013 / 0202684, US2014 / 0141070, US2014 / 0255472, US2014 / 0039032, US2018 / 0028664, U52016 / 0317458, and US2013 / 0195920.;

[0205] 4.4 Co - lipids

[0206] In addition to the ionizable lipid component described herein, the LNPs of the present disclosure may further include one or more co - lipids. In some embodiments, the co - lipid is a neutral lipid. In some embodiments, the neutral lipid is zwitterionic, e.g., having an overall net zero charge.

[0207] Neural lipids include, for example, phospholipids, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides. When selecting neutral lipids for the compositions described herein, it is generally guided by considering, for example, LNP size and the stability of the LNP in the bloodstream. Generally, the LNPs of the present disclosure include an auxiliary lipid component, which includes neutral lipids that are phospholipids. Non-limiting examples of phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine, or dilinoleoyl phosphatidylcholine. In some embodiments, the neutral lipid component is a lipid having two acyl groups (i.e., diacyl phosphatidylcholine and diacyl phosphatidylethanolamine). Lipids with various acyl chain groups of different chain lengths and degrees of saturation are available, or can be isolated or synthesized by well-known techniques. In one embodiment, the neutral lipid includes saturated fatty acids having a carbon chain length in the range of C 10 to C 30 . In one embodiment, neutral lipids with mono- or di-unsaturated fatty acids having a carbon chain length in the range of C 10 to C 30 are used. Additionally, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. The neutral lipid can also be composed of sphingomyelin or dihydrosphingomyelin.

[0208] In some embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG).

[0209] In some embodiments, the phospholipid has a hydrocarbon chain or "tail" having 12 to 24 carbons, such as 16 to 20 carbons, 18 to 22 carbons, 12 to 18 carbons. In some embodiments, the phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. In some embodiments, the phospholipid tail does not contain double bonds, i.e., the bonds are saturated bonds. In some embodiments, the phospholipid tail is unsaturated, i.e., it contains one or more double bonds, such as 1, 2, 3, 4, or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, i.e., it contains one or more triple bonds, such as 1, 2, 3, 4, or 5 triple bonds. In some embodiments, the phospholipid tail contains one or more ring structures. In some embodiments, one or more ring structures are selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclic groups; 5- to 6-membered aryl groups; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclic groups; and 7- to 10-membered bicyclic aryl groups, where each ring structure is independently substituted with 0 to 7 R A groups; each R AIndependently selected from halogen or an optionally substituted group selected from C 1-12 aliphatic groups, phenyl or a 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclic group. In some such cases, the ring structure is cholesterol or a cholesterol derivative. In some embodiments, the phospholipid is symmetric, i.e., all tails of the phospholipid are the same. In other embodiments, the phospholipid is asymmetric, i.e., the phospholipid contains two different hydrocarbon chains.

[0210] In some embodiments, the co-lipid is or comprises a symmetric or asymmetric aliphatic phospholipid moiety, each independently an optionally substituted, branched or straight-chain, partially unsaturated or saturated C9-C 24 aliphatic group.

[0211] In some embodiments, the co-lipid contains one or more optionally substituted and / or optionally bridged ring structures in the hydrophobic tail. Exemplary co-lipids of this type include:

[0212] In some embodiments, the co-lipid includes phosphatidylethanolamine (PE). Phosphatidylethanolamine (PE) is a class of phospholipids incorporating ethanolamine as the head group. In some embodiments, phosphatidylethanolamine is selected from the group consisting of phosphatidylethanolamine, dioleoyl phosphatidylethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (Δ9-cis PE or DOPE), palmitoyl-oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE), dimyristoyl phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) (DMPE), (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), dielaidoyl-phosphatidylethanolamine (DEPE), lysophosphatidylethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE). In certain embodiments, the phosphatidylethanolamine is dioleoyl phosphatidylethanolamine (also referred to as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or (Δ9-cis) PE or DOPE), which has tails of 18 carbons and one saturated bond (“18-1”), as shown below:

[0213]

[0214] In some embodiments, the auxiliary lipid includes phosphatidylcholine (PC). Phosphatidylcholine (PC) is a class of phospholipids incorporating choline as the head group. In some embodiments, phosphatidylcholine is selected from the group consisting of: phosphatidylcholine, distearoyl phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine) (DSPC), dioleoyl phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine) (Δ9-cis PC or DOPC), dipalmitoyl phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (DPPC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di(eicosenoyl)-sn-glycero-3-phosphocholine (“20-1PC” or “20:1PC”), egg phosphatidylcholine (EPC), dimyristoyl phosphatidylcholine (DMPC), dierucoyl phosphatidylcholine (DEPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, 1,2-dicholesteryl succinyl-sn-glycero-3-phosphocholine (DChemsPC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-palmitoyl-2-cholesteryl carbonyl-sn-glycero-3-phosphocholine (PChcPC), and 1-palmitoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (PChemsPC). In certain embodiments, the phosphatidylcholine is distearoyl phosphatidylcholine (DSPC) (also known as 1,2-distearoyl-sn-glycero-3-phosphocholine), which has a tail of 18 carbons and no saturated bonds (“18-0”), as shown below:

[0215]

[0216] In certain embodiments, the phosphatidylcholine is dioleoyl phosphatidylcholine (also known as 1,2-dioleoyl-sn-glycero-3-phosphocholine, (Δ9-cis) PC or DOPC), which has a tail of 18 carbons and one saturated bond (“18-1”), as shown below:

[0217]

[0218] In certain embodiments, the phosphatidylcholine is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Δ9-cis PC), which has a tail of 16 carbons and one saturated bond (“16-1”), as shown below:

[0219]

[0220] In certain embodiments, phosphatidylcholine is an asymmetric lipid having a first tail of 16 carbons and a second tail of 18 carbons. In some such cases, the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e.g., it is 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (also referred to as "16-0 / 18-1PC", "16:0 / 18:1PC", or POPC), as shown below:

[0221]

[0222] In certain embodiments, phosphatidylcholine is 1,2-dicholesteryl succinyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below:

[0223]

[0224] In certain embodiments, phosphatidylcholine is 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:

[0225]

[0226] In certain embodiments, phosphatidylcholine is 1-palmitoyl-2-cholesteryl carbonyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:

[0227]

[0228] In certain embodiments, phosphatidylcholine is 1-palmitoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:

[0229]

[0230] In some embodiments, the helper lipid includes phosphatidylglycerol selected from the group consisting of: phosphatidylglycerol, dioleoyl phosphatidylglycerol (1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), and palmitoyl oleoyl phosphatidylglycerol (POPG).

[0231] In some embodiments, the helper lipid includes phosphatidylserine, e.g., phosphatidylserine or dioleoyl phosphatidylserine (DOPS).

[0232] In some embodiments, the helper lipid includes lecithin, such as phosphatidylcholine or lysophosphatidylcholine.

[0233] In some embodiments, the helper lipid includes sphingomyelin (SM), such as egg sphingomyelin (ESM).

[0234] In some embodiments, the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebroside, or dicetyl phosphate.

[0235] In some aspects, the LNP can further comprise a component such as a sterol, for example, to provide membrane integrity. An exemplary sterol that can be used in the lipid nanoparticle is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Exemplary cholesterol derivatives are described in International Application WO2009 / 127060 and U.S. Patent Publication US2010 / 0130588. The component (such as a sterol) that provides membrane integrity can account for 0 to 50% (mol) of the total lipids present in the lipid nanoparticle. In some embodiments, such a component accounts for 20 to 50% (mol), 30 to 40% (mol) of the total lipid content of the lipid nanoparticle.

[0236] Thus, the neutral lipid component of the LNP can also include cholesterol or its derivatives or analogs. A variety of cholesterol analogs and derivatives can be suitable for the LNP of the present disclosure. In some embodiments, the helper lipid component includes cholesterol.

[0237] In some embodiments, the LNP includes a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or its derivatives or analogs.

[0238] In some embodiments, the LNP includes a neutral lipid component that includes phosphatidylethanolamine phospholipid and cholesterol or its derivatives or analogs.

[0239] In some embodiments, the LNP includes a neutral lipid component that includes DOPE phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DSPC phospholipid and cholesterol. In some embodiments, the LNP includes a neutral lipid component that includes DOPC phospholipid and cholesterol.

[0240] 4.5 Other Compounds

[0241] The LNPs of the present disclosure may also include one or more additional lipid components. Such lipids can be selected to provide desired characteristics of the nanoparticle properties, such as particle stability, delivery efficacy, tolerability, and biodistribution.

[0242] In some aspects, the LNP may further comprise a non-cationic lipid. Non-ionic lipids include amphiphilic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be neutrally uncharged, zwitterionic, or anionic. Non-cationic lipids are generally used to enhance fusibility. Exemplary non-cationic lipids contemplated for use in the methods and compositions are described in International Application PCT / US2018 / 050042, published as WO2019051289A1. Exemplary non-cationic lipids are described in International Application Publication WO2017 / 099823 and U.S. Patent Publication US2018 / 0028664.

[0243] Non-limiting examples of non-cationic lipids include phosphorus-free lipids such as stearylamine, dodecylamine, cetylamine, acetyl palmitate, glycerol ricinoleate, cetyl stearate, isopropyl myristate, amphoacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amide, di-octadecyl dimethyl ammonium bromide, ceramides, sphingomyelin, and the like.

[0244] In some embodiments, the LNP includes one or more lipids capable of reducing aggregation. Generally, lipids capable of reducing aggregation include at least one hydrocarbon tail or chain linked to a hydrophilic group, which can be structured at the surface of the LNP and provide reduced LNP aggregation. Thus, lipids capable of reducing aggregation are sometimes referred to as conjugated lipids or coating lipids.

[0245] Lipids capable of reducing particle aggregation may comprise conjugated lipid molecules such as polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of the lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic-polymer-lipid conjugates (CPL), or mixtures thereof. In one embodiment, the LNP comprises a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used in combination with CPL.

[0246] In some embodiments, the lipid capable of reducing aggregation is a PEG-lipid. A PEG-lipid refers to a lipid having one or more hydrocarbon tails linked to one or more polyethylene glycol (PEG) moieties via an optional linker.

[0247] It should be understood that the PEG moiety may include terminal modifications to provide conjugation to a lipid tail, for example via an optional linker. The PEG moiety may be capped as a hydroxyl or alkyl ether (e.g., a methoxy terminal group). In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxypolyethylene glycol) conjugated lipid. PEG-lipids of interest include, but are not limited to, PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauroxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or mixtures thereof.

[0248] Exemplary PEG-lipid conjugates include, but are not limited to: PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Patent Nos. 5,885,613, 6,287,591, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, and US2017 / 0119904. In some embodiments, the PEG-lipid is the compound disclosed in US2018 / 0028664. In some embodiments, the PEG-lipid is disclosed in US20150376115 or US2016 / 0376224. The PEG-DAA conjugate can be, for example, PEG-dilauroxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilauryl glycerol, PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG-dilauryl glycerol amide, PEG-dimyristyl glycerol amide, PEG-dipalmitoyl glycerol amide, PEG-distearyl glycerol amide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol)), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some instances, the PEG-lipid can be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.

[0249] As described above, lipids conjugated to molecules other than PEG can also be used instead of PEG-lipids. For example, instead of or in addition to PEG-lipids, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer-lipid (CPL) conjugates can be used. Exemplary conjugated lipids, namely PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in the following: International Patent Application Publications WO1996 / 010392, WO1998 / 051278, WO2002 / 087541, WO2005 / 026372, WO2008 / 147438, WO2009 / 086558, WO2012 / 000104, WO2017 / 117528, WO2017 / 099823, WO2015 / 199952, WO2017 / 004143, WO2015 / 095346, WO2012 / 000104, WO2012 / 000104, and WO2010 / 006282; U.S. Patent Application Publications US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2013 / 0303587, US2018 / 0028664, US2015 / 0376115, US2016 / 0376224, US2016 / 0317458, US2013 / 0303587, US2013 / 0303587, and US20110123453; and U.S. Patents No. 5,885,613, No. 6,287,591, No. 6,320,017, and No. 6,586,559.

[0250] 4.6 Targeting Ligands

[0251] In some embodiments, it may be desirable to restrict transfection of nucleic acids to certain cells or tissues. For example, the liver may be the target organ of interest, in part because of its central role in the metabolism and production of proteins and thus diseases caused by defects in liver-specific gene products (such as urea cycle disorders), and may benefit from specific targeting of cells (such as hepatocytes).

[0252] In some embodiments, the LNP further comprises a component that includes a targeting ligand. The targeting ligand can be selected as needed based on the target cell or tissue to which the LNP of the present disclosure is desired to be directed. In some embodiments, the targeting ligand is a ligand of a cell surface receptor. In some embodiments, the cell surface receptor is the asialoglycoprotein receptor (ASGPR). ASGPR is expressed on the surface of hepatocytes.

[0253] In some embodiments, the targeting ligand is a ligand of ASGPR, such as a ligand containing N-acetylgalactosamine (GalNAc). A variety of GalNAc-containing ligands and ligands (including multivalent GalNAc ligands) can be used in the LNPs of the present disclosure, including, for example, those disclosed in WO2021178725, the entire disclosure of which is incorporated herein by reference.

[0254] In some embodiments, the PEG-lipid is linked to the targeting ligand. In some embodiments, the targeting ligand of interest (such as those described herein) is linked to the end of the PEG moiety. For example, a trisGalNac ligand conjugated to a PEG-lipid can provide binding of the LNP to the ASGPR receptor of the target cell and cause endocytosis of the LNP.

[0255] 4.7 Lipid nanoparticles comprising the lipid of formula (I)

[0256] In some embodiments, the LNP comprises an ionizable lipid of formula (I) (such as those described herein); a nucleic acid cargo (such as those described herein); additional ionizable lipids (such as those described herein); phospholipids (such as those described herein), cholesterol (such as those described herein); and lipids capable of reducing aggregation (such as those described herein).

[0257] In some embodiments of the LNP, the nucleic acid cargo comprises DNA, such as oligonucleotides, plasmid DNA, dogbone DNA, minicircle DNA, covalently closed circular DNA, ceDNA, or chemically modified derivatives thereof. In certain cases, the nucleic acid consists essentially of DNA. In some embodiments of the LNP, the nucleic acid cargo comprises RNA, such as siRNA, gRNA, mRNA, circular RNA, or chemically modified derivatives thereof. In certain cases, the nucleic acid consists essentially of RNA. In certain embodiments of the LNP, the nucleic acid cargo comprises DNA, such as oligonucleotides, plasmid DNA, dogbone DNA, minicircle DNA, covalently closed circular DNA, ceDNA, or chemically modified derivatives thereof, and also comprises RNA, such as siRNA, gRNA, mRNA, circular RNA, etc., or chemically modified derivatives thereof.

[0258] In some embodiments of the LNP, the phospholipids are selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), and derivatives thereof. In some cases, the phospholipid is phosphatidylethanolamine (PE). In some cases, the phospholipid is phosphatidylcholine (PC). In certain embodiments of the LNP, the phospholipids include hydrocarbon chains each independently having 12 to 24 carbons. In some cases, the hydrocarbon chains each independently have 16 to 20 carbons. In some cases, the hydrocarbon chains are saturated. In some cases, the hydrocarbon chains are unsaturated. In some cases, the hydrocarbon chains each independently contain 1 to 4 double bonds. In some cases, the phospholipid contains two different hydrocarbon chains. In certain embodiments of the LNP, the phospholipids include dioleoylphosphatidylethanolamine (DOPE, 18:1PE). In some cases, the phospholipid includes 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some cases, the phospholipid includes 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some cases, the phospholipid includes 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (Δ9-cis PC). In some cases, the phospholipid includes 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). In some cases, the phospholipid includes a mixture of dioleoylphosphatidylethanolamine (DOPE, 18-1) and dioleoylphosphatidylcholine (DOPC, 18-1).

[0259] In certain embodiments of the LNP, the lipid capable of reducing aggregation is a PEG-lipid. In some cases, the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0260] In certain embodiments, the LNP further comprises a targeting ligand (such as described herein). In some cases, the targeting ligand comprises GalNac. In certain embodiments, the targeting ligand is linked to a ligand capable of reducing aggregation. In some cases, the lipid capable of reducing aggregation linked to the targeting ligand is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0261] In some embodiments, the LNP includes an ionizable lipid of formula (I) (such as described herein); a phospholipid (DOPE); cholesterol; and a lipid capable of reducing aggregation (PEG-DMG).

[0262] In some embodiments, the LNP comprises an ionizable lipid, which is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid, which is phosphatidylethanolamine (e.g., DOPE); cholesterol; and an anti-aggregation lipid, which is PEG-DMG and / or PEG-DSG-GalNAc or PEG-DSPE-GalNac.

[0263] In some embodiments, the LNP comprises an ionizable lipid, which is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid, which is phosphatidylcholine (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC); cholesterol; and a coating lipid (polyethylene glycol-dimyristoyl glycerol, PEG-DMG), as disclosed, for example, by Tam et al. (2013), Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0264] Typically, lipid particles are prepared with a total lipid to DNA (mass or weight) ratio of about 5:1 to 50:1. This is also referred to as the ratio of the positively charged polymer amine (N = nitrogen) groups to the negatively charged nucleic acid phosphate (P) groups, or the N / P ratio. In some embodiments, the N / P ratio (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 50:1, about 7:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and DNA can be adjusted to provide the desired N / P ratio, e.g., an N / P ratio of 3:1 (“3”), 4:1 (“4”), 5:1 (“5”), 6:1 (“6”), 7:1 (“7”), 8:1 (“8”), 9:1 (“9”), 10:1 (“10”), 11:1 (“11”), 12:1 (“12”), 13:1 (“13”), 14:1 (“14”), or higher. Typically, the total lipid content of the lipid particle formulation can be in the range of about 5 mg / mL to about 30 mg / mL.

[0265] In some embodiments, the N / P ratio is 5 to 30. In certain cases, the N / P ratio is 7. In certain cases, the N / P ratio is 14. In certain cases, the N / P ratio is 28.

[0266] In some embodiments, the average diameter of the lipid nanoparticles is between about 10 nm and about 1000 nm. In some embodiments, the diameter of the lipid nanoparticles is less than 300 nm. In some embodiments, the diameter of the lipid nanoparticles is between about 10 nm and about 300 nm. In some embodiments, the diameter of the lipid nanoparticles is less than 200 nm. In some embodiments, the diameter of the lipid nanoparticles is between about 25 nm and about 200 nm. In some embodiments, a lipid nanoparticle formulation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution in which the average size (e.g., diameter) is from about 70 nm to about 200 nm, more typically the average size is about 100 nm or less.

[0267] In some embodiments, the average diameter of the LNP is 25 to 250 nm, 25 to 240 nm, 25 to 230 nm, 25 to 220 nm, 25 to 210 nm, 25 to 200 nm, 25 to 190 nm, 25 to 180 nm, 25 to 170 nm, 25 to 160 nm, 25 to 150 nm, 25 to 140 nm, 25 to 130 nm, 25 to 120 nm, 25 to 110 nm, 25 to 100 nm, 25 to 90 nm, 25 to 80 nm, 25 to 70 nm, 25 to 60 nm, or 25 to 50 nm.

[0268] In some embodiments, the average diameter of the LNP is 60 to 250 nm, 70 to 250 nm, 80 to 250 nm, 90 to 250 nm, 100 to 250 nm, 110 to 250 nm, 120 to 250 nm, 130 to 250 nm, 140 to 250 nm, 150 to 250 nm, 160 to 250 nm, 170 to 250 nm, 180 to 250 nm, 190 to 250 nm, 200 to 250 nm, 210 to 250 nm, 220 to 250 nm, 230 to 250 nm, or 240 to 250 nm.

[0269] In some embodiments, the average diameter of the LNP is 60 to 250 nm, 70 to 240 nm, 80 to 230 nm, 90 to 220 nm, 100 to 210 nm, 110 to 200 nm, 120 to 190 nm, 130 to 180 nm, 140 to 170 nm, or 150 to 160 nm.

[0270] In some embodiments, the structural features of the target tissue can be utilized to direct the distribution of LNPs to such target tissues. For example, to target hepatocytes, the size of the LNPs can be adjusted such that their size is smaller than the fenestrae of the endothelial lining of the hepatic sinusoids in the liver; thus, the LNPs can easily penetrate through such endothelial fenestrae to reach the target hepatocytes. In some embodiments, the size of the LNPs can be such that the particles have a sufficient diameter to limit or specifically avoid distribution to certain cells or tissues. For example, the size of the LNPs can be set such that their size is larger than the fenestrae of the endothelial lining of the hepatic sinusoids, thereby limiting the distribution of the LNPs to hepatocytes. In such embodiments, the large LNPs will not easily penetrate the endothelial fenestrae but will be cleared by the macrophage Kupffer cells within the hepatic sinusoids. In some embodiments, the size of the LNPs is in the range of about 25 to 250 nm or 25 nm to 100 nm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.

[0271] Non-limitingly, the ionizable lipid can account for 20 to 90% (mol) of the total lipids present in the lipid nanoparticle. For example, the molar content of the ionizable lipid can be 20 to 70% (mol), 30 to 60% (mol), or 40 to 50% (mol) of the total lipids present in the lipid nanoparticle. In some embodiments, the ionizable lipid accounts for about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticle. In some embodiments, the ionizable lipid accounts for about 50 mol% to about 85 mol%, about 50 mol% to about 80 mol%, about 50 mol% to about 75 mol%, about 50 mol% to about 70 mol%, about 50 mol% to about 65 mol%, about 50 mol% to about 60 mol%, about 55 mol% to about 65 mol%, or about 55 mol% to about 70 mol% (or any fraction or range therein) of the total lipids present in the particle. In a specific embodiment, the cationic lipid accounts for 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, or 60 mol% (or any fraction thereof) of the total lipids present in the particle.

[0272] The neutral lipid component can account for 10 to 60% (mol) of the total lipids present in the lipid nanoparticle. For example, the non-cationic lipid content is 10 to 50% (mol) or 20 to 55% (mol) of the total lipids present in the lipid nanoparticle. In some embodiments, the non-cationic lipid accounts for about 10 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 45 mol%, about 37 mol% to about 42 mol% (or any fraction or range therein) of the total lipids present in the particle. In a specific embodiment, the non-cationic lipid accounts for 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46%, 47%, 48%, 49% or 50% (or any fraction or range therein) of the total lipids present in the particle.

[0273] In embodiments where the lipid particle contains a mixture of phospholipid and cholesterol or cholesterol derivative, the mixture can account for at most about 40 mol%, 45 mol%, 50 mol%, 55 mol% or 60 mol% of the total lipids present in the particle. In a specific embodiment, the mixture of phospholipid and cholesterol or cholesterol derivative accounts for at most 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46%, 47%, 48%, 49% or 50% (or any fraction or range therein) of the total lipids present in the particle.

[0274] In some embodiments, the LNP contains a phospholipid component in the mixture in an amount of about 2 mol% to about 20 mol%, about 2 mol% to about 15 mol%, about 2 mol% to about 12 mol%, about 4 mol% to about 15 mol% or about 4 mol% to about 10 mol% (or any fraction or range therein) of the total lipids present in the particle. In some embodiments, the phospholipid component in the mixture accounts for about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, about 5 mol% to about 8 mol%, about 6 mol% to about 9 mol%, about 6 mol% to about 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol% or 10 mol% (or any fraction or range therein) of the total lipids present in the particle.

[0275] In some embodiments, the LNP comprises a cholesterol component in the mixture in an amount of about 25 mol% to about 45 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 27 mol% to about 37 mol%, about 25 mol% to about 30 mol%, or about 35 mol% to about 40 mol% (or any fraction or range therein) of the total lipids present in the particle. In some embodiments, the cholesterol component in the mixture is about 25 mol% to about 35 mol%, about 27 mol% to about 35 mol%, about 29 mol% to about 35 mol%, about 30 mol% to about 35 mol%, about 30 mol% to about 34 mol%, about 31 mol% to about 33 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36%, 37%, 38%, or 39% (or any fraction or range therein) of the total lipids present in the particle.

[0276] It should be understood that the mole percentages of the components described herein in the LNP are target amounts, and the actual amounts of each lipid component present in the formulation can vary, e.g., ±5 mol%.

[0277] In some embodiments, the LNP comprises a lipid capable of reducing aggregation (e.g., a PEG-lipid conjugate) in an amount of about 1.5% to about 4% of the total lipids present in the particle, such as about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25% (or any fraction or range therein). According to some embodiments, the lipid capable of reducing aggregation is present at 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% (or any fraction or range therein) of the total lipids present in the particle.

[0278] In various embodiments, the molar ratio of ionizable lipid to neutral lipid is from about 2:1 to about 8:1. In some embodiments, the lipid nanoparticle does not contain any phospholipids.

[0279] In certain embodiments, the LNP comprises:

[0280] a) an ionizable lipid that is 40 to 60 mol% of the total lipids present;

[0281] b) Phospholipids that account for 6 to 20 mol% of the total lipids present;

[0282] c) Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0283] d) Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0284] In certain embodiments, the LNP comprises:

[0285] a) Ionizable lipids that account for 40 to 60 mol% of the total lipids present;

[0286] b) Phospholipids that account for 10 to 20 mol% of the total lipids present;

[0287] c) Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0288] d) Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0289] In certain embodiments, the LNP comprises:

[0290] a) Ionizable lipids that account for 40 to 49 mol% of the total lipids present;

[0291] b) Phospholipids that account for 10 to 20 mol% of the total lipids present;

[0292] c) Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0293] d) Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0294] In some embodiments, the ratio of ionizable lipid:phospholipid:cholesterol:PEG (as a percentage of the total lipid content) is A:B:C:D, where:

[0295] a. A = 40% - 60%, B = 5% - 20%, C = 25% - 50%, and D = 1.5% - 3.0%, and where A + B + C + D = 100%;

[0296] b. A = 40% - 60%, B = 6% - 20%, C = 35% - 45%, and D = 1.5% - 2.5%, and where A + B + C + D = 100%;

[0297] b. A = 40% - 60%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5%, and where A + B + C + D = 100%;

[0298] c. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5%, and where A + B + C + D = 100%;

[0299] d. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5%, and where A + B + C + D = 100%;

[0300] d. A = 39% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3%, and where A + B + C + D = 100%;

[0301] e. A = 40% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3%, and where A + B + C + D = 100%;

[0302] f. A = 45% - 50%, B = 20% - 25%, C = 25% - 30%, and D = 0% - 1%, and where A + B + C + D = 100%;

[0303] g. A = 40% - 60%, B = 10% - 30%, C = 20% - 45%, and D = 0% - 3%, and where A + B + C + D = 100%;

[0304] h. A = 40% - 60%, B = 10% - 30%, C = 25% - 45%, and D = 0% - 3%, and where A + B + C + D = 100%;

[0305] i. A = 45% - 55%, B = 10% - 20%, C = 30% - 40%, and D = 1% - 2%, and where A + B + C + D = 100%;

[0306] j. A = 45% - 50%, B = 10% - 15%, C = 35% - 40%, and D = 1% - 2%, and where A + B + C + D = 100%;

[0307] k. A = 45% - 65%, B = 5% - 20%, C = 20% - 45%, and D = 0% - 3%, and where A + B + C + D = 100%;

[0308] m. A = 45%, B = 15%, C = 37.5%, and D = 2.5%;

[0309] n. A = 57%, B = 12%, C = 28.5%, and D = 2.5%;

[0310] 1. A = 50% - 60%, B = 5% - 15%, C = 30% - 45%, and D = 0% - 3%, and wherein A + B + C + D = 100%;

[0311] m. A = 55% - 60%, B = 5% - 15%, C = 30% - 40%, and D = 1% - 2%, and wherein A + B + C + D = 100%; or

[0312] n. A = 55% - 60%, B = 5% - 10%, C = 30% - 35%, and D = 1% - 2%, and wherein A + B + C + D = 100%.

[0313] 4.8 Nucleic Acid Carriers

[0314] In many embodiments, a given lipid nanoparticle can include a carrier or payload to be delivered to a cell. Of particular interest in some embodiments are carriers that comprise polynucleotides. In some embodiments, the polynucleotide is DNA. DNA nucleic acid compositions of any structure can be included in the LNPs of the present disclosure. For example, the DNA can be circular, such as a plasmid, nanoplasmid, minicircle, covalently closed circular DNA, circular viral genome, etc. As another example, the DNA can be linear, such as dogbone or other blunt-ended DNA, linear viral genome, etc. As another example, the DNA can be multivalent, such as 3DNA. The DNA can be single-stranded or double-stranded or a hybrid of single-stranded and double-stranded. The DNA can be chemically modified. In some embodiments, the polynucleotide is RNA. RNA nucleic acid compositions of any structure can be included in the LNPs of the present disclosure. For example, the RNA can be linear or it can be circular. It can be mRNA, siRNA, shRNA, guide RNA (gRNA), microRNA (miRNA), circular RNA (circRNA). It can be chemically modified.

[0315] One or more additional compounds can be therapeutic agents. The therapeutic agents can be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agents can be selected from any class suitable for therapeutic purposes. In other words, the therapeutic agents can be selected based on the therapeutic purpose and the desired biological effect. For example, if the DNA within the LNP is used to treat cancer, then the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including but not limited to small molecules, antibodies, or antibody-drug conjugates). In another example, if the LNP containing DNA is used to treat an infection, then the additional compound can be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In yet another example, if the LNP containing DNA is used to treat an immune disease or disorder, then the additional compound can be a compound that modulates the immune response (e.g., an immunosuppressive agent, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In some embodiments, different mixtures of different lipid nanoparticles containing different compounds such as DNA encoding different proteins or different compounds such as therapeutic agents can be used in the compositions and methods of the present invention. In some embodiments, the additional compound is an immunomodulator. For example, the additional compound is an immunosuppressive agent. In some embodiments, the additional compound is an immunostimulant.

[0316] 4.9 Pharmaceutical Compositions

[0317] Also provided herein is a pharmaceutical composition comprising a lipid nanoparticle-encapsulated nucleic acid (e.g., DNA) and a pharmaceutically acceptable carrier or excipient. In some aspects, the present disclosure provides a lipid nanoparticle formulation that further comprises one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose, and / or glycine.

[0318] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0319] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, surfactant, or emulsifying agent that has been approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

[0320] "Pharmaceutically acceptable salts" include acid and base addition salts. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), and these are formed with inorganic acids and organic acids. Examples of the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Examples of the organic acids include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Salts derived from organic bases include, but are not limited to, primary amine salts, secondary amine salts, and tertiary amine salts. Substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0321] 4.10 Preparation Method

[0322] Any suitable method can be used to prepare the LNPs of the present disclosure. The LNP compositions can be prepared by high-energy mixing of ethanol lipids with aqueous DNA at low pH, which protonates the ionizable lipids and provides favorable energetics for DNA / lipid association and particle nucleation. The particles can be further stabilized by dilution with water and removal of the organic solvent. The particles can be concentrated to the desired level.

[0323] 4.11 Method of Use

[0324] As shown in the working examples and figures herein, compared to an industry standard LNP (comprising 50% ionizable lipid ALC-0315, 10% DSPC, 38.5% cholesterol, and 1.5% PEG lipid) administered at the same dose, the LNPs and LNP pharmaceutical compositions of the present disclosure have lower in vivo toxicity when formulated with nucleic acids, e.g., at least 2-fold lower toxicity, e.g., 3-fold, 4-fold, or 5-fold lower toxicity, in some cases 10-fold, 20-fold, or 50-fold lower toxicity, and in certain cases 100-fold lower toxicity. "Less toxic" means eliciting a reduced immune response, e.g., characterized by a decrease in the amount of one or more cytokines after administration to an organism.

[0325] At the same time, it has been observed that the LNPs and LNP pharmaceutical compositions of the present disclosure are effective in delivering their nucleic acid cargo to target cells of interest, including where the LNPs and LNP pharmaceutical compositions of the present disclosure are comparable or more effective in delivering their nucleic acid cargo to target cells of interest compared to the same industry standard LNP administered at the same dose, e.g., having 2-fold or higher efficacy, e.g., 3-fold, 4-fold, or 5-fold or higher efficacy, in some cases 10-fold, 20-fold, or 50-fold efficacy, and in certain cases 100-fold more effective or higher. "More effective" means being able to deliver more nucleic acid cargo to cells, resulting in an increase in the amount of mRNA transcribed from the nucleic acid cargo or an increase in the amount of protein translated, e.g., an increase of 2-fold or more, e.g., an increase of 3-fold, 4-fold, 5-fold, e.g., an increase of 10-fold, 20-fold, 50-fold, and in some cases an increase of 100-fold or more.

[0326] In other words, the LNPs of the present disclosure exhibit improved pharmacokinetic (PK) profiles, which broaden the therapeutic index of the compositions. The therapeutic index or therapeutic ratio means the range of doses at which a drug is effective without unacceptable adverse events, which is calculated as the ratio of the blood concentration at which the drug becomes toxic to the concentration at which the drug is effective. Compared to the existing art, this improvement makes them more suitable for delivering nucleic acids (including DNA) to cells in vitro and in vivo, and thus they have a wide range of uses in many applications, including delivering nucleic acids (including DNA) into cells for research and therapeutic applications.

[0327] In performing such methods, cells are typically contacted with an amount of the composition (e.g., an LNP or its pharmaceutical composition) effective to deliver the reagent into the cytoplasm of the cell. In some embodiments, the contacting is in vitro. In other embodiments, the contacting is in vivo. In some embodiments, the method further comprises measuring the amount of protein produced.

[0328] The present disclosure also provides methods for treating or preventing a disease in an individual in need thereof, wherein an effective amount of a therapeutic composition described herein is administered to the individual. The route of administration will vary naturally with the location and nature of the disease being treated and can include, for example, intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The encapsulated polynucleotide compositions described herein can be used to treat any indication that would benefit from delivery of a therapeutic vehicle to target cells.

[0329] The present disclosure also provides methods for immunizing an individual against a disease, wherein an effective amount of a therapeutic composition described herein is administered to the individual. The route of administration will vary naturally with the location and nature of the immunizing agent and can include, for example, intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.

[0330] The present disclosure also provides the particles of the present disclosure, the carriers of the present disclosure, the recombinant DNA of the present disclosure, or a composition thereof for use as a medicament. In some embodiments, the medicament is used for expressing a protein in a cell. In some embodiments, the expression of the protein is used to treat a disease in which the cell lacks the protein. In some embodiments, the expression of the protein is used to treat a disease in which another cell lacks the protein. In some embodiments, the medicament is used to treat cancer. In some embodiments, the medicament is used for immunizing against a disease.

[0331] 4.12 Utility

[0332] For example, the subject methods and compositions as described above can be used in any application that requires delivery of a nucleic acid vehicle. Applications of interest include research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications, and therapeutic applications. In some cases, the nucleic acid vehicle that can be introduced into a cell and subsequently into the cell nucleus by the methods of the invention includes those encoding research proteins, diagnostic proteins, and therapeutic proteins.

[0333] A research protein is a protein whose activity can be used in a research protocol. Thus, a research protein is a protein employed in an experimental procedure. A research protein can be any protein having such utility, where in some cases, the research protein is a protein domain that is also provided in a research protocol by expressing it in a cell from a coding vector. Examples of specific types of research proteins include, but are not limited to: transcriptional regulators of inducible expression systems, members of signal generation systems (such as enzymes and their substrates), hormones, prohormones, proteases, enzyme activity regulators, perturbing dimers and peptide aptamers, antibodies, regulators of protein-protein interactions, genome modifying proteins (such as CRE recombinase, meganucleases, zinc finger nucleases, CRISPR / Cas-9 nucleases, TAL effector nucleases, etc.), cell reprogramming proteins (such as Oct 3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc.), and so on.

[0334] A diagnostic protein is a protein whose activity can be used in a diagnostic protocol. Thus, a diagnostic protein is a protein employed in a diagnostic procedure. A diagnostic protein can be any protein having such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal generation systems (such as enzymes and their substrates), labeled binding members (such as labeled antibodies and their binding fragments), peptide aptamers, and the like.

[0335] Target proteins further include therapeutic proteins. Therapeutic proteins of interest include, but are not limited to, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.

[0336] Target cells to which nucleic acids can be delivered according to embodiments of the present disclosure can vary widely. Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293, etc., mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples, and the like. Some non-limiting examples of mammalian cells include, but are not limited to, mouse cells, rat cells, hamster cells, rodent cells, and non-human primate cells. In some embodiments, the target cells are human cells. It should also be understood that the target cells can be any cell type. For example, the target cells can be stem cells, which can include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, umbilical cord blood stem cells, or adult stem cells (i.e., tissue-specific stem cells). In other cases, the target cells can be any differentiated cell type found in an individual. Cells of interest include dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, and the like.

[0337] Target cells can include cells at a target location, such as the liver, or cells that are near or adjacent to hepatocytes, such as hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.

[0338] In some cases, the application of interest is a therapeutic application, such as in the treatment of a disease. For example, the compositions and methods of the present application can be used to deliver nucleic acid sequences to cells to correct genetic deficiencies. As a non-limiting example, the compositions of the present application can be used to treat genetic deficiencies that affect hepatocyte function, or genetic deficiencies elsewhere in the body that can be remedied by using hepatocytes as a bioreactor to secrete the defective protein.

[0339] 4.13 Definitions

[0340] Those skilled in the art will understand that, generally speaking, the terms used herein and especially in the appended claims (e.g., the main body of the appended claims) are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "including but not limited to", etc.). Those skilled in the art will further understand that if a specific number of introduced claim recitations is desired, such intent will be explicitly stated in the claim, and in the absence of such a statement, there is no such intent. For example, for purposes of aiding understanding, the appended claims may contain the use of introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that any particular claim that introduces a claim recitation by the indefinite article "a" and / or "an" limits the claim recitation so introduced to only embodiments containing one such recitation, even when the same claim includes an introductory phrase "one or more" or "at least one" and an indefinite article such as "a" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); this also applies to cases where a definite article is used to introduce a claim recitation. In addition, even if a specific number or words of the introduced claim recitation are explicitly recited, those skilled in the art will also recognize that such recitation should be interpreted as meaning at least the recited number (e.g., the unadorned recitation "two recitations" without any other modifiers generally means at least two recitations or two or more recitations). In addition, in those cases where a convention similar to "at least one of A, B, and C" is used, generally such a construction is established in the sense that those skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include but not be limited to systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). In cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such a construction is intended in the sense that those skilled in the art should understand the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include but not be limited to systems having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C all together, etc.). Those skilled in the art will further understand that in fact any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as contemplating the possibility of including one of the terms, any one of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0341] In addition, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any single member or subgroup of members of the Markush group.

[0342] As will be understood by those skilled in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also cover any and all possible subranges and combinations of subranges thereof. Any recited range can be readily identified for the purposes of being well described and can be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. Those skilled in the art will also understand that all language such as “at most,” “at least,” “greater than,” “less than,” etc. includes the recited numbers and refers to ranges that can then be divided into subranges as discussed above. Finally, those skilled in the art will understand that a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0343] The terms “individual,” “subject,” and “host” are used interchangeably herein and refer to any individual for whom diagnosis, treatment, or therapy is desired. In some aspects, the individual is a mammal. In some aspects, the individual is a human. In some aspects, the individual is a patient. In some aspects, the individual is a human patient. In some aspects, the individual may have or be suspected of having a disorder or health condition associated with a gene of interest (GOI). In some aspects, the individual is a human at risk of being diagnosed with a disorder or health condition associated with the GOI at or after diagnosis. In some cases, a diagnosis of risk of having a disorder or health condition associated with the GOI can be determined based on the presence of one or more mutations in the endogenous GOI in the genome or in the genomic sequence proximal to the GOI that may affect GOI expression.

[0344] The term "treatment" as used herein to refer to a disease or medical condition means achieving at least an improvement in symptoms associated with the condition afflicting an individual, where improvement is used in a broad sense to mean at least a reduction in the magnitude of a parameter (e.g., a symptom) associated with the condition being treated (such as hemophilia A). Thus, treatment also includes where the pathological condition or at least the symptoms associated therewith are completely suppressed, e.g., prevented from occurring, or completely eliminated such that the host no longer has the condition, or at least the symptoms that characterize the condition. Accordingly, treatment includes: (i) prevention, i.e., reducing the risk of development of clinical symptoms, including preventing the clinical symptoms from developing, e.g., preventing disease progression; (ii) inhibition, i.e., arresting the development or further development of clinical symptoms, e.g., alleviating or completely suppressing an active disease.

[0345] As used herein, the terms "effective amount", "pharmaceutically effective amount", or "therapeutically effective amount" mean the amount of a composition sufficient to provide the desired utility when administered to an individual suffering from a particular condition. Thus, the term "therapeutically effective amount" refers to the amount of a therapeutic cell or a composition having a therapeutic cell sufficient to effect a particular action when administered to an individual in need of treatment. An effective amount will also include an amount sufficient to prevent or delay the development of symptoms of a disease, alter the course of symptoms of a disease (e.g., (but not limited to) slowing the progression of symptoms of a disease) or reverse the symptoms of a disease. It is understood that for any given situation, the appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.

[0346] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering a compound of interest to an individual. "Pharmaceutically acceptable excipients" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0347] The term "pharmaceutical composition" is intended to encompass compositions suitable for administration to an individual, such as a mammal, particularly a human. Generally, a "pharmaceutical composition" is sterile and preferably free of contaminants capable of causing adverse reactions in the individual (i.e., the compounds in the pharmaceutical composition are of pharmaceutical grade). Pharmaceutical compositions can be designed for administration to an individual or patient in need thereof via a variety of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, etc.

[0348] As used herein, the phrase "having the formula" or "having the structure" is not intended to be limiting and is used in the same manner as the common term "comprising". The term "independently selected from" is used herein to indicate that the recited elements (e.g., R groups, etc.) can be the same or different.

[0349] As used herein, the terms "may", "optional", "optionally" or "optionally may" mean that the subsequently described situation may or may not occur, and the description includes instances where the situation occurs and instances where the situation does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus the description includes structures in which a non-hydrogen substituent is present and structures in which a non-hydrogen substituent is not present.

[0350] "Acyl" means the group H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclic-C(O)- and substituted heterocyclic-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0351] The term "alkyl" means a branched or unbranched saturated hydrocarbon group (i.e., a single group), typically but not necessarily containing from 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, octyl, decyl, etc., and cycloalkyl, such as cyclopentyl, cyclohexyl, etc. Typically, although not necessarily, alkyl herein may contain from 1 to about 18 carbon atoms, and such groups may contain from 1 to about 12 carbon atoms. The term "lower alkyl" means an alkyl having 1 to 6 carbon atoms. "Substituted alkyl" means an alkyl substituted with one or more substituents, and this includes cases where two hydrogen atoms from the same carbon atom in the alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl may include a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" mean an alkyl substituent in which at least one carbon atom is replaced by a heteroatom, as described in further detail below. Unless otherwise indicated, the terms "alkyl" and "lower alkyl" include straight-chain, branched-chain, cyclic, unsubstituted, substituted and / or heteroatom-containing alkyl and lower alkyl, respectively.

[0352] The term "substituted alkyl" means an alkyl as defined herein, wherein one or more carbon atoms in the alkyl chain have been optionally replaced by heteroatoms such as -O-, -N-, -S-, -S(O) n-(where n is from 0 to 2), -NR- (where R is hydrogen or alkyl), and has 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thioketone group, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic group, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NRaRb, where R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic group.

[0353] The term "alkenyl" refers to a straight-chain, branched-chain or cyclic hydrocarbon group having 2 to about 24 carbon atoms and containing at least one double bond, such as vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, etc. Generally, although not necessarily, the alkenyls herein may contain 2 to about 18 carbon atoms and, for example, may contain 2 to about 12 carbon atoms. The term "lower alkenyl" means an alkenyl having 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted with one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl in which at least one carbon atom is replaced by a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" respectively include straight-chain, branched-chain, cyclic, unsubstituted, substituted and / or heteroatom-containing alkenyls and lower alkenyls.

[0354] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having 2 to about 24 carbon atoms and containing at least one triple bond, such as ethynyl, n-propynyl, etc. Generally, although not necessarily, the alkynyls herein may contain 2 to about 18 carbon atoms, and such groups may contain 2 to about 12 carbon atoms. The term "lower alkynyl" means an alkynyl having 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl in which at least one carbon atom is replaced by a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" respectively include straight-chain, branched-chain, cyclic, unsubstituted, substituted and / or heteroatom-containing alkynyls and lower alkynyls.

[0355] Unless otherwise indicated, the term "aryl" refers to an aromatic substituent, typically but not necessarily containing from 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings fused together, directly linked or indirectly linked (such that different aromatic rings are joined to a common group such as a methylene or ethylene moiety). An aryl can contain, for example, from 5 to 20 carbon atoms, and as another example, an aryl can contain from 5 to 12 carbon atoms. For example, an aryl can contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. "Substituted aryl" refers to an aryl moiety substituted with one or more substituents, and as will be described in further detail below, the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom is replaced by a heteroatom. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C 14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thienyl, imidazolyl, pyrimidyl, and oxazolyl; which may be further substituted with one to five members selected from the group consisting of: hydroxy, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, e.g., Katritzky, Handbook of Heterocyclic Chemistry). If not otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0356] The term "alkylene" refers to a diradical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene" refers to an alkylene linkage containing from 1 to 6 carbon atoms. Examples include methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)6-), etc.

[0357] Similarly, the terms "alkenylene", "alkynylene", "arylene", "aralkylen", and "alkarylene" refer to diradical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.

[0358] The term "amino" refers to the group -NRR', where R and R' are independently hydrogen or non-hydrogen substituents, and non-hydrogen substituents include, for example, alkyl, aryl, alkenyl, aralkyl, and their substituted and / or heteroatom-containing variants.

[0359] "Cycloalkyl" means a cycloalkyl group having a single or multiple rings with 3 to 10 carbon atoms, said rings including fused rings, bridged rings and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl, etc.

[0360] The term "substituted cycloalkyl" means a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents, said substituents being selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thione, carboxyl, carboxyalkyl, thioaryloxy, thioheteraryloxy, thioheteracyloxy, thio, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0361] "Heteroaryl" means an aromatic group having from 1 to 15 carbon atoms (such as 1 to 10 carbon atoms) and from 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl may have a single ring (such as pyridyl, imidazolyl, or furyl) or multiple fused rings (such as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl) in the ring system, where at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of the aromatic ring. In certain embodiments, one or more nitrogen and / or sulfur ring atoms of the heteroaryl are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. The term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl, and furyl. Unless otherwise restricted by the definition of the heteroaryl substituent, such heteroaryl may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thio, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azide, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl, and trifluoromethyl.

[0362] The terms "heterocyclic", "heterocyclic", and "heterocyclic group" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused, bridged, and spiro ring systems, and having 3 to 15 ring atoms, including 1 to 4 heteroatoms. These ring heteroatoms are selected from nitrogen, sulfur, and oxygen, where in a fused ring system, one or more rings may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide N-oxide, -S(O)-, or -SO2- moieties.

[0363] Examples of heterocycles and heteroaryls include, but are not limited to: azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinoxaline, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, thiomorpholino (also known as thiamorpholino), 1,1-dioxothiomorpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0364] Unless otherwise restricted by the definition of the heterocyclic substituent, such heterocyclic groups may optionally be substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteraryloxy, thioheterocyloxy, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic group, heterocyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycles.

[0365] As mentioned in some of the foregoing definitions, "substituted" as in "substituted alkyl", "substituted aryl", etc. means that in an alkyl, aryl, or other moiety, at least one hydrogen atom bonded to a carbon (or other) atom is replaced by one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and hydrocarbon moieties C1-C 24 alkyl (including C1-C 18 alkyl, further including C1-C 12 alkyl, and further including C1-C6 alkyl), C2-C 24 alkenyl (including C2-C 18 alkenyl, further including C2-C 12 alkenyl, and further including C2-C6 alkenyl), C2-C 24 alkynyl (including C2-C 18 alkynyl, further including C2-C 12 alkynyl, and further including C2-C6 alkynyl), C5-C 30Aryl (including C5-C 20 aryl, and further including C5-C 12 aryl) and C6-C 30 Aralkyl (including C6-C 20 aralkyl, and further including C6-C 12 aralkyl). The above hydrocarbon moieties may be further substituted by one or more functional groups or additional hydrocarbon moieties, such as those specifically recited. Unless otherwise specified, any group described herein shall be construed to include substituted and / or heteroatom-containing moieties in addition to the unsubstituted group.

[0366] As used in "linking group", "linker moiety", etc., "linking" or "linker" means a linking moiety that connects two groups via a covalent bond. The linker can be straight-chain, branched-chain, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene and linking moieties containing functional groups, said functional groups including but not limited to: amido (-NH-CO-), urea (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), episulfide (-S-), dioxirane (-O-O-), carbonyl dioxylate (-O-CO-O-), alkyl dioxylate (-O-(CH2)n-O-), epoxyimino (-O-NH-), imino (-NH-), carbonyl (-CO-), etc. In some cases, one, two, three, four or five or more carbon atoms of the linker backbone may optionally be substituted by sulfur, nitrogen or oxygen heteroatoms. The bonds between the backbone atoms can be saturated or unsaturated, usually not more than one, two or three unsaturated bonds in the linker backbone. The linker may include one or more substituents, such as having alkyl, aryl or alkenyl. The linker may include but not limited to poly(ethylene glycol) units (e.g., -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g., (C1-C 12 )alkyl), which may be straight-chain or branched-chain, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include cyclic groups, such as aryl, heterocycle or cycloalkyl, where 2 or more atoms of the cyclic group are included in the backbone, such as 2, 3 or 4 atoms. The linker may be cleavable or non-cleavable. Any suitable orientation and / or linkage of the linker with the linking group may be used.

[0367] When the term "substituted" appears before a list of groups that may be substituted, it is intended that the term apply to each member of that group. For example, the phrase "substituted alkyl and aryl" should be construed as "substituted alkyl and substituted aryl".

[0368] In addition to the disclosure herein, when the term "substituted" is used to modify a specified group or moiety, it can also mean that one or more hydrogen atoms of the specified group or moiety are each independently replaced by the same or different substituent groups as defined below.

[0369] In addition to the groups disclosed herein for each term, unless otherwise stated, the substituents used to replace one or more hydrogens on the saturated carbon atoms in the specified group or free radical (any two hydrogens on a single carbon can be replaced by =O, =NR 70 , =N-OR 70 , =N2 or =S) are -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trifluoromethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , wherein R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heteroalkylalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl, each R 70 is independently hydrogen or R 60 ; each R 80 is independently R 70 , or alternatively, two R 80' together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heteroalkyl group, which heteroalkyl group may optionally include 1 to 4 additional heteroatoms the same or different selected from the group consisting of O, N, and S, wherein N may have -H or C1-C3 alkyl substitution; and each M + is a counterion having a net single positive charge. Each M + may independently be, for example, an alkali ion such as K + , Na + , Li + ; an ammonium ion such as + N(R 60 )4; or an alkaline earth metal ion such as [Ca 2+ 0.5 , [Mg 2+ 0.5 or [Ba 2+ 0.5 ("subscript 0.5 means that one of the counterions of such divalent alkaline earth metal ions can be the ionized form of the compounds of the present invention, and another typical counterion (such as chloride) or two ionized compounds disclosed herein can be used as the counterion of such divalent alkaline earth metal ions, or the doubly ionized compounds of the present invention can be used as the counterion of such divalent alkaline earth metal ions). As a specific example, -NR 80 R 80 ​​​Intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

[0370] Except as disclosed herein, unless otherwise specified, the substituents of the hydrogens on the unsaturated carbon atoms in "substituted" alkenes, alkynes, aryls, and heteroaryls are -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2- M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + As previously defined, provided that in the case of a substituted alkene or alkyne, the substituent is not -OM + 、-OR 70 、-SR 70 or -SM + .

[0371] In addition to the groups disclosed herein for each term, unless otherwise specified, the substituents for hydrogen on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are -R 60 , -O - M + 、-OR 70 、-SR 70 , -S - M + 、-NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 、-S(O)2O - M + 、-S(O)2OR 70 、-OS(O)2R 70 、-OS(O)2O - M + 、-OS(O)2OR 70 、-P(O)(O - )2(M + )2、-P(O)(OR 70 ) - M + 、-P(O)(OR 70 )(OR 70 )、-C(O)R 70 、-C(S)R 70, -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + are as previously defined.

[0372] In addition to the disclosure herein, in one embodiment, the substituted group has 1, 2, 3 or 4 substituents, 1, 2 or 3 substituents, 1 or 2 substituents or 1 substituent.

[0373] Unless otherwise specified, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group, followed by naming the adjacent functional group towards the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-O-C(O)-.

[0374] Regarding any group containing one or more substituents disclosed herein, it should of course be understood that such groups do not contain any substitutions or substitution patterns that are sterically unrealistic and / or synthetically infeasible. Additionally, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

[0375] In certain embodiments, substituents can contribute to the optical and / or stereoisomerism of the compounds. Salt, solvate, hydrate, and prodrug forms of the compounds are also of interest. All such forms are encompassed within the present disclosure. Accordingly, the compounds described herein include their salt, solvate, hydrate, prodrug, and isomeric forms, including their pharmaceutically acceptable salt, solvate, hydrate, prodrug, and isomers. In certain embodiments, the compounds can be metabolized into pharmaceutically active derivatives.

[0376] Those skilled in the art will appreciate that, as used herein, a bond designated as in a small molecule structure refers to a bond that is, in some embodiments, a single bond (e.g., a saturated bond) and, in some embodiments, a double bond (e.g., an unsaturated bond). For example, the following structure: is intended to encompass both both.

[0377] Unless otherwise specified, reference to an atom is meant to include isotopes of said atom. By way of example, reference to H is intended to include 1 H, 2 H (i.e., D), and 3 H (i.e., T), and reference to C is intended to include 12 C and all isotopes of carbon (such as 13 C).

[0378] Definitions of other terms and concepts appear throughout the detailed description.

[0379] The following examples are provided in an illustrative rather than a limiting manner.

[0380] 5. Enumerated Embodiments

[0381] Example 1. An ionizable lipid compound of formula (I):

[0382] (Z-L-Y)-W n -(X-R) (n-1)

[0383] (I)

[0384] Wherein:

[0385] a. Z is an ionizable head group;

[0386] b. L is an optionally substituted (C 1- C 12 ) alkylene;

[0387] c. Y is a linking group;

[0388] d. W n is a straight-chain alkyl core having n carbon atoms, where n is from 3 to 6;

[0389] e. X is an optional linking group; and

[0390] f. Each R is independently a lipid tail.

[0391] Example 2. The compound according to Example 1, wherein n is from 4 to 6.

[0392] Example 3. The compound according to Example 2, wherein n is 4.

[0393] Example 4. The compound according to Example 2, wherein n is 5.

[0394] Example 5. The compound according to Example 2, wherein n is 6.

[0395] Example 6. The compound according to Example 1, wherein n is 3.

[0396] Example 7. The compound according to any one of Examples 1 to 6, wherein W n is selected from:[[]]

[0397]

[0398] wherein:[[]]

[0399] * depicts the attachment point to Y;

[0400] Each ** depicts the attachment point to X;

[0401] G 1 is H or a group that is cyclically linked to Y, and the group together with the carbon atom of W to which it is attached n forms a heterocycle; and

[0402] G 2 is H or -CH2OH.

[0403] Example 8. The compound according to any one of Examples 1 to 6, wherein Y is selected from -O-, -C(R 10 )2-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, where R 10 is selected from H and C 1-6 alkyl.

[0404] Example 9. The compound according to Example 8, wherein Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -.

[0405] Example 10. The compound according to Example 8, wherein Y is -CH2-.

[0406] Example 11. The compound according to Example 7, wherein G 1 is a group that is cyclically connected to Y and forms a heterocycle together with the carbon atom of W to which it is attached. n to which it is attached.

[0407] Example 12. The compound according to any one of Examples 1 to 11, wherein L is a (C2-C6) alkylene group or a substituted (C2-C6) alkylene group.

[0408] Example 13. The compound according to Example 12, wherein L is -(CH2)2-.

[0409] Example 14. The compound according to Example 12, wherein L is -(CH2)3-.

[0410] Example 15. The compound according to any one of Examples 1 to 14, wherein Z contains a tertiary amino group.

[0411] Example 16. The compound according to Example 15, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently an alkyl group or a substituted alkyl group.

[0412] Example 17. The compound according to Example 16, wherein R 11 and R 12 are each C 1-6 alkyl.

[0413] Example 18. The compound according to Example 17, wherein R 11 and R 12 are each C 1-3 alkyl.

[0414] Example 19. The compound according to Example 18, wherein R 11 and R 12 are each methyl.

[0415] Example 20. The compound according to Example 18, wherein R 11 and R 12 are each ethyl.

[0416] Example 21. The compound according to any one of Examples 1 to 20, wherein each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O-, -(CH2) s OC(O)O-, -(CH2) s OC(O)NR 10 -, -(CH2) s O-, -(CH2) s SC(O)NR 10 -, -(CH2) s C(O)NR 10 -, -(CH2) s NR 10 C(O)-, -(CH2) s S-, -(CH2) s NR 10 -, -(CH2) s NR 10 C(O)O- and -(CH2) s NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl and s is from 0 to 6.

[0417] Example 22. The compound according to any one of Examples 1 to 21, wherein each X is independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl.

[0418] Example 23. The compound according to Example 22, wherein each X is independently selected from -OC(O)-, -C(O)O- and -OC(O)O-.

[0419] Example 24. The compound according to Example 23, wherein each -X-R is -OC(O)R.

[0420] Example 25. The compound according to any one of Examples 1 to 24, wherein each R is independently an aliphatic hydrocarbon group, and the aliphatic hydrocarbon group is straight-chain or branched-chain, saturated or unsaturated and / or optionally contains a cyclic group.

[0421] Example 26. The compound according to any one of Examples 1 to 25, wherein each R is a straight-chain hydrocarbon group optionally containing one or more cyclic groups.

[0422] Example 27. The compound according to any one of Examples 1 to 26, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

[0423] Example 28. The compound according to Example 27, wherein each R is selected from C6-C 12 alkyl and C6-C 12 alkenyl.

[0424] Example 29. The compound according to Example 26, wherein at least one R is a straight-chain hydrocarbon group containing a cyclic group.

[0425] Example 30. The compound according to Example 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle and heteroaryl, and any one of the monocyclic or bicyclic groups is optionally substituted.

[0426] Example 31. The compound according to any one of Examples 1 to 25, wherein at least one R is a branched-chain hydrocarbon group optionally containing a cyclic group.

[0427] Example 32. The compound according to Example 31, wherein each R is a branched-chain hydrocarbon group.

[0428] Example 33. The compound according to Example 32, wherein the branched-chain hydrocarbon group contains 8 to 20 carbon atoms.

[0429] Example 34. The compound according to any one of Examples 31 to 33, wherein the branched-chain hydrocarbon group is saturated.

[0430] Example 35. The compound according to any one of Examples 31 to 33, wherein the branched-chain hydrocarbon group is unsaturated.

[0431] Example 36. The compound according to any one of Examples 31 to 35, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C12 Alkenyl

[0432] Example 37. The compound according to Example 31, wherein at least one R is a branched hydrocarbon group containing a cyclic group.

[0433] Example 38. The compound according to Example 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, and any one of the monocyclic or bicyclic groups is optionally substituted.

[0434] Example 39. The compound according to Example 1, wherein the compound has the formula (IIA):

[0435]

[0436] Example 40. The compound according to Example 39, wherein Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -, wherein R 10 is selected from H and C 1-6 alkyl.

[0437] Example 41. The compound according to Example 40, wherein Y is -O-.

[0438] Example 42. The compound according to Example 40, wherein Y is -OC(O)-.

[0439] Example 43. The compound according to Example 40, wherein Y is -OC(O)NR 10 -.

[0440] Example 44. The compound according to any one of Examples 39 to 43, wherein L is a (C2-C6) alkylene or a substituted (C2-C6) alkylene.

[0441] Example 45. The compound according to Example 44, wherein L is -(CH2)2-.

[0442] Example 46. The compound according to Example 44, wherein L is -(CH2)3-.

[0443] Example 47. The compound according to Example 44, wherein L is -(CH2)4-.

[0444] Example 48. The compound according to any one of Examples 39 to 47, wherein Z is -NR 11 R 12 wherein R 11 and R 12 are each independently C 1-6 alkyl or substituted C1-6 Alkyl

[0445] Example 49. The compound according to Example 48, wherein R 11 and R 12 are each C 1-3 alkyl

[0446] Example 50. The compound according to Example 49, wherein R 11 and R 12 are each methyl

[0447] Example 51. The compound according to Example 49, wherein R 11 and R 12 are each ethyl

[0448] Example 52. The compound according to any one of Examples 39 to 51, wherein each X is independently selected from -OC(O)-, -C(O)O- and -OC(O)O-

[0449] Example 53. The compound according to any one of Examples 39 to 52, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl

[0450] Example 54. The compound according to any one of Examples 39 to 52, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups

[0451] Example 55. The compound according to Example 54, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl

[0452] Example 56. The compound according to Example 39, wherein the compound has the formula (IIIA):

[0453]

[0454] wherein:

[0455] R 11 and R 12 are each independently C 1-3 alkyl;

[0456] q is from 1 to 4;

[0457] Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -; and

[0458] each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

[0459] Example 57. The compound according to Example 1, wherein the compound has the formula (IIB):

[0460]

[0461] Example 58. The compound according to Example 57, wherein Y is selected from -O-, -OC(O)-, -OC(O)NR 10 -, -NR 10 C(O)-, -NR 10 C(O)O-, and -NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl.

[0462] Example 59. The compound according to Example 58, wherein Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-.

[0463] Example 60. The compound according to any one of Examples 57 to 59, wherein L is (C2-C6) alkylene or substituted (C2-C6) alkylene.

[0464] Example 61. The compound according to Example 60, wherein L is -(CH2)2-.

[0465] Example 62. The compound according to Example 60, wherein L is -(CH2)3-.

[0466] Example 63. The compound according to Example 60, wherein L is -(CH2)4-.

[0467] Example 64. The compound according to any one of Examples 57 to 59, wherein Z is -NR 11 R 12 , wherein R 11 and R12 each independently is C 1-6 alkyl or substituted C 1-6 alkyl.

[0468] Example 65. The compound according to Example 64, wherein R 11 and R 12 each is C 1-3 alkyl.

[0469] Example 66. The compound according to Example 64, wherein R 11 and R 12 each is methyl.

[0470] Example 67. The compound according to any one of Examples 57 to 66, wherein each X is independently selected from -OC(O)-, -C(O)O- and -OC(O)O-.

[0471] Example 68. The compound according to any one of Examples 57 to 67, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

[0472] Example 69. The compound according to any one of Examples 57 to 67, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups.

[0473] Example 70. The compound according to Example 69, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

[0474] Example 71. The compound according to Example 57, wherein the compound has the formula (IIIB):

[0475]

[0476] wherein:

[0477] R 11 and R 12 each independently is C 1-3 alkyl;

[0478] q is 1 to 4;

[0479] Y is selected from -NHC(O)-, -NHC(O)O- and -NHC(O)S-; and

[0480] Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

[0481] Example 72. The compound according to Example 1, wherein the compound has the formula (IIC):

[0482]

[0483] Example 73. The compound according to Example 72, wherein Y is selected from -O-, -OC(O)-, -OC(O)NR 10 -, and -C(R 10 )2-, wherein R 10 is selected from H and C 1-6 alkyl.

[0484] Example 74. The compound according to Example 73, wherein Y is -O-.

[0485] Example 75. The compound according to Example 73, wherein Y is -C(R 10 )2-.

[0486] Example 76. The compound according to any one of Examples 72 to 75, wherein L is (C2-C6) alkylene or substituted (C2-C6) alkylene.

[0487] Example 77. The compound according to any one of Examples 76, wherein L is -(CH2)2-.

[0488] Example 78. The compound according to any one of Examples 76, wherein L is -(CH2)3-.

[0489] Example 79. The compound according to any one of Examples 76, wherein L is -(CH2)4-.

[0490] Example 80. The compound according to any one of Examples 72 to 79, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently C 1-6 alkyl or substituted C1-6 Alkyl

[0491] Example 81. The compound according to Example 80, wherein R 11 and R 12 are each C 1-3 alkyl

[0492] Example 82. The compound according to Example 81, wherein R 11 and R 12 are each methyl

[0493] Example 83. The compound according to any one of Examples 72 to 82, wherein each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O-, -(CH2) s OC(O)O-, where s is from 0 to 6

[0494] Example 84. The compound according to any one of Examples 72 to 82, wherein each s is 0

[0495] Example 85. The compound according to any one of Examples 72 to 82, wherein each s is 1

[0496] Example 86. The compound according to any one of Examples 72 to 82, wherein each s is 3

[0497] Example 87. The compound according to any one of Examples 72 to 86, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl

[0498] Example 88. The compound according to any one of Examples 72 to 86, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups

[0499] Example 89. The compound according to Example 88, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl

[0500] Example 90. The compound according to Example 72, wherein the compound has the formula (IIIC):

[0501]

[0502] Wherein:

[0503] R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl;

[0504] q is from 1 to 4;

[0505] Y is selected from -O- and -C(R 10 )2-;

[0506] Each s is independently 0, 1, or 2;

[0507] W is -O- and -C(R 10 )2-; and

[0508] Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and each of t and u is from 1 to 10.

[0509] Example 91. A compound according to any one of Examples 1 to 90, wherein each R is independently

[0510]

[0511] Cy A and Cy B are each independently a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, said cyclic group being selected from 5- to 12-membered monocyclic, bicyclic, bridged polycyclic and spirocyclic groups;

[0512] R x and R y are each independently a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 20 aliphatic group; and

[0513] r, p, and q are each independently an integer from 0 to 20.

[0514] Example 92. A compound according to Example 91, wherein at least one R comprises a moiety selected from the following:

[0515]

[0516]

[0517] Each # represents an attachment point to X or an attachment point to a straight-chain or branched-chain hydrocarbon chain of R.

[0518] Example 93. A lipid nanoparticle comprising an ionizable lipid compound according to any one of Examples 1 to 92.

[0519] Example 94. The lipid nanoparticle according to Example 93, further comprising a neutral lipid and a lipid capable of reducing aggregation.

[0520] Example 95. The lipid nanoparticle according to Example 94, wherein the neutral lipid comprises a phospholipid.

[0521] Example 96. The lipid nanoparticle according to Example 94 or 95, wherein the neutral lipid comprises cholesterol.

[0522] Example 97. The lipid nanoparticle according to Example 96, comprising:

[0523] a. A nucleic acid,

[0524] b. An ionizable lipid,

[0525] c. A phospholipid,

[0526] d. Cholesterol, and

[0527] e. A lipid capable of reducing aggregation.

[0528] Example 98. The lipid nanoparticle according to Example 97, wherein the nucleic acid comprises DNA.

[0529] Example 99. The lipid nanoparticle according to Example 98, wherein the nucleic acid comprises RNA.

[0530] Example 100. The lipid nanoparticle according to Example 98, wherein the nucleic acid comprises DNA and RNA.

[0531] Example 101. The lipid nanoparticle according to Example 100, wherein the RNA is selected from mRNA, gRNA, and siRNA.

[0532] Example 102. The lipid nanoparticle according to any one of Examples 97 to 101, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), and derivatives thereof.

[0533] Example 103. The lipid nanoparticle according to Example 102, wherein the phospholipid is phosphatidylethanolamine (PE).

[0534] Example 104. The lipid nanoparticle according to Example 103, wherein the phospholipid is phosphatidylcholine (PC).

[0535] Example 105. The lipid nanoparticle according to any one of Examples 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12 to 24 carbons.

[0536] Example 106. The lipid nanoparticle according to Example 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16 to 20 carbons.

[0537] Example 107. The lipid nanoparticle according to Example 105 or 106, wherein the hydrocarbon chains are saturated.

[0538] Example 108. The lipid nanoparticle according to Example 105 or 106, wherein the hydrocarbon chains are unsaturated and / or further comprise carbocyclic groups.

[0539] Example 109. The lipid nanoparticle according to Example 108, wherein the hydrocarbon chains each independently comprise 1 to 4 double bonds.

[0540] Example 110. The lipid nanoparticle according to any one of Examples 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.

[0541] Example 111. The lipid nanoparticle according to Example 103, wherein the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0542] Example 112. The lipid nanoparticle according to Example 103, wherein the phospholipid comprises 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

[0543] Example 113. The lipid nanoparticle according to Example 104, wherein the phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Δ9Δ9-cis PC).

[0544] Example 114. The lipid nanoparticle according to Example 106, wherein the lipid nanoparticle comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0545] Example 115. The lipid nanoparticle according to Example 104, wherein the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0546] Example 116. The lipid nanoparticle according to any one of Examples 103 to 115, wherein the lipid capable of reducing aggregation is a PEG-lipid.

[0547] Example 117. The lipid nanoparticle according to Example 116, wherein the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0548] Example 118. The lipid nanoparticle according to any one of Examples 94 to 117, further comprising a targeting ligand.

[0549] Example 119. The lipid nanoparticle according to Example 118, wherein the targeting ligand comprises GalNAc.

[0550] Example 120. The lipid nanoparticle according to Example 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.

[0551] Example 121. The lipid nanoparticle according to Example 120, wherein the lipid capable of reducing aggregation is PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

[0552] Example 122. The lipid nanoparticle according to any one of Examples 94 to 121, wherein the N / P ratio (the ratio of the number of moles of the amine group of the cationic lipid to the number of moles of the phosphate ester of the DNA) is 5 to 30.

[0553] Example 123. The lipid nanoparticle according to Example 122, wherein the N / P ratio is 7.

[0554] Example 124. The lipid nanoparticle according to Example 122, wherein the N / P ratio is 14.

[0555] Example 125. The lipid nanoparticle according to Example 122, wherein the N / P ratio is 28.

[0556] Example 126. The lipid nanoparticle according to any one of Examples 94 to 125, comprising:

[0557] a. Ionizable lipids that account for 40 to 60 mol% of the total lipids present;

[0558] b. Phospholipids that account for 6 to 20 mol% of the total lipids present;

[0559] c. Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0560] d. Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0561] Example 127. The lipid nanoparticles according to any one of Examples 94 to 125, comprising:

[0562] a. Ionizable lipids that account for 40 to 60 mol% of the total lipids present;

[0563] b. Phospholipids that account for 10 to 20 mol% of the total lipids present;

[0564] c. Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0565] d. Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0566] Example 128. The lipid nanoparticles according to any one of Examples 94 to 125, comprising:

[0567] a.e) Ionizable lipids that account for 40 to 49 mol% of the total lipids present;

[0568] b.f) Phospholipids that account for 10 to 20 mol% of the total lipids present;

[0569] c.g) Cholesterol that accounts for 35 to 45 mol% of the total lipids present; and

[0570] d.h) Lipids capable of reducing aggregation that account for 1.5 to 2.5 mol% of the total lipids present.

[0571] Example 129. A pharmaceutical composition comprising the lipid nanoparticles according to any one of Examples 94 to 128 and a pharmaceutically acceptable excipient, carrier or diluent.

[0572] Example 130. A method for delivering nucleic acid into cells, the method comprising contacting the cells with the lipid nanoparticles according to any one of Examples 94 to 128.

[0573] Example 131. The method according to Example 130, wherein the cells are in vitro.

[0574] Example 132. The method according to Example 130, wherein the cell is in vivo.

[0575] Example 133. A method for delivering a nucleic acid to produce a target protein in vivo, the method comprising:

[0576] Systemically administering to an individual in need the pharmaceutical composition according to Example 129, wherein the nucleic acid encodes the target protein and is encapsulated within the lipid nanoparticle, and the administration of the pharmaceutical composition results in an extended stable expression of the target protein.

[0577] 6. Examples

[0578] The following examples are presented in order to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

[0579] General methods in molecular and cell biochemistry can be found in standard textbooks such as: "Molecular Cloning: A Laboratory Manual", 3rd Edition (Sambrook et al., Harbor Laboratory Press 2001); "Short Protocols in Molecular Biology", 4th Edition (Ausubel et al., John Wiley & Sons 1999); "Protein Methods" (Bollag et al., John Wiley & Sons 1996); "Nonviral Vectors for Gene Therapy" (Wagner et al., Academic Press 1999); "Viral Vectors" (Kaplift and Loewy, Academic Press 1995); "Immunology Methods Manual" (I. Lefkovits, Academic Press 1997); and "Cell and Tissue Culture: Laboratory Procedures in Biotechnology" (Doyle and Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for the methods mentioned in this disclosure or methods related to this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., etc., and repositories such as Addgene, Inc., American Type Culture Collection (ATCC), etc.

[0580] Materials and Methods

[0581] LNP formulations. As described by Prud'homme et al. (J Pharm Sci 2018), LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acids (e.g., DNA only, mRNA only, or a DNA / mRNA mixture). Briefly, a lipid excipient mixture (ionizable lipid, helper lipid, cholesterol, PEG-lipid, and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of nucleic acids is prepared in a low pH buffer in the range of pH 3.0 - 4.0. The lipid mixture is then mixed with the aqueous nucleic acid solution at a flow ratio of 1:3 (V / V) using a commercially available mixer device. The resulting solution is immediately diluted with a buffer having a pH range of 5.0 - 6.5. The diluted LNPs are dialyzed and purified against a secondary buffer having a pH range of 7.0 - 8.0. The LNP solution is concentrated using a 100,000 MWCO Amicon Ultra centrifuge tube (Millipore Sigma) and then filtered through a 0.2 μm PES sterile filter. The particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). The encapsulation efficiency is calculated using a Quant-it RiboGreen assay kit.

[0582] Detection of EPO and cytokines in serum. Blood is collected into serum separator tubes via retro-orbital bleeding and processed into serum. Serum samples can be stored at -80 °C from collection until analysis. The serum levels of human EPO protein driven by DNA payload expression are quantified using the MSD U-PLEX human EPO assay according to the manufacturer's instructions. The serum levels of murine cytokines produced by exposure to DNA-LNP are quantified using a mouse pro-inflammatory 7-plex tissue culture kit from MSD according to the manufacturer's instructions.

[0583] FIX Detection in Plasma. Blood was collected into K2EDTA tubes by retro-orbital bleeding and processed into plasma. Plasma samples could be stored at -80 °C from collection until analysis. The plasma levels of human FIX after administration of LNP were quantified using the U-Plex assay on the MSD platform. Briefly, monoclonal mouse anti-human FIX antibody (Prolytix, clone AHIX-5041) was conjugated with biotin and used as a capture reagent on streptavidin-coated plates. Polyclonal goat anti-human FIX antibody (Cedarlane, clone CL20040AP) was conjugated with Sulfo-TAG and used as a detection reagent, and standard settings for electrochemiluminescence (ECL) signal quantification were performed using the QuickPlex SQ 120MM instrument from MSD. Pooled normal human plasma (Affinity Biologicals, FRNCP0125) (which is a pool of normal citrated human plasma collected from at least 20 donors) was used to generate a standard curve and calculate the % of normal human FIX levels. It was confirmed that the assay was specific for human FIX and did not cross-react with mouse FIX, demonstrating a very low background level in untreated mouse plasma samples.

[0584] Example 1. Synthesis of tetra(decanoate) 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetra-yl ester (L-1)

[0585]

[0586] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-(dimethylamino)butyrate

[0587]

[0588] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1 equiv) and diisopropylethylamine (3 equiv) was added 4-(dimethylamino)butyric acid 2,5-dioxopyrrolidin-1-yl ester (2 equiv) in DMF (4V). The reaction mixture was heated at 90 °C for 16 h, then cooled to room temperature and quenched by the addition of water and MTBE. The organic layer was collected and the aqueous layer was further extracted with MTBE (3×3V). The combined organic extracts were washed with 10% CuSO4 (2V), then with brine (2V), dried over MgSO4, filtered, concentrated, and purified by column chromatography. The recovered starting material was re-subjected to the reaction conditions.

[0589] Synthesis of 1,2,4,5-tetrahydroxypentan-3-yl 4-(dimethylamino)butyrate

[0590]

[0591] HCl aqueous solution (1 M) was added to a solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-(dimethylamino)butyrate in methanol (5V). The completion of the reaction mixture was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure and dried thoroughly with several toluene azeotropes.

[0592] Synthesize 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayl tetra(decanoate)

[0593]

[0594] 1,2,4,5-Tetrahydroxypentan-3-yl 4-(dimethylamino)butyrate (1 equiv), decanoic acid (5.5 equiv), EDCI (6 equiv), DMAP (2 equiv), DIEA (8 equiv) and ACN (10 mL) were mixed at 0 °C and then at room temperature. The resulting mixture was stirred at room temperature for an additional 3 h. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.

[0595] Example 2. Synthesis of 1,4,5-tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yl decanoate (L-2)

[0596]

[0597] Synthesize bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol

[0598]

[0599] A solution of 1,2,3,4,5-pentahydroxypentane (25 g, 1 eq), p-toluenesulfonic acid (2.83 g, 0.1 eq), and 2,2-dimethoxypropane (37.65 g, 2.2 eq) in methanol (250 mL) was stirred overnight at room temperature under a nitrogen atmosphere. K2CO3 (5 g) was added to the reaction mixture and stirred for 1 h. The resulting mixture was filtered and the filter cake was washed with MeOH (2×20 mL). The filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (200 mL) and 50 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. It was concentrated under vacuum until no distillate was obtained while maintaining the temperature below 35 °C. 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w / w.) was loaded into the column, and then the dry silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (gradient from 100:0 to 50:50, collected every 200±10 mL). Samples were taken for TLC (EA:PE = 1:1) analysis and the qualified products were combined. This produced bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (12 g, 31.4%) as a colorless oil.

[0600] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl carbonate (4-nitrophenyl ester)

[0601]

[0602] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (10 g, 1 eq) in THF (100 mL), 4-nitrophenyl chloroformate (9.55 g, 1.1 eq) was added portionwise. TEA (13.07 g, 3 eq) was added dropwise at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 60 min. The resulting mixture was used directly in the next step without further purification.

[0603] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate

[0604]

[0605] To a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-nitrophenyl carbonate (110 mL in THF) was added dropwise dimethylaminopropylamine (6.6 g, 2.0 eq) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred for 60 minutes at room temperature under a nitrogen atmosphere. The resulting mixture was quenched with 50 mL of water and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (2 × 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 15 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. It was concentrated under vacuum until no distillate was obtained while maintaining the temperature below 35 °C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 30 w / w.) was loaded into the column, and then the dried silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with CH2Cl2 / MeOH (gradient from 100:0 to 10:1, collected every 100 ± 10 mL). Samples were taken for TLC (CH2Cl2 / MeOH = 5:1) analysis, and the qualified products were combined. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate was obtained as a yellow oil (7 g, two-step yield 45.1%).

[0606] Synthesis of 1,2,4,5-tetrahydroxypentan-3-yl N-[3-(dimethylamino)propyl]carbamate

[0607]

[0608] A solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate (7 g, 1 equivalent) in HCl (6 M, 70 mL) was stirred overnight at 50 °C under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. This gave 1,2,4,5-tetrahydroxypentan-3-yl N-[3-(dimethylamino)propyl]carbamate as a pale yellow oil (4 g, 73.48%).

[0609] Synthesis of 2-{3-[(dimethylamino)propylcarbamoyloxy]-1,4,5-tris(decanoyloxy)pentyl} decanoate

[0610]

[0611] To a stirred solution of 1,2,4,5-tetrahydroxypentane-3-yl N-[3-(dimethylamino)propyl]carbamate (4 g, 1 eq) and decanoyl chloride (21.77 g, 114.152 mmol, 8 eq) in DCM (100 mL). TEA (14.44 g, 142.690 mmol, 10 eq) was added dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (2 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: MeCN:i-PrOH = 1:1; A: water (0.1% TFA), gradient from 50% to 95% in 15 min; flow rate: 50 mL / min: detector, ELSD. The fractions were concentrated under reduced pressure to remove the organic solvent, the aqueous phase was adjusted to pH = 8 with NaHCO3 (5% aqueous solution); extracted with heptane (50 mL × 2), the combined organic phases were dried over anhydrous Na2SO4, and after filtration, the filtrate was concentrated under reduced pressure. This gave 1,4,5-tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yl decanoate as a pale yellow oil (961.2 mg, 7.51%). LCMS: (ES, m / z): 898 [M+H] + ; 1 1H-NMR: (400 MHz, CDCl3, ppm): δ 5.914 - 5.626 (m, 1H), 5.367 - 5.211 (m, 3H), 4.281 - 4.293 (m, 2H), 4.145 - 4.068 (s, 2H), 3.275 - 3.197 (m, 2H), 2.369 - 2.213 (m, 16H), 1.680 (s, 10H), 1.335 (s, 48H), 0.982 - 0.826 (m, 12H).

[0612] Example 3. Synthesis of tetra(decanoic acid) 3-(4-(dimethylamino)butoxy)pentane-1,2,4,5-tetrayl ester (L-3)

[0613]

[0614] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate

[0615]

[0616] To a 100 mL three-necked round-bottom flask at room temperature was added bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (4 g, 17.221 mmol, 1 equiv), DCM (40 mL), and TEA (3.49 g, 34.442 mmol, 2 equiv). MsCl (2.96 g, 25.831 mmol, 1.5 equiv) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred for an additional 1 h at room temperature. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous MgSO4. After filtration, the filtrate was concentrated under reduced pressure. This gave bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (8.1 g, crude) as a brown oil. LCMS: (ES, m / z): 311 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 4.851 (t, J = 4.5 Hz, 1H), 4.307 - 4.247 (m, 1H), 4.153 - 4.068 (m, 2H), 4.045 - 3.967 (m, 2H), 3.145 - 3.070 (m, 3H), 1.457 - 1.330 (m, 12H).

[0617] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine

[0618]

[0619] At room temperature, NaH (870.29 mg, 21.759 mmol, 1.5 equiv, 60%) and THF (25 mL) were added to a 100 mL three-necked round-bottom flask. 4-(Dimethylamino)butan-1-ol (1.7 g, 14.506 mmol, 1 equiv) and THF (25 mL) were added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at 0 °C for an additional 30 minutes. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (4.95 g, 15.957 mmol, 1.1 equiv) and THF (25 mL) were added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at 60 °C overnight. The reaction was quenched with saturated NH4Cl (aqueous solution) at 0 °C. The mixture was basified to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (5 × 100 mL), dried over anhydrous MgSO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (5:1) to give {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine as a brown solid (4.6 g, 80.6% yield over two steps). LCMS: (ES, m / z): 332 [M+1] + . 1 1H NMR (300 MHz, chloroform-d) δ 4.155 - 4.095 (m, 2H), 4.060 - 4.011 (m, 2H), 3.929 - 3.873 (m, 2H), 3.723 (t, J = 6.1 Hz, 2H), 3.563 (d, J = 4.6 Hz, 1H), 2.742 - 2.689 (m, 2H), 2.545 (s, 6H), 1.809 - 1.707 (m, 2H), 1.653 - 1.586 (m, 2H), 1.429 (s, 6H), 1.339 (s, 6H).

[0620] Synthesis of 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetraol

[0621]

[0622] To a 50 mL three-necked round bottom flask at room temperature was added {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}dimethylamine (2.6 g, 7.844 mmol, 1 equiv), H2O (5 mL), and acetic acid (21 mL). The resulting mixture was stirred at 80 °C for 6 h. The mixture was allowed to cool to room temperature. The resulting mixture was concentrated in vacuo. This gave 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetraol (4.8 g, crude) as a brown oil. LCMS: (ES, m / z): 252 [M+1] + . 1 1H NMR (300 MHz, DMSO-d6) δ 3.634 - 3.582 (m, 2H), 3.547 - 3.483 (m, 5H), 3.393 - 3.334 (m, 3H), 3.238 - 3.196 (m, 1H), 2.219 - 2.161 (m, 2H), 2.106 (s, 6H), 1.442 - 1.383 (m, 4H).

[0623] Synthesis of 1,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yl decanoate

[0624]

[0625] To a 250 mL three-necked round bottom flask at room temperature was added 3-[4-(dimethylamino)butoxy]pentane-1,2,4,5-tetraol (3.2 g, 12.733 mmol, 1 equiv), DCM (160 mL), and TEA (20.62 g, 203.728 mmol, 16 equiv). To the above mixture was added dropwise decanoyl chloride (24.28 g, 127.330 mmol, 10 equiv) at 0 °C. The resulting mixture was stirred at 40 °C for an additional 2 h. The mixture was allowed to cool to room temperature. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with CH2Cl2 (2 × 100 mL), the organic phase was collected and dried over anhydrous MgSO4. After filtration, the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (5:1) to give 4 g (crude). The residue was purified by reverse phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C 185 μm; mobile phase, B: MeCN:i-PrOH = 1:1; A: water (0.1% TFA), gradient from 50% to 95% in 15 min; flow rate: 50 mL / min: detector, ELSD. The residue was dissolved in hexane (100 mL). The combined organic layers were washed with saturated NaHCO3 solution (2 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave decanoic acid 1,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yl ester as a pale yellow oil (699.5 mg, two-step yield 11.07%). LCMS: (ES, m / z): 869 [M+1] + . 1 1H NMR (300 MHz, chloroform-d, ppm): δ 5.229 - 5.179 (m, 2H), 4.429 - 4.378 (m, 2H), 4.163 - 4.101 (m, 2H), 3.639 (t, J = 5.3 Hz, 1H), 3.602 - 3.564 (m, 2H), 2.355 - 2.251 (m, 16H), 1.647 - 1.539 (m, 12H), 1.325 - 1.253 (m, 48H), 0.901 - 0.856 (m, 12H).

[0626] Example 4. Synthesis of tri-dodecanoic acid 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyl ester (L-4)

[0627]

[0628] Synthesis of pent-4-ene-1,2,3-triol

[0629]

[0630] HCl (6 M) (0.13 g, 3.544 mmol, 0.2 eq) was added to a mixture of 1-(2,2-dimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (1 eq) in MeOH (1 V). The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was concentrated under reduced pressure, and the material was dried thoroughly under vacuum and subjected to the next reaction.

[0631] Synthesis of tri-dodecanoic acid pent-4-ene-1,2,3-triyl ester

[0632]

[0633] EDCI (1.44 g, 7.525 mmol, 1 eq) was added to a mixture of pent-4-ene-1,2,3-triol (1 eq), dodecanoic acid (3.4 eq), and DMAP (1 eq) in DCM (10 V). The reaction mixture was stirred at 20 °C for 6 h. The resulting mixture was diluted with DCM (5 V). The resulting mixture was washed with water (3 × 1 V) and brine (1 V). The resulting solution was dried over anhydrous Na2SO4. After filtration, the resulting mixture was concentrated under reduced pressure. The residue was dissolved in DCM (1 V) and silica gel (type: ZCX-2, 100 - 200 mesh, 5.00 w. / w.) was added. TLC (PE / EA = 5:1) analysis was performed.

[0634] Synthesis of tris-dodecanoate 4-oxabutane-1,2,3-triyl ester

[0635]

[0636] K2OsO4·2H2O (0.12 eq) was added to a mixture of tris-dodecanoate pent-4-ene-1,2,3-triyl ester (1 eq) in THF (40 V) and H2O (20 V). The reaction mixture was stirred at 20 °C for 10 min, and NaIO4 (5.0 eq) and 2,6-dimethylpyridine (5.0 eq) were added at 20 °C. The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was diluted with EA (20 V). The resulting mixture was washed with 3 × 20 V of water and brine (30 V). The resulting solution was dried over anhydrous Na2SO4. The resulting mixture was concentrated under reduced pressure. TLC (PE / EA = 10:1) analysis was performed.

[0637] Synthesis of tris-dodecanoate 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyl ester

[0638]

[0639] A mixture of tri-dodecanoic acid 4-oxobutane-1,2,3-triyl ester (1 equivalent), 4-(dimethylamino)butane-1,2-diol (1.5 equivalents), DMAc dimethyl acetal (10 equivalents), and camphorsulfonic acid (2 equivalents) in DCE (40V) was heated under reflux overnight. The reaction mixture was cooled to 0 °C and then poured into a rapidly stirred mixture of EA (30V) and saturated aqueous NaHCO3 (30V). When the pH was higher than 7, the organic layer was collected and the product was further extracted from the aqueous layer (3 × 20V) with EA. The organics were combined, washed with brine (30V), dried over MgSO4, filtered, and concentrated, and then purified by silica gel column chromatography to give tri-dodecanoic acid 1-(4-(2-(dimethylamino)ethyl)-1,3-dioxolan-2-yl)propane-1,2,3-triyl ester.

[0640] Example 5. Synthesis of 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate (L-5)

[0641]

[0642] Synthesis of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol

[0643]

[0644] H2 (3 atm) was introduced into a stirred solution of 2,3,4-trihydroxybutanal (10 g, 1 equivalent), 1-(2,4-dimethoxyphenyl)methanamine (13.92 g, 1 equivalent), H2SO4 (1.63 g, 0.2 equivalent), and Raney Ni (3.57 g, 0.5 equivalent) in EtOH (100 mL) at room temperature. The resulting mixture was stirred at 45 °C for 6 hours. The resulting mixture was filtered and the cake was washed with ethanol (2 × 20 mL). The filtrate was concentrated under reduced pressure. The crude product 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol (16 g) was used directly in the next step without further purification.

[0645] Synthesis of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane

[0646]

[0647] At room temperature under a nitrogen atmosphere, imidazole (16 g, 4.0 eq) was added portionwise to a stirred solution of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-1,2,3-triol (16 g, 1 eq, crude) and TBSCl (28 g, 3.1 eq) in DMF (160 mL). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The mixture was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing MeCN (0.1% TFA), gradient from 40% to 90% in 20 min; detector, UV 220 nm. This gave 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane as a brown oil (6.6 g, 22.3%).

[0648] Synthesis of N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide

[0649]

[0650] A solution of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 1 eq) and 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-1-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 1 eq), HATU (6.02 g, 1.2 eq), DIEA (5.12 g, 3 eq) in DMF (50 mL) was stirred at room temperature under a nitrogen atmosphere for 1 h. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing MeCN (0.1% TFA), gradient from 30% to 90% in 20 min; detector, UV 220 nm. This gave N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide as a brown oil (4.2 g, 51.89%).

[0651] Synthesis of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide

[0652]

[0653] A solution of N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide (4.2 g, 6.852 mmol, 1 equiv) and TBAF (1.79 g, 6.852 mmol, 1 equiv) in THF (40 mL) was stirred at room temperature under a nitrogen atmosphere for 3 h. The resulting mixture was concentrated in vacuo. The crude product N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide (5 g, crude) was used directly in the next step without further purification.

[0654] Synthesis of 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate

[0655]

[0656] A solution of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4-trihydroxybutyl)butanamide (5 g, 13.005 mmol, 1 equiv), lauric acid (9.12 g, 45.518 mmol, 3.5 equiv), EDC·HCl (8.08 g, 52.020 mmol, 4 equiv), and DMAP (1.58 g, 13.0 mmol, 1 equiv) in DCM (50 mL) was stirred at room temperature under a nitrogen atmosphere for 4 h. The resulting mixture was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing MeCN (0.1% TFA), gradient from 30% to 90% in 20 min; detector, UV 220 nm. This gave 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate as a white semi-solid (3 g, 24.77%).

[0657] Synthesis of 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate

[0658]

[0659] A solution of 1-{N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate (2.5 g, 2.684 mmol, 1 equiv) in 1,4-dioxane (25 mL) containing HCl (gas) was stirred at room temperature under a nitrogen atmosphere for 6 h. The resulting mixture was concentrated in vacuo. The residue was purified by reverse-phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: MeCN:i-PrOH = 1:1; A: water (0.1% TFA), gradient from 45% to 85% in 15 min; flow rate: 50 mL / min: detector, ELSD. This gave 1-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate TFA salt as a white semi-solid (683.0 mg, 32.57%). LCMS: (ES, m / z): 782 [M+H] + ; 1 1H-NMR: (400 MHz, CDCl3, ppm): δ 12.182 (s, 1H), 7.283 - 7.193 (m, 1H), 5.261 - 5.187 (m, 2H), 4.365 - 4.336 (m, 1H), 4.156 - 4.110 (m, 1H), 3.635 - 3.607 (m, 1H), 3.402 - 3.367 (m, 1H), 3.367 - 2.893 (m, 2H), 2.864 (s, 1H), 2.693 - 2.298 (m, 12H), 2.112 (s, 2H), 1.613 (s, 6H), 1.272 (s, 48H), 0.992 - 0.910 (m, 9H).

[0660] Example 6. Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayl tetranonanoate (L-6)

[0661]

[0662] 3-((4-(dimethylamino)butanoyl)oxy)pentane-1,2,4,5-tetrayl tetranonanoate was synthesized in the same manner as (L-1), using nonanoic acid instead of decanoic acid.

[0663] Example 7. Synthesis of 3-(((3-(dimethylamino)propyl)carbamoyl)oxy)pentane-1,2,4,5-tetrayl tetranonanoate (L-7)

[0664]

[0665] 3-(((3-(Dimethylamino)propyl)carbamoyl)oxy)pentane-1,2,4,5-tetrayl tetranonanoate was synthesized in the same manner as (L-2), using nonanoyl chloride instead of decanoyl chloride.

[0666] Example 8. Synthesis of 3-(4-(Dimethylamino)butoxy)pentane-1,2,4,5-tetrayl tetranonanoate (L-8)

[0667]

[0668] 3-(4-(Dimethylamino)butoxy)pentane-1,2,4,5-tetrayl tetranonanoate was synthesized in a manner similar to (L-3), using nonanoyl chloride instead of decanoyl chloride.

[0669] Example 9. Synthesis of 3-(3-(Dimethylamino)propoxy)pentane-1,2,4,5-tetrayl tetra(decanoate) (L-9)

[0670]

[0671] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol

[0672]

[0673] A solution of 1,2,3,4,5-pentahydroxypentane (20 g, 131.5 mmol, 1 eq), p-toluenesulfonic acid (2.26 g, 13.15 mmol, 0.1 eq), and 2,2-dimethoxypropane (30.1 g, 289.3 mmol, 2.2 eq) in methanol (200 mL) was stirred at room temperature under a nitrogen atmosphere for 16 h. K2CO3 (5 g) was added to the reaction mixture and stirred at room temperature for 1 h. The resulting mixture was filtered and the cake was washed with MeOH (2 × 20 mL). The filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (200 mL) and 40 g of silica gel (type: ZCX-2, 100-200 mesh, 2 w. / w.) was added. It was concentrated under vacuum until no distillate was left while maintaining the temperature below 35 °C. 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w / w.) was loaded into the column, and then the dried silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash and was subsequently eluted with PE / EA (gradient from 100:0 to 50:50, collected every 200 ± 10 mL). Samples were taken for TLC (EA:PE = 1:1) analysis. The qualified products were combined. This yielded bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as a colorless oil (15 g, 49.1%).

[0674] Methyl bis(2,2-dimethyl-1,3-dioxolan-4-yl) methanesulfonate

[0675]

[0676] At 0 °C under a nitrogen atmosphere, MsCl (3.70 g, 32.289 mmol, 1.5 eq) was added dropwise to a stirred solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (5 g, 21.526 mmol, 1 eq) and TEA (4.36 g, 43.052 mmol, 2 eq) in CH2Cl2 (50 mL). The resulting mixture was stirred for an additional 3 h at room temperature. The resulting mixture was washed with water (2 × 50 mL). The aqueous layer was extracted with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and dried over anhydrous Mg2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave methyl bis(2,2-dimethyl-1,3-dioxolan-4-yl) methanesulfonate as a yellow oil (6 g, 88.01%).

[0677] Synthesis of 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-dimethylpropan-1-amine

[0678]

[0679] At 0 °C under a nitrogen atmosphere, 3-(dimethylamino)-1-propanol (1.5 g, 14.540 mmol, 1.00 equivalent) was added portionwise to a stirred solution of NaH (1.74 g, 43.620 mmol, 3 equivalents) in THF (15 mL). The resulting mixture was stirred at 0 °C for an additional 0.5 h. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (6.77 g, 21.810 mmol, 1.5 equivalents) was added portionwise to the above mixture at 0 °C. The resulting mixture was stirred at 60 °C for an additional 6 h. The reaction was quenched by the addition of NH4Cl (aqueous solution) (15 mL) at room temperature. The aqueous layer was extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The mixture was dissolved in DCM (50 mL) and 15 g of silica gel (type: ZCX-2, 100 - 200 mesh, 3 w. / w.) was added. It was concentrated in vacuo until no distillate remained, while maintaining the temperature below 35 °C. 150 g of silica gel (type: ZCX-2, 100 - 200 mesh, 10 w / w.) was loaded into the column, and then the dried silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash, and then eluted with CH2Cl2 / MeOH (9:1) (gradient from 100:0 to 90:10, collected every 200 ± 10 mL). The sample was analyzed by TLC (CH2Cl2 / MeOH = 9 / 1), and the qualified products were combined. This produced {3-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}dimethylamine (1.07 g, 16.46%).

[0680] Synthesis of 3-(3-(dimethylamino)propoxy)pentane-1,2,4,5-tetraol

[0681]

[0682] {3-[Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]propyl}dimethylamine (1 g, 3.150 mmol, 1 equivalent) and hydrogen chloride (6 M, 10 mL) were added to a 100 mL three-necked round-bottom flask at room temperature. The resulting mixture was stirred at 60 °C for 6 h. The resulting mixture was concentrated under reduced pressure. The crude product mixture was used directly in the next step without further purification.

[0683] Synthesis of 1,4,5-tris(decanoyloxy)-3-[3-(dimethylamino)propoxy]pentan-2-yl decanoate (L-9)

[0684]

[0685] 3-[3-(Dimethylamino)propoxy]pentane-1,2,4,5-tetraol (1 g, 4.214 mmol, 1 equiv), DCM (10 mL), and capric acid (3.99 g, 23.177 mmol, 5.5 equiv) were added to a 100 mL round-bottom flask at room temperature. EDCI (4.85 g, 25.284 mmol, 6 equiv) and DMAP (1.03 g, 8.428 mmol, 2 equiv) were added to the above mixture at room temperature. The resulting mixture was stirred at room temperature for 24 h. The reaction was quenched by the addition of water (50 mL) at room temperature. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous MgSO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water (0.1% TFA) containing CH3CN / IPA (1:1), gradient from 40% to 90% in 20 min; detector, ELSD. The fractions were concentrated to remove CH3CN under reduced pressure and basified to pH 8 with saturated NaHCO3 (aqueous solution). The aqueous layer was extracted with n-heptane (2 × 100 mL) and dried over anhydrous Na2SO4; after filtration, the filtrate was concentrated under reduced pressure. This gave 1,4,5-tris(decanoyloxy)-3-[3-(dimethylamino)propoxy]pentan-2-yl decanoate (0.561 g, 8.47%) as a yellow oil. LCMS: (ES, m / z): 855 [M+1] + . 1 1H NMR (300 MHz, chloroform-d) δ: 5.412 - 5.135 (m, 2H), 4.491 - 4.305 (m, 2H), 4.295 - 4.032 (m, J = 12.1, 6.4 Hz, 2H), 3.753 - 3.373 (m, 3H), 2.591 - 2.061 (m, 16H), 1.853 - 1.496 (m, 10H), 1.274 (d, J = 6.1 Hz, 48H), 0.878 (t, J = 6.7 Hz, 12H).

[0686] Example 10. Synthesis of tetra(capric acid) 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraester (L-10)

[0687]

[0688] Synthesis of (4-bromobut-2-yn-1-yl)diethylamine HBr salt

[0689]

[0690] 4-(Diethylamino)but-2-yn-1-ol (11 g, 78.014 mmol, 1 equiv) and DCM (110 mL) were added to a 250 mL round-bottom flask under a nitrogen atmosphere at room temperature. Thereafter, a solution of PBr3 (63.26 g, 233.688 mmol, 3 equiv) in DCM (100 mL) was added while stirring at 0 °C for 15 minutes. The above mixture was stirred at room temperature for 4 hours. LCMS showed that the reaction was complete. Then the reaction was quenched by adding 300 mL of Na2CO3 (saturated aqueous solution). The resulting solution was extracted with 3 × 100 mL of DCM. The organic layers were combined. The organic phase was washed with 1 × 150 mL of Na2CO3 (saturated aqueous solution) and 1 × 150 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with ethyl acetate / hexane (83 / 17) to give the product (4-bromobut-2-yn-1-yl)diethylamine HBr salt (7.2 g, 32.61%) as a colorless solid.

[0691] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine

[0692]

[0693] Under a nitrogen atmosphere at room temperature, bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol HBr salt (6.9 g, 50.707 mmol, 1.5 equiv) and toluene (80 mL) were added to a 250 mL three-necked round-bottom flask. NaH (8.11 g, 338.050 mmol, 10 equiv, 60%) was added thereto at room temperature. The mixture was stirred at room temperature for 1 hour. (4-Bromobut-2-yn-1-yl)diethylamine (6.9 g, 33.805 mmol, 1 equiv), Na2CO3 (10.75 g, 101.415 mmol, 3 equiv) and toluene (100 mL) were added to another 100 mL round-bottom flask. The mixture was stirred at room temperature for 30 minutes. The mixture was added to the above reaction mixture. The resulting mixture was stirred at 80 °C overnight. LCMS showed that the reaction was complete. Then the reaction was quenched by adding 100 mL of 5% citric acid (aqueous solution). The resulting solution was extracted with 3 × 100 mL of ethyl acetate. The organic layers were combined. The organic phase was washed with 1 × 200 mL of Na2CO3 (saturated aqueous solution) and 1 × 200 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with ethyl acetate / hexane (3 / 2) to give the product {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine (2.66 g, 30.73%) as a light red oily liquid.

[0694] Synthesis of {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}diethylamine

[0695]

[0696] {4-[Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]but-2-yn-1-yl}diethylamine (2.65 g, 11.253 mmol, 1 equiv), MeOH (40 mL) and Pd / C (10%, 1.2 g) were added to a round-bottom flask purged and maintained with an inert nitrogen atmosphere. The flask was evacuated and flushed with nitrogen three times, and then flushed with hydrogen. The mixture was hydrogenated at room temperature under a hydrogen atmosphere (30 psi) for 18 hours. LCMS showed that the reaction was complete. The mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure to give {4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl}diethylamine (1 g, 37.31%) as a yellow oil.

[0697] Synthesis of 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride

[0698]

[0699] At room temperature under a nitrogen atmosphere, 950 mg (9.736 mmol, 1 equiv) of (4-[bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]butyl)diethylamine and 5 mL of THF were added to a 250 mL three-necked round-bottom flask. HCl (6 M, 40 mL) was added dropwise thereto at 0 °C over 15 minutes. The mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated to give 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride (700 mg, 88.64%).

[0700] Synthesis of 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetrayl tetra(decanoate)

[0701]

[0702] 3-(4-(Diethylamino)butoxy)pentane-1,2,4,5-tetraol hydrochloride (700 mg, 3.579 mmol, 1 equiv), decanoic acid (3.39 g, 19.684 mmol, 5.5 equiv), DMAP (0.87 g, 7.158 mmol, 2 equiv), EDCI (4.12 g, 21.474 mmol, 6 equiv) and ACN (21 mL) were added to a 150 mL three-necked round-bottom flask at room temperature. The mixture was stirred at room temperature for 16 hours. The reaction solution was depressurized and concentrated in vacuo. 200 mL of DCM was added, and it was washed with 5% citric acid solution (3×100), then washed three times with saturated brine, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by flash preparative HPLC under the following conditions: C18 silica gel column; mobile phase, A: water containing 0.05% TFA / B: CH3CN (0% CH3CN increased to 95% in 15 min), collected eluent (gradient: A: 0.05% TFA B: CH3CN = 15 / 85); detector, ELSD. The organic solvent was removed under reduced pressure, and it was basified to pH 8 with saturated Na2CO3 (aqueous solution). The aqueous layer was extracted with heptane (3×100 mL). The resulting mixture was concentrated under reduced pressure. This gave 3-(4-(diethylamino)butoxy)pentane-1,2,4,5-tetrayl tetra(decanoate) (0.5145 g, 25.84%). LCMS: (ES, m / z): 896.7 [M+1] +.H-NMR(300MHz,CDCl3)δ:5.360 - 5.189(m,2H),4.434 - 4.357(m,2H),4.163 - 4.101(m,2H),3.658 - 3.502(m,3H),2.562 - 2.491(m,4H),2.452 - 2.409(m,2H),2.352 - 2.245(m,8H),1.606 - 1.463(m,12H),1.266(s,48H),1.044 - 0.977(m,6H),0.900 - 0.856(m,12H).

[0703] Example 11. Synthesis of 3-(4-propylphenyl)propanoic acid 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl ester (L-11)

[0704]

[0705] Synthesis of 3-(4-propylphenyl)acrylic acid

[0706]

[0707] Add 4-propyl-benzaldehyde (10 g, 67.474 mmol, 1 equiv), pyridine (2.67 mL, 33.737 mmol, 0.5 equiv) and malonic acid (7.72 g, 74.221 mmol, 1.1 equiv) to a 40 mL vial at room temperature. Stir the reaction mixture at 80 °C for 12 h. Allow the mixture to cool to room temperature. Collect the precipitated solid by filtration and wash with 3 × 60 mL water and 3 × 60 mL methyl tert-butyl ether / heptane (2:1). After filtration, the obtained solid is dried under infrared light. This gives (2E)-3-(4-propylphenyl)prop-2-enoic acid as a white solid (9.5 g, 72.90%).

[0708] Synthesis of 3-(4-propylphenyl)propanoic acid

[0709]

[0710] Add (2E)-3-(4-propylphenyl)prop-2-enoic acid (9.5 g, 49.936 mmol, 1 equiv), EA (95 mL), MeOH (95 mL) and Pd / C (4.75 g, 46.984 mmol) to a 250 mL round-bottom flask at room temperature. Stir the resulting mixture at room temperature under a hydrogen atmosphere for 12 h. After filtration, concentrate the filtrate under reduced pressure. This gives 3-(4-propylphenyl)propanoic acid as a white solid (8.58 g, 88.48%). LCMS: (ES, m / z): 193 [M+1]+ .

[0711] Synthesis of 3-(4-propylphenyl)propanoic acid 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl ester

[0712]

[0713] To a 40 mL vial at room temperature was added 3-(4-propylphenyl)propanoic acid (4.46 g, 23.177 mmol, 5.5 equiv), 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetraol (1 g, 4.214 mmol, 1.00 equiv), DMAP (1.03 g, 8.428 mmol, 2 equiv), EDCI (6.47 g, 33.712 mmol, 8 equiv) and DCM (10 mL). The resulting mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C18 5 μm; mobile phase, B: CH3CN, A: water (0.1% TFA), gradient from 50% to 95% in 15 min; flow rate: 50 mL / min: detector, ELSD. The organic solvents were removed under reduced pressure and the mixture was basified to pH 8 with saturated Na2CO3 (aqueous solution). The aqueous layer was extracted with heptane (3 × 100 mL). The resulting mixture was concentrated under reduced pressure. This gave 3-(4-propylphenyl)propanoic acid 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(4-propylphenyl)propanoyl]oxy})pentan-2-yl ester as a yellow oil (0.5574 g, 13.33%). LCMS: (ES, m / z): 934.6 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 7.070 (s, 16H), 5.438 (t, J = 5.4 Hz, 1H), 5.281 (d, J = 5.4 Hz, 1H), 5.075 (d, J = 5.9 Hz, 1H), 4.304 - 4.172 (m, 1H), 3.775 (s, 1H), 3.469 - 3.308 (m, 2H), 3.299 - 3.165 (m, 2H), 2.923 - 2.807 (m, 8H), 2.706 - 2.148 (m, 24H), 1.827 (m, 2H), 1.657 - 1.537 (m, 8H), 0.974 - 0.873 (m, 12H).

[0714] Example 12. Synthesis of 6-[3-(dimethylamino)propoxy]undecanedioic acid 1,11-bis(pentadec-8-yl ester) (L-12)

[0715]

[0716] Synthesis of tridec-1,12-dien-7-ol

[0717]

[0718] To a 500 mL three-necked round-bottom flask at room temperature was added Mg (16.40 g, 674.955 mmol, 5 eq) and THF (50 mL). To the above mixture was added dropwise 6-bromo-1-ene (55.03 g, 337.478 mmol, 2.5 eq) at 55 °C. The resulting mixture was stirred for an additional 1 h at 55 °C. Ethyl formate (10 g, 134.991 mmol, 1 eq) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred for an additional 2 h at room temperature. The reaction was quenched with saturated NH4Cl (aqueous solution) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with heptane / EA (10:1) to give tridec-1,12-dien-7-ol as a pale yellow oil (23.5 g, 88.67%).

[0719] Synthesis of N,N-dimethyl-3-(tridec-1,12-dien-7-yloxy)propan-1-amine

[0720]

[0721] At room temperature, trideca-1,12-dien-7-ol (10 g, 50.934 mmol, 1 equiv) and toluene (200 mL) were added to a 500 mL three-necked round-bottom flask. NaH (4.00 g, 166.554 mmol, 3.27 equiv) was added portionwise to the above mixture at 0 °C over 10 min. The resulting mixture was stirred at 85 °C overnight. (3-Chloropropyl)dimethylamine hydrochloride (15.28 g, 101.868 mmol, 2 equiv) was added portionwise to the above mixture at 80 °C. The resulting mixture was stirred at 80 °C for an additional 8 h. The mixture was allowed to cool to room temperature. The resulting mixture was diluted with water (300 mL). The resulting mixture was extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with water (2 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with CH2Cl2 / MeOH (10:1) to give N,N-dimethyl-3-(trideca-1,12-dien-7-yloxy)propan-1-amine (12.5 g, 87.19%) as a pale yellow oil.

[0722] Synthesis of 6-(3-(dimethylamino)propoxy)undecanedioic acid hydrochloride

[0723]

[0724] At room temperature, N,N-dimethyl-3-(trideca-1,12-dien-7-yloxy)propan-1-amine (7.6 g, 27.000 mmol, 1 equiv) and AcOH (140 mL) were added to a 1000 mL four-necked round-bottom flask. KMnO4 (17 g, 107.573 mmol, 3.98 equiv, in 700 mL H2O) was added dropwise to the above mixture at room temperature. The resulting mixture was stirred at 15 °C for an additional 3 h. The reaction was quenched by adding Na2S2O3 (17 g) and NaHSO3 (3 g) at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column, AQ-C18 silica gel; mobile phase, water (0.1% HCl) containing MeCN, gradient from 10% to 35% in 16 min; detector, ELSD. This gave 6-(3-(dimethylamino)propoxy)undecanedioic acid hydrochloride (2.9 g, 30.35%) as a pale yellow oil.

[0725] Synthesis of 6-[3-(dimethylamino)propoxy]undecanedioic acid 1,11-bis(pentadec-8-yl ester)

[0726]

[0727] At room temperature, 6-(3-(dimethylamino)propoxy)undecanedioic acid hydrochloride (850 mg, 2.402 mmol, 1 equiv), pentadec-8-ol (1.4 g, 6.129 mmol, 2.55 equiv), ACN (17 mL), TEA (728 mg, 7.206 mmol, 3.0 equiv) and DMAP (100 mg, 0.819 mmol, 0.34 equiv) were added to a 100 mL round-bottom flask. EDCI (1.4 g, 7.303 mmol, 3.04 equiv) was added portionwise to the above mixture at room temperature. The resulting mixture was stirred at room temperature for an additional 16 h. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with saturated Na2CO3 (2 × 20 mL), MeOH / H2O (4:1, 5 × 20 mL), H2O (3 × 20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, water containing MeCN (0.1% TFA), gradient from 35% to 70% in 10 min; detector, Ms. The product fractions were concentrated under vacuum to remove ACN, and saturated Na2CO3 (30 mL) was added. The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with MeOH / H2O (4:1, 5 × 20 mL), H2O (3 × 20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave 6-[3-(dimethylamino)propoxy]undecanedioic acid 1,11-bis(pentadec-8-yl ester) as a pale yellow oil (680 mg, HPLC: 95.1%, yield: 34.24%). LCMS: (ES, m / z): 738.7 [M+H] + ; 1 1H NMR: (400 MHz, chloroform-d) δ 4.892 - 4.830 (m, 2H), 3.435 (t, J = 6.4 Hz, 2H), 3.208 - 3.183 (m, 1H), 2.363 (br, 2H), 2.301 - 2.245 (m, 10H), 1.761 - 1.726 (m, 2H), 1.709 - 1.693 (m, 4H), 1.658 - 1.586 (m, 14H), 1.511 - 1.412 (m, 42H), 0.877 (t, J = 6.8 Hz, 12H).

[0728] Example 13. Synthesis of bis(heptadec-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate (L-13)

[0729]

[0730] Synthetic nona-1,8-dien-5-ol

[0731]

[0732] At room temperature and under a nitrogen atmosphere, 4-bromo-1-butene (91.12 g, 674.955 mmol, 2.5 equivalents) was added dropwise to a stirred solution of Mg (32.81 g, 1349.910 mmol, 5.0 equivalents) and I2 (200 mg, 0.788 mmol) in THF (100 mL). The mixture was stirred at 55 °C under a nitrogen atmosphere for 1 hour. Ethyl formate (20 g, 269.982 mmol, 1 equivalent) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for an additional 2 hours. The reaction was quenched with saturated NH4Cl (aqueous solution) (400 mL, 20 V) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 200 mL, 20 V). The combined organic layers were washed with brine (200 mL, 10 V) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (volume ratio) (gradient from 100:0 to 90:10, the eluent for the collected product was PE / EA = 92 / 8). The sample was analyzed by TLC (PE / EA = 10:1), and nona-1,8-dien-5-ol (26.0 g, 64.01%) was obtained as a pale yellow oil.

[0733] Synthetic N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine

[0734]

[0735] At room temperature under a nitrogen atmosphere, NaH (22.25 g, 556.245 mmol, 3.0 equivalents, 60%) was added portionwise to a stirred solution of nona-1,8-dien-5-ol (26 g, 185.415 mmol, 1 equivalent) in toluene (500 mL, 20V). The reaction mixture was stirred at 80 °C under a nitrogen atmosphere for 8 hours. At 80 °C, (3-chloropropyl)dimethylamine hydrochloride (58.68 g, 371.428 mmol, 2.0 equivalents) was added to the above mixture. The resulting mixture was stirred at 80 °C for an additional 16 hours. The reaction was quenched with saturated NH4Cl (aqueous solution) (500 mL, 20V) at room temperature. The resulting mixture was extracted with EtOAc (2 × 250 mL, 20V). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the eluent for the collected product CH2Cl2 / MeOH = 90 / 10). Samples were taken for TLC (CH2Cl2 / MeOH = 10:1) analysis to obtain N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine (35 g, 74.96%) as a pale yellow oil.

[0736] Synthesis of 4-(3-(dimethylamino)propoxy)heptanedioic acid

[0737]

[0738] At room temperature, KMnO4 (33.66 g, 212.976 mmol, 4.0 equivalents) dissolved in water (1.2 L, 100V) was added to a stirred solution of N,N-dimethyl-3-(nona-1,8-dien-5-yloxy)propan-1-amine (12 g, 53.244 mmol, 1 equivalent) in AcOH (240 mL, 20V). The resulting mixture was stirred at room temperature for 1 hour. The reaction was quenched with solid Na2S2O3 (33.66 g, 212.976 mmol, 4.0 equivalents) at room temperature and stirred for 30 minutes. The resulting mixture was concentrated under reduced pressure. The residue was purified by slurrying with DMF (1 L, 80V). The crude product was used directly in the next step without further purification.

[0739] Synthesis of bis(heptadec-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate

[0740]

[0741] At room temperature, EDCI (13.20 g, 68.883 mmol, 3.00 eq) was added to a stirred solution of 4-(3-(dimethylamino)propoxy)heptanedioic acid (6.00 g, 22.961 mmol, 1.0 eq), DMAP (5.61 g, 45.922 mmol, 2.00 eq), and 9-heptadecanol (14.72 g, 57.402 mmol, 2.50 eq). The resulting mixture was stirred at room temperature for 16 h. The reaction was quenched by the addition of water (1.0 L, 80 V) at room temperature. The resulting mixture was extracted with EtOAc (2 × 300 mL, 60 V). The combined organic layers were washed with brine (300 mL, 30 V) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the collected product eluent DCM / MeOH = 95 / 5). The sample was analyzed by TLC (DCM:MeOH = 10:1) to give bis(nonadec-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate (601.1 mg, 3.55%) as a yellow oil. LCMS: (ES, m / z): 738.7 [M+H] + ; 1 1H NMR (400 MHz, chloroform-d) δ 4.874 - 4.843 (m, 2H), 3.486 - 3.455 (m, 2H), 3.314 - 3.285 (m, 1H), 2.480 (br, 2H), 2.387 - 2.293 (m, 10H), 1.818 - 1.752 (m, 6H), 1.512 - 1.499 (m, 8H), 1.296 - 1.258 (m, 48H), 0.894 - 0.878 (m, 12H).

[0742] Example 14. Synthesis of (3-{[1,3-bis({[(nonadec-9-yloxy)carbonyl]oxy})propan-2-yl]oxy}propyl)dimethylamine (L-14)

[0743]

[0744] Synthesis of 2,2-dimethyl-1,3-dioxolan-5-ol

[0745]

[0746] Under a nitrogen atmosphere at room temperature, 2,2-dimethyl-1,3-dioxane-5-one (15 g, 115.258 mmol, 1 equiv) and THF (150 mL) were placed in a 500 mL round-bottom flask. LiAlH4 (4.37 g, 115.258 mmol, 1.0 equiv) was added thereto at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction was quenched by adding water (4.5 mL), 4.5 mL (wt% 15% NaOH), and 13.5 mL of water at 0 °C. The resulting solution was diluted with 300 mL of THF and Na2SO4 (30 g) was added thereto. The mixture was heated to room temperature and stirred for 15 min. The resulting mixture was filtered; the filter cake was washed with EA (2 × 100 mL). The filtrate was concentrated under reduced pressure to give 2,2-dimethyl-1,3-dioxane-5-ol as a colorless oil (12 g, 78.78%).

[0747] Synthesis of {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine

[0748]

[0749] Under a nitrogen atmosphere at room temperature, 2,2-dimethyl-1,3-dioxane-5-ol (5 g, 37.833 mmol, 1 equiv) and DMF (100 mL) were placed in a 100 mL round-bottom flask. NaH (2.27 g, 94.582 mmol, 2.5 equiv) was added thereto at 0 °C. The mixture was stirred at room temperature for 1 h. (3-Chloropropyl)dimethylamine (5.52 g, 45.400 mmol, 1.2 equiv, in 10 mL of DMF) was added thereto at room temperature. The mixture was stirred at 50 °C for 16 h. The reaction was quenched by adding NH4Cl (saturated 50 mL) at 5 °C. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with (MeOH / DCM = 1 / 1) to give {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine as a yellow solid (2.1 g, 25.54%).

[0750] Synthesis of 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride

[0751]

[0752] Under a nitrogen atmosphere at room temperature, {3-[(2,2-dimethyl-1,3-dioxan-5-yl)oxy]propyl}dimethylamine (1.0 g, 4.602 mmol, 1 equiv) and DCM (20 mL) were placed in a 40 mL vial. 1,4-Dioxane containing HCl (gas) (4 M, 5 mL) was added thereto at 0 °C. The mixture was stirred at 0 °C for 1 h. The resulting mixture was concentrated in vacuo to give 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride as a yellow oil (780 mg, crude). The crude product was used directly in the next step without further purification.

[0753] Synthesis of 4-nitrophenyl heptadecane-9-yl carbonate

[0754]

[0755] Under a nitrogen atmosphere at room temperature, 9-heptadecanol (5 g, 19.495 mmol, 1 equiv), DMAP (0.95 g, 7.798 mmol, 0.4 equiv), TEA (3.95 g, 38.990 mmol, 2 equiv) and THF (100 mL) were placed in a 250 mL round-bottomed flask. 4-Nitrophenyl chloroformate (4.32 g, 21.445 mmol, 1.1 equiv, in 20 mL THF) was added thereto at 0 °C. The mixture was stirred at 70 °C for 2 h. The resulting mixture was diluted with THF (100 mL). The resulting mixture was filtered; the filter cake was washed with THF (2 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with Hep / EA (volume ratio) (gradient from 100:0 to 90:10, and the product eluent collected was Hep / EA = 97 / 3). The sample was analyzed by TLC (Hep:EA = 10:1) to give 4-nitrophenyl heptadecane-9-yl carbonate as a pale yellow oil (3.5 g, 41.52%).

[0756] Synthesis of (3-{[1,3-bis({[(heptadecane-9-yloxy)carbonyl]oxy})propane-2-yl]oxy}propyl)dimethylamine

[0757]

[0758] At room temperature under a nitrogen atmosphere, 2-[3-(dimethylamino)propoxy]propane-1,3-diol hydrochloride (0.79 g, 3.736 mmol, 0.45 eq), DMAP (0.23 g, 1.868 mmol, 0.5 eq), TEA (1.51 g, 14.944 mmol, 4 eq) and DMF (16 mL) were placed in a 250 mL round-bottom flask. To this was added 4-nitrophenyl heptadec-9-yl carbonate (3.5 g, 8.302 mmol, 1 eq in 30 mL DMF) at room temperature. The mixture was stirred at 80 °C for 16 h. The resulting mixture was diluted with EA (300 mL) and washed with 5% wt citric acid (1×50 mL), NaHCO3 (1×50 mL) and water (2×150 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the product eluent collected was DCM / MeOH = 97 / 3). The sample was analyzed by TLC (DCM:MeOH = 10:1 0.2). (3-{[1,3-Bis({[(heptadec-9-yloxy)carbonyl]oxy})propane-2-yl]oxy}propyl)dimethylamine was obtained as a yellow oil (800 mg). The product was dissolved in n-heptane (100 mL). The n-heptane was washed with MeOH / H2O (4:1) (2×10 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give (3-{[1,3-Bis({[(heptadec-9-yloxy)carbonyl]oxy})propane-2-yl]oxy}propyl)dimethylamine as a yellow oil (584.1 mg, 9.07%). LCMS: (ES, m / z): 742.18 [M+1] + ; 1 H NMR (400 MHz, chloroform-d) δ 4.732 - 4.671 (m, 2H), 4.272 - 4.177 (m, 4H), 3.781 - 3.768 (m, 1H), 3.677 - 3.645 (m, 2H), 2.406 (s, 2H), 2.273 (s, 6H), 1.799 - 1.766 (m, 2H), 1.623 - 1.517 (m, 8H), 1.338 - 1.285 (m, 48H), 0.918 - 0.855 (m, 12H).

[0759] Example 15. Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1,9-bis(octadec-8-yl ester) (L-15)

[0760]

[0761] Synthesis of methyl 3-(2,6-dioxocyclohexyl)propionate

[0762]

[0763] Place 1,3-cyclohexanedione (25 g, 222.959 mmol, 1 equiv), DMF (50 mL), Cs2CO3 (43.59 g, 133.775 mmol, 0.6 equiv) and methyl acrylate (23.03 g, 267.551 mmol, 1.2 equiv) into a 250 mL round-bottom flask. Stir the reaction mixture at 80 °C for 12 h. Then quench the reaction by adding 200 mL of water / ice. Adjust the pH of the solution to 6 with HCl (1 mol / L). Extract the resulting solution with 3 × 200 mL of ethyl acetate and combine the organic layers. Wash the resulting mixture with 200 mL of NaCl. Dry the mixture over anhydrous sodium sulfate and concentrate it in vacuo. This gives methyl 3-(2,6-dioxocyclohexyl)propionate (35 g, crude) as a yellow oil.

[0764] Synthesis of 5-oxononanoic acid

[0765]

[0766] Place methyl 3-(2,6-dioxocyclohexyl)propionate (35 g, 176.573 mmol, 1 equiv) and HCl (1 M) (70 mL) into a 250 mL round-bottom flask. Stir the reaction mixture at 110 °C for 12 h. Concentrate the resulting mixture in vacuo. Dissolve the residue in 200 mL of MTBE and stir for 1 h. Filter to collect the solid. This gives 5-oxononanoic acid (15 g, 42.01%) as a brown solid.

[0767] Synthesis of pentadecan-8-ol

[0768]

[0769] Place 8-pentadecanone (15 g, 66.253 mmol, 1 equiv), THF (450 mL), MeOH (150 mL) and NaBH4 (7.52 g, 198.759 mmol, 3.0 equiv) into a 1.0 L round-bottom flask. Stir the reaction mixture at 20 °C for 3 h. Pour the reaction mixture into 500 mL of ice water. Extract the resulting mixture with EA (3 × 500 mL). Wash the combined organic layers with water (3 × 100 mL) and NaCl (100 mL, aqueous solution), and dry over anhydrous Na2SO4. After filtration, concentrate the filtrate under reduced pressure to give pentadecan-8-ol (10 g, crude) as a white solid.

[0770] Synthesis of 5-oxononanedioic acid 1,9-bis(pentadec-8-yl ester)

[0771]

[0772] 5-oxononanedioic acid (5.0 g, 24.727 mmol, 1 equiv), pentadec-8-ol (10.17 g, 44.509 mmol, 1.8 equiv), DCM (100 mL), DMAP (3.02 g, 24.727 mmol, 1 equiv) and EDCI (10.43 g, 54.399 mmol, 2.2 equiv) were placed into a 250 mL round-bottom flask. The reaction mixture was stirred at room temperature for 12 h. The resulting mixture was diluted with DCM (100 mL). The reaction was quenched by adding citric acid (5% aqueous solution) (60 mL) at 5 °C and washed with 2 × 30 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with Hep / EA (volume ratio) (gradient from 100:0 to 90:10, and the eluent for the collected product Hep / EA = 95 / 5). The sample was analyzed by TLC (Hep:EA = 10:1 0.2). The resulting mixture was concentrated under reduced pressure to give 5-oxononanedioic acid 1,9-bis(pentadec-8-yl ester) as a pale yellow oil (10.2 g, 66.21%).

[0773] Synthesis of 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1,9-bis(pentadec-8-yl ester)

[0774]

[0775] 5-oxononanedioic acid 1,9-bis(pentadec-8-yl ester) (5.5 g, 8.828 mmol, 1 equiv) and THF (44 mL) were placed into a 250 mL round-bottom flask under N2 atmosphere at room temperature. [3-(Chloromagnesio)propyl]dimethylamine (88.28 mL, 88.280 mmol, 10 equiv) was added dropwise thereto at -60 °C. The mixture was stirred at -60 °C for 2 h. The reaction was quenched by adding NH4Cl (aqueous solution) (80 mL) at 5 °C and washed with 2 × 40 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (volume ratio) (gradient from 100:0 to 70:30, and the eluent for the collected product DCM / MeOH = 82 / 18). The sample was analyzed by TLC (DCM:MeOH = 2:1 0.1). The resulting mixture was concentrated under reduced pressure to give 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1,9-bis(pentadec-8-yl ester) as a pale yellow oil (2.7 g, 43.07%).

[0776] Synthesis of 5-[3-(dimethylamino)propylidene]nonanedioic acid 1,9-bis(octadec-8-yl) ester

[0777]

[0778] Under a nitrogen atmosphere at room temperature, 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1,9-bis(octadec-8-yl) ester (2.5 g, 3.520 mmol, 1 equiv) and DCM (5 mL) were placed in a 40 mL vial. Et3SiH (4.09 g, 35.200 mmol, 10 equiv) and BF3·Et2O (5.00 g, 35.200 mmol, 10 equiv) were added thereto at room temperature. The mixture was stirred at 45 °C for 2 h. The resulting mixture was diluted with DCM (100 mL). The reaction was quenched by adding NaHCO3 (aqueous solution) (25 mL) at 5 °C and washed with 2 × 25 mL of water. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the product eluent collected was DCM / MeOH = 94 / 6). Samples were taken for TLC (DCM:MeOH = 10:1 0.15) analysis. The resulting mixture was concentrated under reduced pressure to give 5-[3-(dimethylamino)propylidene]nonanedioic acid 1,9-bis(octadec-8-yl) ester as a pale yellow oil (1.6 g, 62.51%).

[0779] Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1,9-bis(octadec-8-yl) ester (L-15)

[0780]

[0781] At room temperature, 5-[3-(dimethylamino)propylidene]nonanedioic acid 1,9-bis(pentadec-8-yl ester) (1.6 g, 2.312 mmol, 1 equiv) and EtOH (32 mL) were placed in a 100 mL vial. Pd / C (0.49 g, 0.462 mmol, 0.2 equiv, 10%) was added thereto. The mixture was stirred at room temperature under a H2 (30 psi) atmosphere for 2 h. The resulting mixture was filtered; the filter cake was washed with EA (2 × 32 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the product eluent collected was DCM / MeOH = 95 / 5). The sample was analyzed by TLC (DCM:MeOH = 10:1 0.2). The filtrate was concentrated under reduced pressure. The residue was dissolved in n-heptane (160 mL, 100V). Subsequently, the n-heptane phase was washed with MeOH / H2O (4:1) (2 × 32 mL, 10V), MeCN / H2O (4:1) (2 × 32 mL, 10V), and water (32 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give 5-[3-(dimethylamino)propyl]nonanedioic acid 1,9-bis(pentadec-8-yl ester) as a pale yellow oil (1.0640 g, 66.31%). LCMS: (ES, m / z): 694.9 [M+H] + . 1 H NMR (400 MHz, chloroform-d) δ 4.934 - 4.832 (m, 2H), 2.333 - 2.172 (m, 12H), 1.681 - 1.564 (m, 4H), 1.558 - 1.477 (m, 8H), 1.471 - 1.399 (m, 2H), 1.389 - 1.203 (m, 47H), 0.896 (t, J = 6.8 Hz, 12H).

[0782] Example 16. Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris(dodecanoyloxy)pentan-2-yl dodecanoate (L-16)

[0783]

[0784] 3-[3-(Dimethylamino)propoxy]pentane-1,2,4,5-tetraol (1 g, 4.214 mmol, 1 equiv), lauric acid (4.64 g, 23.177 mmol, 5.5 equiv), EDCI (4.85 g, 25.284 mmol, 6 equiv), DMAP (1.03 g, 8.428 mmol, 2 equiv), DIEA (4.36 g, 33.712 mmol, 8 equiv) and ACN (10 mL) were added to a 40 mL vial at room temperature. The resulting mixture was stirred for an additional 3 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in water (100 mL). The resulting mixture was extracted with heptane (3 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C 18 5 um; mobile phase, B: CH3CN, A: water (0.1% TFA), gradient from 50% to 95% in 15 min; flow rate: 50 mL / min: detector, ELSD. The fractions were concentrated to remove CH3CN under reduced pressure and basified to pH 8 with saturated NaHCO3 (aqueous solution). The aqueous layer was extracted with n-heptane (2 × 100 mL) and dried over anhydrous Na2SO4; after filtration, the filtrate was concentrated under reduced pressure. This yielded 3-[3-(dimethylamino)propoxy]-1,4,5-tris(dodecanoyloxy)pentan-2-yl dodecanoate as a yellow oil (0.6852 g, 20.24%). LCMS: (ES, m / z): 967 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 5.469 (t, J = 5.5 Hz, 1H), 5.381 - 5.255 (m, 1H), 5.225 - 5.110 (m, 1H), 4.373 - 4.261 (m, 1H), 4.073 - 3.925 (m, 1H), 3.557 - 3.353 (m, 4H), 2.456 - 2.265 (m, 10H), 2.258 - 2.104 (m, 6H), 1.735 - 1.675 (m, 2H), 1.650 - 1.532 (m, 8H), 1.267 (d, J = 4.9 Hz, 64H), 0.880 (t, J = 6.6 Hz, 12H).

[0785] Example 17. Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl]9-pentadec-8-yl ester (L-17)

[0786]

[0787] Synthesis of 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid

[0788]

[0789] A solution of 5-oxononanoic acid (4 g, 19.782 mmol, 1 equiv) in DCM (60 mL) was treated with DMAP (0.48 g, 3.956 mmol, 0.2 equiv) and pentadec-8-ol (2.94 g, 12.858 mmol, 0.65 equiv) at 25 °C under a nitrogen atmosphere, followed by the addition of EDCI (4.17 g, 21.760 mmol, 1.1 equiv) portionwise at 25 °C. The resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 18 h. The mixture was acidified to pH 5 with 0.05 M HCl. The resulting mixture was washed with 1×50 mL of 0.05 M HCl and 1×100 mL of brine. The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 15 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w / w) was added. It was concentrated to no distillate under vacuum while maintaining the temperature below 35 °C. 160 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (2:1) (gradient from 10:1 to 2:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 1:1) analysis, and the qualified products were combined. This gave 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid as a pale yellow oil (4 g, 49.01%). LCMS: (ES, m / z): 413.2 [M+H] + . 1 1H NMR: (400 MHz, chloroform-d) δ 4.939 - 4.830 (m, 2H), 2.560 - 2.464 (m, 4H), 2.409 (t, J = 7.213 Hz, 2H), 2.332 (t, J = 7.242 Hz, 2H), 1.978 - 1.862 (m, 4H), 1.577 - 1.462 (m, 4H), 1.347 - 1.211 (m, 20H), 0.934 - 0.863 (m, 6H).

[0790] Synthesis of 2-(bromomethyl)-octahydro-1H-indene

[0791]

[0792] A solution of octahydro-1H-inden-2-ylmethanol (16 g, 103.726 mmol, 1 eq) in DCM (300 mL) was treated with triphenylphosphine (40.81 g, 155.589 mmol, 1.5 eq) at 0 °C under a nitrogen atmosphere, and then carbon tetrabromide (51.60 g, 155.589 mmol, 1.5 eq) was added portionwise at 0 °C. The resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 18 h. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 15 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated in vacuo until no distillate remained while maintaining the temperature below 35 °C. 160 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (100:1) (gradient from 1:0 to 30:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 20:1) analysis, and the qualified products were combined. This produced 2-(bromomethyl)-octahydro-1H-indene (22 g, 97.67%) as a colorless oil. 1 H NMR: (400 MHz, chloroform-d) δ 3.480 - 3.378 (m, 2H), 2.663 - 2.368 (m, 1H), 2.072 - 1.691 (m, 4H), 1.644 - 0.887 (m, 10H).

[0793] Synthesis of 2-[2-isocyano-2-(4-methylphenylsulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene

[0794]

[0795] A mixture of NaH (5.80 g, 144.898 mmol, 2.3 equiv, 60%) in DMSO (200 mL) was stirred at 25 °C under a nitrogen atmosphere for 1 h. TosMIC (12.3 g, 62.999 mmol, 1.00 equiv) and TBAI (2327 mg, 6.300 mmol, 0.1 equiv) were added portionwise to the above mixture at 25 °C. The resulting mixture was stirred at 25 °C for an additional 3 h. 2-(Bromomethyl)-octahydro-1H-indene (21.89 g, 100.798 mmol, 1.6 equiv) in DMSO (100 mL) was added dropwise to the above mixture at 25 °C over 20 min. The resulting mixture was stirred at 25 °C for an additional 18 h. The reaction was quenched with saturated NH4Cl (aqueous solution) at 25 °C. The resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (3 × 300 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 50 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated under vacuum until no distillate remained while maintaining the temperature below 35 °C. 500 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (20:1) (gradient from 50:0 to 10:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 10:1) analysis, and the qualified products were combined. This gave 2-[2-isocyano-2-(4-methylphenylsulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene as a yellow oil (9 g, 30.54%). LCMS: (ES, m / z): 468.4 [M + H] + . 1 H NMR: (400 MHz, chloroform-d) δ 7.932 - 7.853 (m, 2H), 7.480 - 7.392 (m, 2H), 2.512 (s, 3H), 2.273 - 1.769 (m, 14H), 1.567 - 1.407 (m, 8H), 1.390 - 1.068 (m, 12H).

[0796] Synthesis of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one

[0797]

[0798] A solution of 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-1H-inden-2-yl)propyl]-octahydro-1H-indene (9 g, 19.243 mmol, 1 equiv) in DCM (60 mL) was stirred at 25 °C. HCl (5 mL, 4 M in MeOH) was added dropwise to the above mixture at 25 °C. The resulting mixture was stirred at 25 °C for an additional 3 h. Brine (100 mL) was added. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine (2 × 200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 30 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated to no distillate under vacuum while maintaining the temperature below 35 °C. 300 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (30:1) (gradient from 50:0 to 10:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 10:1) analysis, and the qualified products were combined. The residue was purified by silica gel column chromatography, eluted with PE / EA (30:1), to give 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one as a pale yellow solid (5 g, 85.90%). LCMS-:(ES,m / z):303.4[M+H] + . 1 H NMR: (400 MHz, chloroform-d) δ 2.668 - 2.308 (m, 6H), 2.039 - 1.690 (m, 8H), 1.633 - 0.949 (m, 20H).

[0799] Synthesis of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol

[0800]

[0801] A solution of 1,3-bis(octahydro-1H-inden-2-yl)propan-2-one (5 g, 16.529 mmol, 1 equiv) in tetrahydrofuran (50 mL) was treated with NaBH4 (1.38 g, 36.364 mmol, 2.2 equiv) at 0 °C, and then methanol (50 mL) was added dropwise at 0 °C. The resulting mixture was stirred at 25 °C for 3 h. The reaction was quenched by adding saturated NH4Cl (aqueous solution) (20 mL) at 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with brine (2 × 200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 10 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated to no distillate under vacuum while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dried silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (30:1) (gradient from 50:0 to 20:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 20:1) analysis, and the qualified products were combined. This produced 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol (4.6 g, 91.39%) as a colorless oil. LCMS: (ES, m / z): 287.3 [M - 18 + H] + . 1 H NMR: (400 MHz, chloroform-d) δ 3.698 - 3.574 (m, 1H), 2.394 - 2.044 (m, 2H), 1.970 - 1.701 (m, 8H), 1.605 - 1.090 (m, 25H).

[0802] Synthesis of 5-oxononanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester

[0803]

[0804] A solution of 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid (6 g, 14.542 mmol, 1 equiv) in DCM (120 mL) was treated at 25 °C under a nitrogen atmosphere with 1,3-bis(octahydro-1H-inden-2-yl)propan-2-ol (5.31 g, 17.450 mmol, 1.2 equiv) and DMAP (1.79 g, 14.620 mmol, 1 equiv), followed by the addition portionwise of EDCI (3.64 g, 18.913 mmol, 1.3 equiv) at 25 °C. The resulting mixture was stirred at 45 °C under a N2 atmosphere for 18 h. The resulting mixture was diluted with DCM (120 mL). The combined organic layers were washed with 0.05 M HCl (1×100 mL) and brine (1×200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (50 mL) and 30 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated in vacuo until no distillate was obtained while maintaining the temperature below 35 °C. 300 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dried silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with PE / EA (5:1) (gradient from 20:1 to 4:1, collected every 200 ± 10 mL). Samples were taken for TLC (PE / EA = 3:1) analysis, and the qualified products were combined. This gave 5-oxononanoic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester as a pale yellow oil (3.7 g, 36.40%). LCMS: (ES, m / z): 721.5 [M+Na] + . 1 1H NMR: (400 MHz, chloroform-d) δ 5.015 - 4.907 (m, 1H), 4.913 - 4.831 (m, 1H), 2.483 (t, J = 7.2 Hz, 4H), 2.320 (t, J = 7.2 Hz, 4H), 2.145 - 2.005 (m, 1H), 1.984 - 1.741 (m, 12H), 1.747 - 1.607 (m, 3H), 1.607 - 1.414 (m, 3H), 1.408 - 1.194 (m, 28H), 1.199 - 1.048 (m, 3H), 0.941 - 0.839 (m, 6H).

[0805] Synthesis of 5-[3-(dimethylamino)propyl]-5-hydroxynonanoic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester

[0806]

[0807] At -60 °C under a nitrogen atmosphere, [3-(chloromagnesio)propyl]dimethylamine (75.10 mL, 75.090 mmol, 15 eq) was added dropwise to a stirred solution of 5-oxononanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester (3.5 g, 5.006 mmol, 1 eq) in tetrahydrofuran (50 mL). The resulting mixture was stirred at -60 °C under a nitrogen atmosphere for 2 h. The reaction was quenched by the addition of saturated NH4Cl (aqueous solution) (50 mL) at -60 °C. The mixture was acidified to pH 6 with 0.05 M HCl. The resulting mixture was extracted with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with brine (1 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and 10 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated in vacuo until no more distillate was obtained while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that had absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. It was eluted with CH2Cl2 / MeOH (20:1) (gradient from 50:1 to 10:1, collected every 200 ± 10 mL). Samples were taken for TLC (CH2Cl2 / MeOH = 10:1) analysis, and the qualified products were combined. This gave 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester as a pale yellow oil (2.5 g, 63.51%). LCMS: (ES, m / z): 786.7 [M + 1] + . 1 H NMR: (400 MHz, chloroform-d) δ 5.046 - 4.780 (m, 2H), 3.087 - 2.944 (m, 2H), 2.758 (s, 6H), 2.439 - 2.222 (m, 4H), 2.205 - 2.015 (m, 1H), 1.999 - 1.750 (m, 10H), 1.744 - 1.568 (m, 9H), 1.574 - 1.405 (m, 18H), 1.405 - 1.043 (m, 33H), 0.946 - 0.835 (m, 6H).

[0808] Synthesis of (5Z)-5-[3-(dimethylamino)propylidene]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester

[0809]

[0810] At 0 °C under a nitrogen atmosphere, BF3·Et2O (4.33 g, 30.520 mmol, 10 equiv) was added dropwise to a stirred solution of 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester (2.4 g, 3.052 mmol, 1 equiv) and triethylsilane (3.55 g, 30.520 mmol, 10 equiv) in DCM (50 mL). The resulting mixture was stirred at 40 °C under a nitrogen atmosphere for 2 h. The combined organic layers were washed with NaHCO3 (2 × 50 mL), brine (1 × 50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (10 mL) and 10 g of silica gel (type: ZCX-2, 100 - 200 mesh, 2.00 w. / w.) was added. It was concentrated under vacuum until no distillate remained while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100 - 200 mesh, 20.00 w / w.) was loaded into the column, and then the dry silica gel that absorbed the reaction mixture prepared in the last step. The product was purified using CombiFlash. Elution was carried out with CH2Cl2 / MeOH (20:1) (gradient from 50:1 to 10:1, collecting every 200 ± 10 mL). Samples were taken for TLC (CH2Cl2 / MeOH = 10:1) analysis, and the qualified products were combined. This gave (5Z)-5-[3-(dimethylamino)propylidene]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester as a pale yellow oil (1.5 g, 63.97%). LCMS: (ES, m / z): 768.7 [M+H] + . 1 1H NMR: (400 MHz, chloroform-d) δ 5.228 - 5.086 (m, 1H), 5.021 - 4.786 (m, 2H), 2.563 - 2.176 (m, 13H), 1.182 - 1.992 (m, 5H), 1.992 - 1.773 (m, 8H), 1.773 - 1.588 (m, 7H), 1.593 - 1.054 (m, 45H), 1.653 - 0.658 (m, 7H).

[0811] Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester (L-17)

[0812]

[0813] At 25 °C under a nitrogen atmosphere, Pd / C (0.26 g, 2.443 mmol, 1.44 equivalents) was added to a stirred solution of (5Z)-5-[3-(dimethylamino)propylidene]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester (1.3 g, 1.692 mmol, 1 equivalent) in EtOH (40 mL). The flask was evacuated, flushed with nitrogen three times, and then flushed with hydrogen. The mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. The resulting mixture was filtered, and the filter cake was washed with EA (2 × 100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in heptane (150 mL). The resulting mixture was washed with 2 × 100 mL ACN / H2O (5:1, 50 mL) and 1 × 100 mL ACN (50 mL). The heptane phase was concentrated under reduced pressure. This yielded 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,3-bis(octahydro-1H-inden-2-yl)propan-2-yl ester] 9-pentadec-8-yl ester as a pale yellow oil (0.5205 g, 38.46%). LCMS: (ES, m / z): 770.7 [M+H] + . 1 1H NMR: (400 MHz, chloroform-d) δ 5.008 - 4.822 (m, 2H), 2.333 - 2.082 (m, 12H), 1.961 - 1.734 (m, 8H), 1.726 - 1.397 (m, 23H), 1.392 - 1.197 (m, 36H), 1.193 - 1.005 (m, 4H), 0.935 - 0.800 (m, 6H).

[0814] Example 18. Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl ester] 9-pentadec-8-yl ester (L-18)

[0815]

[0816] Synthesis of 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid

[0817]

[0818] At room temperature, 5-oxononanoic acid (11 g, 54.400 mmol, 1 equiv), DCM (200 mL), pentadec-8-ol (11.80 g, 51.680 mmol, 0.95 equiv), DMAP (1.33 g, 10.880 mmol, 0.2 equiv) and EDCI (11.47 g, 59.840 mmol, 1.1 equiv) were added to a 500 mL round-bottom flask. The resulting mixture was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with PE / EA (10:1) to give 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid as a yellow oil (10 g, yield: 44.55%).

[0819] Synthesis of methyl 2-(1,3-dihydroinden-2-ylidene)acetate

[0820]

[0821] Methyl 2-(dimethoxyphosphoryl)acetate (103.35 g, 567.485 mmol, 2.5 equiv) and THF (900 mL) were added to a 2000 mL three-necked round-bottom flask at room temperature. NaH (22.70 g, 567.485 mmol, 2.5 equiv, 60%) was added portionwise to the above mixture at 0 °C. The resulting mixture was stirred at 0 °C for an additional 30 minutes. Methyl 2-indanone (30 g, 226.994 mmol, 1 equiv, in 200 mL THF) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by the addition of saturated NH4Cl (aqueous solution) (300 mL) at 0 °C. The resulting mixture was extracted with heptane (3 × 200 mL). The combined organic layers were washed with saturated aqueous Na2CO3 (2 × 50 mL), water (2 × 50 mL), MeOH / H2O (4:1, 4 × 50 mL), water (2 × 50 mL) and brine (1 × 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (98:2) to give methyl 2-(1,3-dihydroinden-2-ylidene)acetate as an orange oil (25.6 g, yield: 59.68%).

[0822] Synthesis of methyl 2-(octahydro-1H-inden-2-yl)acetate

[0823]

[0824] In a 1 L pressure tank reactor, Pd / C (10%, 12.5 g) was added to a solution of methyl 2-(1,3-dihydroinden-2-ylidene)acetate (25 g, 132.819 mmol, 1 equiv) in 750 mL of AcOH. The mixture was hydrogenated at 110 °C and 40 atm hydrogen pressure for 24 h. The resulting mixture was cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated. The resulting mixture was diluted with water (250 mL). The resulting mixture was extracted with heptane (3 × 200 mL). The combined organic layers were washed with water (2 × 500 mL), saturated NaHCO3 (2 × 300 mL), MeOH (4 × 200 mL), and brine (1 × 300 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave methyl 2-(octahydro-1H-inden-2-yl)acetate (21 g, yield: 80.55%) as a pale yellow solid.

[0825] Synthesis of 2-(octahydro-1H-inden-2-yl)ethanol

[0826]

[0827] Methyl 2-(octahydro-1H-inden-2-yl)acetate (20 g, 101.890 mmol, 1 equiv) and THF (200 mL) were added to a 500 mL four-necked round-bottom flask at room temperature. LiAlH4 (40 mL, 80.000 mmol, 0.79 equiv) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by adding water (3 mL) at 0 °C. Aqueous NaOH solution (3 mL, 15% w / w) and water (9 mL) were added dropwise to the resulting mixture. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in heptane (500 mL). The resulting mixture was washed with 2 × 200 mL of water, 3 × 200 mL of water / MeOH (1:4), 2 × 200 mL of aqueous citric acid solution (5% w / w), 2 × 200 mL of saturated NaHCO3, and brine (200 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (10:1) to give 2-(octahydro-1H-inden-2-yl)ethanol (14.3 g, yield: 83.40%) as a pale yellow oil.

[0828] Synthesis of 2-(2-bromoethyl)-octahydro-1H-indene

[0829]

[0830] To a 500 mL three-necked round-bottom flask at room temperature was added 2-(octahydro-1H-inden-2-yl)ethanol (14 g, 83.195 mmol, 1 equiv) and DCM (280 mL). To the above mixture was added portionwise PPh3 (32.73 g, 124.792 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred for an additional 10 minutes at room temperature. To the above mixture was added dropwise CBr4 (41.38 g, 124.792 mmol, 1.5 equiv, in 200 mL DCM) at 10 °C. The resulting mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with heptane (500 mL). The solid was filtered off; the filter cake was washed with heptane (2 × 50 mL). The filtrate was washed with 2 × 200 mL water, 3 × 200 mL water / MeOH (1:4), 2 × 200 mL aqueous citric acid solution (5% w / w), 2 × 200 mL saturated NaHCO3 and brine (200 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with heptane, to give 2-(2-bromoethyl)-octahydro-1H-indene as a pale yellow oil (15.1 g, yield: 77.96%).

[0831] Synthesis of 2-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene

[0832]

[0833] Add DMSO (30 mL) and NaH (1.13 g, 28.252 mmol, 2.31 equiv, 60%) to a 250 mL three-necked round-bottom flask at room temperature. Stir the resulting mixture at room temperature for an additional 1 h. Add TosMIC (2.39 g, 12.241 mmol, 1 equiv) portionwise to the above mixture at room temperature over 10 min. Stir the resulting mixture at room temperature for an additional 1 h. Add TBAB (0.45 g, 1.224 mmol, 0.1 equiv) and 2-(2-bromoethyl)-octahydro-1H-indene (6.00 g, 25.706 mmol, 2.1 equiv, 99.3%) dropwise to the above mixture at 20 °C over 1 h. Stir the resulting mixture at room temperature overnight. Dilute the resulting mixture with saturated NH4Cl (120 mL). Extract the resulting mixture with hexane (3 × 60 mL). Wash the combined organic layers with water (2 × 150 mL), dry over anhydrous Na2SO4. After filtration, concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography, eluting with PE / EA (95:5) to give 2-[3-isocyano-3-(4-methylphenylsulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene as a pale yellow oil (4.8 g, yield: 49.62%).

[0834] Synthesis of 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one

[0835]

[0836] Add 2-[3-isocyano-3-(4-methylphenylsulfonyl)-5-(octahydro-1H-inden-2-yl)pentyl]-octahydro-1H-indene (4 g, 7.592 mmol, 1 equiv, 94.1%) and HCl (gas) in 1,4-dioxane to a 250 mL three-necked round-bottom flask at 0 °C. Stir the resulting mixture at room temperature for an additional 3 h. Dilute the resulting mixture with water (100 mL). Extract the resulting mixture with hexane (3 × 30 mL). Wash the combined organic layers with saturated NaHCO3 (2 × 50 mL), water (2 × 50 mL) and brine (1 × 50 mL), dry over anhydrous Na2SO4. After filtration, concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography, eluting with PE / EA (95:5) to give 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one as a pale yellow oil (2.8 g, yield: 95.13%).

[0837] Synthesis of 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol

[0838]

[0839] At 0 °C, 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-one (2.5 g, 7.351 mmol, 1 eq., 97.2%) and MeOH (25 mL) were added to a 100 mL three-necked round-bottom flask. NaBH4 (0.28 g, 7.351 mmol, 1 eq.) was added portionwise to the above mixture at 0 °C within 2 minutes. The resulting mixture was stirred at 0 °C for an additional 2 hours. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with hexane (3 × 30 mL). The combined organic layers were washed with saturated NaHCO3 (2 × 50 mL), water (2 × 50 mL), and brine (1 × 50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (95:5), to give 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol as an off-white solid (2.3 g, yield: 91.72%).

[0840] Synthesis of 5-oxononanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl ester] 9-pentadec-8-yl ester

[0841]

[0842] At room temperature, 1,5-bis(octahydro-1H-inden-2-yl)pentan-3-ol (1.8 g, 5.277 mmol, 1 eq., 97.5%), 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid (2.61 g, 6.332 mmol, 1.2 eq.), DCM (36 mL), DIEA (1.36 g, 10.554 mmol, 2 eq.), and DMAP (128.94 mg, 1.055 mmol, 0.2 eq.) were added to a 100 mL three-necked round-bottom flask. EDCI (1.52 g, 7.915 mmol, 1.5 eq.) was added portionwise to the above mixture at room temperature. The resulting mixture was stirred at room temperature for an additional 3 hours. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (30 mL). The resulting mixture was extracted with heptane (3 × 20 mL). The combined organic layers were washed with saturated Na2CO3 (30 mL), MeOH / H2O (4:1, 2 × 50 mL), water (50 mL), and brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (9:1), to give 5-oxononanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl ester] 9-pentadec-8-yl ester as a pale yellow oil (3.6 g, yield: 93.25%).

[0843] Synthesis of 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] ester 9-pentadec-8-yl ester

[0844]

[0845] To a 250 mL three-necked round-bottom flask at room temperature was added 5-oxononanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] ester 9-pentadec-8-yl ester (3 g, 4.101 mmol, 1 equiv, 99.4%) and THF (30 mL). To the above mixture was added dropwise [3-(chloromagnesio)propyl]dimethylamine (41.01 mL, 41.010 mmol, 10 equiv) at -50 °C. The resulting mixture was stirred for an additional 2 h at -40 °C. The reaction was quenched by adding saturated NH4Cl (aqueous solution) (20 mL) at -30 °C. The mixture was warmed to room temperature. The resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (1 × 20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (13:87), to give 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] ester 9-pentadec-8-yl ester as a pale yellow oil (2.1 g, yield: 59.11%).

[0846] Synthesis of 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] ester 9-pentadec-8-yl ester

[0847]

[0848] At room temperature, 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadec-8-yl ester (2 g, 2.309 mmol, 1 equiv, 94%) and DCM (20 mL) were added to a 50 mL round-bottom flask. Et3SiH (2.68 g, 23.090 mmol, 10 equiv) and BF3·Et2O (3.28 g, 23.090 mmol, 10 equiv) were added dropwise to the above mixture at room temperature. The resulting mixture was stirred at 40 °C for an additional 2 h. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed with brine (1 × 20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (13:87) to give 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadec-8-yl ester as a pale yellow oil (1.28 g, yield: 49.05%).

[0849] Synthesis of 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadec-8-yl ester

[0850]

[0851] At room temperature, (4Z)-5-[3-(dimethylamino)propyl]non-4-enedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadec-8-yl ester (650 mg, 0.789 mmol, 1 equiv, 96.7%) and EtOH (13 mL) were added to a 50 mL round-bottom flask. Pd / C (150 mg) was added to the above mixture at room temperature. The mixture was hydrogenated at room temperature under a hydrogen pressure of 30 psi for 3 h. After filtration, the filtrate was concentrated under reduced pressure. The residue was diluted with heptane (80 mL). The solid was filtered off. The filtrate was washed with MeOH / H2O (4:1, 2 × 50 mL), water (50 mL) and brine (1 × 50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave 5-[3-(dimethylamino)propyl]nonanedioic acid 1-[1,5-bis(octahydro-1H-inden-2-yl)pentan-3-yl] 9-pentadec-8-yl ester as a colorless oil (504.9 mg, yield: 45.01%). LCMS: (ES, m / z): 798.7 [M+H] + . 11H NMR: (400 MHz, chloroform-d, ppm) δ 5.4.894 - 4.818 (m, 2H), 2.275 - 2.013 (m, 12H), 1.906 - 1.556 (m, 13H), 1.512 - 1.468 (m, 16H), 1.386 - 1.166 (m, 40H), 1.115 - 1.050 (m, 3H), 0.903 - 0.869 (m, 8H).

[0852] Example 19. 5-[3-(Dimethylamino)propyl]azelate 1-[1,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadec-8-yl ester (L-19)

[0853]

[0854] Synthesis of 1-[3-isocyano-3-(4-methylphenylsulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene

[0855]

[0856] Add DMSO (120 mL) and NaH (1.74 g, 72.329 mmol, 2.4 equiv) to a 500 mL four-necked round-bottom flask at room temperature. Add TosMIC (5.88 g, 30.137 mmol, 1 equiv) portionwise to the above mixture at room temperature over 10 minutes. Stir the resulting mixture at room temperature for an additional 0.5 hour. Add TBAI (1.11 g, 3.014 mmol, 0.1 equiv) portionwise to the above mixture at room temperature. Stir the resulting mixture at room temperature for an additional 15 minutes. Add 1-(2-bromoethyl)-4-methylbenzene (6 g, 30.137 mmol, 1 equiv) dropwise to the above mixture at room temperature over 15 minutes. Stir the resulting mixture at room temperature for an additional 18 hours. Quench the reaction with 300 mL of saturated NH4Cl (aqueous solution) at 0 °C. Extract the resulting mixture with EtOAc (2 × 200 mL). Wash the combined organic layers with water (2 × 200 mL) and brine (1 × 200 mL), and dry over anhydrous Na2SO4. After filtration, concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography, eluting with PE / EA (4:1) to obtain 1-[3-isocyano-3-(4-methylphenylsulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene as a pale yellow oil (5 g, 38.44%).

[0857] Synthesis of 1,5-bis(4-methylphenyl)pentan-3-one

[0858]

[0859] 1-[3-Isocyano-3-(4-methylphenylsulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene (5 g, 11.585 mmol, 1 equiv) and MeOH (100 mL, 4 M) containing HCl(g) were added to a 250 mL round-bottom flask at room temperature. The resulting mixture was stirred at room temperature for 18 h. LCMS showed the reaction was complete. The resulting mixture was concentrated in vacuo. The resulting mixture was diluted with 60 mL of sodium carbonate (5.0%, aqueous solution). The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (4:1) to give 1,5-bis(4-methylphenyl)pentan-3-one (2.5 g, 81.01%) as a pale yellow oil.

[0860] Synthesis of 1,5-bis(4-methylphenyl)pentan-3-ol

[0861]

[0862] 1,5-Bis(4-methylphenyl)pentan-3-one (2.5 g, 9.385 mmol, 1 equiv), MeOH (4 mL) and THF (8 mL) were placed in a 250-mL three-neck round-bottom flask at 0 °C. NaBH4 (0.99 g, 26.041 mmol, 1.5 equiv) was then added portionwise at 0 °C. The resulting solution was stirred at 0 °C for 10 min. LCMS showed the reaction was complete. The resulting mixture was quenched by adding citric acid (5%, 50 mL) at 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (1 × 200 mL), brine (1 × 200 mL). Dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with EA:heptane (1:8) to give 1,5-bis(4-methylphenyl)pentan-3-ol (2.3 g, 91.31%).

[0863] Synthesis of 5-oxononanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadec-8-yl ester

[0864]

[0865] 1,5-bis(4-methylphenyl)pentan-3-ol (2.3 g, 8.569 mmol, 1 equiv), 5,9-dioxo-9-(pentadec-8-yloxy)nonanoic acid (3.54 g, 8.569 mmol, 1 equiv), EDCI (2.46 g, 12.854 mmol, 1.5 equiv), DMAP (1.05 g, 8.569 mmol, 1 equiv) and DCM (23 mL) were added to a 250 mL three-necked round-bottom flask at room temperature. The mixture was stirred at room temperature for 18 h. LCMS showed the reaction was complete. The resulting solution was diluted with 250 mL of DCM. The resulting solution was washed with 1×200 mL of citric acid (5%, aqueous solution), 2×200 mL of water and 1×200 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with Hep / EA (volume ratio) (gradient from 100:0 to 90:10, and the eluent for the collected product Hep / EA = 95 / 5) to give 5-oxononanoic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl] ester 9-pentadec-8-yl ester as a colorless oil (5.1 g, 89.77%).

[0866] Synthesis of 5-[3-(dimethylamino)propyl]-5-hydroxynonanoic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl] ester 9-pentadec-8-yl ester

[0867]

[0868] 5-Oxononanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl] ester 9-pentadec-8-yl ester (3.5 g, 5.279 mmol, 1 equiv) and tetrahydrofuran (10.5 mL) were placed in a 250 mL round-bottom flask under N2 atmosphere at room temperature. The mixture was cooled to -60 °C. [3-(Chloromagnesio)propyl]dimethylamine (52.79 mL, 52.790 mmol, 10 equiv) was added dropwise thereto at -60 °C. The mixture was stirred at -60 °C for 2 h. LCMS showed the reaction was complete. The reaction was quenched by adding 80 mL of NH4Cl (saturated) at 0 °C. The resulting mixture was extracted with EA (2 × 100 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (volume ratio) (gradient from 100:0 to 70:30, and the eluent for the collected product was DCM / MeOH = 82 / 18), to give 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl] ester 9-pentadec-8-yl ester as a colorless oil (1.3 g, 32.83%).

[0869] Synthesis of inseparable (Z)-5-(3-(dimethylamino)propylidene)nonanedioic acid 1-(1,5-di-p-tolylpentan-3-yl) ester 9-(pentadec-8-yl) ester, (E)-5-(3-(dimethylamino)propyl)non-4-enedioic acid 1-(1,5-di-p-tolylpentan-3-yl) ester 9-(pentadec-8-yl) ester, and (E)-5-(3-(dimethylamino)propyl)non-4-enedioic acid 9-(1,5-di-p-tolylpentan-3-yl) ester 1-(pentadec-8-yl) ester

[0870]

[0871] 5-[3-(Dimethylamino)propyl]-5-hydroxynonanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl ester] 9-pentadec-8-yl ester (1.3 g, 1.733 mmol, 1 equiv) and DCM (0.55 mL, 8.665 mmol, 5 equiv) were placed in a 50 mL round-bottom flask under N2 atmosphere at room temperature. BF3·Et2O (2.46 g, 17.330 mmol, 10 equiv) and Et3SiH (2.02 g, 17.330 mmol, 10 equiv) were added thereto at room temperature. The mixture was stirred at 45 °C for 2 h. LCMS showed that the reaction was complete. The reaction was quenched by adding 25 mL of saturated NaHCO3 at 0 °C. The resulting mixture was extracted with DCM (2 × 50 mL). The combined organic layers were washed with water (2 × 100 mL) and brine (1 × 100 mL), and dried over anhydrous Na2SO4. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (volume ratio) (gradient from 100:0 to 90:10, and the collected product eluent DCM / MeOH = 94 / 6) to give an inseparable mixture of product isomers as a colorless oil (1.1 g, 86.82%).

[0872] Synthesis of 5-[3-(Dimethylamino)propyl]nonanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl ester] 9-pentadec-8-yl ester

[0873]

[0874] (1.1 g, 1.505 mmol, 1 equiv) and EtOH (22 mL) were placed in a 100 mL round-bottom flask. Pd / C (0.22 g, 10%) was added thereto at room temperature. The mixture was stirred at room temperature under H2 (30 psi) atmosphere for 4 h. LCMS showed that the reaction was complete. The resulting mixture was filtered; the filter cake was washed with EA (2 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in n-heptane (110 mL, 100V). Subsequently, the n-heptane phase was washed with 2 × 30 mL of MeOH / H2O (4:1), 2 × 30 mL of MeCN / H2O (4:1), and 1 × 30 mL of water, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give 5-[3-(Dimethylamino)propyl]nonanedioic acid 1-[1,5-bis(4-methylphenyl)pentan-3-yl ester] 9-pentadec-8-yl ester as a colorless oil (539.5 mg, 48.84%). LCMS: (ES, m / z): 733.6 [M+H] + . 11H NMR (400 MHz, chloroform-d, ppm) δ 7.122 - 7.050 (m, 8H), 5.031 - 5.007 (m, 1H), 4.990 - 4.862 (m, 1H), 2.686 - 2.615 (m, 4H), 2.585 - 2.268 (m, 18H), 1.989 - 1.811 (m, 4H), 1.668 - 1.534 (m, 10H), 1.360 - 1.282 (m, 27H), 0.921 - 0.876 (m, 6H).

[0875] Example 20. Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayl tetra(decanoate) (L-20)

[0876]

[0877] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol

[0878]

[0879] Add xylitol (40 g, 262.905 mmol, 1 eq), MeOH (400 mL, 10V), 2,2-dimethoxypropane (600 mL, 15v), and TsOH·H2O (10 g, 52.581 mmol, 0.2 eq) to a 2 L three-necked round-bottom flask at room temperature. Stir the resulting mixture overnight at room temperature under a nitrogen atmosphere. Add K2CO3 (7.263 g, 0.2 eq) portionwise to the above mixture at room temperature. Stir the resulting mixture for an additional 30 minutes at room temperature. Filter the resulting mixture and concentrate the filtrate under reduced pressure. Add 150 g of silica gel (type: ZCX-2, 100 - 200 mesh, 1.5 w. / w.) to the mixture. Subsequently, concentrate this mixture under vacuum until no distillate remains, while maintaining the temperature below 35 °C. Pack 700 g of silica gel (type: ZCX-2, 100 - 200 mesh, 9.0 w. / w.) into the column, followed by the prepared dry silica gel, which has absorbed the reaction mixture from the last step. Purify the product using combi-flash. Elute with n-heptane / EA (gradient from 100:0 to 90:10). Take samples for TLC (EA: n-heptane = 1:4) analysis and combine the qualified products. Obtain bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as a pale yellow oil (36.1 g, yield: 59.12%).

[0880] Synthesis of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate

[0881]

[0882] A solution of bis(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (25.8 g, 111.075 mmol, 1 equiv) in DCM (300 mL) was treated with TEA (33.72 g, 333.225 mmol, 3 equiv) for 10 min at room temperature under a nitrogen atmosphere. Subsequently, MsCl (19.08 g, 166.613 mmol, 1.5 equiv) was added dropwise at 0 °C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by adding a saturated NH4Cl solution at room temperature. The resulting mixture was extracted with DCM (3 × 300 mL). The combined organic layers were washed with brine (2 × 300 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate as a brown solid (35.8 g crude).

[0883] Synthesis of 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan-1-amine

[0884]

[0885] To a 1 L three-necked round-bottom flask was added 3-(diethylamino)propan-1-ol (10.57 g, 80.552 mmol, 2.5 equiv), THF (300 mL), and NaH (1.93 g, 80.552 mmol, 2.5 equiv) at 0 °C. The resulting mixture was stirred for 30 min at 0 °C under a nitrogen atmosphere. Bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl methanesulfonate (10 g, crude) was added portionwise to the above mixture at 0 °C. The resulting mixture was stirred for 18 h at room temperature under a nitrogen atmosphere. The reaction was quenched with saturated NH4Cl (aqueous solution) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 300 mL). The combined organic layers were concentrated under reduced pressure. 20 g of silica gel (type: ZCX-2, 100 - 200 mesh, 1.5 w. / w.) was added to the mixture. Subsequently, this mixture was concentrated under vacuum to no distillate while maintaining the temperature below 35 °C. 200 g of silica gel (type: ZCX-2, 100 - 200 mesh, 9.0 w. / w.) was added to the column, followed by the prepared dry silica gel that had absorbed the reaction mixture of the last step. The product was purified using combi-flash. Elution was carried out with DCM / MeOH (gradient from 100:0 to 90:10). Samples were taken for TLC (DCM / MeOH (10:1)) analysis and the qualified products were combined. 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan-1-amine was obtained as a pale yellow oil (1.9 g).

[0886] Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetraol

[0887]

[0888] Add 3-(bis(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-N,N-diethylpropane-1-amine (1.9 g, 5.500 mmol, 1 equiv) and HCl (6 M) (20 mL) to a 100 mL round-bottom flask at room temperature. Stir the resulting mixture at 60 °C under a nitrogen atmosphere for 3 h. Concentrate the resulting mixture in vacuo. The crude product (1.8 g) is used directly in the next step without further purification.

[0889] Synthesis of 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayl tetra(decanoate)

[0890]

[0891] Add 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetraol (1.8 g, 6.783 mmol, 1 equiv), ACN (100 mL), decanoic acid (7.01 g, 40.698 mmol, 6 equiv), DMAP (1.66 g, 13.566 mmol, 2 equiv) and EDCI (7.80 g, 40.698 mmol, 6 equiv) to a 250 mL round-bottom flask at room temperature. Stir the resulting mixture at room temperature overnight. Concentrate the mixture under reduced pressure. Dissolve the residue in water (200 mL). Extract the resulting mixture with heptane (3 × 200 mL). Dry the combined organic layers over anhydrous Na2SO4. After filtration, concentrate the filtrate under reduced pressure. The residue is purified by reverse-phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, CH3CN and water (0.05% TFA), gradient from 50% CH3CN to 95% in 15 min; detector, UV 200 nm. Collect the product eluent and concentrate. Dilute the resulting mixture with n-heptane (300 mL) and basify to pH 8 - 9 with saturated Na2CO3 (3%). Wash the organic layer with brine (2 × 100 mL) and H2O / MeOH = 1 / 5 (1 × 100 mL). Dry and concentrate the organic layer over anhydrous Na2SO4. This gives 3-(3-(diethylamino)propoxy)pentane-1,2,4,5-tetrayl tetra(decanoate) as a pale yellow oil (526.8 mg, yield: 8.80%). LCMS: (ES, m / z): 883 [M+1] + . 11H NMR (300 MHz, CDCl3, ppm) δ 5.484 (t, J = 5.4 Hz, 1H), 5.347 - 5.311 (m, 1H), 5.205 - 5.171 (m, 1H), 4.352 - 4.299 (m, 1H), 4.027 - 3.966 (m, 1H), 3.507 - 3.394 (m, 4H), 2.550 - 2.452 (m, 6H), 2.344 - 2.262 (m, 8H), 1.741 - 1.586 (m, 10H), 1.390 - 1.210 (m, 48H), 1.011 (t, J = 7.2 Hz, 6H), 0.879 (t, J = 6.9 Hz, 12H).

[0892] Example 21. Synthesis of 3-(octahydro-1H-inden-2-yl)propanoic acid 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl ester

[0893]

[0894] Synthesis of octahydro-1H-inden-2-carboxylic acid

[0895]

[0896] To a solution of 2,3-dihydro-1H-inden-2-carboxylic acid (25 g, 154.142 mmol, 1 equiv) in 500 mL of AcOH in a pressure vessel was added Pd / C (25 g). The mixture was hydrogenated at 120 °C under 30 psi hydrogen pressure for 24 h. The mixture was filtered through a Celite pad and the filtrate was concentrated under reduced pressure. The resulting mixture was diluted with water (250 mL). The mixture was extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with brine (1 × 250 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave octahydro-1H-inden-2-carboxylic acid (22 g, 75.08%) as a brown oil.

[0897] Synthesis of octahydro-1H-inden-2-ylmethanol

[0898]

[0899] At room temperature under a nitrogen atmosphere, LiAlH4 (6.59 g, 173.595 mmol, 1.5 eq) was added dropwise to a stirred mixture of octahydro-1H-indene-2-carboxylic acid (22 g, 115.730 mmol, 1 eq, 88.5%) in THF. The resulting mixture was stirred at room temperature for 4 h. The reaction was quenched by the addition of hydrochloric acid (2 M) (100 mL) at 0 °C. The resulting mixture was extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine (1 × 200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave octahydro-1H-indene-2-ylmethanol (16 g, 79.68%) as a yellow oil.

[0900] Synthesis of octahydro-1H-indene-2-carbaldehyde

[0901]

[0902] Octahydro-1H-indene-2-ylmethanol (15 g, 97.243 mmol, 1 eq) and DCM (150 mL) were added to a 250 mL three-necked round-bottom flask at room temperature. Dess-Martin (45.37 g, 106.967 mmol, 1.1 eq) was added portionwise to the above mixture at 0 °C. The resulting mixture was stirred at room temperature for an additional 2 h. The reaction was quenched by the addition of Na2S2O3 (aqueous solution) (100 mL) at room temperature. The resulting mixture was extracted with CH2Cl2 (2 × 200 mL). The combined organic layers were washed with NaHCO3 (aqueous solution) (3 × 200 mL), dried over anhydrous MgSO4. After filtration, the filtrate was concentrated under reduced pressure. This gave octahydro-1H-indene-2-carbaldehyde (5.95 g, 26.69%) as a yellow oil.

[0903] Synthesis of methyl (2E)-3-(octahydro-1H-indene-2-yl)prop-2-enoate

[0904]

[0905] Add octahydro-1H-indene-2-carbaldehyde (5.7 g, 37.442 mmol, 1 equiv), 2-MeTHF (60 mL), and methyl 2-(triphenyl-λ5-phosphanylidene)acetate (15.02 g, 44.930 mmol, 1.2 equiv) to a 250 mL three-necked round-bottom flask at room temperature. Stir the resulting mixture at room temperature for 2 h. Combine with the previous 200 mg of reactant. Concentrate the resulting mixture under reduced pressure. Dissolve the mixture in DCM (100 mL) and add 24 g of silica gel (type: ZCX-2, 100-200 mesh, 4 w. / w.). Concentrate to dryness under vacuum while maintaining the temperature below 35 °C. Load 200 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w / w.) into the column, and then the dry silica gel that absorbed the reaction mixture prepared in the last step. Purify the product using combi-flash. Elute with PE / EA (gradient from 100:0 to 90:10, collect every 200 ± 10 mL). Take samples for TLC (EA:PE = 5:1) analysis and combine the qualified products. This gives methyl (2E)-3-(octahydro-1H-inden-2-yl)prop-2-enoate (5.51 g, 63.00%) as a colorless oil.

[0906] Synthesis of methyl 3-(octahydro-1H-inden-2-yl)propionate

[0907]

[0908] Add Pd / C (2.5 g) to a solution of methyl (2E)-3-(octahydro-1H-inden-2-yl)prop-2-enoate (5 g, 24.004 mmol, 1 equiv) in 50 mL of MeOH in a pressure vessel. Hydrogenate the mixture at room temperature under a hydrogen pressure of 30 psi for 12 h. Combine with the previous 500 mg of reactant. Filter through a Celite pad and concentrate the filtrate under reduced pressure. Filter the resulting mixture and wash the filter cake with MeOH (2 × 20 mL). Concentrate the filtrate under reduced pressure. This gives methyl 3-(octahydro-1H-inden-2-yl)propionate (5.1 g, 72.19%) as a colorless oil.

[0909] Synthesis of 3-(octahydro-1H-inden-2-yl)propanoic acid

[0910]

[0911] Methyl 3-(octahydro-1H-inden-2-yl)propionate (5 g, 23.774 mmol, 1 equiv), THF (50 mL), MeOH (50 mL), H2O (50 mL), and LiOH·H2O (2.00 g, 47.548 mmol, 2 equiv) were added to a 250 mL three-necked round-bottom flask at room temperature. The resulting mixture was stirred at room temperature for 2 h. The organic solvents were removed under reduced pressure. The resulting mixture was extracted with EtOAc (2 × 50 mL) and the aqueous phase was collected. The aqueous phase was acidified to pH = 2 with HCl (6 M). The resulting mixture was extracted with EtOAc (2 × 100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This gave 3-(octahydro-1H-inden-2-yl)propionic acid (3.941 g, 74.10%) as a white solid.

[0912] Synthesis of 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl 3-(octahydro-1H-inden-2-yl)propionate

[0913]

[0914] 3-(Octahydro-1H-inden-2-yl)propionic acid (0.85 g, 4.330 mmol, 1 equiv), ACN (8.5 mL), 3-[3-(dimethylamino)propoxy]pentane-1,2,4,5-tetraol (5.14 g, 21.650 mmol, 5 equiv), EDCI (4.98 g, 25.980 mmol, 6 equiv), DMAP (1.06 g, 8.660 mmol, 2 equiv), and DIEA (4.48 g, 34.640 mmol, 8 equiv) were added to an 80 mL vial at room temperature. The resulting mixture was stirred at room temperature for an additional 3 h. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with heptane (3 × 150 mL). The collected organic phase was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase HP flash chromatography under the following conditions: column, XSelect CSH Prep C 185 μm; mobile phase, B: CH3CN, A: water (0.1% TFA), gradient from 50% to 95% in 15 min; flow rate: 50 mL / min; detector, ELSD. The mixture obtained by concentration under reduced pressure was basified to pH 8 with saturated Na2CO3 (aqueous solution). The aqueous layer was extracted with heptane (3 × 100 mL). The mixture obtained by concentration under reduced pressure. This gave 3-(octahydro-1H-inden-2-yl)propanoic acid 3-[3-(dimethylamino)propoxy]-1,4,5-tris({[3-(octahydro-1H-inden-2-yl)propanoyl]oxy})pentan-2-yl ester as a yellow oil (0.5272 g, 12.90%). LCMS: (ES, m / z): 950.9 [M+1] + . 1 H NMR (300 MHz, chloroform-d) δ 5.456 (t, J = 5.5 Hz, 1H), 5.342 (t, J = 5.5 Hz, 1H), 5.145 (q, J = 5.0 Hz, 1H), 4.395 - 4.285 (m, 1H), 4.000 - 3.875 (m, 1H), 3.584 - 3.377 (m, 4H), 2.413 - 2.252 (m, 10H), 2.243 - 2.184 (m, 6H), 1.999 - 1.799 (m, 14H), 1.763 - 1.584 (m, 15H), 1.540 - 1.418 (m, 14H), 1.352 - 1.245 (m, 21H), 1.161 - 1.027 (m, 6H).

[0915] Example 22. Synthesis of bis(decanoic acid) 5-(dimethylamino)-1-((2-heptylnonanoyl)oxy)pentane-2,3-diyl ester (L-22)

[0916]

[0917] Synthesis of 2-(5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)acetaldehyde

[0918]

[0919] At room temperature, 3,4,5-trihydroxypentanal (20 g, 149.108 mmol, 1 equiv), pTsOH (1.28 g, 7.455 mmol, 0.05 equiv), and acetone (200 mL) were added to a 500 mL three-necked round-bottom flask. 2,2-Dimethoxypropane (18.64 g, 178.930 mmol, 1.2 equiv) was added dropwise to the above mixture at 0 °C. The resulting mixture was stirred at 0 °C for an additional 30 minutes. The mixture was basified to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was concentrated under reduced pressure to remove acetone. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was conc...

Claims

1. An ionizable lipid compound of formula (I): (Z-L-Y)-W n -(X-R) (n-1) (I) Wherein: Z is an ionizable head group; L is an optionally substituted (C 1- C 12 ) alkylene; Y is a linking group; W n is a straight-chain alkyl core having n carbon atoms, where n is from 3 to 6; X is an optional linking group; and Each R is independently a lipid tail.

2. The compound according to claim 1, wherein n is from 4 to 6.

3. The compound according to claim 2, wherein n is 4.

4. The compound according to claim 2, wherein n is 5.

5. The compound according to claim 2, wherein n is 6.

6. The compound according to claim 1, wherein n is 3.

7. The compound according to any one of claims 1 to 6, wherein W n is selected from: Wherein: * depicts the attachment point to Y; Each ** depicts the attachment point to X; G 1 is H or a group that is linked to Y in a ring form, and said group, together with the carbon atom of W to which it is attached, forms a heterocycle; and n ​ G 2 is H or -CH2OH.

8. A compound according to any one of claims 1 to 6, wherein Y is selected from -O-, -C(R 10 )2-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl.

9. The compound according to claim 8, wherein Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -.

10. The compound according to claim 8, wherein Y is -CH2-.

11. The compound according to claim 7, wherein G 1 is a group that is cyclically linked to Y and, together with the carbon atom of W to which it is attached n forms a heterocycle.

12. The compound according to any one of claims 1 to 11, wherein L is (C2-C6) alkylene or substituted (C2-C6) alkylene.

13. The compound according to claim 12, wherein L is -(CH2)2-.

14. The compound according to claim 12, wherein L is -(CH2)3-.

15. The compound according to any one of claims 1 to 14, wherein Z contains a tertiary amino group.

16. The compound according to claim 15, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently alkyl or substituted alkyl.

17. The compound according to claim 16, wherein R 11 and R 12 are each C 1-6 alkyl.

18. The compound according to claim 17, wherein R 11 and R 12 are each C 1-3 alkyl.

19. The compound according to claim 18, wherein R 11 and R 12 are each methyl.

20. The compound according to claim 18, wherein R 11 and R 12 are each ethyl.

21. A compound according to any one of claims 1 to 20, wherein each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O-, -(CH2) s OC(O)O-, -(CH2) s OC(O)NR 10 -, -(CH2) s O-, -(CH2) s SC(O)NR 10 -, -(CH2) s C(O)NR 10 -, -(CH2) s NR 10 C(O)-, -(CH2) s S-, -(CH2) s NR 10 -, -(CH2) s NR 10 C(O)O- and -(CH2) s NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl and s is from 0 to 6.

22. The compound according to any one of claims 1 to 21, wherein each X is independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -O-, -OC(O)NR 10 -, -SC(O)NR 10 -, -C(O)NR 10 -, -NR 10 C(O)-, -S-, -NR 10 -, -NR 10 C(O)O- and -NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl groups.

23. The compound according to claim 22, wherein each X is independently selected from -OC(O)-, -C(O)O- and -OC(O)O-.

24. The compound according to claim 23, wherein each -X-R is -OC(O)R.

25. The compound according to any one of claims 1 to 24, wherein each R is independently an aliphatic hydrocarbon group, the aliphatic hydrocarbon group being straight-chain or branched, saturated or unsaturated and / or optionally containing a cyclic group.

26. The compound according to any one of claims 1 to 25, wherein each R is a straight-chain hydrocarbon group optionally containing one or more cyclic groups.

27. A compound according to any one of claims 1 to 26, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

28. The compound according to claim 27, wherein each R is selected from C6-C 12 alkyl and C6-C 12 alkenyl.

29. The compound according to claim 26, wherein at least one R is a straight-chain hydrocarbon group containing a cyclic group.

30. The compound according to claim 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle and heteroaryl, wherein any one of the monocyclic or bicyclic groups is optionally substituted.

31. The compound according to any one of claims 1 to 25, wherein at least one R is a branched hydrocarbon group optionally containing a cyclic group.

32. The compound according to claim 31, wherein each R is a branched hydrocarbon group.

33. The compound according to claim 32, wherein the branched hydrocarbon group contains 8 to 20 carbon atoms.

34. The compound according to any one of claims 31 to 33, wherein the branched hydrocarbon group is saturated.

35. The compound according to any one of claims 31 to 33, wherein the branched hydrocarbon group is unsaturated.

36. The compound according to any one of claims 31 to 35, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

37. The compound according to claim 31, wherein at least one R is a branched hydrocarbon group containing a cyclic group.

38. The compound according to claim 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, and any one of the monocyclic or bicyclic groups is optionally substituted.

39. The compound according to claim 1, wherein the compound has the formula (IIA):

40. The compound according to claim 39, wherein Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -, wherein R 10 is selected from H and C 1-6 alkyl.

41. The compound according to claim 40, wherein Y is -O-.

42. The compound according to claim 40, wherein Y is -OC(O)-.

43. The compound according to claim 40, wherein Y is -OC(O)NR 10 -.

44. The compound according to any one of claims 39 to 43, wherein L is a (C2-C6) alkylene group or a substituted (C2-C6) alkylene group.

45. The compound according to claim 44, wherein L is -(CH2)2-.

46. The compound according to claim 44, wherein L is -(CH2)3-.

47. The compound according to claim 44, wherein L is -(CH2)4-.

48. A compound according to any one of claims 39 to 47, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently C 1-6 alkyl or substituted C 1-6 alkyl.

49. The compound according to claim 48, wherein R 11 and R 12 are each C 1-3 alkyl.

50. The compound according to claim 49, wherein R 11 and R 12 are each methyl.

51. The compound according to claim 49, wherein R 11 and R 12 are each ethyl.

52. The compound according to any one of claims 39 to 51, wherein each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

53. A compound according to any one of claims 39 to 52, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

54. The compound according to any one of claims 39 to 52, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups.

55. The compound according to claim 54, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

56. The compound according to claim 39, wherein the compound has the formula (IIIA): wherein: R 11 and R 12 each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl; q is from 1 to 4; Y is selected from -O-, -OC(O)-, and -OC(O)NR 10 -; and Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently from 1 to 10.

57. The compound according to claim 1, wherein the compound has the formula (IIB):

58. The compound according to claim 57, wherein Y is selected from -O-, -OC(O)-, -OC(O)NR 10 -, -NR 10 C(O)-, -NR 10 C(O)O- and -NR 10 C(O)S-, wherein R 10 is selected from H and C 1-6 alkyl.

59. The compound according to claim 58, wherein Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-.

60. The compound according to any one of claims 57 to 59, wherein L is a (C2-C6) alkylene group or a substituted (C2-C6) alkylene group.

61. The compound according to claim 60, wherein L is -(CH2)2-.

62. The compound according to claim 60, wherein L is -(CH2)3-.

63. The compound according to claim 60, wherein L is -(CH2)4-.

64. A compound according to any one of claims 57 to 59, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently C 1-6 alkyl or substituted C 1-6 alkyl.

65. The compound according to claim 64, wherein R 11 and R 12 are each C 1-3 alkyl.

66. The compound according to claim 64, wherein R 11 and R 12 are each methyl.

67. The compound according to any one of claims 57 to 66, wherein each X is independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

68. A compound according to any one of claims 57 to 67, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

69. The compound according to any one of claims 57 to 67, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups.

70. The compound according to claim 69, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

71. The compound according to claim 57, wherein the compound has the formula (IIIB): wherein: R 11 and R 12 each independently selected from C 1-3 alkyl or C 1-4 heteroalkyl; q is from 1 to 4; Y is selected from -NHC(O)-, -NHC(O)O-, and -NHC(O)S-; and Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and t and u are each independently 1 to 10.

72. The compound according to claim 1, wherein the compound has the formula (IIC):

73. The compound according to claim 72, wherein Y is selected from -O-, -OC(O)-, -OC(O)NR 10 -, and -C(R 10 )2-, wherein R 10 is selected from H and C 1-6 alkyl.

74. The compound according to claim 73, wherein Y is -O-.

75. The compound according to claim 73, wherein Y is -C(R 10 )2-.

76. A compound according to any one of claims 72 to 75, wherein L is a (C2-C6) alkylene group or a substituted (C2-C6) alkylene group.

77. A compound according to any one of claims 76, wherein L is -(CH2)2-.

78. A compound according to any one of claims 76, wherein L is -(CH2)3-.

79. A compound according to any one of claims 76, wherein L is -(CH2)4-.

80. A compound according to any one of claims 72 to 79, wherein Z is -NR 11 R 12 , wherein R 11 and R 12 are each independently C 1-6 alkyl or substituted C 1-6 alkyl.

81. The compound according to claim 80, wherein R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl.

82. The compound according to claim 81, wherein R 11 and R 12 are each methyl.

83. A compound according to any one of claims 72 to 82, wherein each X is independently selected from -(CH2) s OC(O)-, -(CH2) s C(O)O-, -(CH2) s OC(O)O-, where s is from 0 to 6.

84. A compound according to any one of claims 72 to 82, wherein each s is 0.

85. A compound according to any one of claims 72 to 82, wherein each s is 1.

86. A compound according to any one of claims 72 to 82, wherein each s is 3.

87. A compound according to any one of claims 72 to 86, wherein each R is selected from C5-C 20 alkyl, C5-C 20 alkenyl and C5-C 20 alkynyl.

88. A compound according to any one of claims 72 to 86, wherein at least one R is a branched hydrocarbon group containing 8 to 20 carbon atoms, optionally further containing one or more cyclic groups.

89. The compound according to claim 88, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl.

90. A compound according to claim 72, wherein the compound has the formula (IIIC): Wherein: R 11 and R 12 are each independently selected from C 1-3 alkyl and C 1-4 heteroalkyl; q is 1 to 4; Y is selected from -O- and -C(R 10 )2-; Each s is independently 0, 1 or 2; W is -O- and -C(R 10 )2-; and Each R is independently selected from C5-C 20 alkyl, C5-C 20 alkenyl, -CH(R 7 )2 and -(CH2) t J(CH2) u , where each R 7 is independently C5-C 12 alkyl or C5-C 12 alkenyl, J is a cyclic group, and each of t and u is from 1 to 10.

91. A compound according to any one of claims 1 to 90, wherein each R is independently Cy A and Cy B each independently is a bond or an optionally substituted saturated, partially unsaturated or aromatic cyclic group, and the cyclic group is selected from a 5- to 12-membered monocyclic group, bicyclic group, bridged polycyclic group and spirocyclic group; R x and R y each independently is a bond or an optionally substituted straight-chain or branched-chain, saturated or partially unsaturated C1-C 20 aliphatic group; and r, p and q are each independently an integer from 0 to 20.

92. A compound according to claim 91, wherein at least one R contains a moiety selected from the following: Each # represents an attachment point to X or to a straight or branched hydrocarbon chain of R.

93. A lipid nanoparticle comprising an ionizable lipid compound according to any one of claims 1 to 92.

94. A lipid nanoparticle according to claim 93, further comprising a neutral lipid and an aggregation-reducing lipid.

95. A lipid nanoparticle according to claim 94, wherein the neutral lipid comprises a phospholipid.

96. A lipid nanoparticle according to claim 94 or 95, wherein the neutral lipid comprises cholesterol.

97. A lipid nanoparticle according to claim 96, comprising: a) nucleic acid, b) ionizable lipid, c) phospholipid, d) cholesterol, and e) an aggregation-reducing lipid.

98. A lipid nanoparticle according to claim 97, wherein the nucleic acid comprises DNA.

99. A lipid nanoparticle according to claim 98, wherein the nucleic acid comprises RNA.

100. A lipid nanoparticle according to claim 98, wherein the nucleic acid comprises DNA and RNA.

101. A lipid nanoparticle according to claim 100, wherein the RNA is selected from mRNA, gRNA and siRNA.

102. A lipid nanoparticle according to any one of claims 97 to 101, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG), and derivatives thereof.

103. The lipid nanoparticle according to claim 102, wherein the phospholipid is phosphatidylethanolamine (PE).

104. The lipid nanoparticle according to claim 103, wherein the phospholipid is phosphatidylcholine (PC).

105. The lipid nanoparticle according to any one of claims 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12 to 24 carbons.

106. The lipid nanoparticle according to claim 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16 to 20 carbons.

107. The lipid nanoparticle according to claim 105 or 106, wherein the hydrocarbon chains are saturated.

108. The lipid nanoparticle according to claim 105 or 106, wherein the hydrocarbon chains are unsaturated and / or further comprise carbocyclic groups.

109. The lipid nanoparticle according to claim 108, wherein the hydrocarbon chains each independently comprise 1 to 4 double bonds.

110. The lipid nanoparticle according to any one of claims 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.

111. The lipid nanoparticle according to claim 103, wherein the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

112. The lipid nanoparticle according to claim 103, wherein the phospholipid comprises 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

113. The lipid nanoparticle according to claim 104, wherein the phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Δ9Δ9-cis PC).

114. The lipid nanoparticle according to claim 106, wherein the lipid nanoparticle comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

115. The lipid nanoparticle according to claim 104, wherein the lipid nanoparticle comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

116. The lipid nanoparticle according to any one of claims 103 to 115, wherein the lipid capable of reducing aggregation is a PEG-lipid.

117. The lipid nanoparticle according to claim 116, wherein the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

118. The lipid nanoparticle according to any one of claims 94 to 117, which further comprises a targeting ligand.

119. The lipid nanoparticle according to claim 118, wherein the targeting ligand comprises GalNAc.

120. The lipid nanoparticle according to claim 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.

121. The lipid nanoparticle according to claim 120, wherein the lipid capable of reducing aggregation is PEG-1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).

122. The lipid nanoparticle according to any one of claims 94 to 121, wherein the N / P ratio (the ratio of the number of moles of amine groups of the cationic lipid to the number of moles of phosphate esters of DNA) is from 5 to 30.

123. The lipid nanoparticle according to claim 122, wherein the N / P ratio is 7.

124. The lipid nanoparticle according to claim 122, wherein the N / P ratio is 14.

125. The lipid nanoparticle according to claim 122, wherein the N / P ratio is 28.

126. The lipid nanoparticle according to any one of claims 94 to 125, comprising: a) an ionizable lipid accounting for 40 to 60 mol% of the total lipids present; b) a phospholipid accounting for 6 to 20 mol% of the total lipids present; c) cholesterol accounting for 35 to 45 mol% of the total lipids present; and d) a lipid capable of reducing aggregation accounting for 1.5 to 2.5 mol% of the total lipids present.

127. The lipid nanoparticle according to any one of claims 94 to 125, comprising: a) an ionizable lipid accounting for 40 to 60 mol% of the total lipids present; b) a phospholipid accounting for 10 to 20 mol% of the total lipids present; c) cholesterol accounting for 35 to 45 mol% of the total lipids present; and d) a lipid capable of reducing aggregation accounting for 1.5 to 2.5 mol% of the total lipids present.

128. The lipid nanoparticle according to any one of claims 94 to 125, comprising: e) an ionizable lipid accounting for 40 to 49 mol% of the total lipids present; f) a phospholipid accounting for 10 to 20 mol% of the total lipids present; g) cholesterol accounting for 35 to 45 mol% of the total lipids present; and h) a lipid capable of reducing aggregation accounting for 1.5 to 2.5 mol% of the total lipids present.

129. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 94 to 128 and a pharmaceutically acceptable excipient, carrier or diluent.

130. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with the lipid nanoparticle according to any one of claims 94 to 128.

131. The method according to claim 130, wherein the cell is in vitro.

132. The method according to claim 130, wherein the cell is in vivo.

133. A method for delivering a nucleic acid to produce a target protein in vivo, the method comprising: systemically administering to an individual in need the pharmaceutical composition according to claim 129, wherein the nucleic acid encodes the target protein and is encapsulated within the lipid nanoparticle, and the administration of the pharmaceutical composition enables prolonged stable expression of the target protein.

Images