WO2024107906A2 - Ionizable lipids and lipid nanoparticle compositions for the delivery of nucleic acids - Google Patents

Ionizable lipids and lipid nanoparticle compositions for the delivery of nucleic acids Download PDF

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WO2024107906A2
WO2024107906A2 PCT/US2023/079923 US2023079923W WO2024107906A2 WO 2024107906 A2 WO2024107906 A2 WO 2024107906A2 US 2023079923 W US2023079923 W US 2023079923W WO 2024107906 A2 WO2024107906 A2 WO 2024107906A2
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compound
lipid
alkyl
independently
mol
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WO2024107906A3 (en
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Amit Sagi
Rob BURKE
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Seawolf Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/24—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane

Definitions

  • nucleic acid there are many instances in which delivery of a nucleic acid is desired, where such instances include 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. Genetic disorders, although individually rare, collectively represent a significant disease burden, particularly for children, resulting in substantial disability and mortality.
  • viral vectors such as vectors based on AAV
  • AAV vectors are commonly employed to deliver genes into cells.
  • AAV vectors are limited in the size of genetic cargo that can be packaged. Accordingly, any genetic cargo greater than 4.7kB is not suitable for delivery with AAV vectors, which limits the utility of such vectors for many indications.
  • viral vectors, such as AAV induce an antibody response, limiting redosing, which is not suitable for some indications.
  • expression from successfully transduced cells can be reduced or lost with cell division and turnover, requiring redosing - which may not be possible or effective due to immune memory.
  • many subjects have pre-existing immunity to commonly used viral vectors such as AAV, which can limit even initial treatment with an AAV gene therapy.
  • viral vector such as AAV can be toxic at the doses that would be required to achieve therapeutic benefit in some indications.
  • novel ionizable lipids having an ionizable headgroup connected to lipid tails via a linear alkyl core.
  • the linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails.
  • novel lipid nanoparticle (LNP) compositions for the delivery of nucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using compositions of the invention in research and as therapeutics.
  • FIGs. 1 A-C describe studies performed to assess how the structure of the ionizable lipid impacts the efficacy and toxicity of DNA-LNPs.
  • FIG. 1 A Formulation details for the test articles. The ionizable lipid was varied in all formulations.
  • Ionizable cationic lipids tested were ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate), MC3 (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate, and two ionizable cationic lipids of the present disclosure, L-2 and L-3.
  • the phospholipid in all formulations is DSPC.
  • the nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed of all test articles.
  • npDNA nanoplasmid DNA
  • FIG. IB EPO serum levels in wild type BALB/c mice were measured 4 hours post-i.v. administration of test articles at a dose of 1 mg/kg.
  • FIG. 1C IL-6 cytokine levels in serum were measured 4 hours after i.v. administration of test articles at a dose of 1 mg/kg to wild type BALB/c mice.
  • FIGs. 2A-0 describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs.
  • FIG. 2 A - 2B Formulation details for the test articles. The ionizable lipid was varied in these formulations. Two benchmark ionizable lipids, ALC-0315 and MC3 are 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).
  • FIG. 2D-I The levels of IL-6, IFNy, TNFa, IL-1 , IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 2A (formulated with the DSPC phospholipid) at a dose of 1 or 0.3 mg/kg.
  • FIG. 2J-O The levels of IL-6, IFNy, TNFa, IL-10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 2B (formulated with the DOPE phospholipid).
  • FIGs. 3 A-F describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs.
  • FIG. 3A-3B Formulation details for the test articles.
  • the ionizable lipid was varied in these formulations.
  • the benchmark ionizable lipid, ALC-0315 is described above.
  • Other benchmark 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).
  • the phospholipid in these formulations is DSPC (FIG. 3 A) or DOPE (FIG. 3B).
  • the nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles.
  • npDNA nanoplasmid DNA
  • FIG. 3C EPO protein levels in serum of wild type BALB/c mice were measured 7 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
  • 3D-I The levels of IL-6, IFNy, TNFa, IL-10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 3A (formulated with the DSPC phospholipid) at a dose of 1 or 0.3 mg/kg.
  • FIG. 3J-O The levels of IL-6, IFNy, TNFa, IL- 10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 3B (formulated with the DOPE phospholipid).
  • FIG. 4B EPO protein levels in serum of wild type BALB/c mice were measured 3 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
  • FIG. 4C IL-6 cytokine levels in serum of wild type BALB/c mice were measured 4 hours after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
  • FIGs 5 A-C describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs.
  • FIG. 5 A Formulation details for the test articles. The ionizable lipid was varied in these formulations. Two benchmark ionizable lipids, ALC-0315 and ARCT, are described above. One 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 cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles.
  • npDNA nanoplasmid DNA
  • FIG. 5B EPO protein levels in serum of wild type BALB/c mice were measured 3 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg to wild type balb/c mice.
  • FIG. 5C IL-6 cytokine levels in serum of wild type BALB/c mice were measured 4 hours after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
  • the nucleic acid cargo used in all formulations comprised nanoplasmid DNA (npDNA) and mRNA mixed at a 1 :3 (w/w) ratio of DNA:mRNA, where the npDNA comprises a TTR promoter driving the expression of a human Factor IX (FIX) transgene.
  • FIX human Factor IX
  • FIG. 6B Human FIX protein levels in plasma of wild type balb/c mice 21 days after i.v. administration of test articles at a dose of 0.5 mg/kg DNA (1.5 mg/kg mRNA).
  • FIG. 6C - K Cytokine levels in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles at a dose of 0.5 mg/kg DNA (1.5 mg/kg mRNA). 4. DETAILED DESCRIPTION
  • Novel lipid nanoparticle compositions are provided for the delivery of nucleic acid to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the lipid nanoparticle compositions of this disclosure in research and as therapeutics.
  • a “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), a protein, a small molecule, and the like to a target site of interest.
  • an active agent or therapeutic agent such as a nucleic acid (e.g., DNA and/or RNA), a protein, a small molecule, and the like.
  • a nucleic acid agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.
  • the lipid nanoparticle includes several lipid components, including e.g., an ionizable lipid, one or more helper lipid(s) (e.g., non-cationic lipid(s)), and a lipid that prevents aggregation of the nanoparticle (also referred to as a coat lipid or conjugated lipid e.g., a PEG-lipid).
  • a lipid nanoparticle (LNP) composition as described herein comprising a nucleic acid, where the nucleic acid is substantially encapsulated by the lipid components of the LNP.
  • the lipid nanoparticles (LNPs) of this disclosure can include ionizable lipids.
  • ionizable lipid is typically employed in lipid nanoparti cles(LNPs) to condense its nucleic acid cargo, e.g., DNA or RNA, at low pH and to drive membrane association and fusogenicity.
  • the term “ionizable lipid” refers to a lipid comprising an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or less), but which can remain neutral at, e.g., a higher pH, such as physiological pH.
  • Ionizable lipids can have less interactions with cell membranes when neutral, and then become charged when internalized into endosomes in a target cell, where the pH is lower than in the extracellular environment. Ionizable lipids which are protonated and, therefore, become positively charged, may promote membrane destabilization and facilitate endosomal escape of the nanoparticle.
  • 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., a pH of 6.5 or less).
  • the ionizable lipids are cationic lipids including at least one ionizable amino group that is positively charged or becomes protonated at a selected pH, for example at pH of 6.5 or lower.
  • the cationic lipid includes one or more tertiary amino groups, e.g., a trialkyl amino group.
  • the ionizable lipid includes an ionizable headgroup (e.g., an ionizable amino group) connected to the lipid tails via a linear alkyl core.
  • the linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails.
  • the linear alkyl core has 3 carbon atoms and 2 lipid tails.
  • the linear alkyl core has 4 carbon atoms and 3 lipid tails.
  • the linear alkyl core has 5 carbon atoms and 4 lipid tails.
  • the linear alkyl core has 6 carbon atoms and 5 lipid tails.
  • the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, a linear alkyl core, hydrocarbon chains (e.g., C8-C20 carbon chains, such as Cis alkyl chains), ether linkages between the linear alkyl core and hydrocarbon chains, and 0 to 3 double bonds per hydrocarbon chain.
  • the cationic lipid comprises the same number of hydrocarbon chains as ether linkages.
  • the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and ester linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ester linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and carbonate linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as carbonate linkages. 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.
  • aspects of this disclosure include an ionizable lipid compound of formula (I): (Z-L-Y)-W n -(X-R) ( n-i)
  • Z is an ionizable head group
  • L is an optionally substituted (Ci-Ci2)alkylene
  • Y is a linking group
  • W n is a linear alkyl core of n carbon atoms, wherein n is 3 to 6; X is an optional linking group; and each R is independently a lipid tail.
  • n is 4 to 6, such that the linear alkyl core has 4 to 6 carbon atoms.
  • n is 4, such that the linear alkyl core has 4 carbon atoms.
  • n is 5, such that the linear alkyl core has 5 carbon atoms.
  • n is 6, such that the linear alkyl core has 6 carbon atoms.
  • n is 3, such that the linear alkyl core has 3 carbon atoms.
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and each G 2 is independently H or -CH2OH.
  • at least one G 2 is H.
  • both G 2 groups are H.
  • at least one G 2 is -CH2OH.
  • both G 2 groups are -CH2OH.
  • one G 2 is H and the other is -CH2OH.
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and G 2 is H or -CH2OH. In certain cases, G 2 is H. In certain cases, G 2 is -CH2OH.
  • W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to
  • G 2 is H or -CH2OH. In certain cases, G 2 is H. In certain cases, G 2 is -CH2OH.
  • the linear alky core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle.
  • G 1 is cyclically linked with Y to form a 5-membered heterocycle.
  • G 1 is cyclically linked with Y to form a 6-membered heterocycle.
  • G 1 is H.
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle; and G 2 is H or -CH2OH.
  • G 1 is cyclically linked with Y to form a 5-membered heterocycle.
  • G 1 is cyclically linked with Y to form a 6-membered heterocycle.
  • G 1 is H.
  • G 2 is H.
  • G 2 is -CH2OH.
  • G 1 and G 2 are H. In certain cases, G 1 and G 2 are both H. In certain cases, G 1 is H and G 2 is - CH2OH. In certain cases, G 1 is cyclically linked with Y to provide a heterocycle, and G 2 is H. In certain cases, G 1 is a cyclically linked with Y to provide a heterocycle, and G 2 is - CH2OH.
  • the linear alkyl core W n is: where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle; and G 2 is H or -CH2OH.
  • G 1 is cyclically linked with Y to form a 5-membered heterocycle.
  • G 1 is cyclically linked with Y to form a 6-membered heterocycle.
  • G 1 is H.
  • G 2 is H.
  • G 2 is -CH2OH.
  • G 1 and G 2 are H. In certain cases, G 1 and G 2 are both H. In certain cases, G 1 is H and G 2 is - CH2OH. In certain cases, G 1 is cyclically linked with Y to provide a heterocycle, and G 2 is H. In certain cases, G 1 is a cyclically linked with Y to provide a heterocycle, and G 2 is - CH2OH.
  • linking group is meant a linking moiety that connects two groups via covalent bonds.
  • the linking group Y may be linear, branched, cyclic, a single atom, or a covalent bond.
  • linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, carbonate, alkyldioxy, oxyimino, amino, carbonyl, heterocycle (e.g., cyclic acetal) etc.
  • 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 C1-6 alkyl.
  • Y is selected from — O — , - OC(O) — , — C(0)0 — , and — OC(O)NR 10 — .
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • Y is — C(R 10 )2 — , where each R 10 is H.
  • Y is — C(O)O — .
  • Y is — OC(O)O — .
  • 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.
  • W n comprises a group G 1 adjacent to the point of attachment to linking group Y.
  • G 1 is cyclically linked with the linking group Y to provide a heterocycle.
  • G 1 is cyclically linked with Y to provide a 5-membered heterocycle.
  • the 5-membered heterocycle is a cyclic acetal.
  • G 1 is cyclically linked with Y to provide a 6-membered heterocycle.
  • the 6-membered heterocycle is a cyclic acetal.
  • the linking group Y is linked to an ionizable head group Z through an optionally substituted (Ci-Ci2)alkylene L.
  • L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene.
  • L is (C2-C4)alkylene or substituted (C2-C4)alkylene.
  • L is C2- alkylene or substituted C2-alkylene.
  • L is Cs-alkylene or substituted C3- alkylene.
  • L is C4-alkylene or substituted C4-alkylene.
  • L is Cs-alkylene or substituted Cs-alkylene. In certain cases, L is Ce-alkylene or substituted Ce- 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 -(CJfcje-
  • -Y-L-Z is of the formula — O(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)NH(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — CH2(CH2)rZ, where r is 2-6. In some cases, r is 2- 4. In some cases, r is 2. In some cases, r is 3. In some cases, r is 4.
  • the ionizable lipid of formula (I) includes an ionizable head group.
  • the ionizable head group includes a primary, secondary or tertiary amine that may be protonated at physiological pH.
  • the ionizable head group comprises a tertiary amino group.
  • the ionizable head group is of the formula -NR n R 12 , wherein R 11 and R 12 are each independently alkyl or substituted alkyl.
  • R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each independently C1-3 alkyl or substituted C1-3 alkyl.
  • R 11 and R 12 are each C1-3 alkyl. In some embodiments, R 11 and R 12 are each methyl. In some embodiments, R 11 and R 12 are each ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted secbutyl.
  • both R 11 and R 12 is independently an optionally substituted butyl.
  • 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.
  • each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, Ci-4 heteroalkyl, and C1-3 heteroalkyl.
  • formula (I) includes 2-5 lipid tails R, that are linked to the core W n , optionally via an additional linking group X (e.g., -(X-R)(n-i>).
  • the ionizable lipid of formula (I) includes a linking group X.
  • the linking group X may be linear, branched, cyclic or a single atom. Examples of such linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, thiocarbamate, carbonate, alkyldioxy, oxyimino, amino, carbonyl etc.
  • each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , — (CH 2 ) S OC(O)NR 10 — , — (CH 2 ) S O— , — (CH 2 ) s SC(O)NR 10 — , — (CH 2 ) S C(O)NR 10 — , — (CH 2 ) s NR 10 C(O)— , — (CH 2 )sS— , — (CH 2 ) S NR 10 — , — (CH 2 ) s NR 10 C(O)O— , and — (CH2)SNR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl and s is 0-6.
  • each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , and — (CH2) S OC(O)O — .
  • each X is — (CH2)sOC(O) — , where s is 0, 1 or 2.
  • each X is — (CH2)sC(O)O — , where s is 0, 1 or 2.
  • each X is — (CH2)sOC(O)O — , where s is 0, 1 or 2.
  • at least one X group is — (CH2)sO — , where s is 0, 1 or 2.
  • At least one X group is — (CH2)SOC(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)sSC(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)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)sNR 10 C(O) — , where R 10 is H and s is 0, 1 or 2.
  • At least one X group is — (CH2)sS — , where s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 10 — , where R 10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR 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)sNR 10 C(O)S — , where R 10 is H and s is 0, 1 or 2.
  • 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 Ci-6 alkyl.
  • 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.
  • 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.
  • each — X-R is independently selected from — (CH 2 )SOC(O)R, — (CH 2 )SC(O)OR, — (CH 2 )SOC(O)OR, — (CH 2 )SOR, — (CH 2 ) S OC(O)NR 10 R, — (CH 2 ) S SC(O)NR 10 R, — (CH 2 ) S C(O)NR 10 R, — (CH 2 ) S NR 10 C(O)R, — (CH 2 )SSR, — (CH 2 )SNR 10 R, — (CH 2 )SNR 10 C(O)OR, and — (CH 2 ) s NR 10 C(O)SR, wherein R 10 is selected from H and Ci-6 alkyl, s is 0-6 and each R is independently a lipid tail.
  • each — X-R is — (CH2)sOC(O)R. In some embodiments, each — X-R is — (CH2)SC(O)OR. In some embodiments, each — X-R is — (CH2)sOC(O)OR. In some embodiments, each — X-R is — (CH2)sOR. In some embodiments, each — X-R is — (CH2)SOC(O)NR 10 R. In some embodiments, each — X-R is — (CH2)sSC(O)NR 10 R. In some embodiments, each — X-R is — (CH2)sC(O)NR 10 R.
  • each — X-R is — (CH2)SNR 10 C(O)R. In some embodiments, each — X-R is — (CH2)sSR. In some embodiments, each — X-R is — (CH2)sNR 10 R. In some embodiments, each — X-R is — (CH2)SNR 10 C(O)OR. In some embodiments, each — X-R is — (CH2)sNR 10 C(O)SR.
  • 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 Ci-6 alkyl and each R is independently a lipid tail.
  • each — X-R is OC(O)R.
  • each — X-R is C(O)OR.
  • 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.
  • each — X-R is — NR 10 C(O)SR.
  • each lipid tail is independently an aliphatic hydrocarbon group that is straight chain or branched, saturated or unsaturated and/or optionally comprises a cyclic group.
  • each R is a linear hydrocarbon group optionally comprising one or more cyclic groups.
  • each R is a linear hydrocarbon group independently selected from a C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • each R is a linear hydrocarbon group independently selected from a C6-C12 alkyl, and C6-C12 alkenyl.
  • at least one R is a linear hydrocarbon group comprising a cyclic group.
  • the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
  • At least one R is a branched hydrocarbon group optionally comprising one or more cyclic groups.
  • each R is a branched hydrocarbon group optionally comprising one or more cyclic groups.
  • the branched hydrocarbon group comprises 8-20 carbon atoms.
  • the branched hydrocarbon group comprises 8 carbon atoms.
  • the branched hydrocarbon group comprises 9 carbon atoms.
  • the branched hydrocarbon group comprises 10 carbon atoms.
  • the branched hydrocarbon group comprises 11 carbon atoms.
  • the branched hydrocarbon group comprises 12 carbon atoms.
  • the branched hydrocarbon group comprises 13 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 14 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 15 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 16 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 17 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 18 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 19 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 20 carbon atoms. In some embodiments, the branched hydrocarbon group is saturated. In some embodiments, the branched hydrocarbon group is unsaturated.
  • R is of the formula - CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In some embodiments each R 7 is Cs-alkyl, or Cs-alkenyl. In some embodiments each R 7 is Ce-alkyl, or Ce-alkenyl. In some embodiments each R 7 is C?-alkyl, or C?-alkenyl. In some embodiments each R 7 is Cs-alkyl, or Cs-alkenyl. In some embodiments each R 7 is C9-alkyl, or C9-alkenyl. In some embodiments each R 7 is Cio-alkyl, or Cio-alkenyl.
  • each R 7 is Cn-alkyl, or Cn-alkenyl. In some embodiments each R 7 is Ci2-alkyl, or C12- alkenyl. In some embodiments, at least one R is a branched hydrocarbon group comprising a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
  • R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • the cyclic group is an optionally substituted monocyclic cycloalkyl.
  • the cyclic group is an optionally substituted bicyclic cycloalkyl.
  • the cyclic group is an optionally substituted monocyclic aryl group.
  • the cyclic group is an optionally substituted bicyclic aryl group.
  • the cyclic group is an optionally substituted monocyclic or bicyclic heterocyclic group.
  • the cyclic group is an optionally substituted monocyclic or bicyclic heteroaryl group.
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; r, p, and q is each independently an integer from 0 to 20.
  • R is wherein, Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group; r, p, and q is each independently an integer from 0 to 10.
  • R is wherein,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, Ci-Ce aliphatic group; r, p, and q is each independently an integer from 0 to 6.
  • R represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is , where each
  • # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain
  • R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. of R. In some embodiments, each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • R is each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain ofR.
  • the compound of formula (I) is of the formula (IIA):
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 H and Ci-6 alkyl.
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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 - (CH 2 )4-.
  • Z is a tertiary amine.
  • Z is -NR n R 12 , wherein R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each C1-3 alkyl.
  • R 11 and R 12 are each methyl.
  • R 11 and R 12 are each ethyl.
  • both R 11 and R 12 are propyl.
  • both R 11 and R 12 are n-propyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
  • each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • at least one X is — OC(O) — .
  • each X is — OC(O) — .
  • at least one X is — C(O)O — .
  • each X is — C(O)O — .
  • at least one X is — OC(O)O — .
  • each X is — OC(O)O— .
  • each -X-R is of the formula — OC(O)R. In certain embodiments, each -X-R is of the formula — C(O)OR. In certain embodiments, each -X-R is of the formula — OC(O)OR.
  • each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl.
  • each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl.
  • each R is C5-C12 alkyl.
  • each R is C5 alkyl.
  • each R is Ce alkyl.
  • each R is C7 alkyl.
  • each R is Cs alkyl.
  • each R is C9 alkyl.
  • each R is C10 alkyl.
  • each R is C11 alkyl.
  • each R is C12 alkyl.
  • At least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein).
  • R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • At least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • R is -(CH2)tJ(CH 2 )u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t is 1 to 5.
  • u is 1 to 5.
  • the compound is of formula (IIIA): UA) wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH2)tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • formula (IIA) the compound is of formula (IIIA), wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 is selected from H and C1-6 alkyl.
  • Y is — O — .
  • Y is — OC(O) — .
  • Y is — OC(O)NR 10 — , where R 10 is H.
  • q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl..
  • each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl.
  • each R is C11 alkyl. In certain cases, each R is C12 alkyl. [0060] In certain embodiments of formula (IIIA), at least one R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • 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 C1-6 alkyl.
  • Y is selected from — NHC(O)— , — NHC(O)O— , and — NHC(O)S— .
  • Y is — NHC(O)— .
  • Y is — NHC(O)O — .
  • Y is — NHC(O)S — .
  • L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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 - (CH 2 )4-.
  • Z is a tertiary amine.
  • Z is -NR n R 12 , wherein R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each C1-3 alkyl.
  • R 11 and R 12 are each methyl.
  • R 11 and R 12 are each ethyl.
  • both R 11 and R 12 are propyl.
  • both R 11 and R 12 are n-propyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl.
  • each -X-R is of the formula — OC(O)R. In certain embodiments of formula (IIB), each -X-R is of the formula — C(O)OR. In certain embodiments of formula (IIB), each -X-R is of the formula — OC(O)OR.
  • each R is selected from C5-C20 alkyl, Cs- C20 alkenyl, and a C5-C20 alkynyl.
  • each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl.
  • each R is C5-C12 alkyl.
  • each R is Cs alkyl.
  • each R is Ce alkyl.
  • each R is C7 alkyl.
  • each R is Cs alkyl.
  • each R is C9 alkyl.
  • each R is C10 alkyl.
  • each R is C11 alkyl.
  • each R is C12 alkyl.
  • At least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein).
  • R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a Cs-C 12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • R is -(CH2)tJ(CH 2 )u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t is 1 to 5.
  • u is 1 to 5.
  • the compound is of formula (IIIB): wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and - CH(R 7 )2, and -(CH2)tJ(CH2)u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group and t and u are each independently 1 to 10.
  • the compound is of formula (IIIB), wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Y is — NHC(O) — . In certain cases, Y is — NHC(O)O — . In certain cases, Y is — NHC(O)S — .
  • q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl.
  • each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
  • At least one R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkyl. In certain cases, each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t and u are each 1 to 3. In some cases, t is 2 and u is 3.
  • the compound of formula (I) is of the formula (IIC):
  • Y is selected from — C(R 10 )2 — and —
  • R 10 is selected from H and Ci-6 alkyl.
  • Y is — O — .
  • Y is — C(R 10 )2 — , where R 10 is H.
  • L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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 - (CH 2 )4-.
  • Z is a tertiary amine.
  • Z is -NR n R 12 , wherein R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • R 11 and R 12 are each C1-3 alkyl.
  • R 11 and R 12 are each methyl.
  • R 11 and R 12 are each ethyl.
  • both R 11 and R 12 are propyl.
  • both R 11 and R 12 are n-propyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 are isopropyl.
  • both R 11 and R 12 is independently an optionally substituted butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
  • each X is independently selected from — (CH2) S OC(O) — , — (CH2) S C(O)O — , — (CH2) S OC(O)O — , where s is 0-6. In some cases, at least one X is — (CH2)sOC(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) S C(O)O — , where s is 0, 1 or 2.
  • each X is — (CH2)sC(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)sOC(O)O — , where s is 0, 1 or 2. In certain cases, each X is — (CH2)sOC(O)O — , where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 0 and X is — OC(O)O — .
  • each -X-R is of the formula — (CH2)SOC(O)R, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2)SC(O)OR, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2) S OC(O)OR, where s is 0-6.
  • each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl.
  • each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl.
  • each R is C5-C12 alkyl.
  • each R is C5 alkyl.
  • each R is Ce alkyl.
  • each R is C7 alkyl.
  • each R is Cs alkyl.
  • each R is C9 alkyl.
  • each R is C10 alkyl.
  • each R is C11 alkyl.
  • each R is C12 alkyl.
  • At least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein).
  • R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C 12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C6-C9 alkenyl.
  • At least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups.
  • R is -(CH2)tJ(CH 2 )u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t is 1 to 5.
  • u is 1 to 5.
  • the compound is of formula (IIIC): wherein:
  • R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-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 — or — CH2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and - (CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
  • the compound is of formula (IIIC), wherein: R 11 and R 12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
  • Y is selected from — O — , and — C(R 10 )2 — ; s is 0 to 2;
  • W is O or CH2; and each R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Y is selected from — C(R 10 )2 — and — O — , wherein R 10 is selected from H and C1-6 alkyl.
  • Y is — O — .
  • Y is — C(R 10 )2 — , where R 10 is H.
  • q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
  • R 11 and R 12 are different. In certain cases, at least one of R 11 and R 12 is methyl. In certain cases, R 11 and R 12 are the same. In certain cases, both R 11 and R 12 are methyl. In certain cases, at least one of R 11 and R 12 is ethyl. In certain cases, both R 11 and R 12 are ethyl. In certain cases, both R 11 and R 12 are propyl. In certain cases, both R 11 and R 12 are n-propyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 are isopropyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl.
  • both R 11 and R 12 is independently an optionally substituted n-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted sec-butyl. In certain cases, both R 11 and R 12 is independently an optionally substituted butyl. 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(CH 3 )CH 2 OH, -CH 2 CH(OH)CH 3 , and -CH2CH2CH2OH. In certain cases, each R 11 and R 12 is independently selected from an optionally substituted Ci-4 alkyl, Ci- 3 alkyl, C 1-4 heteroalkyl, and Ci- 3 heteroalkyl.
  • W is — CH2 — . In certain embodiments of formula (IIIC), W is — O — .
  • s is 0. In certain embodiments, s is 1.
  • W is — CH2 — and s is 0. In certain cases,
  • W is — CH2 — and s is 1. In certain cases, W is — O — and s is 0.
  • each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
  • At least one R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C5-C12 alkenyl.
  • each R is -CH(R 7 )2 and each R 7 is a C6-C9 alkyl.
  • each R is -CH(R 7 )2 and each R 7 is a C6-C9 alkenyl.
  • R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10.
  • J is an aryl group.
  • J is phenyl.
  • t and u are each 1 to 3. In some cases, t is 2 and u is 3.
  • each R is independently wherein,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; r, p, and q is each independently an integer from 0 to 20.
  • R is wherein,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group;
  • r, p, and q is each independently an integer from 0 to 10.
  • R is wherein,
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, Ci-Ce aliphatic group; r, p, and q is each independently an integer from 0 to 6.
  • the lipid is selected from a compound of TableTable 1 :
  • the lipid nanoparticle compositions can include one or more additional ionizable lipid components in addition to the ionizable lipid of formula (I) (e.g., as described above). Any convenient lipid that carries a net positive charge at or around physiological pH may find use as an additional ionizable lipid in the compositions described herein.
  • Non-limiting examples of cationic lipids are described in detail herein.
  • Cationic lipids and related analogs which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. 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 herein incorporated by reference in their entirety for all purposes.
  • Additional cationic lipids of interest include, but are not limited to, l,2-distearydoxy-N,N- dimethyl-3-aminopropane (DSDMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDM ), l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDM ), 1,2- di ol ey I oxy -N,N-d methyl -3 -am nopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N — N- trieth
  • DC-Chol N-(l-(2,3-dioleyloxy)propyl)-
  • cationic lipids can be used, such as, e g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • a cationic lipid is an amino lipid including one or two fatty acyl or fatty alkyl chains.
  • LNPs of this disclosure can also include one or more helper lipid(s), in addition to the ionizable lipid component described herein.
  • the helper lipid is a neutral lipid.
  • the neutral lipid is zwitterionic, e.g., has an overall net zero charge.
  • Neural lipids include, for example, phospholipids, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
  • the selection of neutral lipids for use in the compositions described herein is generally guided by consideration of, e.g., LNP size and stability of the LNPs in the bloodstream.
  • the LNPs of this disclosure includes a helper lipid component that includes a neutral lipid that is a phospholipid.
  • Non-limiting examples of phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.
  • the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well- known techniques.
  • the neutral lipids include saturated fatty acids with carbon chain lengths in the range of Cio to C30.
  • neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Cio to C30 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • the neutral lipids may also be composed of sphingomyelin, or dihydrosphingomyeline.
  • the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG).
  • the phospholipid has a hydrocarbon chain, or “tail” having 12-24 carbons, e.g., 16-20 carbons, 18-22 carbons, 12-18 carbons.
  • phospholipid has a carbon tail of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons.
  • the phospholipid tail comprises no double bonds, i.e. the bonds are saturated bonds.
  • the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g., 1, 2, 3, 4 or 5 double bonds.
  • the phospholipid tail is unsaturated, that is, it comprises one or more triple bonds, e.g. 1, 2, 3, 4 or 5 triple bonds.
  • the phospholipid tail comprises one or more ring structures.
  • the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 R A groups; each R A is independently selected from halogen, or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl.
  • the ring structure is a cholesterol or cholesterol derivative.
  • 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 comprises two different hydrocarbon chains.
  • a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24 aliphatic.
  • the helper lipid comprises one or more optionally substituted and/or optionally bridged ring structures in the hydrophobic tail.
  • exemplary helper lipids of this class include:
  • the helper lipid includes a phosphatidylethanolamine (PE).
  • PE Phosphatidylethanolamines
  • the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (A9-Cis PE, or DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) (DPPE), dimyristoylphosphoethanol
  • 16-0-monom ethyl PE dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), di elaidoyl-phosphatidyl ethanolamine (DEPE), lysophosphatidyl ethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE).
  • SOPE l-stearoyl-2-oleoyl-phosphatidy ethanolamine
  • DEPE di elaidoyl-phosphatidyl ethanolamine
  • DLPE 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine
  • DiPPE diphytanoyl-sn-glycero-3
  • the phosphatidylethanolamine is dioleoylphosphatidylethanolamine (also referred to as l,2-dioleoyl- w-glycero-3- phosphoethanolamine, or (A9-Cis) PE, or DOPE), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:
  • the helper lipid includes a phosphatidylcholine (PC).
  • PC Phosphatidylcholines
  • phosphatidylcholine is selected from the group consisting of phosphatidylcholine, distearoylphosphatidylcholine (l,2-distearoyl-sn-glycero-3- phosphocholine) (DSPC), dioleoylphosphatidylcholine (l,2-dioleoyl-sn-glycero-3- phosphocholine) (A9-Cis PC, or DOPC), dipalmitoylphosphatidylcholine (1,2-dipalmitoyl- sn-glycero-3 -phosphocholine) (DPPC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoylphosphatid
  • the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as 1 ,2-distearoyl-.s//-glycero-3-phosphocholine), having a tail of 18 carbons and no saturated bonds (“18-0”) as shown below:
  • DSPC distearoylphosphatidylcholine
  • the phosphatidylcholine is dioleoylphosphatidycholine (also referred to as l,2-dioleoyl-sn-glycero-3-phosphocholine, (A9-Cis) PC or DOPC), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:
  • the phosphatidylcholine is l,2-dipalmitoyl-sn-glycero-3- phosphocholine (delta9-Cis PC), having a tail of 16 carbons and one saturated bond (“16-1”) as shown below:
  • the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons.
  • the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is l-palmitoyl-2- oleoyl-glycero-3 -phosphocholine (also referred to as “16-0/18-1 PC”, “16:0/18: 1 PC” or POPC) as shown below:
  • the phosphatidylcholine is 1,2- dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below:
  • the phosphatidylcholine is l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:
  • the phosphatidylcholine is l-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:
  • the phosphatidylcholine is l-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:
  • the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2- dioleoyl-sn-glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG).
  • the helper lipid includes a phosphatidylserine, e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
  • DOPS dioleoylphosphatidylserine
  • the helper lipid includes a lecithin, e.g. lecithin or lysolecithin.
  • the helper lipid includes a sphingomyelin (SM), e.g. egg sphingomyelin (ESM).
  • SM sphingomyelin
  • ESM egg sphingomyelin
  • the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate.
  • the LNP can further comprise a component, such as a sterol, to provide membrane integrity.
  • a component such as a sterol
  • a sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)- ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the component providing membrane integrity such as a sterol
  • the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof.
  • the helper lipid component includes cholesterol.
  • the LNP includes a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or a derivative or analog thereof.
  • the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.
  • 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. 4.5 Other Components
  • LNPs of this disclosure can also include one or more additional lipid components.
  • Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.
  • the LNP can further comprise a non-cationic lipid.
  • Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the noncationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid.
  • Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned 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 US patent publication US2018/0028664.
  • Non-limiting examples of non-cationic lipids include, nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, is
  • the LNP includes one or more lipids capable of reducing aggregation.
  • a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation.
  • the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.
  • a lipid capable of reducing aggregation of particles may comprise a conjugated lipid molecule, such as a polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization.
  • exemplary conjugated lipids include, but are not limited to, polyethyleneglycol (PEG)-lipid conjugate, polyoxazoline (POZ)-lipid conjugates, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof.
  • the LNPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
  • the lipid capable of reducing aggregation is a PEG-lipid.
  • a PEG-lipid refers to a lipid having one or more hydrocarbon tail(s) linked to one or more polyethylene glycol (PEG) moiety(ies) via an optional linker.
  • PEG polyethylene glycol
  • the PEG moieties may include terminal modification(s) to provide for e.g., conjugation to the lipid tails via the optional linker.
  • the PEG moiety may be terminated in as a hydroxyl group, or an alkyl ether (e.g., a methoxy terminal group).
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • PEG-lipids of interest include, but are not limited to, a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C 16), a PEG-distearyloxypropyl (C 18), or mixtures thereof.
  • Exemplary PEG-lipid conjugates include, but are not limited to, PEG- diacylglycerol (DAG) (such as 1 -(monomethoxy -poly ethyleneglycol)-2, 3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn
  • PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, and US2017/0119904.
  • a PEG- lipid is a compound disclosed in US2018/0028664.
  • a PEG-lipid is disclosed in US20150376115 or in US2016/0376224.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG-DMG, PEG- dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- omegal-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(poly ethylene glycol
  • the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • PEG-lipid conjugates polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • POZ polyoxazoline
  • CPL cationic-polymer lipid
  • conjugated lipids i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the International patent application publications WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO20 17/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO20 10/006282, US 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
  • the liver can be a target organ of interest in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).
  • the LNP further includes a component including a targeting ligand.
  • the targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure.
  • the targeting ligand is a ligand of a cell surface receptor.
  • the cell surface receptor is asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells.
  • the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand.
  • GalNAc N- acetylgalactosamine
  • a variety of GalNAc containing ligands and ligands, including multivalent GalNAc ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference
  • the PEG-lipid is linked to the targeting ligand.
  • the targeting ligand of interest e.g., as described herein
  • a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP.
  • the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a nucleic acid cargo (e.g., as described herein); an additional ionizable lipid (e.g., as described herein); a phospholipid (e.g., as described herein), cholesterol (e.g., as described herein); and a lipid capable of reducing aggregation (e.g., as described herein).
  • an ionizable lipid of Formula (I) e.g., as described herein
  • a nucleic acid cargo e.g., as described herein
  • an additional ionizable lipid e.g., as described herein
  • a phospholipid e.g., as described herein
  • cholesterol e.g., as described herein
  • a lipid capable of reducing aggregation e.g., as described herein.
  • the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof.
  • the nucleic acid consists essentially of DNA.
  • the nucleic acid cargo comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, or a chemically modified derivative thereof.
  • the nucleic acid consists essentially of RNA.
  • the nucleic acid cargo includes DNA, e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof, and further includes RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.
  • DNA e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof
  • RNA e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.
  • the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
  • the phospholipid is phosphatidylethanolamine (PE).
  • the phospholipid is a phosphatidylcholine (PC).
  • the phospholipid includes hydrocarbon chains each independently having 12-24 carbons. In some cases, the hydrocarbon chains each independently have 16-20 carbons. In certain cases, the hydrocarbon chains are saturated.
  • the hydrocarbon chains are unsaturated. In certain cases, the hydrocarbon chains each independently comprise 1-4 double bonds. In certain cases, the phospholipid comprises two different hydrocarbon chains. In certain embodiments of the LNP, the phospholipid includes dioleoylphosphatidylethanolamine (DOPE, 18: 1 PE). In certain cases, the phospholipid includes l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain cases, the phospholipid includes l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DOPE dioleoylphosphatidylethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3-phosphocholine
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • the phospholipid includes l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (delta9-Cis PC). In certain cases, the phospholipid includes l-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE). In certain cases, the phospholipid includes a mixture of di oleoylphosphatidylethanolamine (DOPE, 18-1) and di oleoylphosphatidy choline (DOPC, 18-1).
  • DOPE di oleoylphosphatidylethanolamine
  • DOPC di oleoylphosphatidy choline
  • the lipid capable of reducing aggregation is a PEG-lipid.
  • the PEG lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • the LNP further comprises a targeting ligand (e.g., as described herein).
  • the targeting ligand comprises GalNac.
  • the targeting ligand is linked to the ligand capable of reducing aggregation.
  • the lipid capable of reducing aggregation linked to the targeting ligand is PEG- l,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and/or PEG-DSG-GalNAc or PEG-DSPE-GalNac.
  • a ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group
  • DOPE phosphatidylethanolamine
  • the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g. l,2-distearoyl-sn-glycero-3 -phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.
  • the N/P ratio can be in the range of from about 1 : 1 to about 50: 1, from about 7: 1 to about 25:1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
  • the amounts of lipids and DNA can be adjusted to provide a desired N/P ratio, for example, 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.
  • N/P ratio 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.
  • the lipid particle formulation's overall lipid content can range from about 5 mg/mL to about 30 mg/mL.
  • the N/P ratio is from 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.
  • a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm.
  • a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.
  • the mean size e.g., diameter
  • an LNP has a mean diameter of 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.
  • an LNP has a mean diameter of 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
  • an LNP has a mean diameter of 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.
  • the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues.
  • a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly, the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes.
  • large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids.
  • the size of the LNPs is within the range of about 25 to 250 nm or 25nm to lOOnm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.
  • ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle.
  • ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle.
  • the ionizable lipid comprises from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cationic lipid comprises 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 lipid present in the particle.
  • the neutral lipid components can comprise 10-60% (mol) of the total lipid present in the lipid nanoparticle.
  • the non-cationic lipid content is 10-50% (mol) or 20- 55% (mol) of the total lipid present in the lipid nanoparticle.
  • the noncationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the non-cationic lipid comprises 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 thereof or range therein) of the total lipid present in the particle.
  • the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative
  • the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the mixture of phospholipid and cholesterol or a cholesterol derivative comprises up to 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 thereof or range therein) of the total lipid present in the particle.
  • the LNP comprises a phospholipid component in the mixture in an amount of from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the LNP includes a cholesterol component in the mixture in an amount of from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from 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 thereof or range therein) of the total lipid present in the particle.
  • mol percentage of components described herein in the LNP is a target amount, and that the actual amount of each lipid component present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the LNP includes a lipid capable of reducing aggregation (e.g., a PEG-lipid conjugate) in an amount of about 1.5% to about 4%, for example 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 thereof or range therein) of the total lipid present in the particle.
  • a lipid capable of reducing aggregation e.g., a PEG-lipid conjugate
  • 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 thereof or range therein) of the total lipid present in the particle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.
  • the lipid nanoparticles do not comprise any phospholipids.
  • the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • the LNP comprises: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • A+B+C+D 100%; d.
  • A+B+C+D 100% g.
  • A 40% - 60%
  • B 10% - 30%
  • C 20% - 45%
  • D 0% - 3%
  • A+B+C+D 100%; k.
  • a given lipid nanoparticle may include a cargo, or payload, to be delivered to cells.
  • cargos that comprise a polynucleotide.
  • the polynucleotide is a DNA.
  • DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the DNA may be circular, e.g., a plasmid, a nanoplasmid, a mini circle, a covalently closed circular DNA, a circular viral genome, and the like.
  • the DNA may be linear, e.g., a doggybone or other closed-end DNA, a linear viral genome, and the like.
  • the DNA may be multivalent, e.g., a 3DNA.
  • the DNA may be single stranded or double stranded or a hybrid of single and double stranded.
  • the DNA may be chemically modified.
  • the polynucleotide is an RNA.
  • RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure.
  • the RNA may be linear or it may be circular. It may be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), a circular RNA (circRNA). It may be chemically modified.
  • the one or more additional compounds can be a therapeutic agent.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected from any class suitable for the therapeutic objective.
  • the therapeutic agent can be selected according to the treatment objective and biological action desired.
  • the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate).
  • the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound).
  • the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways).
  • an immunosuppressant e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways.
  • different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention.
  • the additional compound is an immune modulating agent.
  • the additional compound is an immunosuppressant.
  • the additional compound is immune stimulatory agent.
  • lipid nanoparticle-encapsulated nucleic acid e.g., DNA
  • a pharmaceutically acceptable carrier or excipient e.g., water
  • the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients.
  • the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, 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, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, 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
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but 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, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxye
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methyl
  • the LNP compositions can be prepared by high energy mixing of ethanolic lipids with aqueous DNA at low pH which protonates the ionizable lipid and provides favorable energetics for DNA / lipid association and nucleation of particles.
  • the particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.
  • the LNPs and LNP pharmaceutical composition of the present disclosure when formulated with nucleic acids, are less toxic in vivo as 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, e.g. at least 2-fold less toxic, e.g. 3-fold, 4-fold or 5-fold less toxic, in some instances 10-fold, 20-fold, or 50-fold less toxic, in certain instances 100-fold less toxic.
  • less toxic it is meant eliciting a reduced immune response, e.g., characterized in a reduced amount of one or more cytokines upon administration to an organism.
  • the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be efficacious at delivering their nucleic acid cargo to the target cell of interest, including where LNPs and LNP pharmaceutical composition of the present disclosure are equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same industry standard LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g. 3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more.
  • more efficacious it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g. 10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more.
  • the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition.
  • PK pharmacokinetics
  • a therapeutic index or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective.
  • This improvement over the art makes them more amenable to delivering nucleic acids, including DNA, to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids, including DNA, to cells for research and for therapeutic applications.
  • the cells are typically contacted with the composition, e.g., LNP or pharmaceutical composition thereof, in amount effective to deliver the agent into the cytoplasm of the cell.
  • 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.
  • the present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of the disease being treated, and may 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 are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.
  • the present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject.
  • the route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
  • the present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament.
  • the medicament is for expressing a protein in a cell.
  • the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein.
  • the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein.
  • the medicament is for the treatment of a cancer.
  • the medicament is for immunization against a disease. 4.12 Utility
  • the subject methods and compositions can be used in any application where delivery of a cargo nucleic acid is desired.
  • Applications of interest include both research and therapeutic applications.
  • Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications.
  • cargo nucleic acids that may be introduced into a cell, and subsequently a nucleus, via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.
  • Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure.
  • the research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR/Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3/4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.
  • transcription modulators of inducible expression systems include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and
  • Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure.
  • the diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.
  • Proteins of interest further include therapeutic proteins.
  • Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.
  • Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely.
  • Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like.
  • Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell.
  • the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type.
  • the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells).
  • the target cell may be any differentiated cell type found in a subject.
  • Cells of interest include both 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, etc.
  • Targeted cells may include the cells of a targeted location, such as, e.g., the liver, or cells near or adjacent to hepatocytes, e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.
  • hepatocytes e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.
  • HSCs hepatic stellate cells
  • KCs Kupffer cells
  • LSECs liver sinusoidal endothelial cells
  • ductal cells or combinations thereof.
  • the application of interest is a therapeutic application, for example, in the treatment of a disease.
  • the compositions and methods of the present application may be used to deliver a nucleic acid sequence to a cell to complement a genetic deficiency.
  • compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.
  • the terms "individual,” “subject” and “host” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • the subject is a patient.
  • the subject is a human patient.
  • the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI).
  • GOI gene-of-interest
  • the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later.
  • the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.
  • treatment used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated.
  • a parameter e.g., a symptom
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
  • an effective amount means a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition.
  • the term “therapeutically effective amount” therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment.
  • An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount” can be determined by one of ordinary skill in the art using routine experimentation.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject.
  • pharmaceutically acceptable excipient can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
  • the terms “may,” “optional,” “optionally,” or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
  • 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 wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
  • Acyl refers to the groups 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)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl-C(
  • alkyl refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
  • alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms.
  • lower alkyl intends an alkyl group of 1 to 6 carbon atoms.
  • heteroatom-containing alkyl and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
  • substituted alkyl is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 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, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thio
  • alkenyl refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like.
  • alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms.
  • lower alkenyl intends an alkenyl group of 2 to 6 carbon atoms.
  • substituted alkenyl refers to alkenyl substituted with one or more substituent groups
  • heteroatom-containing alkenyl and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom.
  • alkenyl and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
  • alkynyl refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms.
  • substituted alkynyl refers to alkynyl substituted with one or more substituent groups
  • heteroatomcontaining alkynyl and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom.
  • alkynyl and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
  • aryl refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety).
  • Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms.
  • aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl -substituted aryl, etc.).
  • substituted aryl refers to an aryl moiety substituted with one or more substituent groups
  • heteroatomcontaining aryl and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra.
  • Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, Ci-Cs alkoxy, Ci-Cs 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.
  • alkylene refers to a di-radical 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 alkylene linkages containing from 1 to 6 carbon atoms. Examples include, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)e-), and the like.
  • alkenylene refers to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
  • amino refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,
  • Heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring.
  • the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N— >0), sulfinyl, or sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, 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, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thio
  • heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms.
  • ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -SO2- moieties.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, 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,
  • heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
  • substituted as in “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents.
  • substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-Ce alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2- Ce alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl).
  • C1-C24 alkyl including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-Ce al
  • hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
  • linking or "linker” as in “linking group,” “linker moiety,” etc., is meant a linking moiety that connects two groups via covalent bonds.
  • the linker may be linear, branched, cyclic or a single atom.
  • linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH- CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (- O-(CH2)n-O-), epoxyimino (-0-NH-), epimino (-NH-), carbonyl (-CO-), etc.
  • one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
  • the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g., (Ci-Ci2)alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), and the like.
  • poly(ethylene glycol) unit(s) e.g., -(CH2-CH2-O)-
  • ethers e.g., thioethers, amines
  • alkyls e.g., (Ci-Ci2)alkyl
  • alkyls e.g., (Ci-Ci2)alkyl
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
  • a linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used.
  • substituted When the term "substituted" appears prior or after a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase “substituted alkyl and aryl” is to be interpreted as “substituted alkyl and substituted aryl.”
  • substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
  • 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 ion, such as [Ca 2+ ]o.5, [Mg 2+ ]o.5, or [Ba 2+ ]o.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 )4
  • -NR 80 R 80 is meant to include -NH2, -NH-alkyl, A-pyrrolidinyl, 7V-piperazinyl, 47V- methyl-piperazin-l-yl and 7V-morpholinyl.
  • substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -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 , -
  • R 60 , R 70 , R 80 and M + are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O'M + , -OR 70 , -SR 70 , or -S“M + .
  • substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O M + , -OR 70 , -SR 70 , -S'M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O) 2 R 70 , -S(O)2O'M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS( O) 2 O'M + , -OS(O) 2 OR 70 , -P(O)(O-) 2 (M + )2, -P(O)(OR 70 )O'M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , - C
  • R 70 , R 80 and M + are as previously defined.
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • arylalkyloxycarbonyl refers to the group (aryl)-(alkyl)-O-C(O)-.
  • any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • a substituent may contribute to optical isomerism and/or stereo isomerism of a compound.
  • Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure.
  • the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof.
  • a compound may be a metabolized into a pharmaceutically active derivative.
  • a bond designated as — in a small molecule structure refers to a bond that, in some embodiments, is a single (e.g., saturated) bond, and in some embodiments, is a double (e.g., unsaturated) bond.
  • the following structure is intended to encompass both and
  • reference to an atom is meant to include isotopes of that atom.
  • reference to H is meant to include 1 H, 2 H (i.e., D) and 3 H (i.e., T)
  • reference to C is meant to include 12 C and all isotopes of carbon (such as 13 C).
  • Embodiment 1 An ionizable lipid compound of formula (I): (Z-L-Y)-W n -(X-R) ( n-i)
  • Embodiment 2 The compound of embodiment 1, wherein n is 4 to 6.
  • Embodiment 3 The compound of embodiment 2, wherein n is 4.
  • Embodiment 4 The compound of embodiment 2, wherein n is 5.
  • Embodiment 5 The compound of embodiment 2, wherein n is 6.
  • Embodiment 6 The compound of embodiment 1, wherein n is 3.
  • Embodiment 7 The compound of any one of embodiments 1 to 6, wherein W selected from: wherein:
  • G 1 is H, or a group cyclically linked with Y that together with the carbon atom of W n to which they are attached provide a heterocycle; and G 2 is H or -CH2OH.
  • Embodiment 8 The compound of any one of embodiments 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 C1-6 alkyl.
  • Embodiment 9 The compound of embodiment 8, wherein Y is selected from — O — , — OC(O)— , and — OC(O)NR 10 — .
  • Embodiment 10 The compound of embodiment 8, wherein Y is — CH2 — .
  • Embodiment 11 The compound of embodiment 7, wherein G 1 is a group that is cyclically linked with Y and together with the carbon atom of W n to which they are attached provides a heterocycle.
  • Embodiment 12 The compound of any one of embodiments 1 to 11, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
  • Embodiment 13 The compound of embodiment 12, wherein L is -(CH 2 ) 2 -.
  • Embodiment 14 The compound of embodiment 12, wherein L is -(CH 2 )3- Embodiment 15. The compound of any one of embodiments 1 to 14, wherein Z comprises a tertiary amino group.
  • Embodiment 16 The compound of embodiment 15, wherein Z is -NR U R 12 , wherein R 11 and R 12 are each independently alkyl or substituted alkyl.
  • Embodiment 17 The compound of embodiment 16, wherein R 11 and R 12 are each C1-6 alkyl.
  • Embodiment 18 The compound of embodiment 17, wherein R 11 and R 12 are each C1-3 alkyl.
  • Embodiment 19 The compound of embodiment 18, wherein R 11 and R 12 are each methyl.
  • Embodiment 20 The compound of embodiment 18, wherein R 11 and R 12 are each ethyl.
  • Embodiment 21 The compound of any one of embodiments 1 to 20, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH 2 )sOC(O)O— ,— (CH 2 ) S OC(O)NR 10 — , — (CH 2 )sO— , — (CH 2 ) s SC(O)NR 10 — , — (CH 2 ) S C(O)NR 10 — , — (CH 2 ) s NR 10 C(O)— , — (CH 2 ) S S— , — (CH 2 ) S NR 10 — , — (CH2)SNR 10 C(O)O — , and — (CH2)sNR 10 C(O)S — , wherein R 10 is selected from H and C1-6 alkyl and s is 0-6.
  • Embodiment 22 The compound of any one of embodiments 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 C1-6 alkyl.
  • Embodiment 23 The compound of embodiment 22, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— .
  • Embodiment 24 The compound of embodiment 23, wherein each — X-R is — OC(O)R.
  • Embodiment 25 The compound of any one of embodiments 1 to 24, wherein each R is independently an aliphatic hydrocarbon group that is straight chain or branched, saturated or unsaturated and/or optionally comprises a cyclic group.
  • Embodiment 26 The compound of any one of embodiments 1 to 25, wherein each R is a linear hydrocarbon group optionally comprising one or more cyclic groups.
  • Embodiment 27 The compound of any one of embodiments 1 to 26, wherein each R is selected from a C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 28 The compound of embodiment 27, wherein each R is selected from a C6-C12 alkyl, and C6-C12 alkenyl.
  • Embodiment 29 The compound of embodiment 26, wherein at least one R is a linear hydrocarbon group comprising a cyclic group.
  • Embodiment 30 The compound of embodiment 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
  • Embodiment 31 The compound of any one of embodiments 1 to 25, wherein at least one R is a branched hydrocarbon group optionally comprising a cyclic group.
  • Embodiment 32 The compound of embodiment 31, wherein each R is a branched hydrocarbon group.
  • Embodiment 33 The compound of embodiment 32, wherein the branched hydrocarbon group comprises 8-20 carbon atoms.
  • Embodiment 34 The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is saturated.
  • Embodiment 35 The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is unsaturated.
  • Embodiment 36 The compound of any one of embodiments 31 to 35, wherein R is - CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 37 The compound of embodiment 31, wherein at least one R is a branched hydrocarbon group comprising a cyclic group.
  • Embodiment 38 The compound of embodiment 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
  • Embodiment 39 The compound of embodiment 1, wherein the compound is of formula (IIA):
  • Embodiment 40 The compound of embodiment 39, wherein Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — , wherein R 10 is selected from H and Ci-6 alkyl.
  • Embodiment 41 The compound of embodiment 40, wherein Y is — O — .
  • Embodiment 42 The compound of embodiment 40, wherein Y is — OC(O) — ,
  • Embodiment 43 The compound of embodiment 40, wherein Y is — OC(O)NR 10 — .
  • Embodiment 44 The compound of any one of embodiments 39 to 43, wherein L is (C2-
  • Embodiment 45 The compound of embodiment 44, wherein L is -(CH2)2-
  • Embodiment 46 The compound of embodiment 44, wherein L is -(CEb)?-.
  • Embodiment 47 The compound of embodiment 44, wherein L is -(CH2)4-
  • Embodiment 48 The compound of any one of embodiments 39 to 47, wherein Z is -
  • R 11 and R 12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
  • Embodiment 49 The compound of embodiment 48, wherein R 11 and R 12 are each Ci -3 alkyl.
  • Embodiment 50 The compound of embodiment 49, wherein R 11 and R 12 are each methyl.
  • Embodiment 51 The compound of embodiment 49, wherein R 11 and R 12 are each ethyl.
  • Embodiment 52 The compound of any one of embodiments 39 to 51, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • Embodiment 53 The compound of any one of embodiments 39 to 52, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 54 The compound of any one of embodiments 39 to 52, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
  • Embodiment 55 The compound of embodiment 54, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 56 The compound of embodiment 39, wherein the compound is of formula (IIIA): wherein:
  • R 11 and R 12 are each independently C1-3 alkyl; q is 1 to 4;
  • Y is selected from — O — , — OC(O) — , and — OC(O)NR 10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • Embodiment 57 The compound of embodiment 1, wherein the compound is of formula (IIB):
  • Embodiment 58 The compound of embodiment 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 C1-6 alkyl.
  • Embodiment 59 The compound of embodiment 58, wherein Y is selected from — NHC(O)— , — NHC(O)O— , and — NHC(O)S— .
  • Embodiment 60 The compound of any one of embodiments 57 to 59, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
  • Embodiment 61 The compound of embodiment 60, wherein L is -(CH2)2- Embodiment 62.
  • Embodiment 63 The compound of embodiment 60, wherein L is -(CH2)4-
  • Embodiment 64 The compound of any one of embodiments 57 to 59, wherein Z is -
  • R 11 and R 12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
  • Embodiment 65 The compound of embodiment 64, wherein R 11 and R 12 are each C1-3 alkyl.
  • Embodiment 66 The compound of embodiment 64, wherein R 11 and R 12 are each methyl.
  • Embodiment 67 The compound of any one of embodiments 57 to 66, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
  • Embodiment 68 The compound of any one of embodiments 57 to 67, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 69 The compound of any one of embodiments 57 to 67, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic groups.
  • Embodiment 70 The compound of embodiment 69, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 71 The compound of embodiment 57, wherein the compound is of formula (IIIB): wherein:
  • R 11 and R 12 are each independently C1-3 alkyl; q is 1 to 4; Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
  • Embodiment 72 The compound of embodiment 1, wherein the compound is of formula (IIC):
  • Embodiment 73 The compound of embodiment 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 Ci -6 alkyl.
  • Embodiment 74 The compound of embodiment 73, wherein Y is — O — .
  • Embodiment 75 The compound of embodiment 73, wherein Y is — C(R 10 )2 — . — .
  • Embodiment 76 The compound of any one of embodiments 72 to 75, wherein L is (C2-
  • Embodiment 77 The compound of any one of embodiments 76, wherein L is -(CH2)2-
  • Embodiment 78 The compound of any one of embodiments 76, wherein L is -(CTb)?-.
  • Embodiment 79 The compound of any one of embodiments 76, wherein L is -(CEb ⁇ -.
  • Embodiment 80 The compound of any one of embodiments 72 to 79, wherein Z is -
  • R 11 and R 12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
  • Embodiment 81 The compound of embodiment 80, wherein R 11 and R 12 are each C1-3 alkyl.
  • Embodiment 82 The compound of embodiment 81, wherein R 11 and R 12 are each methyl.
  • Embodiment 83 The compound of any one of embodiments 72 to 82, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , wherein s is 0-6.
  • Embodiment 84 The compound of any one of embodiments 72 to 82, wherein each s is Embodiment 85.
  • Embodiment 86 The compound of any one of embodiments 72 to 82, wherein each s is 3.
  • Embodiment 87 The compound of any one of embodiments 72 to 86, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
  • Embodiment 88 The compound of any one of embodiments 72 to 86, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
  • Embodiment 89 The compound of embodiment 88, wherein R is -CH(R 7 )2, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
  • Embodiment 90 The compound of embodiment 72, wherein the compound is of formula (IIIC): wherein:
  • R 11 and R 12 are each independently selected from C1-3 alkyl and Ci-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-C20 alkyl, C5-C20 alkenyl, -CH(R 7 )2, and -(CH 2 )tJ(CH 2 )u, wherein each R 7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
  • Embodiment 91 The compound of any one of embodiments 1 to 90, wherein each R is independently
  • Cy A and Cy B is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
  • R x and R y is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; and r, p, and q is each independently an integer from 0 to 20.
  • Embodiment 92 The compound of embodiment 91, wherein at least one R comprises a
  • each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
  • Embodiment 93 A lipid nanoparticle comprising an ionizable lipid compound according to any one of embodiments 1 to 92.
  • Embodiment 94 The lipid nanoparticle of embodiment 93, further comprising a neutral lipid and a lipid capable of reducing aggregation.
  • Embodiment 95 The lipid nanoparticle of embodiment 94, wherein the neutral lipid comprises a phospholipid.
  • Embodiment 96 The lipid nanoparticle of embodiment 94 or 95, wherein the neutral lipid comprises cholesterol.
  • Embodiment 97 The lipid nanoparticle of embodiment 96, comprising: a. a nucleic acid, b. an ionizable lipid, c. a phospholipid, d. cholesterol, and e. a lipid capable of reducing aggregation.
  • Embodiment 98 The lipid nanoparticle of embodiment 97, wherein the nucleic acid comprises DNA.
  • Embodiment 99 The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises RNA.
  • Embodiment 100 The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises DNA and RNA.
  • Embodiment 101 The lipid nanoparticle of embodiment 100, wherein the RNA is selected from mRNA, gRNA, and siRNA.
  • Embodiment 102 The lipid nanoparticle of any one of embodiments 97 to 101, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • Embodiment 103 The lipid nanoparticle of embodiment 102, wherein the phospholipid is a phosphatidylethanolamine (PE).
  • PE phosphatidylethanolamine
  • Embodiment 104 The lipid nanoparticle of embodiment 103, wherein the phospholipid is a phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • Embodiment 105 The lipid nanoparticle of any one of embodiments 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
  • Embodiment 106 The lipid nanoparticle of embodiment 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
  • Embodiment 107 The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are saturated.
  • Embodiment 108 The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are unsaturated and/or further comprise a carbocyclyl.
  • Embodiment 109. The lipid nanoparticle of embodiment 108, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
  • Embodiment 110 The lipid nanoparticle of any one of embodiments 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.
  • Embodiment 111 The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l,2-dioleyl-sn-glycero-3 -phosphoethanolamine (DOPE).
  • DOPE l,2-dioleyl-sn-glycero-3 -phosphoethanolamine
  • Embodiment 112. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE).
  • SOPE l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine
  • Embodiment 113 The lipid nanoparticle of embodiment 104, wherein the phospholipid comprises l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (A9A9-Cis PC).
  • Embodiment 114 The lipid nanoparticle of embodiment 106, wherein the lipid nanoparticle comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • Embodiment 115 The lipid nanoparticle of embodiment 104, wherein the lipid nanoparticle comprises l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • Embodiment 116 The lipid nanoparticle of any one of embodiments 103 to 115 wherein the lipid capable of reducing aggregation is a PEG-lipid.
  • Embodiment 117 The lipid nanoparticle of embodiment 116, wherein the PEG- lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG- DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG- DSG[2K]).
  • Embodiment 118 The lipid nanoparticle of any one of embodiments 94 to 117, further comprising a targeting ligand.
  • Embodiment 119 The lipid nanoparticle of embodiment 118, wherein the targeting ligand comprises GalNAc.
  • Embodiment 120 The lipid nanoparticle of embodiment 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.
  • Embodiment 121 The lipid nanoparticle of embodiment 120, wherein the lipid capable of reducing aggregation is PEG-l,2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
  • Embodiment 122 The lipid nanoparticle of any one of embodiments 94 to 121, wherein the N/P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30.
  • Embodiment 123 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 7.
  • Embodiment 124 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 14.
  • Embodiment 125 The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 28.
  • Embodiment 126 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 6 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 127 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 128 The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. e) an ionizable lipid at 40 to 49 mol % of the total lipid present; b. f) a phospholipid at 10 to 20 mol % of the total lipid present; c. g) cholesterol at 35 to 45 mol % of the total lipid present; and d. h) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
  • Embodiment 129 A pharmaceutical composition comprising a lipid nanoparticle of any one of embodiments 94 to 128 and a pharmaceutically acceptable excipient, carrier, or diluent.
  • Embodiment 130 A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of embodiments 94 to 128.
  • Embodiment 132 The method according to embodiment 130, wherein the cell is in vivo.
  • Embodiment 133 A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of embodiment 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.
  • LNP formulation LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA/mRNA mixtures) as described in Prud’Neill et al. (J Pharm Sci 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG- lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range pH 3.0 - 4.0.
  • nucleic acid e.g., DNA only, mRNA only, or DNA/mRNA mixtures
  • 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 pH range of 5.0-6.5.
  • the diluted LNP is subjected to dialysis purification against a secondary buffer with the pH range of 7.0-8.0.
  • the LNP solution is concentrated by using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through 0.2 pm PES sterilizing-grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.
  • EPO and cytokine detection in serum Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis.
  • the serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions.
  • the serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse Prolnflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.
  • the plasma samples may be stored at -80C from collection until analysis.
  • the plasma levels of human FIX following administration of LNPs were quantified using a U-Plex assay on the MSD platform. Briefly, a monoclonal mouse anti-human FIX antibody (Prolytix, clone AHIX-5041) was conjugated to biotin and used as the capture reagent on streptavidin-coated plates.
  • a polyclonal goat anti-human FIX antibody (Cedarlane, clone CL20040AP) was conjugated to Sulfo-TAG and used as the detection reagent with the standard setup for quantification of electrochemiluminescence (ECL) signal using the QuickPlex SQ 120MM instrument from MSD.
  • ECL electrochemiluminescence
  • Pooled normal human plasma (Affinity Biologicals, FRNCP0125), which is a pool of normal citrated human plasma collected from a minimum of 20 donors, was used to generate a standard curve and calculate % of normal human FIX levels. The assay was confirmed to be specific for human FIX and not to cross-react with mouse FIX, demonstrating very low levels of background in untreated mouse plasma samples.
  • silica gel type: ZCX-2, 100-200 mesh, 20 w/w.
  • silica gel type: ZCX-2, 100-200 mesh, 10 w/w.
  • the product was purified using CombiFlash, then eluted with CH2CI2 / MeOH (9:1) (gradient from 100:0 to 90: 10, collected every 200 ⁇ 10 mL).
  • the reaction solution was decompressed and vacuum concentrated, 200 mL DCM was added, and washed with 5% citric acid solution (3*100), then washed with saturated salt water three times, dried with anhydrous sodium sulfate and concentrated.
  • the organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.).
  • the resulting mixture was extracted with heptane (3 xl50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5 pm; mobile phase, B: C LCN, A: Water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.). The aqueous layer was extracted with heptane (3x100 mL). The resulting mixture was concentrated under reduced pressure.
  • Trideca-l,12-dien-7-ol (10 g, 50.934 mmol, 1 equiv) and Toluene (200 mL) at room temperature.
  • NaH 4.00 g, 166.554 mmol, 3.27 equiv
  • the resulting mixture was stirred for an additional overnight at 85°C.
  • (3- chloropropyl) dimethylamine hydrochloride (15.28 g, 101.868 mmol, 2 equiv) in portions at 80°C. The resulting mixture was stirred for an additional 8 h at 80°C.
  • the resulting mixture was stirred for an additional 16 h at room temperature.
  • the resulting mixture was diluted with water (50 mL).
  • the resulting mixture was extracted with heptane (3x20 mL).
  • the combined organic layers were washed with sat. Na2CCh (2x20 mL), Me0H/H20 (4: 1, 5x20 mL), H2O (3x20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% TFA), 35% to 70% gradient in 10 min; detector, Ms.
  • the resulting mixture was stirred for 18 h at 45°C under N2 atmosphere.
  • the resulting mixture was diluted with DCM (120 mL).
  • the combined organic layers were washed with 0.05M HC1 (1x100 mL) and Brine (1x200 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C.
  • silica gel type: ZCX-2, 100-200 mesh, 20.00 w/w.
  • the last step prepared dry silica gel which absorbed the reaction mixture.
  • Eluted with PE / EA (5: 1) grade from 20: 1 to 4: 1, collected every 200 ⁇ 10 mL).
  • silica gel type: ZCX-2, 100-200 mesh, 20.00 w/w.
  • the last step prepared dry silica gel which absorbed the reaction mixture.
  • Eluted with CH2CI2 / MeOH (20: 1) gradient from 50: 1 to 10: 1, collected every 200 ⁇ 10 mL).
  • the resulting mixture was filtered, the filter cake was washed with EA (2x100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in Heptane (150 mL). The resulting mixture was washed with 2x100 mL of ACN/H2O (5: 1, 50 mL), 1x100 mL of ACN (50 mL). The heptane phase was concentrated under reduced pressure.
  • the reaction was quenched by the addition of sat. NH4Q (aq.) (300 mL) at 0°C.
  • the resulting mixture was extracted with Heptane (3 x 200 mL).
  • the combined organic layers were washed with aqueous sat. Na2CCh (2x50 mL), water (2x50 mL), MeOH / H2O (4: 1, 4x50 mL), water (2x50 mL) and brine (1x50 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting mixture was concentrated under reduced pressure.
  • the resulting mixture was diluted with heptane (500 mL).
  • the solid was filtered out; the filter cake was washed with heptane (2x50 mL).
  • the filtrate was washed with 2x200 mL of water, 3x200 mL of water/MeOH (1 :4), 2x200 mL of aqueous citric acid (5% w/w), 2x200 mL of sat. NaHCCh and brine (200 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting mixture was stirred for an additional 2 h at 40°C.
  • the resulting mixture was diluted with water (20 mL).
  • the resulting mixture was extracted with CH2CI2 (3 x 20 mL).
  • the combined organic layers were washed with brine (1 x 20 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • the resulting solution was diluted with 250 mL of DCM.
  • the resulting solution was washed with 1 x 200 mL of citric acid (5%, aq.), 2 x 200 mL of water, and 1 x 200 mL of brine, dried with anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
  • silica gel type: ZCX-2, 100-200 mesh, 1.5 w./w.
  • This mixture was then concentrated to no fraction under vacuum while maintaining the temperature below 35 °C.
  • 700 g silica gel type: ZCX-2, 100- 200 mesh, 9.0 w./w.
  • combi-flash to purify the product.
  • Eluted with n-heptane/EA (Gradient from 100:0 to 90: 10).
  • Example 21 Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris( ⁇ [3-(octahydro-lH- inden-2-yl)propanoyl]oxy ⁇ )pentan-2-yl 3-(octahydro-lH-inden-2-yl)propanoate oxylic acid
  • reaction mixture was stirred at 20 °C for 10 min, NalCb (3.09 g, 14.430 mmol, 5.0 equiv) and 2,6-lutidine (1.55 g, 14.430 mmol, 5.0 equiv) was added at 20 °C.
  • the reaction mixture was stirred at 20 °C for 18 h.
  • the resulting mixture was diluted with EA (200 mL).
  • the resulting mixture was washed with 3 x 60 mL of water, brine (60 mL).
  • the resulting solution was dried over anhydrous Na2SO4.
  • the resulting mixture was concentrated under reduced pressure.
  • the resulting mixture was washed with 3x28 mL of water, brine (28 mL), dried over anhydrous Na2SO4. After filtration. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.05% CF3COOH), 30% to 90% gradient in 26 min; detector, ELSD. The collect liquid was added 28 mL NaHCOs (2M, aq). The resulting mixture was extracted with n-Heptane (3 x 70 mL). The combined organic layers were washed with 3 x 28 mL of MeOH/H2O(4/l), water (28 mL).
  • L-24 is synthesized in a manner similar to L-18, substituting 4-oxoheptanedioic acid instead of 5-oxononanedioic acid.
  • Example 25 Synthesis of 3-(3-(dimethylamino)propoxy)-l,5-bis((3-(octahydro-lH- inden-2-yl)propanoyl)oxy)pentane-2,4-diyl bis(decanoate) (L-25)
  • L-25 is synthesized in a manner similar to L-21, but instead the xylitol core is acylated first with 3-(octahydro-lH-inden-2-yl)propanoic acid at primary then the secondary alcohols are acylated with decanoyl chloride to provide the final product.
  • L-26 is synthesized in a manner similar to L-5 and L-23.
  • Example 27 Preparation and analysis of lipid nanoparticle formulations with varying ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above and detailed in Fig. 1 A.
  • LNPs lipid nanoparticles
  • Wild type female BALB/c mice (approximately 7-8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 5 mL/kg body weight.
  • the DNA-LNPs were administered at 1 mg/kg based on the weight of the DNA payload.
  • blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. IB.
  • the DNA-LNP formulation with ionizable lipid L-3 produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC-0315 (FIG. IB).
  • the DNA-LNP formulation with ionizable lipid L-2 produced measurable but somewhat lower EPO expression levels, as did the exemplary ionizable lipid MC3 (FIG. IB).
  • blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 1C.
  • the DNA-LNP formulations with ionizable lipids L-2 and L-3 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid MC3, which were higher than the IL-6 levels generated by formulations with the exemplary ionizable lipid ALC-0315 (FIG. 1C).
  • Example 28 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 2A.
  • the LNP formulations using DOPE as the phospholipid are detailed in Fig. 2B.
  • Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Seven days after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 2C.
  • the DNA-LNP with ionizable lipid L-15 When formulated with DSPC, the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315, MC3, and LP01, and comparable to the exemplary ionizable lipids SM102 and ARCT (FIG. 2C).
  • the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids MC3, LP01, and SM102, and comparable to the exemplary ionizable lipids ALC-0315 and ARCT (FIG. 2C).
  • the DNA-LNPs with ionizable lipid L-9 When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-9 produced somewhat lower EPO expression levels than L-15, comparable to several of the exemplary ionizable lipids (FIG. 2C). When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-5 produced substantially lower EPO expression levels, below the levels of the exemplary ionizable lipids (FIG. 2C). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 2D.
  • the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid ALC-0315, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2D).
  • the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of IL-6 that were generally similar compared to all the exemplary ionizable lipids (FIG. 2D).
  • the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipid ALC-0315, which were typically lower than the cytokine levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2E).
  • the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of cytokines that were generally similar compared to all the exemplary ionizable lipids (FIG. 2F).
  • Example 29 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 3 A.
  • the LNP formulations using DOPE as the phospholipid are detailed in Fig. 3B.
  • Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload.
  • blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 3C.
  • the DNA-LNP with ionizable lipid L-12 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C).
  • the DNA-LNPs with ionizable lipids L-13 and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipid ssOP, and comparable to the exemplary ionizable lipid ALC-0315 (FIG. 3C).
  • the DNA- LNPs with ionizable lipids L-12, L-13, and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC-0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C).
  • IL-6 levels in serum determined as presented in FIG. 3D.
  • the DNA-LNP with ionizable lipid L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D).
  • the DNA-LNPs with ionizable lipids L-12 and L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid A9, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids ALC-0315 and ssOP (FIG. 3D).
  • the DNA-LNPs with ionizable lipids L- 12 and L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D).
  • the DNA-LNP with ionizable lipid L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP (FIG. 3D).
  • blood was collected via a retro-orbital bleed and serum levels of multiple cytokines determined as presented in FIG. 3E and FIG. 3F.
  • the DNA-LNPs with ionizable lipids L-12, L-13, and L-14 produced generally similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP, although for several cytokines L-14 produced the lowest levels and ssOP produced the highest levels (FIG. 3E).
  • the DNA-LNPs with ionizable lipids L-12, L-13, and L-14 produced generally similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP, although for several cytokines L-14 produced the lowest levels (FIG. 3F).
  • Example 30 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 4A.
  • Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs are administered at 1 or 0.3 mg/kg based on the weight of the DNA payload.
  • blood is collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 4B.
  • the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipid ALC-0315 (FIG. 4B).
  • the DNA-LNPs with ionizable lipids L-9 and L-ll produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC- 0315 (FIG. 4B).
  • the DNA-LNPs with ionizable lipids L-16 and L-10 produced measurable EPO expression levels, somewhat lower than the exemplary ionizable lipid ALC-0315 (FIG. 4B).
  • blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 4C.
  • the DNA-LNPs with ionizable lipids L-15, L-9, L-16, L-10, and L- 11 produced generally similar serum levels of IL-6 compared to the exemplary ionizable lipid ALC-0315, with L-16 resulting in the highest IL-6 levels (FIG. 4C).
  • Example 31 Preparation and analysis of further LNP formulations with varying additional ionizable lipids
  • DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above.
  • LNPs lipid nanoparticles
  • the LNP formulations using DSPC as the phospholipid are detailed in Fig. 5A.
  • Wild type adult BALB/c mice were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the DNA-LNPs are administered at 1 or 0.3 mg/kg based on the weight of the DNA payload.
  • blood is collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 5B.
  • the DNA-LNP with ionizable lipid L-21 produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC-0315 (FIG. 5B).
  • the DNA-LNPs with ionizable lipids L-17 and L-20 produced robust EPO expression levels, comparable to the exemplary ionizable lipids ARCT and CL1 and higher than the exemplary lipid ALC-0315 (FIG. 5B).
  • the DNA-LNPs with ionizable lipid L-19 produced robust EPO expression levels, higher than all three exemplary ionizable lipids (FIG. 5B).
  • blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 5C.
  • the DNA-LNPs with ionizable lipids L-17, L-19, and L-21 produced generally similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, ARCT, and CL1 (FIG. 5C).
  • Example 32 Preparation and analysis of LNPs comprising varying ionizable lipids coformulated with both DNA and mRNA
  • DNA and mRNA payloads were co-formulated into lipid nanoparticles (LNPs) comprising ionizable lipids of the present disclosure, using a 1 :3 (w/w) ratio of DNA:mRNA, as described above.
  • LNPs lipid nanoparticles
  • DOPE DOPE
  • Wild type adult BALB/c mice were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight.
  • the LNPs are administered at 0.5 mg/kg based on the weight of the DNA payload (e.g., 1.5 mg/kg based on the weight of the mRNA payload).
  • Twenty-one days after dosing blood was collected via a retro-orbital bleed and human FIX levels in plasma determined as presented in FIG. 6B.
  • the LNPs with ionizable lipid L-15 produced FIX expression levels about 2-fold higher compared to exemplary ionizable lipid CL1 (FIG.

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Abstract

Novel ionizable lipids are provided. Also provided are novel lipid nanoparticle compositions for the delivery of nucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the compositions in research and as therapeutics.

Description

IONIZABLE LIPIDS AND LIPID NANOPARTICLE COMPOSITIONS FOR THE DELIVERY OF NUCLEIC ACIDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/425,969, filed November 16, 2022, and U.S. Provisional Application No. 63/455,243, filed March 28, 2023, the entirety of each of which is hereby incorporated by reference.
1. INTRODUCTION
[0002] There are many instances in which delivery of a nucleic acid is desired, where such instances include 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. Genetic disorders, although individually rare, collectively represent a significant disease burden, particularly for children, resulting in substantial disability and mortality.
[0003] In the field of gene therapy, viral vectors, such as vectors based on AAV, are commonly employed to deliver genes into cells. However, AAV vectors are limited in the size of genetic cargo that can be packaged. Accordingly, any genetic cargo greater than 4.7kB is not suitable for delivery with AAV vectors, which limits the utility of such vectors for many indications. In addition, viral vectors, such as AAV, induce an antibody response, limiting redosing, which is not suitable for some indications. Moreover, in indications where the target cells are dividing, such as the liver, expression from successfully transduced cells can be reduced or lost with cell division and turnover, requiring redosing - which may not be possible or effective due to immune memory. In addition, many subjects have pre-existing immunity to commonly used viral vectors such as AAV, which can limit even initial treatment with an AAV gene therapy. Furthermore, viral vector such as AAV can be toxic at the doses that would be required to achieve therapeutic benefit in some indications.
[0004] Lipid nanoparticles (LNPs) provide an alternative to viral gene therapy. While lipid nanoparticles have been developed and employed for delivery of many RNA therapeutics, lipid-nanoparticle-delivered RNA has limited therapeutic longevity. DNA delivered by lipid nanoparticles designed for RNA delivery suffers from poor efficiency and significant innate immune response activation in treated subjects. New delivery vehicles for delivering nucleic acids, such as DNA, to cells, stand to significantly advance numerous scientific pursuits, particularly in vivo for patients in need of gene therapy but also in vitro for research applications.
2. SUMMARY
[0005] Provided herein are novel ionizable lipids having an ionizable headgroup connected to lipid tails via a linear alkyl core. The linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails. Also provided herein are novel lipid nanoparticle (LNP) compositions for the delivery of nucleic acid material to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using compositions of the invention in research and as therapeutics.
3. BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGs. 1 A-C describe studies performed to assess how the structure of the ionizable lipid impacts the efficacy and toxicity of DNA-LNPs. (FIG. 1 A) Formulation details for the test articles. The ionizable lipid was varied in all formulations. Ionizable cationic lipids tested were ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate), MC3 (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate, and two ionizable cationic lipids of the present disclosure, L-2 and L-3. The phospholipid in all formulations is DSPC. The nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed of all test articles. (FIG. IB) EPO serum levels in wild type BALB/c mice were measured 4 hours post-i.v. administration of test articles at a dose of 1 mg/kg. (FIG. 1C) IL-6 cytokine levels in serum were measured 4 hours after i.v. administration of test articles at a dose of 1 mg/kg to wild type BALB/c mice.
[0007] FIGs. 2A-0 describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs. (FIG. 2 A - 2B) Formulation details for the test articles. The ionizable lipid was varied in these formulations. Two benchmark ionizable lipids, ALC-0315 and MC3 are 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 was DSPC (FIG. 2B) or DOPE (FIG. 2C). The nucleic acid cargo used in all formulations was a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (FIG. 2B) (FIG. 2C) EPO protein levels in serum of wild type BALB/c mice were measured 7 days after i.v. administration of test articles in FIG. 2A and 2B at a dose of 1 or 0.3 mg/kg to wild type BALB/c mice. (FIG. 2D-I) The levels of IL-6, IFNy, TNFa, IL-1 , IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 2A (formulated with the DSPC phospholipid) at a dose of 1 or 0.3 mg/kg. (FIG. 2J-O) The levels of IL-6, IFNy, TNFa, IL-10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 2B (formulated with the DOPE phospholipid).
[0008] FIGs. 3 A-F describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs. (FIG. 3A-3B) Formulation details for the test articles. The ionizable lipid was varied in these formulations. The benchmark ionizable lipid, ALC-0315, is described above. Other benchmark 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 (FIG. 3 A) or DOPE (FIG. 3B). The nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (FIG. 3C) EPO protein levels in serum of wild type BALB/c mice were measured 7 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg. (FIG. 3D-I) The levels of IL-6, IFNy, TNFa, IL-10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 3A (formulated with the DSPC phospholipid) at a dose of 1 or 0.3 mg/kg. (FIG. 3J-O) The levels of IL-6, IFNy, TNFa, IL- 10, IL-12, and KC were measured in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles in FIG. 3B (formulated with the DOPE phospholipid).
[0009] FIGs. 4A-C describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs. (FIG. 4 A) Formulation details for the test articles. The ionizable lipid was varied in these formulations. The benchmark ionizable lipid, ALC-0315, is described above. Five ionizable lipids of the present disclosure, L-9, L-10, and L-ll, L-15, and L-16, were also tested. The phospholipid in these formulations is DSPC. The nucleic acid cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (FIG. 4B) EPO protein levels in serum of wild type BALB/c mice were measured 3 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg. (FIG. 4C) IL-6 cytokine levels in serum of wild type BALB/c mice were measured 4 hours after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
[0010] FIGs 5 A-C describe further studies performed to assess how the structures of additional ionizable lipids impact the efficacy and toxicity of DNA-LNPs. (FIG. 5 A) Formulation details for the test articles. The ionizable lipid was varied in these formulations. Two benchmark ionizable lipids, ALC-0315 and ARCT, are described above. One 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 cargo used in all formulations is a nanoplasmid DNA (npDNA) comprising a hAAT promoter driving the expression of an EPO transgene. Good encapsulation efficiency and small size were observed for all test articles. (FIG. 5B) EPO protein levels in serum of wild type BALB/c mice were measured 3 days after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg to wild type balb/c mice. (FIG. 5C) IL-6 cytokine levels in serum of wild type BALB/c mice were measured 4 hours after i.v. administration of test articles at a dose of 1 or 0.3 mg/kg.
[0011] FIGs 6A-K describe studies performed to assess how the structures of ionizable lipids impact the efficacy and toxicity of LNPs co-formulated with both DNA and mRNA. (FIG. 6A) Formulation details for the test articles. The ionizable lipid was varied in these formulations. The benchmark ionizable lipid, CL1, is 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 cargo used in all formulations comprised nanoplasmid DNA (npDNA) and mRNA mixed at a 1 :3 (w/w) ratio of DNA:mRNA, where the npDNA comprises a TTR promoter driving the expression of a human Factor IX (FIX) transgene. Good encapsulation efficiency and small size were observed for all test articles. (FIG. 6B) Human FIX protein levels in plasma of wild type balb/c mice 21 days after i.v. administration of test articles at a dose of 0.5 mg/kg DNA (1.5 mg/kg mRNA). (FIG. 6C - K) Cytokine levels in serum of wild type BALB/c mice 4 hours after i.v. administration of test articles at a dose of 0.5 mg/kg DNA (1.5 mg/kg mRNA). 4. DETAILED DESCRIPTION
4.1 Lipid Nanoparticle Compositions
[0012] Novel lipid nanoparticle compositions are provided for the delivery of nucleic acid to cells in vitro and in vivo with different and improved pharmacokinetic profiles as compared to what is typically observed in the art. Also provided are methods for using the lipid nanoparticle compositions of this disclosure in research and as therapeutics.
[0013] A “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), a protein, a small molecule, and the like to a target site of interest. In the lipid nanoparticle, a nucleic acid agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.
[0014] In general, the lipid nanoparticle includes several lipid components, including e.g., an ionizable lipid, one or more helper lipid(s) (e.g., non-cationic lipid(s)), and a lipid that prevents aggregation of the nanoparticle (also referred to as a coat lipid or conjugated lipid e.g., a PEG-lipid). In some aspects, this disclosure provides for a lipid nanoparticle (LNP) composition as described herein comprising a nucleic acid, where the nucleic acid is substantially encapsulated by the lipid components of the LNP.
4.2 Ionizable Lipids
[0015] The lipid nanoparticles (LNPs) of this disclosure can include ionizable lipids. As summarized above, provided herein are novel ionizable lipids. The ionizable lipid is typically employed in lipid nanoparti cles(LNPs) to condense its nucleic acid cargo, e.g., DNA or RNA, at low pH and to drive membrane association and fusogenicity. The term “ionizable lipid” refers to a lipid comprising an ionizable group that carries a net charge at a selected pH (e.g., a pH of 6.5 or less), but which can remain neutral at, e.g., a higher pH, such as physiological pH. The pH-sensitivity of such ionizable lipids can be desirable to provide for intracellular delivery of nucleic acid cargo. Ionizable lipids can have less interactions with cell membranes when neutral, and then become charged when internalized into endosomes in a target cell, where the pH is lower than in the extracellular environment. Ionizable lipids which are protonated and, therefore, become positively charged, may promote membrane destabilization and facilitate endosomal escape of the nanoparticle.
[0016] 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., a pH of 6.5 or less). In some embodiments, the ionizable lipids are cationic lipids including at least one ionizable amino group that is positively charged or becomes protonated at a selected pH, for example at pH of 6.5 or lower. In some embodiments, the cationic lipid includes one or more tertiary amino groups, e.g., a trialkyl amino group.
[0017] As disclosed herein, the ionizable lipid includes an ionizable headgroup (e.g., an ionizable amino group) connected to the lipid tails via a linear alkyl core. The linear alkyl core can have n carbon atoms, where n-1 carbon atoms in the linear alkyl core are linked to lipid tails. In some embodiments, the linear alkyl core has 3 carbon atoms and 2 lipid tails. In some embodiments, the linear alkyl core has 4 carbon atoms and 3 lipid tails. In some embodiments, the linear alkyl core has 5 carbon atoms and 4 lipid tails. In some embodiments, the linear alkyl core has 6 carbon atoms and 5 lipid tails.
[0018] In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, a linear alkyl core, hydrocarbon chains (e.g., C8-C20 carbon chains, such as Cis alkyl chains), ether linkages between the linear alkyl core and hydrocarbon chains, and 0 to 3 double bonds per hydrocarbon chain. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ether linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and ester linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as ester linkages. In some embodiments, the cationic lipid comprises a protonatable tertiary amine headgroup, a linear alkyl core, hydrocarbon chains (e.g., as described herein), and carbonate linkages between the linear alkyl core and hydrocarbon chains. In some embodiments, the cationic lipid comprises the same number of hydrocarbon chains as carbonate linkages. 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.
[0019] Aspects of this disclosure include an ionizable lipid compound of formula (I): (Z-L-Y)-Wn-(X-R)(n-i)
(I) wherein:
Z is an ionizable head group;
L is an optionally substituted (Ci-Ci2)alkylene;
Y is a linking group;
Wn is a linear alkyl core of n carbon atoms, wherein n is 3 to 6; X is an optional linking group; and each R is independently a lipid tail.
[0020] In some embodiments of formula (I), n is 4 to 6, such that the linear alkyl core has 4 to 6 carbon atoms. In some cases, n is 4, such that the linear alkyl core has 4 carbon atoms. In some cases, n is 5, such that the linear alkyl core has 5 carbon atoms. In some cases, n is 6, such that the linear alkyl core has 6 carbon atoms. In some embodiments, n is 3, such that the linear alkyl core has 3 carbon atoms.
[0021] In some embodiments of formula (I), the linear alkyl core Wn is:
Figure imgf000008_0001
where * depicts the point of attachment to Y and each ** depicts the point of attachment to
[0022] In some embodiments of formula (I), the linear alkyl core Wn is:
Figure imgf000008_0002
where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and each G2 is independently H or -CH2OH. In certain cases, at least one G2 is H. In certain cases, both G2 groups are H. In certain cases, at least one G2 is -CH2OH. In certain cases, both G2 groups are -CH2OH. In certain cases, one G2 is H and the other is -CH2OH. [0023] In some embodiments of formula (I), the linear alkyl core Wn is:
Figure imgf000008_0003
where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and G2 is H or -CH2OH. In certain cases, G2 is H. In certain cases, G2 is -CH2OH.
[0024] In some embodiments of formula (I), Wn is:
Figure imgf000009_0001
where * depicts the point of attachment to Y and each ** depicts the point of attachment to
X; and G2 is H or -CH2OH. In certain cases, G2 is H. In certain cases, G2 is -CH2OH.
[0025] In some embodiments of formula (I), the linear alky core Wn is:
Figure imgf000009_0002
where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; and G1 is H, or a group cyclically linked with Y that together with the carbon atom of Wn to which they are attached provide a heterocycle. In certain cases, G1 is cyclically linked with Y to form a 5-membered heterocycle. In certain cases, G1 is cyclically linked with Y to form a 6-membered heterocycle. In certain cases, G1 is H.
[0026] In some embodiments of formula (I), the linear alkyl core Wn is:
Figure imgf000009_0003
where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; G1 is H, or a group cyclically linked with Y that together with the carbon atom of Wn to which they are attached provide a heterocycle; and G2 is H or -CH2OH. In certain cases, G1 is cyclically linked with Y to form a 5-membered heterocycle. In certain cases, G1 is cyclically linked with Y to form a 6-membered heterocycle. In certain cases, G1 is H. In certain cases, G2 is H. In certain cases, G2 is -CH2OH. In certain cases, at least one of G1 and G2 are H. In certain cases, G1 and G2 are both H. In certain cases, G1 is H and G2 is - CH2OH. In certain cases, G1 is cyclically linked with Y to provide a heterocycle, and G2 is H. In certain cases, G1 is a cyclically linked with Y to provide a heterocycle, and G2 is - CH2OH.
[0027] In some embodiments of formula (I), the linear alkyl core Wn is:
Figure imgf000010_0001
where * depicts the point of attachment to Y and each ** depicts the point of attachment to X; G1 is H, or a group cyclically linked with Y that together with the carbon atom of Wn to which they are attached provide a heterocycle; and G2 is H or -CH2OH. In certain cases, G1 is cyclically linked with Y to form a 5-membered heterocycle. In certain cases, G1 is cyclically linked with Y to form a 6-membered heterocycle. In certain cases, G1 is H. In certain cases, G2 is H. In certain cases, G2, is -CH2OH. In certain cases, at least one of G1 and G2 are H. In certain cases, G1 and G2 are both H. In certain cases, G1 is H and G2 is - CH2OH. In certain cases, G1 is cyclically linked with Y to provide a heterocycle, and G2 is H. In certain cases, G1 is a cyclically linked with Y to provide a heterocycle, and G2 is - CH2OH.
[0028] As described herein above, in formula (I) the alkyl linear core Wn is linked to an ionizable head group Z through a linking group Y. By “linking group” is meant a linking moiety that connects two groups via covalent bonds. The linking group Y may be linear, branched, cyclic, a single atom, or a covalent bond. Examples of such linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, carbonate, alkyldioxy, oxyimino, amino, carbonyl, heterocycle (e.g., cyclic acetal) etc.
[0029] In some embodiments of formula (I), Y is selected from — O — , — C(R10)2 — , — OC(O)— , — C(O)O— , — OC(O)O— , — OC(O)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O— , and — NR10C(O)S— , wherein R10 is selected from H and C1-6 alkyl. In some cases, Y is selected from — O — , - OC(O) — , — C(0)0 — , and — OC(O)NR10 — . In some cases, Y is — O — . In some cases, Y is — OC(O) — . In some cases, Y is — OC(O)NR10 — , where R10 is H. In some cases, Y is — C(R10)2 — , where each R10 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)NR10 — where each R10 is H. In some cases, Y is — C(O)NR10 — where each R10 is H. In some cases, Y is — NR10C(O) — where each R10 is H. In some cases, Y is — S — . In some cases, Y is — NR2 — . In some cases, Y is — NR10C(O)O — , where each R10 is H. In some cases, Y is — NR10C(O)S — where each R10 is H.
[0030] In some embodiments, Wn comprises a group G1 adjacent to the point of attachment to linking group Y. In some embodiments, G1 is cyclically linked with the linking group Y to provide a heterocycle. In some embodiments, G1 is cyclically linked with Y to provide a 5-membered heterocycle. In some embodiments, the 5-membered heterocycle is a cyclic acetal. In some embodiments, G1 is cyclically linked with Y to provide a 6-membered heterocycle. In some embodiments, the 6-membered heterocycle is a cyclic acetal.
[0031] As described herein above, in formula (I) the linking group Y is linked to an ionizable head group Z through an optionally substituted (Ci-Ci2)alkylene L. In some embodiments of formula (I), L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene. In some embodiments, L is (C2-C4)alkylene or substituted (C2-C4)alkylene. In certain cases, L is C2- alkylene or substituted C2-alkylene. In certain cases, L is Cs-alkylene or substituted C3- alkylene. In certain cases, L is C4-alkylene or substituted C4-alkylene. In certain cases, L is Cs-alkylene or substituted Cs-alkylene. In certain cases, L is Ce-alkylene or substituted Ce- 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 -(CJfcje-
[0032] In some embodiments, -Y-L-Z is of the formula — O(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — OC(O)NH(CH2)rZ, where r is 2-6. In some embodiments, -Y-L-Z is of the formula — CH2(CH2)rZ, where r is 2-6. In some cases, r is 2- 4. In some cases, r is 2. In some cases, r is 3. In some cases, r is 4.
[0033] As described herein, the ionizable lipid of formula (I) includes an ionizable head group. In some embodiments the ionizable head group includes a primary, secondary or tertiary amine that may be protonated at physiological pH. In some embodiments, the ionizable head group comprises a tertiary amino group. In certain embodiments, the ionizable head group is of the formula -NRnR12, wherein R11 and R12 are each independently alkyl or substituted alkyl. In some embodiments, R11 and R12 are each independently C1-6 alkyl or substituted C1-6 alkyl. In some embodiments, R11 and R12 are each independently C1-3 alkyl or substituted C1-3 alkyl. In some embodiments, R11 and R12 are each C1-3 alkyl. In some embodiments, R11 and R12 are each methyl. In some embodiments, R11 and R12 are each ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted secbutyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, Ci-4 heteroalkyl, and C1-3 heteroalkyl.
[0034] As described herein, formula (I) includes 2-5 lipid tails R, that are linked to the core Wn, optionally via an additional linking group X (e.g., -(X-R)(n-i>).
[0035] In some embodiments, the ionizable lipid of formula (I) includes a linking group X. The linking group X may be linear, branched, cyclic or a single atom. Examples of such linking groups include but are not limited to, alkyl, alkenylene, alkynylene, arylene, alkarylene, aralykylene, amido, ureylene, imide, ether, thioether, thiocarbamate, carbonate, alkyldioxy, oxyimino, amino, carbonyl etc. In some embodiments of formula (I), each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , — (CH2)SOC(O)NR10— , — (CH2)SO— , — (CH2)sSC(O)NR10— , — (CH2)SC(O)NR10— , — (CH2)sNR10C(O)— , — (CH2)sS— , — (CH2)SNR10— , — (CH2)sNR10C(O)O— , and — (CH2)SNR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl and s is 0-6. In some embodiments, each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , and — (CH2)SOC(O)O — . In some embodiments, each X is — (CH2)sOC(O) — , where s is 0, 1 or 2. In some embodiments, each X is — (CH2)sC(O)O — , where s is 0, 1 or 2. In some embodiments, each X is — (CH2)sOC(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)SOC(O)NR10 — , where R10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sSC(O)NR10 — , where R10 is H. and s is 0, 1, or 2 In some embodiments, at least one X group is — (CH2)sC(O)NR10 — , where R10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR10C(O) — , where R10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sS — , where s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR10 — , where R10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR10C(O)O — , where R10 is H and s is 0, 1 or 2. In some embodiments, at least one X group is — (CH2)sNR10C(O)S — , where R10 is H and s is 0, 1 or 2.
[0036] In some embodiments of formula (I), each X is independently selected from — OC(O)— , — C(O)O— , — OC(O)O— , — O— , — OC(O)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O— , and — NR10C(O)S— , wherein R10 is selected from H and Ci-6 alkyl. 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)NR10 — , where R10 is H. In some embodiments, at least one X group is — SC(O)NR10 — , where R10 is H. In some embodiments, at least one X group is — C(O)NR10 — , where R10 is H. In some embodiments, at least one X group is — NR10C(O) — , where R10 is H. In some embodiments, at least one X group is — S — . In some embodiments, at least one X group is — NR10 — , where R10 is H. In some embodiments, at least one X group is — NR10C(O)O — , where R10 is H. In some embodiments, at least one X group is — NR10C(O)S — , where R10 is H.
[0037] In some embodiments of formula (I), each — X-R is independently selected from — (CH2)SOC(O)R, — (CH2)SC(O)OR, — (CH2)SOC(O)OR, — (CH2)SOR, — (CH2)SOC(O)NR10R, — (CH2)SSC(O)NR10R, — (CH2)SC(O)NR10R, — (CH2)SNR10C(O)R, — (CH2)SSR, — (CH2)SNR10R, — (CH2)SNR10C(O)OR, and — (CH2)sNR10C(O)SR, wherein R10 is selected from H and Ci-6 alkyl, s is 0-6 and each R is independently a lipid tail. In some embodiments, each — X-R is — (CH2)sOC(O)R. In some embodiments, each — X-R is — (CH2)SC(O)OR. In some embodiments, each — X-R is — (CH2)sOC(O)OR. In some embodiments, each — X-R is — (CH2)sOR. In some embodiments, each — X-R is — (CH2)SOC(O)NR10R. In some embodiments, each — X-R is — (CH2)sSC(O)NR10R. In some embodiments, each — X-R is — (CH2)sC(O)NR10R. In some embodiments, each — X-R is — (CH2)SNR10C(O)R. In some embodiments, each — X-R is — (CH2)sSR. In some embodiments, each — X-R is — (CH2)sNR10R. In some embodiments, each — X-R is — (CH2)SNR10C(O)OR. In some embodiments, each — X-R is — (CH2)sNR10C(O)SR.
[0038] 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)NR10R, SC(O)NR10R, C(O)NR10R, NR10C(O)R, SR, NR10R, NR10C(O)OR, and NR10C(O)SR, wherein R10 is selected from H and Ci-6 alkyl 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)NR10R. In some embodiments, each — X-R is SC(O)NR10R. In some embodiments, each — X-R is — C(O)NR10R. In some embodiments, each — X-R is — NR10C(O)R. In some embodiments, each — X-R is — SR. In some embodiments, each — X-R is — NR10R. In some embodiments, each — X-R is — NR10C(O)OR. In some embodiments, each — X-R is — NR10C(O)SR. In some embodiments of formula (I), each lipid tail is independently an aliphatic hydrocarbon group that is straight chain or branched, saturated or unsaturated and/or optionally comprises a cyclic group.
[0039] In some embodiments of formula (I), each R is a linear hydrocarbon group optionally comprising one or more cyclic groups. In some embodiments, each R is a linear hydrocarbon group independently selected from a C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In some embodiments, each R is a linear hydrocarbon group independently selected from a C6-C12 alkyl, and C6-C12 alkenyl. In some embodiments, at least one R is a linear hydrocarbon group comprising a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
[0040] In some embodiments of formula (I), at least one R is a branched hydrocarbon group optionally comprising one or more cyclic groups. In some embodiments, each R is a branched hydrocarbon group optionally comprising one or more cyclic groups. In some embodiments, the branched hydrocarbon group comprises 8-20 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 8 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 9 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 10 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 11 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 12 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 13 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 14 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 15 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 16 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 17 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 18 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 19 carbon atoms. In some embodiments, the branched hydrocarbon group comprises 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 is of the formula - CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In some embodiments each R7 is Cs-alkyl, or Cs-alkenyl. In some embodiments each R7 is Ce-alkyl, or Ce-alkenyl. In some embodiments each R7 is C?-alkyl, or C?-alkenyl. In some embodiments each R7 is Cs-alkyl, or Cs-alkenyl. In some embodiments each R7 is C9-alkyl, or C9-alkenyl. In some embodiments each R7 is Cio-alkyl, or Cio-alkenyl. In some embodiments each R7 is Cn-alkyl, or Cn-alkenyl. In some embodiments each R7 is Ci2-alkyl, or C12- alkenyl. In some embodiments, at least one R is a branched hydrocarbon group comprising a cyclic group. In some embodiments, the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
[0041] In some embodiments R is a linear or branched hydrocarbon group comprising one or more cyclic groups. In some embodiments, the cyclic group is an optionally substituted monocyclic cycloalkyl. In some embodiments, the cyclic group is an optionally substituted bicyclic cycloalkyl. 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.
[0042] In some embodiments of Formula
Figure imgf000015_0001
wherein,
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; r, p, and q is each independently an integer from 0 to 20.
[0043] In some embodiments, R is
Figure imgf000015_0002
wherein, CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group; r, p, and q is each independently an integer from 0 to 10.
[0044] In some embodiments, R is
Figure imgf000016_0001
wherein,
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, Ci-Ce aliphatic group; r, p, and q is each independently an integer from 0 to 6.
Figure imgf000016_0002
Figure imgf000017_0001
represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
Figure imgf000017_0002
, where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
Figure imgf000017_0003
, where each
# represents the point of attachment to X, or the point of attachment to a linear or branched
Figure imgf000017_0004
hydrocarbon chain of R. In some embodiments, R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched
Figure imgf000018_0001
hydrocarbon chain of R. In some embodiments, R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000018_0002
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
In some embodiments,
Figure imgf000018_0003
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000018_0004
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000018_0005
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000018_0006
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000019_0001
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000019_0002
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000019_0003
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R
Figure imgf000019_0004
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000019_0005
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000020_0001
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain
#
JQI of R. In some embodiments, R is , where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000020_0002
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000020_0003
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
#
Figure imgf000020_0004
, where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000020_0005
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
Figure imgf000021_0001
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
Figure imgf000021_0002
of R. In some embodiments,
Figure imgf000021_0003
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000021_0004
where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000022_0001
, where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments, R is
Figure imgf000022_0002
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000022_0003
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R. In some embodiments,
Figure imgf000022_0004
each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain ofR.
[0045] In some embodiments, the compound of formula (I) is of the formula (IIA):
Figure imgf000022_0005
(IIA), wherein X, R, Y, L and Z are as defined herein above.
[0046] In certain embodiments of formula (IIA), Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — , wherein R10H and Ci-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)NR10 — , where R10 is H.
[0047] In certain embodiments of formula (IIA), L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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-.
[0048] In certain embodiments of formula (IIA), Z is a tertiary amine. In certain cases, Z is -NRnR12, wherein R11 and R12 are each independently C1-6 alkyl or substituted C1-6 alkyl. In certain cases, R11 and R12 are each C1-3 alkyl. In certain cases, R11 and R12 are each methyl. In certain cases, R11 and R12 are each ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
[0049] 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 certain cases, each X is — C(O)O — . In certain embodiments of formula (IIA), at least one X is — OC(O)O — . In certain cases, each X is — OC(O)O— .
[0050] In certain embodiments of formula (IIA), each -X-R is of the formula — OC(O)R. In certain embodiments, each -X-R is of the formula — C(O)OR. In certain embodiments, each -X-R is of the formula — OC(O)OR.
[0051] In certain embodiments of formula (IIA), each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
[0052] In certain embodiments of formula (IIA), at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of formula (IIA), R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C 12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl.
[0053] In certain embodiments of formula (IIA), at least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.
[0054] In certain embodiments of formula (IIA), the compound is of formula (IIIA):
Figure imgf000024_0001
UA) wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and -(CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10. [0055] In some embodiments of formula (IIA), the compound is of formula (IIIA), wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
[0056] In certain embodiments of formula (IIIA), Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — , wherein R10 is selected from H and C1-6 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)NR10 — , where R10 is H.
[0057] 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.
[0058] In certain embodiments of formula (IIIA), R11 and R12 are different. In certain cases, at least one of R11 and R12 is methyl. In certain cases, R11 and R12 are the same. In certain cases, both R11 and R12 are methyl. In certain cases, at least one of R11 and R12 is ethyl. In certain cases, both R11 and R12 are ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, Ci-3 alkyl, C 1-4 heteroalkyl, and Ci-3 heteroalkyl..
[0059] In certain embodiments of formula (IIIA), each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl. [0060] In certain embodiments of formula (IIIA), at least one R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl.
[0061] In certain embodiments of formula (IIIA), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 5. In some cases, t is 2 and u is 3.
[0062] In certain embodiments of the compound of formula (I), the compound is of formula (IIB):
Figure imgf000026_0001
(IIB), wherein X, R, Y, L and Z are as defined herein.
[0063] In certain embodiments of formula (IIB), Y is selected from — O — , — OC(O) — , — OC(O)NR10— , — NR10C(O)— , — NR10C(O)O— , and — NR10C(O)S— , wherein R10 is selected from H and C1-6 alkyl. 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 — .
[0064] In certain embodiments of formula (IIB), L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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-.
[0065] In certain embodiments of formula (IIB), Z is a tertiary amine. In certain cases, Z is -NRnR12, wherein R11 and R12 are each independently C1-6 alkyl or substituted C1-6 alkyl. In certain cases, R11 and R12 are each C1-3 alkyl. In certain cases, R11 and R12 are each methyl. In certain cases, R11 and R12 are each ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, Ci-3 alkyl, C 1-4 heteroalkyl, and Ci-3 heteroalkyl.
[0066] 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— .
[0067] In certain embodiments of formula (IIB), each -X-R is of the formula — OC(O)R. In certain embodiments of formula (IIB), each -X-R is of the formula — C(O)OR. In certain embodiments of formula (IIB), each -X-R is of the formula — OC(O)OR.
[0068] In certain embodiments of formula (IIB), each R is selected from C5-C20 alkyl, Cs- C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
[0069] In certain embodiments of formula (IIB), at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of formula (IIB), R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a Cs-C 12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl.
[0070] In certain embodiments of formula (IIB), at least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5. [0071] In certain embodiments of formula (IIB), the compound is of formula (IIIB):
Figure imgf000028_0001
wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and - CH(R7)2, and -(CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group and t and u are each independently 1 to 10.
[0072] In some embodiments of formula (IIB), the compound is of formula (IIIB), wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, and -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
[0073] In certain embodiments of formula (IIIB), Y is — NHC(O) — . In certain cases, Y is — NHC(O)O — . In certain cases, Y is — NHC(O)S — .
[0074] In certain embodiments of formula (IIIB), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
[0075] In certain embodiments of formula (IIIB), R11 and R12 are different. In certain cases, at least one of R11 and R12 is methyl. In certain cases, R11 and R12 are the same. In certain cases, both R11 and R12 are methyl. In certain cases, at least one of R11 and R12 is ethyl. In certain cases, both R11 and R12 are ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, Ci-3 alkyl, C 1-4 heteroalkyl, and Ci-3 heteroalkyl.
[0076] In certain embodiments of formula (IIIB), each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
[0077] In certain embodiments of formula (IIIB), at least one R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl.
[0078] In certain embodiments of formula (IIIB), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 3. In some cases, t is 2 and u is 3.
[0079] In some embodiments, the compound of formula (I) is of the formula (IIC):
Figure imgf000029_0001
QIC), wherein X, R, Y, L and Z are as defined herein above. [0080] In certain embodiments of formula (IIC), Y is selected from — C(R10)2 — and —
O — , wherein R10 is selected from H and Ci-6 alkyl. In certain embodiments of formula (IIC), Y is — O — . In certain embodiments of formula (IIC), Y is — C(R10)2 — , where R10 is H.
[0081] In certain embodiments of formula (IIC), L is (C2-Ce)alkylene or substituted (C2- Ce)alkylene. In certain cases, L is (C2-Ce)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-.
[0082] In certain embodiments of formula (IIC), Z is a tertiary amine. In certain cases, Z is -NRnR12, wherein R11 and R12 are each independently C1-6 alkyl or substituted C1-6 alkyl. In certain cases, R11 and R12 are each C1-3 alkyl. In certain cases, R11 and R12 are each methyl. In certain cases, R11 and R12 are each ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, C1-3 alkyl, C 1-4 heteroalkyl, and C1-3 heteroalkyl.
[0083] In certain embodiments of formula (IIC), each X is independently selected from — (CH2)SOC(O) — , — (CH2)SC(O)O — , — (CH2)SOC(O)O — , where s is 0-6. In some cases, at least one X is — (CH2)sOC(O) — , where s is 0, 1 or 2. In some cases, each X is — (CH2)SOC(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 certain cases, each X is — (CH2)sC(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)sOC(O)O — , where s is 0, 1 or 2. In certain cases, each X is — (CH2)sOC(O)O — , where s is 0, 1 or 2. In certain embodiments of formula (IIC), s is 0 and X is — OC(O)O — .
[0084] In certain embodiments of formula (IIC), each -X-R is of the formula — (CH2)SOC(O)R, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2)SC(O)OR, where s is 0-6. In certain embodiments, each -X-R is of the formula — (CH2)SOC(O)OR, where s is 0-6.
[0085] In certain embodiments of formula (IIC), each R is selected from C5-C20 alkyl, C5- C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
[0086] In certain embodiments of formula (IIC), at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms, optionally further comprising one or more cyclic groups (e.g., as described herein). In certain embodiments of formula (IIC), R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C 12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C6-C9 alkenyl.
[0087] In certain embodiments of formula (IIC), at least one R is a linear or branched hydrocarbon group comprising one or more cyclic groups. In certain embodiments of formula (IIA), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In some embodiments, t is 1 to 5. In some embodiments, u is 1 to 5.
[0088] In certain embodiments of formula (IIC), the compound is of formula (IIIC):
Figure imgf000031_0001
wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , and — C(R10)2 — ; each s is independently 0, 1 or 2;
W is — O — or — CH2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and - (CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
[0089] In some embodiments of formula (IIC), the compound is of formula (IIIC), wherein: R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , and — C(R10)2 — ; s is 0 to 2;
W is O or CH2; and each R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. [0090] In certain embodiments of formula (IIIC), Y is selected from — C(R10)2 — and — O — , wherein R10 is selected from H and C1-6 alkyl. In certain embodiments of formula (IIIC), Y is — O — . In certain embodiments of formula (IIIC), Y is — C(R10)2 — , where R10 is H.
[0091] In certain embodiments of formula (IIIC), q is 1. In certain cases, q is 2. In certain cases, q is 3. In certain cases, q is 4.
[0092] In certain embodiments of formula (IIIC), R11 and R12 are different. In certain cases, at least one of R11 and R12 is methyl. In certain cases, R11 and R12 are the same. In certain cases, both R11 and R12 are methyl. In certain cases, at least one of R11 and R12 is ethyl. In certain cases, both R11 and R12 are ethyl. In certain cases, both R11 and R12 are propyl. In certain cases, both R11 and R12 are n-propyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 are isopropyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, both R11 and R12 is independently an optionally substituted n-butyl. In certain cases, both R11 and R12 is independently an optionally substituted sec-butyl. In certain cases, both R11 and R12 is independently an optionally substituted butyl. In certain cases, each R11 and R12 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 R11 and R12 is independently selected from an optionally substituted Ci-4 alkyl, Ci-3 alkyl, C 1-4 heteroalkyl, and Ci-3 heteroalkyl.
[0093] In certain embodiments of formula (IIIC), W is — CH2 — . In certain embodiments of formula (IIIC), W is — O — .
[0094] In certain embodiments of formula (IIIC), s is 0. In certain embodiments, s is 1.
In certain embodiments s is 2. [0095] In certain embodiments of formula (IIIC), W is — CH2 — and s is 0. In certain cases,
W is — CH2 — and s is 1. In certain cases, W is — O — and s is 0.
[0096] In certain embodiments of formula (IIIC), each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. In certain cases, each R is selected from C5-C12 alkyl, C5-C12 alkenyl, and a C5-C12 alkynyl. In certain cases, each R is C5-C12 alkyl. In certain cases, each R is C5 alkyl. In certain cases, each R is Ce alkyl. In certain cases, each R is C7 alkyl. In certain cases, each R is Cs alkyl. In certain cases, each R is C9 alkyl. In certain cases, each R is C10 alkyl. In certain cases, each R is C11 alkyl. In certain cases, each R is C12 alkyl.
[0097] In certain embodiments of formula (IIIC), at least one R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C5-C12 alkenyl. In certain cases, each R is -CH(R7)2 and each R7 is a C6-C9 alkyl. In certain cases, each R is -CH(R7)2 and each R7 is a C6-C9 alkenyl.
[0098] In certain embodiments of formula (IIIC), R is -(CH2)tJ(CH2)u, where J is a cyclic group and t and u are each independently 1-10. In some embodiments, J is an aryl group. In some embodiments, J is phenyl. In certain embodiments, t and u are each 1 to 3. In some cases, t is 2 and u is 3.
[0099] In some embodiments of Formula (I), (IIA), (IIIA), (IIB), (IIIB), (IIC), and (IIIC), each R is independently
Figure imgf000033_0001
wherein,
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; r, p, and q is each independently an integer from 0 to 20.
[0100] In some embodiments, R is
Figure imgf000033_0002
wherein,
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl; Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C10 aliphatic group; r, p, and q is each independently an integer from 0 to 10.
[0101] In some embodiments, R is
Figure imgf000034_0001
wherein,
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, Ci-Ce aliphatic group; r, p, and q is each independently an integer from 0 to 6.
[0102] In certain embodiments, the lipid is selected from a compound of TableTable 1 :
Figure imgf000034_0002
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
4.3 Additional Ionizable Lipids
[0103] In some embodiments, the lipid nanoparticle compositions can include one or more additional ionizable lipid components in addition to the ionizable lipid of formula (I) (e.g., as described above). Any convenient lipid that carries a net positive charge at or around physiological pH may find use as an additional ionizable lipid in the compositions described herein.
[0104] Non-limiting examples of cationic lipids are described in detail herein. Cationic lipids and related analogs, which are useful in the lipid nanoparticles of the present disclosure, include but are not limited to, those lipids described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. 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 herein incorporated by reference in their entirety for all purposes. Additional cationic lipids of interest include, but are not limited to, l,2-distearydoxy-N,N- dimethyl-3-aminopropane (DSDMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDM ), l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDM ), 1,2- di ol ey I oxy -N,N-d methyl -3 -am nopropane (DODMA), and heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (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”); l,2-Dioleyloxy-3 -trimethylaminopropane chloride salt (“DOTAP. Cl”); 3P-(N — (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(l-(2,3-dioleyloxy)propyl)-N- 2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (“DOSPA”), di octadecyl amidoglycyl carboxy spermine (“DOGS”), l,2-dioleoyl-3 -dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic lipids can be used, such as, e g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL). In particular embodiments, a cationic lipid is an amino lipid including one or two fatty acyl or fatty alkyl chains.
[0105] Further exemplary ionizable lipids which can be adapted for use in the lipid nanoparticles of the present disclosure are described in International PCT patent publications W02015/095340, WO2015/199952, W02018/011633, WO2017/049245, WO2015/061467, WO20 12/040184, WO2012/000104, W02015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO201 1/153120, WO2012/044638, WO2012/054365, WO2011/090965, W02013/016058, W02012/162210, W02008/042973, W02010/129709, W02010/144740, WO2012/099755, WO20 13/049328, WO2013/086322, WO2013/086373, WO2011/071860, W02009/132131, W02010/048536, W02010/088537, WO2010/054401, WO2010/054406, WO2010/054405, WO20 10/054384, WO2012/016184, W02009/086558, WO2010/042877, WO2011/000106, WO20 11/000107, W02005/120152, WO2011/141705, WO2013/126803, W02006/007712, WO20 11/038160, WO2005/121348, WO2011/066651, W02009/127060, WO2011/141704, W02006/069782, WO2012/031043, W02013/006825, WO2013/033563, W02013/089151, WO20 17/099823, WO2015/095346, and WO2013/086354, and US 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.
4.4 Helper Lipids
[0106] LNPs of this disclosure can also include one or more helper lipid(s), in addition to the ionizable lipid component described herein. In some embodiments, the helper lipid is a neutral lipid. In some embodiments, the neutral lipid is zwitterionic, e.g., has an overall net zero charge.
[0107] Neural lipids include, for example, phospholipids, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the compositions described herein is generally guided by consideration of, e.g., LNP size and stability of the LNPs in the bloodstream. In general, the LNPs of this disclosure includes a helper lipid component that includes a neutral lipid that is a phospholipid. Non-limiting examples of phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. In some embodiments, the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well- known techniques. In one embodiment, the neutral lipids include saturated fatty acids with carbon chain lengths in the range of Cio to C30. In one embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Cio to C30 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. The neutral lipids may also be composed of sphingomyelin, or dihydrosphingomyeline. [0108] In some embodiments, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG). [0109] In some embodiments, the phospholipid has a hydrocarbon chain, or “tail” having 12-24 carbons, e.g., 16-20 carbons, 18-22 carbons, 12-18 carbons. In some embodiments, 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 comprises no double bonds, i.e. the bonds are saturated bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more double bonds, e.g., 1, 2, 3, 4 or 5 double bonds. In some embodiments, the phospholipid tail is unsaturated, that is, it comprises one or more triple bonds, e.g. 1, 2, 3, 4 or 5 triple bonds. In some embodiments, the phospholipid tail comprises one or more ring structures. In some embodiments, the one or more ring structures is selected from 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl; 5- to 6- membered aryl; 7- to 10-membered saturated or partially unsaturated bicyclic carbocyclyl; and 7- to 10-membered bicyclic aryl wherein each ring structure is independently substituted with 0-7 RA groups; each RA is independently selected from halogen, or an optionally substituted group selected from C1-12 aliphatic, phenyl, or 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl. In some such instances, the ring structure is a cholesterol or 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 comprises two different hydrocarbon chains.
[0110] In some embodiments, a helper lipid is or comprises symmetric or asymmetric aliphatic phospholipid moieties that are each independently optionally substituted, branched or straight, partially unsaturated or saturated C9-C24 aliphatic.
[0111] In some embodiments, the helper lipid comprises one or more optionally substituted and/or optionally bridged ring structures in the hydrophobic tail. Exemplary helper lipids of this class include:
Figure imgf000045_0001
Figure imgf000045_0002
[0112] In some embodiments, the helper lipid includes a phosphatidylethanolamine (PE). Phosphatidylethanolamines (PE) are a class of phospholipids that incorporate ethanolamine as a headgroup. In some embodiments, the phosphatidylethanolamine selected from the group consisting of phosphatidylethanolamine, dioleoylphosphatidylethanolamine (1,2- dioleyl-sn-glycero-3-phosphoethanolamine) (A9-Cis PE, or DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine) (DPPE), dimyristoylphosphoethanolamine (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) (DMPE), (l,2-distearoyl-sn-glycero-3-phosphoethanolamine) (DSPE), monomethylphosphatidyl -ethanolamine (e.g. 16-0-monom ethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), di elaidoyl-phosphatidyl ethanolamine (DEPE), lysophosphatidyl ethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (DiPPE). In certain embodiments, the phosphatidylethanolamine is dioleoylphosphatidylethanolamine (also referred to as l,2-dioleoyl- w-glycero-3- phosphoethanolamine, or (A9-Cis) PE, or DOPE), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:
Figure imgf000046_0001
[0113] In some embodiments, the helper lipid includes a phosphatidylcholine (PC).). Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. In some embodiments, phosphatidylcholine is selected from the group consisting of phosphatidylcholine, distearoylphosphatidylcholine (l,2-distearoyl-sn-glycero-3- phosphocholine) (DSPC), dioleoylphosphatidylcholine (l,2-dioleoyl-sn-glycero-3- phosphocholine) (A9-Cis PC, or DOPC), dipalmitoylphosphatidylcholine (1,2-dipalmitoyl- sn-glycero-3 -phosphocholine) (DPPC), hydrogenated soy phosphatidylcholine (HSPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-dieicosenoyl-sn-glycero-3 -phosphocholine (“20-1 PC” or “20: 1 PC”), egg phosphatidylcholine (EPC), dimyristoyl phosphatidylcholine (DMPC), dierucoylphosphatidylcholine (DEPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, l,2-dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-palmitoyl-2-cholesterylcarbonoyl-sn-glycero-3 -phosphocholine (PChcPC), and l-palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC). In certain embodiments, the phosphatidylcholine is distearoylphosphatidylcholine (DSPC) (also referred to as 1 ,2-distearoyl-.s//-glycero-3-phosphocholine), having a tail of 18 carbons and no saturated bonds (“18-0”) as shown below:
Figure imgf000047_0001
[0114] In certain embodiments, the phosphatidylcholine is dioleoylphosphatidycholine (also referred to as l,2-dioleoyl-sn-glycero-3-phosphocholine, (A9-Cis) PC or DOPC), having a tail of 18 carbons and one saturated bond (“18-1”) as shown below:
Figure imgf000047_0002
[0115] In certain embodiments, the phosphatidylcholine is l,2-dipalmitoyl-sn-glycero-3- phosphocholine (delta9-Cis PC), having a tail of 16 carbons and one saturated bond (“16-1”) as shown below:
Figure imgf000047_0003
[0116] In certain embodiments, the phosphatidylcholine is an asymmetric lipid, having one tail of 16 carbons and a second tail of 18 carbons. In some such instances, the tail of the phosphatidylcholine having 18 carbons has one saturated bond, e,g, it is l-palmitoyl-2- oleoyl-glycero-3 -phosphocholine (also referred to as “16-0/18-1 PC”, “16:0/18: 1 PC” or POPC) as shown below:
Figure imgf000047_0004
[0117] In certain embodiments, the phosphatidylcholine is 1,2- dicholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (DChemsPC), as shown below:
Figure imgf000048_0001
[0118] In certain embodiments, the phosphatidylcholine is l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), as shown below:
Figure imgf000048_0002
[0119] In certain embodiments, the phosphatidylcholine is l-palmitoyl-2- cholesterylcarbonoyl-sn-glycero-3-phosphocholine (PChcPC), as shown below:
Figure imgf000048_0003
[0120] In certain embodiments, the phosphatidylcholine is l-palmitoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (PChemsPC), as shown below:
Figure imgf000048_0004
[0121] In some embodiments, the helper lipid includes a phosphatidylglycerol selected from the group consisting of phosphatidylglycerol, dioleoylphosphatidylglycerol (1,2- dioleoyl-sn-glycero-3- phospho-(l’-rac-glycerol) (DOPG), dipalmitoylphosphatidylglycerol, (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), and palmitoyloleyolphosphatidylglycerol (POPG). [0122] In some embodiments, the helper lipid includes a phosphatidylserine, e.g. phosphatidyl serine or dioleoylphosphatidylserine (DOPS).
[0123] In some embodiments, the helper lipid includes a lecithin, e.g. lecithin or lysolecithin.
[0124] In some embodiments, the helper lipid includes a sphingomyelin (SM), e.g. egg sphingomyelin (ESM).
[0125] In some embodiments, the helper lipid is cephalin, cardiolipin, phosphatidic acid, cerebrosides, or dicetylphosphate.
[0126] In some aspects, the LNP can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)- ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof. Exemplary cholesterol derivatives are described in International application W02009/127060 and US patent publication US2010/0130588. The component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
[0127] Accordingly, the neutral lipid component of the LNPs can further include cholesterol or a derivative or analog thereof. A variety of cholesterol analogs and derivatives can be adapted form use in the LNPs of this disclosure. In some embodiments, the helper lipid component includes cholesterol.
[0128] In some embodiments, the LNP includes a neutral lipid component that includes a mixture of one or more phospholipids and cholesterol or a derivative or analog thereof.
[0129] In some embodiments, the LNP includes a neutral lipid component that includes a phosphatidylethanolamine phospholipid and cholesterol or a derivative or analog thereof.
[0130] 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. 4.5 Other Components
[0131] LNPs of this disclosure can also include one or more additional lipid components. Such lipids can be selected to provide for a desirable profile of nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution.
[0132] In some aspects, the LNP can further comprise a non-cationic lipid. Non-ionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, the noncationic lipid can be a neutral uncharged, zwitterionic, or anionic lipid. Non-cationic lipids are typically employed to enhance fusogenicity. Exemplary non-cationic lipids envisioned 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 US patent publication US2018/0028664.
[0133] Non-limiting examples of non-cationic lipids include, nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
[0134] In some embodiments, the LNP includes one or more lipids capable of reducing aggregation. In general, a lipids capable of reducing aggregation includes at least a hydrocarbon tail or chain linked to a hydrophilic group which is capable of being configured at the surface of the LNP and provide for reduced LNP aggregation. Thus, the lipid capable of reducing aggregation is sometimes referred to as a conjugated lipid or coat lipid.
[0135] A lipid capable of reducing aggregation of particles may comprise a conjugated lipid molecule, such as a polyethylene glycol (PEG). Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, polyethyleneglycol (PEG)-lipid conjugate, polyoxazoline (POZ)-lipid conjugates, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof. In one embodiment, the LNPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
[0136] 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 tail(s) linked to one or more polyethylene glycol (PEG) moiety(ies) via an optional linker. [0137] It is understood that the PEG moieties may include terminal modification(s) to provide for e.g., conjugation to the lipid tails via the optional linker. The PEG moiety may be terminated in as a hydroxyl group, or an alkyl ether (e.g., a methoxy terminal group). In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid. PEG-lipids of interest include, but are not limited to, a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C 16), a PEG-distearyloxypropyl (C 18), or mixtures thereof.
[0138] Exemplary PEG-lipid conjugates include, but are not limited to, PEG- diacylglycerol (DAG) (such as 1 -(monomethoxy -poly ethyleneglycol)-2, 3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a PEGylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. 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, a PEG- lipid is a compound disclosed in US2018/0028664. In some embodiments, a PEG-lipid is disclosed in US20150376115 or in US2016/0376224. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG- dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- omegal-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(poly ethylene glycol)-2000]. In some examples, the PEG-lipid can be selected from the group consisting of PEG-DMG, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], PEG-DSG.
[0139] As described above, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (CPL) conjugates can be used in place of or in addition to the PEG-lipid. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the International patent application publications WO 1996/010392, WO1998/051278, W02002/087541, W02005/026372, WO2008/147438, W02009/086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO20 17/004143, WO2015/095346, WO2012/000104, WO2012/000104, and WO20 10/006282, US 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 US patents U.S. Pat. Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559.
4.6 Targeting ligand
[0140] In some embodiments, it may be desirable to limit transfection of the nucleic acids to certain cells or tissues. For example, the liver can be a target organ of interest in part due to its central role in metabolism and production of proteins and accordingly diseases which are caused by defects in liver-specific gene products (e.g., the urea cycle disorders) and may benefit from specific targeting of cells (e.g., hepatocytes).
[0141] In some embodiments, the LNP further includes a component including a targeting ligand. The targeting ligand can be selected as desired based on a target sell or tissue to which it is desired to direct the LNPs of this disclosure. In some embodiments, the targeting ligand is a ligand of a cell surface receptor. In some embodiments, the cell surface receptor is asialoglycoprotein receptor (ASGPR). The ASGPR is expressed on the surface of hepatocyte cells.
[0142] In some embodiments, the targeting ligand is a ligand for ASGPR, such as a N- acetylgalactosamine (GalNAc) containing ligand. A variety of GalNAc containing ligands and ligands, including multivalent GalNAc ligands are available for use in the LNP of this disclosure, including, e.g. those disclosed in WO2021178725, the full disclosure of which is incorporated herein by reference
[0143] In some embodiments, the PEG-lipid is linked to the targeting ligand. In some embodiments, the targeting ligand of interest (e.g., as described herein) is linked to a terminal of the PEG moiety. For example, a trisGalNac ligand conjugated to a PEG-lipid can provide for binding of the LNP to the ASGPR receptor of a target cell and result in endocytosis of the LNP.
4.7 Lipid Nanoparticles including lipids of Formula (I)
[0144] In some embodiments, the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a nucleic acid cargo (e.g., as described herein); an additional ionizable lipid (e.g., as described herein); a phospholipid (e.g., as described herein), cholesterol (e.g., as described herein); and a lipid capable of reducing aggregation (e.g., as described herein).
[0145] In some embodiments of the LNP, the nucleic acid cargo comprises DNA, e.g., an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of DNA. In some embodiments of the LNP, the nucleic acid cargo comprises RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, or a chemically modified derivative thereof. In certain cases, the nucleic acid consists essentially of RNA. In certain embodiments of the LNP, the nucleic acid cargo includes DNA, e.g,. an oligonucleotide, a plasmid DNA, a doggybone DNA, a minicircle DNA, a covalently closed circular DNA, a ceDNA, or a chemically modified derivative thereof, and further includes RNA, e.g., an siRNA, a gRNA, an mRNA, a circular RNA, and the like, or a chemically modified derivative thereof.
[0146] In some embodiments of the LNP, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof. In certain cases, the phospholipid is phosphatidylethanolamine (PE). In certain cases, the phospholipid is a phosphatidylcholine (PC). In certain embodiments of the LNP, the phospholipid includes hydrocarbon chains each independently having 12-24 carbons. In some cases, the hydrocarbon chains each independently have 16-20 carbons. In certain cases, the hydrocarbon chains are saturated. In certain cases, the hydrocarbon chains are unsaturated. In certain cases, the hydrocarbon chains each independently comprise 1-4 double bonds. In certain cases, the phospholipid comprises two different hydrocarbon chains. In certain embodiments of the LNP, the phospholipid includes dioleoylphosphatidylethanolamine (DOPE, 18: 1 PE). In certain cases, the phospholipid includes l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain cases, the phospholipid includes l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In certain cases, the phospholipid includes l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (delta9-Cis PC). In certain cases, the phospholipid includes l-stearoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (SOPE). In certain cases, the phospholipid includes a mixture of di oleoylphosphatidylethanolamine (DOPE, 18-1) and di oleoylphosphatidy choline (DOPC, 18-1).
[0147] In certain embodiments of the LNP, the lipid capable of reducing aggregation is a PEG-lipid. In certain cases, the PEG lipid is l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
[0148] In certain embodiments, the LNP further comprises a targeting ligand (e.g., as described herein). In certain cases, the targeting ligand comprises GalNac. In certain embodiments, the targeting ligand is linked to the ligand capable of reducing aggregation. In certain cases, the lipid capable of reducing aggregation linked to the targeting ligand is PEG- l,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]).
[0149] In some embodiments, the LNPs include an ionizable lipid of Formula (I) (e.g., as described herein); a phospholipid that is DOPE, cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG.
[0150] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group; a phospholipid that is a phosphatidylethanolamine, (e.g. DOPE), cholesterol, and a lipid capable of reducing aggregation that is PEG-DMG, and/or PEG-DSG-GalNAc or PEG-DSPE-GalNac.
[0151] In some embodiments, the LNPs include an ionizable lipid that is a cationic lipid comprising a tertiary amino ionizable group, a phospholipid that is a phosphatidylcholine (e.g. l,2-distearoyl-sn-glycero-3 -phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507. [0152] Generally, the lipid particles are prepared that include a total lipid to DNA (mass or weight) ratio of from about 5 : 1 to 50: 1. This is also referred to as the ratio of positively- chargeable polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups, or N/P ratio. In some embodiments, the N/P ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 : 1 to about 50: 1, from about 7: 1 to about 25:1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. The amounts of lipids and DNA can be adjusted to provide a desired N/P ratio, for example, 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. Generally, the lipid particle formulation's overall lipid content can range from about 5 mg/mL to about 30 mg/mL.
[0153] In some embodiments, the N/P ratio is from 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.
[0154] In some embodiments, a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm. In some embodiments, a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.
[0155] In some embodiments, an LNP has a mean diameter of 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.
[0156] In some embodiments, an LNP has a mean diameter of 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
[0157] In some embodiments, an LNP has a mean diameter of 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.
[0158] In some embodiments, the structural characteristics of the target tissue may be exploited to direct the distribution of the LNPs to such target tissues. For example, to target hepatocytes a LNP may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly, the LNP can readily penetrate such endothelial fenestrations to reach the target hepatocytes. In some embodiments, a LNP may be sized such that the dimensions of the particles are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a LNP may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the LNPs to hepatocytes. In such an embodiment, large LNPs will not easily penetrate the endothelial fenestrations, and would instead be cleared by the macrophage Kupffer cells that line the liver sinusoids. In some embodiments, the size of the LNPs is within the range of about 25 to 250 nm or 25nm to lOOnm, preferably less than 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, or less than 100 nm.
[0159] Without limitations, ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid nanoparticle. For example, ionizable lipid molar content can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, ionizable lipid comprises from about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable lipid comprises from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In particular embodiments, the cationic lipid comprises 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 lipid present in the particle.
[0160] The neutral lipid components can comprise 10-60% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 10-50% (mol) or 20- 55% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the noncationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In particular embodiments, the non-cationic lipid comprises 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 thereof or range therein) of the total lipid present in the particle.
[0161] In embodiments where the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle. In particular embodiments, the mixture of phospholipid and cholesterol or a cholesterol derivative comprises up to 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 thereof or range therein) of the total lipid present in the particle.
[0162] In some embodiments, the LNP comprises a phospholipid component in the mixture in an amount of from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
[0163] In some embodiments, the LNP includes a cholesterol component in the mixture in an amount of from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. In some embodiments, the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from 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 thereof or range therein) of the total lipid present in the particle.
[0164] It should be understood that the mol percentage of components described herein in the LNP is a target amount, and that the actual amount of each lipid component present in the formulation may vary, for example, by ±5 mol %.
[0165] In some embodiments, the LNP includes a lipid capable of reducing aggregation (e.g., a PEG-lipid conjugate) in an amount of about 1.5% to about 4%, for example 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 thereof or range therein) of the total lipid present in the particle. 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 thereof or range therein) of the total lipid present in the particle.
[0166] In various embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2: 1 to about 8: 1. In some embodiments, the lipid nanoparticles do not comprise any phospholipids.
[0167] In certain embodiments, the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
In certain embodiments, the LNP comprises: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
In certain embodiments, the LNP comprises: a) an ionizable lipid at 40 to 49 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
[0168] In some embodiments, the ratio of ionizable lipid:phospholipid:Cholesterol:PEG (as a percentage of total lipid content) is A:B:C:D, wherein: a. A = 40% - 60%, B = 5% - 20%, C = 25% - 50%, and D = 1.5% - 3.0% and wherein A+B+C+D = 100% b. A = 40% - 60%, B = 6% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein A+B+C+D
= 100%; b. A = 40% - 60%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein
A+B+C+D = 100%; c. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein
A+B+C+D = 100%; d. A = 40% - 49%, B = 10% - 20%, C = 35% - 45%, and D = 1.5% - 2.5% and wherein
A+B+C+D = 100%; d. A = 39% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein
A+B+C+D = 100%; e. A = 40% - 60%, B = 10% - 25%, C = 20% - 30%, and D = 0% - 3% and wherein
A+B+C+D = 100%; f. A = 45% - 50%, B = 20% - 25%, C = 25% - 30%, and D = 0% - 1% and wherein
A+B+C+D = 100% g. A = 40% - 60%, B = 10% - 30%, C = 20% - 45%, and D = 0% - 3% and wherein
A+B+C+D = 100%; h. A = 40% - 60%, B = 10% - 30%, C = 25% - 45%, and D = 0% - 3% and wherein
A+B+C+D = 100%; i. A = 45% - 55%, B = 10% - 20%, C = 30% - 40%, and D = 1% - 2% and wherein
A+B+C+D = 100%; j. A = 45% - 50%, B = 10% - 15%, C = 35% - 40%, and D = 1% - 2% and wherein
A+B+C+D = 100%; k. A = 45% - 65%, B = 5% - 20%, C = 20% - 45%, and D = 0% - 3% and wherein
A+B+C+D = 100%; m. A = 45%, B = 15%, C = 37.5%, and D = 2.5%; n. A = 57%, B = 12%, C = 28.5%, and D = 2.5% l. A = 50% - 60%, B = 5% - 15%, C = 30% - 45%, and D = 0% - 3% and wherein A+B+C+D = 100%; m. A = 55% - 60%, B = 5% - 15%, C = 30% - 40%, and D = 1% - 2% and wherein A+B+C+D = 100%; or n. A = 55% - 60%, B = 5% - 10%, C = 30% - 35%, and D = 1% - 2% and wherein A+B+C+D = 100%.
4.8 Nucleic Acid Cargo
[0169] In many embodiments, a given lipid nanoparticle may include a cargo, or payload, to be delivered to cells. Of particular interest in some embodiments are cargos that comprise a polynucleotide. In some embodiments, the polynucleotide is a DNA. DNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the DNA may be circular, e.g., a plasmid, a nanoplasmid, a mini circle, a covalently closed circular DNA, a circular viral genome, and the like. As another example, the DNA may be linear, e.g., a doggybone or other closed-end DNA, a linear viral genome, and the like. As another example, the DNA may be multivalent, e.g., a 3DNA. The DNA may be single stranded or double stranded or a hybrid of single and double stranded. The DNA may be chemically modified. In some embodiments, the polynucleotide is an RNA. RNA nucleic acid compositions of any structure may be included in the LNPs of the present disclosure. For example, the RNA may be linear or it may be circular. It may be an mRNA, an siRNA, an shRNA, a guide RNA (gRNA), a microRNA (miRNA), a circular RNA (circRNA). It may be chemically modified.
[0170] The one or more additional compounds can be a therapeutic agent. The therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected according to the treatment objective and biological action desired. For example, if the DNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNP containing the DNA is useful for treating an infection, the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In yet another example, if the LNP containing the DNA is useful for treating an immune disease or disorder, the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention. In some embodiments, the additional compound is an immune modulating agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is immune stimulatory agent.
4.9 Pharmaceutical Compositions
[0171] Also provided herein is a pharmaceutical composition comprising the lipid nanoparticle-encapsulated nucleic acid (e.g., DNA) and a pharmaceutically acceptable carrier or excipient. In some aspects, the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and/or glycine.
[0172] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0173] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, 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, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, 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; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.
[0174] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but 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, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric 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, naphthal ene- 1,5- disulfonic acid, naphthal ene-2-sulfonic acid, l-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, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. 4.10 Methods of Preparation
[0175] Any convenient methods can be used to prepare the LNPs of this disclosure. The LNP compositions can be prepared by high energy mixing of ethanolic lipids with aqueous DNA at low pH which protonates the ionizable lipid and provides favorable energetics for DNA / lipid association and nucleation of particles. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentrated to the desired level.
4.11 Methods of Use
[0176] As illustrated in the working examples and figures here, the LNPs and LNP pharmaceutical composition of the present disclosure, when formulated with nucleic acids, are less toxic in vivo as 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, e.g. at least 2-fold less toxic, e.g. 3-fold, 4-fold or 5-fold less toxic, in some instances 10-fold, 20-fold, or 50-fold less toxic, in certain instances 100-fold less toxic. By “less toxic”, it is meant eliciting a reduced immune response, e.g., characterized in a reduced amount of one or more cytokines upon administration to an organism.
[0177] At the same time, the LNPs and LNP pharmaceutical composition of the present disclosure have been observed to be efficacious at delivering their nucleic acid cargo to the target cell of interest, including where LNPs and LNP pharmaceutical composition of the present disclosure are equally or more efficacious at delivering their nucleic acid cargo to the target cell of interest as that same industry standard LNP administered at the same dose, e.g. having 2-fold the efficacy or more, e.g. 3-fold, 4-fold or 5-fold the efficacy or more, in some instances 10-fold, 20-fold or 50-fold the efficacy, in certain instances 100-fold more efficacious or more. By “more efficacious”, it is meant able to deliver more nucleic acid cargo to the cell, resulting in an increase in the amount of mRNA transcribed from that nucleic acid cargo or an increase in the amount of protein translated, for example a 2-fold increase or more, e.g. a 3-fold, 4-fold, 5-fold increase, e.g. 10-fold, 20-fold, 50-fold increase, in some instances a 100-fold increase or more.
[0178] Put another way, the LNPs of the present disclosure demonstrate an improved pharmacokinetics (PK) profile that broadens the therapeutic index of the composition. By a therapeutic index, or therapeutic ratio, it is meant the range of doses at which a medication is effective without unacceptable adverse events, calculated as the ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. This improvement over the art makes them more amenable to delivering nucleic acids, including DNA, to cells in vitro and in vivo, and accordingly they find many uses in many applications, including in the delivery of nucleic acids, including DNA, to cells for research and for therapeutic applications.
[0179] In performing such methods, the cells are typically contacted with the composition, e.g., LNP or pharmaceutical composition thereof, in amount effective to deliver the agent 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.
[0180] The present disclosure further provides methods of treating or preventing diseases in a subject in need thereof wherein an effective amount of the therapeutic compositions described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of the disease being treated, and may 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 are useful in the treatment of any of any indication in which it is beneficial to deliver a therapeutic cargo into the target cell.
[0181] The present disclosure further provides methods of immunizing a subject against a disease wherein an effective amount of a therapeutic composition described herein is administered to the subject. The route of administration will vary, naturally, with the location and nature of immunization agent, and may include, for example intradermal, transdermal, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration.
[0182] The present disclosure further provides a particle of the disclosure, a vector of the disclosure, a recombinant DNA of the disclosure, or compositions thereof, for use as a medicament. In some embodiments, the medicament is for expressing a protein in a cell. In some embodiments, the expressing of a protein is for the treatment of a disease in which the cell is deficient for the protein. In some embodiments, the expressing of a protein is for the treatment of a disease in which another cell is deficient for the protein. In some embodiments, the medicament is for the treatment of a cancer. In some embodiments, the medicament is for immunization against a disease. 4.12 Utility
[0183] The subject methods and compositions, e.g., as described above, can be used in any application where delivery of a cargo nucleic acid is desired. Applications of interest include both research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications. In some instances, cargo nucleic acids that may be introduced into a cell, and subsequently a nucleus, via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.
[0184] Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure. The research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector. Examples of specific types of research proteins include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zinc-finger nucleases, CRISPR/Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3/4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.
[0185] Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure. The diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.
[0186] Proteins of interest further include therapeutic proteins. Therapeutic proteins of interest include without limitation, hormones and growth and differentiation factors, fibrinolytic proteins, transcription factors, and enzymes.
[0187] Target cells to which nucleic acids may be delivered in accordance with embodiments of this disclosure may vary widely. Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like. Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type. For example, the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. Cells of interest include both 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, etc.
[0188] Targeted cells may include the cells of a targeted location, such as, e.g., the liver, or cells near or adjacent to hepatocytes, e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.
[0189] In some instances, the application of interest is a therapeutic application, for example, in the treatment of a disease. For example, the compositions and methods of the present application may be used to deliver a nucleic acid sequence to a cell to complement a genetic deficiency. As one nonlimiting example, compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.
4.13 Definitions
[0190] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “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 “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either 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.”
[0191] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [0192] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
[0193] The terms "individual," "subject" and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being. In some aspects, the subject is a patient. In some aspects, the subject is a human patient. In some aspects, the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI). In some aspects, the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later. In some cases, the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.
[0194] The term "treatment" used referring to a disease or condition means that at least an amelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia A) being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
[0195] The terms "effective amount," "pharmaceutically effective amount," or "therapeutically effective amount" as used herein mean a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition. The term "therapeutically effective amount" therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.
[0196] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. "Pharmaceutically acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
[0197] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
[0198] As used herein, the phrase "having the formula" or "having the structure" is not intended to be limiting and is used in the same way that the term "comprising" is commonly used. The term "independently selected from" is used herein to indicate that the recited elements, e.g., R groups or the like, can be identical or different.
[0199] As used herein, the terms “may,” "optional," "optionally," or “may optionally” mean that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. 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 wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.
[0200] “Acyl” refers to the groups 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)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(0)-.
[0201] The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although not necessarily, alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms. The term "lower alkyl" intends an alkyl group of 1 to 6 carbon atoms. "Substituted alkyl" refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a -C(=O)- moiety). The terms "heteroatom-containing alkyl" and "heteroalkyl" refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
[0202] The term “substituted alkyl” is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 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, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-aryl, -SO-heteroaryl, -SCh-alkyl, -SCh-aryl, -SCh-heteroaryl, and -NRaRb, wherein R’ and R” may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
[0203] The term "alkenyl" refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally, although again not necessarily, alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms. The term "lower alkenyl" intends an alkenyl group of 2 to 6 carbon atoms. The term "substituted alkenyl" refers to alkenyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
[0204] The term "alkynyl" refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to alkynyl substituted with one or more substituent groups, and the terms "heteroatomcontaining alkynyl" and "heteroalkynyl" refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkynyl" and "lower alkynyl" include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
[0205] The term "aryl", unless otherwise specified, refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms. For example, aryl groups may 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, diphenylether, diphenylamine, benzophenone, and the like. "Substituted aryl" refers to an aryl moiety substituted with one or more substituent groups, and the terms "heteroatomcontaining aryl" and "heteroaryl" refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, Ci-Cs alkoxy, Ci-Cs 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.
[0206] The term "alkylene" refers to a di-radical 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 alkylene linkages containing from 1 to 6 carbon atoms. Examples include, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)e-), and the like.
[0207] Similarly, the terms “alkenylene”, “alkynylene”, “arylene”, “aralkylene”, and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
[0208] The term "amino" refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
[0209] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
[0210] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SCh-alkyl, - SCh-substituted alkyl, -SCh-aryl and -SCh-heteroaryl.
[0211] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N— >0), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, 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, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SCh-alkyl, -SCh-substituted alkyl, -SCh-aryl and -SCh-heteroaryl, and trihalomethyl.
[0212] The terms “heterocycle,” “heterocyclic” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -SO2- moieties.
[0213] 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, quinolizine, 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, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.
[0214] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SCh-alkyl, -SCh-substituted alkyl, -SCh-aryl, -SCh-heteroaryl, and fused heterocycle.
[0215] By "substituted" as in "substituted alkyl," "substituted aryl," and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation, functional groups, and the hydrocarbyl moieties C1-C24 alkyl (including Ci-Cis alkyl, further including C1-C12 alkyl, and further including Ci-Ce alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, and further including C2- Ce alkynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
[0216] By "linking" or "linker" as in "linking group," "linker moiety," etc., is meant a linking moiety that connects two groups via covalent bonds. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH- CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (- O-(CH2)n-O-), epoxyimino (-0-NH-), epimino (-NH-), carbonyl (-CO-), etc. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); ethers, thioethers, amines, alkyls (e.g., (Ci-Ci2)alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used.
[0217] When the term "substituted" appears prior or after a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" is to be interpreted as "substituted alkyl and substituted aryl."
[0218] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
[0219] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =0, =NR70, =N-OR70, =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =0, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2,
=N2, -N3, -SO2R70, -SO2O-M+ -SO2OR70, -OSO2R70, -OSO2O-M+ -OSO2OR70, -P(O)(CF )2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70) 2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O- M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC( O)O'M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2- M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80 s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counter ion with 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(R60)4; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]o.5, or [Ba2+]o.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80 is meant to include -NH2, -NH-alkyl, A-pyrrolidinyl, 7V-piperazinyl, 47V- methyl-piperazin-l-yl and 7V-morpholinyl.
[0220] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O'M+, -OR70, -SR70, -S“M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3- M+, -SO3R70, -OSO2R70, -OSO3-M+ -OSO3R70, -PO3'2(M+)2, -P(O)(OR70)O- M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2-
M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2- M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2-
M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C( R70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O'M+, -OR70, -SR70, or -S“M+.
[0221] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O M+, -OR70, -SR70, -S'M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O'M+, -S(O)2OR70, -OS(O)2R70, -OS( O)2O'M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O'M+, -P(O)(OR70)(OR70), -C(O)R70, - C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(0)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR7 °C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60,
R70, R80 and M+ are as previously defined. [0222] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
[0223] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
[0224] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
[0225] In certain embodiments, a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure. Thus the compounds described herein include salts, solvates, hydrates, prodrug and isomer forms thereof, including the pharmaceutically acceptable salts, solvates, hydrates, prodrugs and isomers thereof. In certain embodiments, a compound may be a metabolized into a pharmaceutically active derivative.
[0226] Those skilled in the art will appreciate that a bond designated as — in a small molecule structure, as used herein, refers to a bond that, in some embodiments, is a single (e.g., saturated) bond, and in some embodiments, is a double (e.g., unsaturated) bond. For example the following structure:
Figure imgf000077_0001
is intended to encompass both
Figure imgf000077_0002
and
Figure imgf000077_0003
[0227] Unless otherwise specified, reference to an atom is meant to include isotopes of that atom. For example, reference to H is meant to include 1H, 2H (i.e., D) and 3H (i.e., T), and reference to C is meant to include 12C and all isotopes of carbon (such as 13C).
[0228] Definitions of other terms and concepts appear throughout the detailed description.
[0229] The following example(s) is/are offered by way of illustration and not by way of limitation. ENUMERATED EMBODIMENTS
Embodiment 1. An ionizable lipid compound of formula (I): (Z-L-Y)-Wn-(X-R)(n-i)
(I) wherein: a. Z is an ionizable head group; b. L is an optionally substituted (Ci-Ci2)alkylene; c. Y is a linking group; d. Wn is a linear alkyl core of n carbon atoms, wherein n is 3 to 6; e. X is an optional linking group; and f. each R is independently a lipid tail.
Embodiment 2. The compound of embodiment 1, wherein n is 4 to 6.
Embodiment 3. The compound of embodiment 2, wherein n is 4.
Embodiment 4. The compound of embodiment 2, wherein n is 5.
Embodiment 5. The compound of embodiment 2, wherein n is 6.
Embodiment 6. The compound of embodiment 1, wherein n is 3.
Embodiment 7. The compound of any one of embodiments 1 to 6, wherein W
Figure imgf000078_0001
selected from:
Figure imgf000078_0002
Figure imgf000079_0001
wherein:
*depicts the point of attachment to Y; each ** depicts a point of attachment to X;
G1 is H, or a group cyclically linked with Y that together with the carbon atom of Wn to which they are attached provide a heterocycle; and G2 is H or -CH2OH.
Embodiment 8. The compound of any one of embodiments 1 to 6, wherein Y is selected from — O— , — C(R10)2— , — OC(O)— , — C(O)O— , — OC(O)O— , — OC(O)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O — , and — NR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl.
Embodiment 9. The compound of embodiment 8, wherein Y is selected from — O — , — OC(O)— , and — OC(O)NR10— .
Embodiment 10. The compound of embodiment 8, wherein Y is — CH2 — .
Embodiment 11. The compound of embodiment 7, wherein G1 is a group that is cyclically linked with Y and together with the carbon atom of Wn to which they are attached provides a heterocycle.
Embodiment 12. The compound of any one of embodiments 1 to 11, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
Embodiment 13. The compound of embodiment 12, wherein L is -(CH2)2-.
Embodiment 14. The compound of embodiment 12, wherein L is -(CH2)3- Embodiment 15. The compound of any one of embodiments 1 to 14, wherein Z comprises a tertiary amino group.
Embodiment 16. The compound of embodiment 15, wherein Z is -NRUR12, wherein R11 and R12 are each independently alkyl or substituted alkyl.
Embodiment 17. The compound of embodiment 16, wherein R11 and R12 are each C1-6 alkyl.
Embodiment 18. The compound of embodiment 17, wherein R11 and R12 are each C1-3 alkyl.
Embodiment 19. The compound of embodiment 18, wherein R11 and R12 are each methyl.
Embodiment 20. The compound of embodiment 18, wherein R11 and R12 are each ethyl.
Embodiment 21. The compound of any one of embodiments 1 to 20, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O— ,— (CH2)SOC(O)NR10— , — (CH2)sO— , — (CH2)sSC(O)NR10— , — (CH2)SC(O)NR10— , — (CH2)sNR10C(O)— , — (CH2)SS— , — (CH2)SNR10— , — (CH2)SNR10C(O)O — , and — (CH2)sNR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl and s is 0-6.
Embodiment 22. The compound of any one of embodiments 1 to 21, wherein each X is independently selected from — OC(O) — , — C(O)O — , — OC(O)O — , — O — , — OC(O)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O — , and — NR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl.
Embodiment 23. The compound of embodiment 22, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— .
Embodiment 24. The compound of embodiment 23, wherein each — X-R is — OC(O)R.
Embodiment 25. The compound of any one of embodiments 1 to 24, wherein each R is independently an aliphatic hydrocarbon group that is straight chain or branched, saturated or unsaturated and/or optionally comprises a cyclic group.
Embodiment 26. The compound of any one of embodiments 1 to 25, wherein each R is a linear hydrocarbon group optionally comprising one or more cyclic groups.
Embodiment 27. The compound of any one of embodiments 1 to 26, wherein each R is selected from a C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
Embodiment 28. The compound of embodiment 27, wherein each R is selected from a C6-C12 alkyl, and C6-C12 alkenyl. Embodiment 29. The compound of embodiment 26, wherein at least one R is a linear hydrocarbon group comprising a cyclic group.
Embodiment 30. The compound of embodiment 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
Embodiment 31. The compound of any one of embodiments 1 to 25, wherein at least one R is a branched hydrocarbon group optionally comprising a cyclic group.
Embodiment 32. The compound of embodiment 31, wherein each R is a branched hydrocarbon group.
Embodiment 33. The compound of embodiment 32, wherein the branched hydrocarbon group comprises 8-20 carbon atoms.
Embodiment 34. The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is saturated.
Embodiment 35. The compound of any one of embodiments 31 to 33, wherein the branched hydrocarbon group is unsaturated.
Embodiment 36. The compound of any one of embodiments 31 to 35, wherein R is - CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
Embodiment 37. The compound of embodiment 31, wherein at least one R is a branched hydrocarbon group comprising a cyclic group.
Embodiment 38. The compound of embodiment 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted.
Embodiment 39. The compound of embodiment 1, wherein the compound is of formula (IIA):
Figure imgf000081_0001
(IIA). Embodiment 40. The compound of embodiment 39, wherein Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — , wherein R10 is selected from H and Ci-6 alkyl.
Embodiment 41. The compound of embodiment 40, wherein Y is — O — .
Embodiment 42. The compound of embodiment 40, wherein Y is — OC(O) — ,
Embodiment 43. The compound of embodiment 40, wherein Y is — OC(O)NR10 — .
Embodiment 44. The compound of any one of embodiments 39 to 43, wherein L is (C2-
Ce)alkylene or substituted (C2-Ce)alkylene.
Embodiment 45. The compound of embodiment 44, wherein L is -(CH2)2-
Embodiment 46. The compound of embodiment 44, wherein L is -(CEb)?-.
Embodiment 47. The compound of embodiment 44, wherein L is -(CH2)4-
Embodiment 48. The compound of any one of embodiments 39 to 47, wherein Z is -
NRnR12, wherein R11 and R12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
Embodiment 49. The compound of embodiment 48, wherein R11 and R12 are each Ci -3 alkyl.
Embodiment 50. The compound of embodiment 49, wherein R11 and R12 are each methyl.
Embodiment 51. The compound of embodiment 49, wherein R11 and R12 are each ethyl.
Embodiment 52. The compound of any one of embodiments 39 to 51, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
Embodiment 53. The compound of any one of embodiments 39 to 52, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
Embodiment 54. The compound of any one of embodiments 39 to 52, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
Embodiment 55. The compound of embodiment 54, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
Embodiment 56. The compound of embodiment 39, wherein the compound is of formula (IIIA):
Figure imgf000083_0001
wherein:
R11 and R12 are each independently C1-3 alkyl; q is 1 to 4;
Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and -(CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
Embodiment 57. The compound of embodiment 1, wherein the compound is of formula (IIB):
Figure imgf000083_0002
Embodiment 58. The compound of embodiment 57, wherein Y is selected from — O — , — OC(O)— , — OC(O)NR10— , — NR10C(O)— , — NR10C(O)O— , and — NR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl.
Embodiment 59. The compound of embodiment 58, wherein Y is selected from — NHC(O)— , — NHC(O)O— , and — NHC(O)S— .
Embodiment 60. The compound of any one of embodiments 57 to 59, wherein L is (C2- Ce)alkylene or substituted (C2-Ce)alkylene.
Embodiment 61. The compound of embodiment 60, wherein L is -(CH2)2- Embodiment 62. The compound of embodiment 60, wherein L is -(CH2)3-
Embodiment 63. The compound of embodiment 60, wherein L is -(CH2)4-
Embodiment 64. The compound of any one of embodiments 57 to 59, wherein Z is -
NRnR12, wherein R11 and R12 are each independently C1-6 alkyl or substituted C1-6 alkyl.
Embodiment 65. The compound of embodiment 64, wherein R11 and R12 are each C1-3 alkyl.
Embodiment 66. The compound of embodiment 64, wherein R11 and R12 are each methyl.
Embodiment 67. The compound of any one of embodiments 57 to 66, wherein each X is independently selected from — OC(O) — , — C(O)O — , and — OC(O)O — .
Embodiment 68. The compound of any one of embodiments 57 to 67, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
Embodiment 69. The compound of any one of embodiments 57 to 67, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic groups.
Embodiment 70. The compound of embodiment 69, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
Embodiment 71. The compound of embodiment 57, wherein the compound is of formula (IIIB):
Figure imgf000084_0001
wherein:
R11 and R12 are each independently C1-3 alkyl; q is 1 to 4; Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and -(CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
Embodiment 72. The compound of embodiment 1, wherein the compound is of formula (IIC):
Z L V
R'X X'R
(IIC).
Embodiment 73. The compound of embodiment 72, wherein Y is selected from — O — , — OC(O) — , — OC(O)NR10 — , and — C(R10)2 — , wherein R10 is selected from H and Ci -6 alkyl.
Embodiment 74. The compound of embodiment 73, wherein Y is — O — .
Embodiment 75. The compound of embodiment 73, wherein Y is — C(R10)2 — . — .
Embodiment 76. The compound of any one of embodiments 72 to 75, wherein L is (C2-
Ce)alkylene or substituted (C2-Ce)alkylene.
Embodiment 77. The compound of any one of embodiments 76, wherein L is -(CH2)2-
Embodiment 78. The compound of any one of embodiments 76, wherein L is -(CTb)?-.
Embodiment 79. The compound of any one of embodiments 76, wherein L is -(CEb^-.
Embodiment 80. The compound of any one of embodiments 72 to 79, wherein Z is -
NRnR12, wherein R11 and R12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
Embodiment 81. The compound of embodiment 80, wherein R11 and R12 are each C1-3 alkyl.
Embodiment 82. The compound of embodiment 81, wherein R11 and R12 are each methyl.
Embodiment 83. The compound of any one of embodiments 72 to 82, wherein each X is independently selected from — (CH2)sOC(O) — , — (CH2)sC(O)O — , — (CH2)sOC(O)O — , wherein s is 0-6.
Embodiment 84. The compound of any one of embodiments 72 to 82, wherein each s is Embodiment 85. The compound of any one of embodiments 72 to 82, wherein each s is
1.
Embodiment 86. The compound of any one of embodiments 72 to 82, wherein each s is 3.
Embodiment 87. The compound of any one of embodiments 72 to 86, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
Embodiment 88. The compound of any one of embodiments 72 to 86, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
Embodiment 89. The compound of embodiment 88, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
Embodiment 90. The compound of embodiment 72, wherein the compound is of formula (IIIC):
Figure imgf000086_0001
wherein:
R11 and R12 are each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , and — C(R10)2 — ; each s is independently 0, 1 or 2;
W is — O — , and — C(R10)2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and -(CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
Embodiment 91. The compound of any one of embodiments 1 to 90, wherein each R is independently
Figure imgf000087_0001
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; and r, p, and q is each independently an integer from 0 to 20.
Embodiment 92. The compound of embodiment 91, wherein at least one R comprises a
Figure imgf000087_0002
Figure imgf000088_0001
where each # represents the point of attachment to X, or the point of attachment to a linear or branched hydrocarbon chain of R.
Embodiment 93. A lipid nanoparticle comprising an ionizable lipid compound according to any one of embodiments 1 to 92.
Embodiment 94. The lipid nanoparticle of embodiment 93, further comprising a neutral lipid and a lipid capable of reducing aggregation.
Embodiment 95. The lipid nanoparticle of embodiment 94, wherein the neutral lipid comprises a phospholipid. Embodiment 96. The lipid nanoparticle of embodiment 94 or 95, wherein the neutral lipid comprises cholesterol.
Embodiment 97. The lipid nanoparticle of embodiment 96, comprising: a. a nucleic acid, b. an ionizable lipid, c. a phospholipid, d. cholesterol, and e. a lipid capable of reducing aggregation.
Embodiment 98. The lipid nanoparticle of embodiment 97, wherein the nucleic acid comprises DNA.
Embodiment 99. The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises RNA.
Embodiment 100. The lipid nanoparticle of embodiment 98, wherein the nucleic acid comprises DNA and RNA.
Embodiment 101. The lipid nanoparticle of embodiment 100, wherein the RNA is selected from mRNA, gRNA, and siRNA.
Embodiment 102. The lipid nanoparticle of any one of embodiments 97 to 101, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof.
Embodiment 103. The lipid nanoparticle of embodiment 102, wherein the phospholipid is a phosphatidylethanolamine (PE).
Embodiment 104. The lipid nanoparticle of embodiment 103, wherein the phospholipid is a phosphatidylcholine (PC).
Embodiment 105. The lipid nanoparticle of any one of embodiments 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons.
Embodiment 106. The lipid nanoparticle of embodiment 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons.
Embodiment 107. The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are saturated.
Embodiment 108. The lipid nanoparticle of embodiment 105 or 106, wherein the hydrocarbon chains are unsaturated and/or further comprise a carbocyclyl. Embodiment 109. The lipid nanoparticle of embodiment 108, wherein the hydrocarbon chains each independently comprise 1-4 double bonds.
Embodiment 110. The lipid nanoparticle of any one of embodiments 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains.
Embodiment 111. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l,2-dioleyl-sn-glycero-3 -phosphoethanolamine (DOPE).
Embodiment 112. The lipid nanoparticle of embodiment 103, wherein the phospholipid comprises l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE).
Embodiment 113. The lipid nanoparticle of embodiment 104, wherein the phospholipid comprises l,2-dipalmitoleoyl-sn-glycero-3 -phosphocholine (A9A9-Cis PC).
Embodiment 114. The lipid nanoparticle of embodiment 106, wherein the lipid nanoparticle comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
Embodiment 115. The lipid nanoparticle of embodiment 104, wherein the lipid nanoparticle comprises l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
Embodiment 116. The lipid nanoparticle of any one of embodiments 103 to 115 wherein the lipid capable of reducing aggregation is a PEG-lipid.
Embodiment 117. The lipid nanoparticle of embodiment 116, wherein the PEG- lipid is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG- DMG[2K]) or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG- DSG[2K]).
Embodiment 118. The lipid nanoparticle of any one of embodiments 94 to 117, further comprising a targeting ligand.
Embodiment 119. The lipid nanoparticle of embodiment 118, wherein the targeting ligand comprises GalNAc.
Embodiment 120. The lipid nanoparticle of embodiment 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation.
Embodiment 121. The lipid nanoparticle of embodiment 120, wherein the lipid capable of reducing aggregation is PEG-l,2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (PEG-DSG[2K]).
Embodiment 122. The lipid nanoparticle of any one of embodiments 94 to 121, wherein the N/P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30. Embodiment 123. The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 7.
Embodiment 124. The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 14.
Embodiment 125. The lipid nanoparticle of embodiment 122, wherein the N/P ratio is 28.
Embodiment 126. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 6 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
Embodiment 127. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. an ionizable lipid at 40 to 60 mol % of the total lipid present; b. a phospholipid at 10 to 20 mol % of the total lipid present; c. cholesterol at 35 to 45 mol % of the total lipid present; and d. a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
Embodiment 128. The lipid nanoparticle of any one of embodiments 94 to 125, comprising: a. e) an ionizable lipid at 40 to 49 mol % of the total lipid present; b. f) a phospholipid at 10 to 20 mol % of the total lipid present; c. g) cholesterol at 35 to 45 mol % of the total lipid present; and d. h) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present.
Embodiment 129. A pharmaceutical composition comprising a lipid nanoparticle of any one of embodiments 94 to 128 and a pharmaceutically acceptable excipient, carrier, or diluent.
Embodiment 130. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of embodiments 94 to 128. Embodiment 131. The method according to embodiment 130, wherein the cell is in vitro.
Embodiment 132. The method according to embodiment 130, wherein the cell is in vivo.
Embodiment 133. A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of embodiment 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.
6. EXAMPLES
[0230] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[0231] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.
Materials and Methods
[0232] LNP formulation. LNPs encapsulating nucleic acid payloads are prepared by mixing an organic solution of lipids with an aqueous solution of nucleic acid (e.g., DNA only, mRNA only, or DNA/mRNA mixtures) as described in Prud’homme et al. (J Pharm Sci 2018). Briefly, the lipidic excipients mixture (ionizable lipid, helper lipid, cholesterol, PEG- lipid and potentially other targeting moieties) is dissolved in an organic solvent. An aqueous solution of the nucleic acid is prepared in a low pH buffer of range 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 pH range of 5.0-6.5. The diluted LNP is subjected to dialysis purification against a secondary buffer with the pH range of 7.0-8.0. The LNP solution is concentrated by using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma) followed by filtration through 0.2 pm PES sterilizing-grade filter. Particle size is determined by dynamic light scattering (Horiba nanoPartica SZ-100). Encapsulation efficiency is calculated by using Quant-it RiboGreen assay kit.
[0233] EPO and cytokine detection in serum. Blood is collected via a retro-orbital bleed into serum separator tubes and processed to serum. The serum samples may be stored at -80C from collection until analysis. The serum levels of human EPO protein driven by expression from the DNA payload are quantified using the U-PLEX Human EPO Assay from MSD according to the manufacturer’s instructions. The serum levels of mouse cytokines resulting from exposure to DNA-LNPs were quantified using the Mouse Prolnflammatory 7-Plex Tissue Culture Kit from MSD according to the manufacturer’s instructions.
[0234] FIX detection in plasma. Blood is collected via a retro-orbital bleed into
K2EDTA tubes and processed to plasma. The plasma samples may be stored at -80C from collection until analysis. The plasma levels of human FIX following administration of LNPs were quantified using a U-Plex assay on the MSD platform. Briefly, a monoclonal mouse anti-human FIX antibody (Prolytix, clone AHIX-5041) was conjugated to biotin and used as the capture reagent on streptavidin-coated plates. A polyclonal goat anti-human FIX antibody (Cedarlane, clone CL20040AP) was conjugated to Sulfo-TAG and used as the detection reagent with the standard setup for quantification of electrochemiluminescence (ECL) signal 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 a minimum of 20 donors, was used to generate a standard curve and calculate % of normal human FIX levels. The assay was confirmed to be specific for human FIX and not to cross-react with mouse FIX, demonstrating very low levels of background in untreated mouse plasma samples.
Example 1. Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (L-l)
Figure imgf000094_0001
[0235] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl 4- (dimethylamino)butanoate
Figure imgf000095_0001
[0236] To a stirred solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol (1 equiv) and diisopropylethylamine (3 equiv) is added 2,5-dioxopyrrolidin-l-yl 4- (dimethylamino)butanoate (2 equiv) in DMF (4 V). The reaction mixture is heated at 90 °C for 16 hours then cooled room temperature and is quenched by the addition of water and MTBE. The organic layer is collected and the aqueous layer is further extracted with MTBE (3 x 3 V). The combined organic extracts are washed with 10% CuSCh (2 V) then brine (2 V), dried over MgSCU, then filtered and concentrated and purified by column chromatography. Recovered starting material is resubjected to the reaction conditions. [0237] Synthesis of l,2,4,5-tetrahydroxypentan-3-yl 4-(dimethylamino)butanoate
Figure imgf000095_0002
[0238] To a solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl 4- (dimethylamino)butanoate in methanol (5 V) is added aqueous HC1 (IM). The reaction mixture is monitored for completion by LCMS. When complete, the reaction mixture is concentrated under reduced pressure, with several toluene azeotropes for thorough drying. [0239] Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-l,2,4,5-tetrayl tetraki s(decanoate)
Figure imgf000096_0001
[0240] l,2,4,5-tetrahydroxypentan-3-yl 4-(dimethylamino)butanoate (1 equiv), decanoic acid (5.5 equiv), EDCI (6 equiv) , DMAP (2 equiv), DIE A (8 equiv) and ACN (10 mL) are mixed at 0 °C then at room temperature. The resulting mixture was stirred for 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 x 150mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure.
Example 2. Synthesis of l,4,5-tris(decanoyloxy)-3-({[3- (dimethylamino)propyl]carbamoyl}oxy)pentan-2-yl decanoate (L-2)
Figure imgf000096_0002
[0241] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol
Figure imgf000097_0001
Step 1
[0242] A solution of 1,2,3,4,5-pentahydroxypentane (25 g, 1 equiv), p-toluenesulfonic acid (2.83 g, 0.1 equiv) and 2,2-dimethoxypropane (37.65 g, 2.2 equiv) in methanol (250 mL) was stirred for overnight at room temperature under nitrogen atmosphere. Added K2CO3 (5 g) into reaction mixture and stirred for 1 h. The resulting mixture was filtered, the filter cake was washed with MeOH (2x20 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. Charged 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (gradient from 100:0 to 50:50, collected every 200 ± 10 mL). Took sample for TLC analysis (EA:PE = 1 :1), combined qualified products. This resulted in (12 g, 31.4%) bis(2,2-dimethyl- l,3-dioxolan-4-yl)methanol as colorless oil.
[0243] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl (4-nitrophenyl) carbonate
Figure imgf000097_0002
[0244] To a stirred solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol (10 g, 1 equiv) in THF (100 mL) was added 4-nitrophenyl carb onochlori date (9.55 g, 1.1 equiv) in portions. Added TEA (13.07 g, 3 equiv) dropwise at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 60 min at room temperature under nitrogen atmosphere. The resulting mixture was used in the next step directly without further purification.
[0245] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl N-[3- (dimethylamino)propyl]carbamate
Figure imgf000098_0001
[0246] To a stirred solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl 4-nitrophenyl carbonate (110 mL THF solution) was added dimethylaminopropylamine (6.6 g, 2.0 eq) dropwises at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for 60 min at room temperature under nitrogen atmosphere. The resulting mixture was quenched with 50 mL of water and extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with water (2x50 mL), 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. Charged 200 g of silica gel (type: ZCX-2, 100-200 mesh, 30 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with CH2CI2 / MeOH (gradient from 100:0 to 10: 1, collected every 100 ± 10 mL). Took sample for TLC analysis (CH2CI2 / MeOH = 5: 1), combined qualified products. Afforded bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl N-[3-(dimethylamino)propyl]carbamate (7 g, 45.1% two steps) as a yellow oil.
[0247] Synthesis of l,2,4,5-tetrahydroxypentan-3-yl N-[3- (dimethylamino)propyl]carbamate
Figure imgf000098_0002
[0248] A solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl N-[3-
(dimethylamino)propyl]carbamate (7 g, 1 equiv) in HC1 (6 M, 70 mL) was stirred for overnight at 50°C under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. This resulted in l,2,4,5-tetrahydroxypentan-3-yl N-[3- (dimethylamino)propyl]carbamate (4 g, 73.48%) as a light yellow oil.
[0249] Synthesis of l,4,5-tris(decanoyloxy)-3-({[3-
(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yl decanoate
Figure imgf000099_0001
[0250] To a stirred solution of l,2,4,5-tetrahydroxypentan-3-yl N-[3- (dimethylamino)propyl]carbamate (4 g, 1 equiv) and decanoyl chloride (21.77 g, 114.152 mmol, 8 equiv) in DCM (100 mL). Added TEA (14.44 g, 142.690 mmol, 10 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. The resulting mixture was extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with water (2x100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP -flash chromatography with the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: MeCN:i-PrOH=l : l; A: Water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50mL/min: detector, ELSD. The fraction removed the organic solvent under reduce pressure and the aqueous phase was adjusted pH=8 with NaHCOs (5% aqueous); extracted with heptane (50 mL *2), combined organic phase and dried over anhydrous Na2SO4, after filtration, the filtrate was concentrated under reduced pressure. This resulted in 1,4,5- tris(decanoyloxy)-3-({[3-(dimethylamino)propyl]carbamoyl}oxy)pentan-2-yl decanoate (961.2 mg, 7.51%) as a light yellow oil. LCMS: (ES, m/z): 898 [M+H]+; XH-NMR: (400 MHz, CDCh,/2pm): 5 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). Example 3. Synthesis of 3-(4-(dimethylamino)butoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (L-3)
Figure imgf000100_0001
[0251] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl methanesulfonate
Figure imgf000100_0002
Into a 100 mL 3-necked round-bottom flask were added bis(2,2-dimethyl-l,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) at room temperature. To the above mixture was added MsCI (2.96 g, 25.831 mmol, 1.5 equiv) dropwise at 0°C. The resulting mixture was stirred for additional Ih at room temperature. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with CH2CI2 (2 x 100 mL). The combined organic layers were washed with water (2x100 mL), dried over anhydrous MgSCh. After filtration, the filtrate was concentrated under reduced pressure. This resulted in bis(2,2-dimethyl-l,3-dioxolan-4- yl)methyl methanesulfonate (8.1 g, crude) as a brown oil. LCMS: (ES, m/z): 311 [M+l]+. XH NMR (300 MHz, Chloroforms/) 54.851 (t, J= 4.5 Hz, IH), 4.307-4.247 (m, IH), 4.153- 4.068 (m, 2H), 4.045-3.967 (m, 2H), 3.145-3.070 (m, 3H), 1.457-1.330 (m, 12H).
[0252] Synthesis of {4-[bis(2,2-dimethyl-l,3-dioxolan-4- yl)methoxy]butyl} dimethylamine
Figure imgf000101_0001
[0253] Into a 100 mL 3-necked round-bottom flask were added NaH (870.29 mg, 21.759 mmol, 1.5 equiv, 60%) and THF (25 mL) at room temperature. To the above mixture was added 4-(dimethylamino)butan-l-ol (1.7 g, 14.506 mmol, 1 equiv) and THF (25 mL) dropwise at 0°C. The resulting mixture was stirred for additional 30 min at 0°C. To the above mixture was added bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl methanesulfonate (4.95 g, 15.957 mmol, 1.1 equiv) and THF (25 mL) dropwise at 0°C. The resulting mixture was stirred for additional overnight at 60°C. The reaction was quenched with sat. NH4Q (aq.) at 0°C. The mixture was basified to pH 8 with saturated NaHCOs (aq.). The resulting mixture was extracted with EtOAc (5 x 100 mL), dried over anhydrous MgSCU. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2CI2 / MeOH (5: 1) to afford {4-[bis(2,2-dimethyl- l,3-dioxolan-4-yl)methoxy]butyl}dimethylamine (4.6 g, 80.6% two step yield) as a brown solid. LCMS: (ES, m/z): 332 [M+l]+. XH NMR (300 MHz, Chloroform-;/) 54.155-4.095 (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).
[0254] Synthesis of 3-[4-(dimethylamino)butoxy]pentane-l,2,4,5-tetrol
Figure imgf000101_0002
[0255] Into a 50mL 3-necked round-bottom flask were added {4-[bis(2,2-dimethyl-l,3- dioxolan-4-yl)methoxy]butyl} dimethylamine (2.6 g, 7.844 mmol, 1 equiv), H2O (5 mL) and acetic acid (21 mL) at room temperature. The resulting mixture was stirred for 6 h at 80°C. The mixture was allowed to cool down to room temperature. The resulting mixture was concentrated under vacuum. This resulted in 3-[4-(dimethylamino)butoxy]pentane-l, 2,4,5- tetrol (4.8 g, crude) as a brown oil. LCMS: (ES, m/z): 252 [M+l]+. XH NMR (300 MHz, DMSO-tA) 5 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).
[0256] Synthesis of l,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yl decanoate
Figure imgf000102_0001
[0257] Into a 250 mL 3-necked round-bottom flask were added 3-[4- (dimethylamino)butoxy]pentane-l,2,4,5-tetrol (3.2 g, 12.733 mmol, 1 equiv), DCM (160 mL) and TEA (20.62 g, 203.728 mmol, 16 equiv) at room temperature. To the above mixture was added decanoyl chloride (24.28 g, 127.330 mmol, 10 equiv) dropwise at 0°C. The resulting mixture was stirred for additional 2 h at 40°C. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with CH2CI2 (2 x 100 mL), the organic phase was collected and dried over anhydrous MgSCh. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2CI2 / MeOH (5: 1) to afford 4 g (crude). The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5 pm; mobile phase, B: MeCN:i-PrOH=l : l;A: Water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The residue was dissolved in hexane (100 mL). The combined organic layers were washed with Saturated NaHCOs solution (2x100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in l,4,5-tris(decanoyloxy)-3-[4-(dimethylamino)butoxy]pentan-2-yl decanoate (699.5 mg, 11.07% two step yield) as a light yellow oil. LCMS: (ES, m/z): 869 [M+l]+. ’H NMR (300 MHz, Chloroforms/, ppm) 5 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).
Example 4. Synthesis of l-(4-(2-(dimethylamino)ethyl)-l,3-dioxolan-2-yl)propane-l,2,3- triyl tridodecanoate (L-4)
Figure imgf000103_0001
[0258] Synthesis of pent-4-ene-l,2,3-triol
Figure imgf000103_0002
[0259] To a mixture of l-(2,2-dimethyl-l,3-dioxolan-4-yl)prop-2-en-l-ol (1 equiv) in MeOH (1 V) is added HC1 (6 M) (0.13 g, 3.544 mmol, 0.2 equiv) . The reaction mixture is stirred at 20 °C for 18 h. The resulting mixture is concentrated under reduced pressure and the material is thoroughly dried under vacuum and subject to the next reaction.
[0260] Synthesis of pent-4-ene-l,2,3-triyl tridodecanoate
Figure imgf000103_0003
[0261] To a mixture of pent-4-ene-l,2,3-triol (1 equiv), dodecanoic acid (3.4 equiv) and DMAP (1 equiv) in DCM (10 V) is added EDCI (1.44 g, 7.525 mmol, 1 equiv). The reaction mixture is stirred at 20 °C for 6 h. The resulting mixture is diluted with DCM (5 V). The resulting mixture is washed with (3 x IV) of water, brine (1 V). The resulting solution is dried over anhydrous Na2SO4. After filtration, the resulting mixture is concentrated under reduced pressure. The residue is dissolved in DCM (1 V) and silica gel (type: ZCX-2, 100- 200 mesh, 5.00 w./w.) is added. TLC analysis (PE / EA = 5: 1). [0262] Synthesis of 4-oxobutane-l,2,3-triyl tridodecanoate
Figure imgf000104_0001
[0263] To a mixture of pent-4-ene-l,2,3-triyl tridodecanoate (1 equiv) in THF (40 V) and H2O (20 V) is added K2OSO4 2H2O (0.12 equiv). The reaction mixture is stirred at 20 °C for 10 min, NalCh (5.0 equiv) and 2,6-lutidine (5.0 equiv) is added at 20 °C. The reaction mixture is stirred at 20 °C for 18 h. The resulting mixture is diluted with EA (20 V). The resulting mixture is washed with 3 x 20 V of water, brine (30 V). The resulting solution is dried over anhydrous Na2SO4. The resulting mixture is concentrated under reduced pressure. TLC analysis (PE / EA = 10: 1).
[0264] Synthesis of l-(4-(2-(dimethylamino)ethyl)-l,3-dioxolan-2-yl)propane-l,2,3-triyl tridodecanoate
Figure imgf000104_0002
A mixture of 4-oxobutane-l,2,3-triyl tridodecanoate (1 equiv.), 4-(dimethylamino)butane- 1,2-diol (1.5 equiv), DMAc dimethyl acetal (10 equiv.), and camphorsulfonic acid (2 equiv) is heated in DCE (40 V) at reflux overnight. The reaction mixture is cooled to 0 °C then poured into a rapidly stirring mixture of EA (30 V) and saturated aqueous NaHCOs (30 V). When the pH is above 7, the organic layer is collected and the product is further extracted with EA from the aqueous layer (3 x 20 V). The organic extracts are combined and washed with brine (30 V), dried over MgSCh, filtered and concentrated then purified by silica column chromatography to provide l-(4-(2-(dimethylamino)ethyl)- 1,3 -di oxolan-2-yl)propane- 1,2,3 - triyl tridodecanoate.
Example 5. Synthesis of l-[4-(dimethylamino)butanamido]-3,4- bis(dodecanoyloxy)butan-2-yl dodecanoate (L-5)
Figure imgf000105_0001
[0265] Synthesis of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-l,2,3-triol
Figure imgf000105_0002
Step 1
PH-SWL-5-1
[0266] To a stirred solution of 2,3,4-trihydroxybutanal (10 g, 1 equiv) and l-(2,4- dimethoxyphenyl)methanamine (13.92 g, 1 equiv), H2SO4 (1.63 g, 0.2 equiv), RaneyNi (3.57 g, 0.5 equiv) in EtOH (100 mL) was introduced H2 (3 atm) at room temperature. The resulting mixture was stirred for 6 h at 45°C. The resulting mixture was filtered, the filter cake was washed with ethanol (2x20 mL). The filtrate was concentrated under reduced pressure. The crude product 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-l,2,3-triol (16 g) was used in the next step directly without further purification.
[0267] Synthesis of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-l-hydroxyethyl)- 2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane
Figure imgf000105_0003
[0268] To a stirred solution of 4-{[(2,4-dimethoxyphenyl)methyl]amino}butane-l,2,3- triol (16 g, 1 equiv, crude) and TBSC1 (28 g, 3.1 eq) in DMF (160 mL) was added Imidazole (16 g, 4.0 eq) in portions at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 3 h at room temperature under nitrogen atmosphere. The mixture was purified by reversed-phase flash chromatography with the following conditions: column, Cl 8 silica gel; mobile phase, MeCN in Water (0.1% TFA), 40% to 90% gradient in 20 min; detector, UV 220 nm. This resulted in 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-l- hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 22.3%) as a brown oil.
[0269] Synthesis of N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2-hydroxybutyl}-N-[(2,4- dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide
Figure imgf000106_0001
[0270] A solution of 5-(2-{[(2,4-dimethoxyphenyl)methyl]amino}-l-hydroxyethyl)- 2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecane (6.6 g, 1 equiv) and 5-(2-{[(2,4- dimethoxyphenyl)methyl]amino}-l-hydroxyethyl)-2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8- disiladecane (6.6 g, 1 equiv), HATU (6.02 g, 1.2 equiv) , DIEA (5.12 g, 3 equiv) in DMF (50 mL) was stirred for 1 h at room temperature under nitrogen atmosphere. The residue was purified by reversed-phase flash chromatography with the following conditions: column, Cl 8 silica gel; mobile phase, MeCN in Water (0.1% TFA), 30% to 90% gradient in 20 min; detector, UV 220 nm. This resulted in N-{3,4-bis[(tert-butyldimethylsilyl)oxy]-2- hydroxybutyl}-N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)butanamide (4.2 g, 51.89%) as a brown oil.
[0271] Synthesis of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4- trihydroxybutyl)butanamide
Figure imgf000106_0002
[0272] 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 for 3h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The crude product N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2, 3,4- trihydroxybutyl)butanamide (5 g, crude) was used in the next step directly without further purification.
[0273] Synthesis of l-{N-[(2,4-dimethoxyphenyl)methyl]-4- (dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate
Figure imgf000107_0001
[0274] A solution of N-[(2,4-dimethoxyphenyl)methyl]-4-(dimethylamino)-N-(2,3,4- trihydroxybutyl)butanamide (5 g, 13.005 mmol, 1 equiv) and lauric acid (9.12 g, 45.518 mmol, 3.5 equiv), EDC.HC1 (8.08 g, 52.020 mmol, 4 equiv), DMAP (1.58 g, 13.0 mmol, 1 equiv) in DCM (50 mL) was stirred for 4 h at room temperature under nitrogen atmosphere. The resulting mixture was extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with water (2x100 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% TFA), 30% to 90% gradient in 20 min; detector, UV 220 nm. This resulted in l-{N-[(2,4-dimethoxyphenyl)methyl]-4- (dimethylamino)butanamido}-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate (3 g, 24.77%) as a white semi-solid.
[0275] Synthesis of l-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate
Figure imgf000107_0002
[0276] A solution of l-{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 HC1 (gas)in 1,4-di oxane (25 mL) was stirred for 6 h at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase HP -flash chromatography with the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: MeCN:i-PrOH=l : l; A: Water (0.1% TFA), 45% to 85% gradient in 15 min; Flow: 50mL/min: detector, ELSD. This resulted in l-[4-(dimethylamino)butanamido]-3,4-bis(dodecanoyloxy)butan-2-yl dodecanoate TFA salt (683.0 mg, 32.57%) as a white semi-solid. LCMS: (ES, m/z . 782 [M+H]+; ’H- NMR: (400 MHz, CDCh,/?pm): 5 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).
Example 6. Synthesis of 3-((4-(dimethylamino)butanoyl)oxy)pentane-l,2,4,5-tetrayl tetranonanoate (L-6)
Figure imgf000108_0001
[0277] 3-((4-(dimethylamino)butanoyl)oxy)pentane-l,2,4,5-tetrayl tetranonanoate is synthesized in the same manner as (L-l) using nonanoic acid instead of decanoic acid.
Example 7. Synthesis of 3-(((3-(dimethylamino)propyl)carbamoyl)oxy)pentane-l, 2,4,5- tetrayl tetranonanoate (L-7)
Figure imgf000109_0001
[0278] 3-(((3-(dimethylamino)propyl)carbamoyl)oxy)pentane-l,2,4,5-tetrayl tetranonanoate is synthesized in the same manner as (L-2), using nonanoyl chloride instead of decanoyl chloride.
Example 8. Synthesis of 3-(4-(dimethylamino)butoxy)pentane-l,2,4,5-tetrayl tetranonanoate (L-8)
Figure imgf000109_0002
[0279] 3-(4-(dimethylamino)butoxy)pentane-l,2,4,5-tetrayl tetranonanoate is synthesized in a manner similar to (L-3), using nonanoyl chloride instead of decanoyl chloride.
Example 9. Synthesis of 3-(3-(dimethylamino)propoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (L-9)
Figure imgf000110_0001
[0280] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol
Figure imgf000110_0002
Step 1
[0281] A solution of 1,2,3,4,5-pentahydroxypentane (20 g, 131.5 mmol, 1 equiv), p- toluenesulfonic acid (2.26 g, 13.15 mmol, 0.1 equiv) and 2,2-dimethoxypropane (30.1 g,
289.3 mmol, 2.2 equiv) in methanol (200 mL) was stirred for 16 h at room temperature under nitrogen atmosphere. K2CO3 (5 g) was added into reaction mixture and stirred for 1 h at room temperature. The resulting mixture was filtered, the filter cake was washed with MeOH (2x20 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. Charged 400 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. CombiFlash was used to purify the product, which was then eluted with PE / EA (gradient from 100:0 to 50:50, collected every 200 ± 10 mL). Took sample for TLC analysis (EA:PE = 1 : 1). The qualified products were combined. This resulted in (15 g, 49.1%) bis(2,2-dimethyl-l,3- dioxolan-4-yl)methanol as colorless oil.
[0282] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl) methyl methanesulfonate
Figure imgf000111_0001
[0283] To a stirred solution of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol (5 g, 21.526 mmol, 1 equiv) and TEA (4.36 g, 43.052 mmol, 2 equiv) in CH2CI2 (50 mL) was added MsCI (3.70 g, 32.289 mmol, 1.5 equiv) dropwise at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for an additional 3 h at room temperature. The resulting mixture was washed with water (2x50 mL). The aqueous layer was extracted with CH2CI2 (2x100 mL). The combined organic layers were washed with water (2x100 mL) and dried over anhydrous Mg2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in bis(2,2-dimethyl-l,3-dioxolan-4-yl) methyl methanesulfonate (6 g, 88.01%) as a yellow oil.
[0284] Synthesis of 3-(bis(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy)-N,N- dimethylpropan- 1 -amine
Figure imgf000111_0002
[0285] To a stirred solution of NaH (1.74 g, 43.620 mmol, 3 equiv) in THF (15 mL) was added 1-propanol, 3 -(dimethylamino)- (1.5 g, 14.540 mmol, 1.00 equiv) in portions at 0 °C under nitrogen atmosphere. The resulting mixture was stirred for additional 0.5 h at 0°C. To the above mixture was added bis(2,2-dimethyl-l,3-dioxolan-4-yl) methyl methanesulfonate (6.77 g, 21.810 mmol, 1.5 equiv) in portions at 0 °C. The resulting mixture was stirred for an additional 6 h at 60 °C. The reaction was quenched by the addition of NH4Q (aq.) (15 mL) at room temperature. The aqueous layer was extracted with EtOAc (3x100 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. Charged 150 g of silica gel (type: ZCX-2, 100-200 mesh, 10 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. The product was purified using CombiFlash, then eluted with CH2CI2 / MeOH (9:1) (gradient from 100:0 to 90: 10, collected every 200 ± 10 mL). The sample was analyzed by TLC (CELCh/MeOH = 9/1), and the qualified products combined. This resulted in {3-[bis(2,2-dimethyl-l,3-dioxolan-4- yl)methoxy]propyl} dimethylamine (1.07 g, 16.46%).
[0286] Synthesis of 3-(3-(dimethylamino)propoxy)pentane-l,2,4,5-tetraol
Figure imgf000112_0001
[0287] Into a 100 mL 3-necked round-bottom flask were added {3-[bis(2,2-dimethyl-l,3- dioxolan-4-yl)methoxy]propyl} dimethylamine (1 g, 3.150 mmol, 1 equiv) and hydrogen chloride (6M, 10 mL) at room temperature. The resulting mixture was stirred for 6 h at 60 °C. The resulting mixture was concentrated under reduced pressure. The crude product mixture was used in the next step directly without further purification.
[0288] Synthesis of l,4,5-tris(decanoyloxy)-3-[3-(dimethylamino)propoxy]pentan-2-yl decanoate (L-9)
Figure imgf000112_0002
[0289] Into a 100 mL round-bottom flask were added 3-[3-(dimethylamino) propoxy] pentane- 1,2, 4, 5-tetrol (1 g, 4.214 mmol, 1 equiv), DCM (10 mL), capric acid (3.99 g, 23.177 mmol, 5.5 equiv) at room temperature. To the above mixture was added EDCI (4.85 g, 25.284 mmol, 6 equiv) and DMAP (1.03 g, 8.428 mmol, 2 equiv) at room temperature. The resulting mixture was stirred for 24 h at room temperature. The reaction was quenched by the addition of water (50 mL) at room temperature. The resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were dried over anhydrous Mg2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, CH3CN/IPA (1 : 1) in water (0.1% TFA), 40% to 90% gradient in 20 min; detector, ELSD. The fraction was removed CEECN under reduce pressure and was basified to pH 8 with saturated NaHCOs (aq.). The aqueous layer was extracted with n-Heptane (2x100 mL) and dried over anhydrous Na2SO4; After filtration, the filtrate was concentrated under reduced pressure. This resulted in l,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+l]+. 'H NMR (300 MHz, Chloroform-d) 5: 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).
Example 10. Synthesis of 3-(4-(diethylamino)butoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (L-10)
Figure imgf000113_0001
[0290] Synthesis of (4-bromobut-2-yn-l-yl)di ethylamine HBr salt
Figure imgf000113_0002
[0291] Into a 250 mL round-bottom flask were added 4-(diethylamino)but-2-yn-l-ol (11 g, 78.014 mmol, 1 equiv) and DCM (110 mL) at room temperature under nitrogen atmosphere. To this was followed by the addition of a solution of PBn (63.26 g, 233.688 mmol, 3 equiv) in DCM (100 mL) with stirring at 0 degrees C over 15 min. The above mixture was stirred for 4 hours at room temperature. LCMS showed the reaction was completed. The reaction was then quenched by the addition of 300 mL of Na2CCh (sat. aq). The resulting solution was extracted with 3 x 100 mL of DCM. The organic layers were combined. The organic phase was washed with 1 x 150 mL of Na2CCh (sat. aq) and 1 x 150 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied on a silica gel column and eluted with ethyl acetate/hexane (83/17) to give product (4-bromobut-2-yn-l-yl)di ethylamine HBr salt (7.2 g, 32.61%) as a colorless solid.
[0292] Synthesis of {4-[bis(2,2-dimethyl-l,3-dioxolan-4-yl) m ethoxy ]but-2 -yn- 1- yl} di ethylamine
Figure imgf000114_0001
[0293] Into a 250 mL three neck round-bottom flask were added bis(2,2-dimethyl-l,3- dioxolan-4-yl)methanol HBr salt (6.9 g, 50.707 mmol, 1.5 equiv) and Toluene (80 mL) at room temperature under nitrogen atmosphere. To this was added NaH (8.11 g, 338.050 mmol, 10 equiv, 60%) at room temperature. The mixture was stirred for 1 h at room temperature. Into another 100 mL round-bottom flask were added (4-bromobut-2-yn-l- yl)diethylamine (6.9 g, 33.805 mmol, 1 equiv), Na2CO3 (10.75 g, 101.415 mmol, 3 equiv) and Toluene (100 mL). The mixture was stirred for 30 min at room temperature. The mixture was added into the above reaction mixture. The resulting mixture was stirred overnight at 80°C. LCMS showed the reaction was completed. The reaction was then quenched by the addition of 100 mL of 5% citric acid (aq). The resulting solution was extracted with 3 x 100 mL of ethyl acetate. The organic layers were combined. The organic phase was washed with 1 x 200 mL of Na2CO3 (sat. aq) and 1 x 200 mL of brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied on a silica gel column and eluted with ethyl acetate/hexane (3/2) to give product {4-[bis(2,2- dimethyl-l,3-dioxolan-4-yl) methoxy]but-2-yn-l-yl} di ethylamine (2.66 g, 30.73%) as a light red oily liquid. [0294] Synthesis of {4-[bis(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy]butyl}diethylamine
Figure imgf000115_0001
[0295] Into a 250 mL round bottom flask purged and maintained with an inert atmosphere of nitrogen, was added {4-[bis(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy]but-2- yn-l-yl] di ethylamine (2.65 g, 11.253 mmol, 1 equiv), MeOH (40 mL) and Pd/C (10%, 1.2g). The flask was evacuated and flushed three times with nitrogen, followed by flushing with hydrogen. The mixture was hydrogenated at room temperature for 18 h under hydrogen atmosphere (30 psi). LCMS showed the reaction was completed. The mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure, to afford {4- [bis(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy]butyl}diethylamine (1 g, 37.31%) as a yellow oil.
[0296] Synthesis of 3-(4-(diethylamino)butoxy)pentane-l,2,4,5-tetraol hydrochloride salt
Figure imgf000115_0002
[0297] Into a 250 mL three neck round-bottom flask were added (4-[bis(2,2-dimethyl- l,3-dioxolan-4-yl)methoxy]butyldiethylamine 950 mg, 9.736 mmol, 1 equiv) and THF (5 mL) at room temperature under nitrogen atmosphere. To this was added HC1 (6 M, 40 mL) dropwise at 0 degrees over 15 min. The mixture was stirred for 2 h at room temperature. LCMS showed the reaction was completed. The reaction mixture was concentrated to obtain 3-(4-(diethylamino)butoxy)pentane-l,2,4,5-tetraol hydrochloride (700 mg, 88.64%).
[0298] Synthesis of 3-(4-(diethylamino)butoxy)pentane-l,2,4,5-tetrayl tetraki s(decanoate)
Figure imgf000116_0001
[0299] Into a 150 mL 3-necked round bottom flask was added 3-(4- (diethylamino)butoxy)pentane-l,2,4,5-tetraol hydrochloride (700 mg, 3.579 mmol, 1 equiv), capric acid (3.39 g, 19.684 mmol, 5.5 equiv) and DMAP (0.87 g, 7.158 mmol, 2 equiv), EDCI (4.12 g, 21.474 mmol, 6 equiv) and ACN (21 mL) at room temperature. The mixture was stirred for 16 h at room temperature. The reaction solution was decompressed and vacuum concentrated, 200 mL DCM was added, and washed with 5% citric acid solution (3*100), then washed with saturated salt water three times, dried with anhydrous sodium sulfate and concentrated. The crude product was purified by Flash-Prep-HPLC under the following conditions: C18 silica gel column; Mobile phase, A 0.05%TFA in water/fkCFLCN (0% CFLCN increased to 95% within 15 min), eluent collected (gradient :A:0.05%TFA B:CH3CN=15/85); detector, ELSD. The organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.). The aqueous layer was extracted with heptane (3x100 mL). The resulting mixture was concentrated under reduced pressure. This resulted in 3-(4-(diethylamino)butoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (0.5145 g, 25.84%). LCMS: (ES, m/z): 896.7 [M+l]+. H-NMR (300 MHz, CDCh) 5: 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). Example 11. Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(4- propylphenyl)propanoyl]oxy})pentan-2-yl 3-(4-propylphenyl)propanoate (L-ll)
Figure imgf000117_0001
[0300] Synthesis of 3-(4-propylphenyl)acrylic acid
Figure imgf000117_0002
[0301] Into a 40 mL vial were added benzaldehyde, 4-propyl- (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) at room temperature. The reaction mixture was stirred for 12 h at 80 °C. The mixture was allowed to cool down to room temperature. The precipitated solids were collected by filtration and washed with 3x60 mL of water and 3x60 mL of methyl t-butyl ether /heptane (2: 1). After filtration, the resulting solid was dried under infrared light. This resulted in (2E)-3-(4-propylphenyl)prop-2-enoic acid (9.5 g, 72.90%) as a white solid.
[0302] Synthesis of 3-(4-propylphenyl)propanoic acid
Figure imgf000118_0001
[0303] Into a mL 250 mL round-bottom flask were added (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 (4.75g, 46.984 mmol) at room temperature. The resulting mixture was stirred for 12 h at room temperature under hydrogen atmosphere. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 3-(4-propylphenyl)propanoic acid (8.58 g, 88.48%) as a white solid.. LCMS: (ES, m/z): 193 [M+l]+.
[0304] Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(4- propylphenyl)propanoyl] oxy } )pentan-2-yl 3 -(4-propylphenyl)propanoate
Figure imgf000118_0002
[0305] Into a 40 mL vial were added 3-(4-propylphenyl)propanoic acid (4.46 g, 23.177 mmol, 5.5 equiv), 3-[3-(dimethylamino)propoxy]pentane-l,2,4,5-tetrol (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) at room temperature. 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 xl50 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5 pm; mobile phase, B: C LCN, A: Water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The organic solvent was removed under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.). The aqueous layer was extracted with heptane (3x100 mL). The resulting mixture was concentrated under reduced pressure. This resulted in 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(4- propylphenyl)propanoyl]oxy})pentan-2-yl 3-(4-propylphenyl)propanoate (0.5574 g, 13.33%) as a yellow oil. LCMS: (ES, m/z): 934.6 [M+l]+. ’H NMR (300 MHz, Chloroform-d) 5 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).
Example 12. Synthesis of l,ll-bis(pentadecan-8-yl) 6-[3-(dimethyl lamino)propoxy] undecanedioate (L-12)
Figure imgf000119_0001
[0306] Synthesis of trideca-l,12-dien-7-ol
Figure imgf000119_0002
[0307] Into a 500 mL 3-necked round-bottom flask were added Mg (16.40 g, 674.955 mmol, 5 equiv) and THF (50 mL) at room temperature. To the above mixture was added 6- Bromohex-l-ene (55.03 g, 337.478 mmol, 2.5 equiv) dropwise min at 55 °C. The resulting mixture was stirred for an additional 1 h at 55°C. To the above mixture was added Ethyl formate (10 g, 134.991 mmol, 1 equiv) dropwise at 0°C. The resulting mixture was stirred for an additional 2 h at room temperature. The reaction was quenched with sat. NH4Q (aq.) at 0°C. The resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with water (2x100 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 afford trideca-l,12-dien-7-ol (23.5 g, 88.67%) as a light yellow oil.
[0308] Synthesis of N,N-dimethyl-3 -(trideca- 1, 12-dien-7-yloxy)propan-l-amine
Figure imgf000120_0001
Into a 500 mL 3-necked round-bottom flask were added Trideca-l,12-dien-7-ol (10 g, 50.934 mmol, 1 equiv) and Toluene (200 mL) at room temperature. To the above mixture was added NaH (4.00 g, 166.554 mmol, 3.27 equiv) in portions over 10 min at 0°C. The resulting mixture was stirred for an additional overnight at 85°C. To the above mixture was added (3- chloropropyl) dimethylamine hydrochloride (15.28 g, 101.868 mmol, 2 equiv) in portions at 80°C. The resulting mixture was stirred for an additional 8 h at 80°C. The mixture was allowed to cool down to room temperature. The resulting mixture was diluted with water (300 mL). The resulting mixture was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with water (2x200 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 CH2CI2 / MeOH (10: 1) to N, N-dimethyl-3 -(trideca- 1,12- dien-7-yloxy)propan-l-amine (12.5 g, 87.19%) as a light yellow oil.
[0309] Synthesis of 6-(3-(dimethylamino)propoxy)undecanedioic acid hydrochloride salt
Figure imgf000121_0001
[0310] Into a 1000 mL 4-necked round-bottom flask were added N,N-dimethyl-3- (trideca-l,12-dien-7-yloxy)propan-l-amine (7.6 g, 27.000 mmol, 1 equiv) and AcOH (140 mL) at room temperature. To the above mixture was added KMnCU (17 g, 107.573 mmol, 3.98 equiv, in 700 mL H2O) dropwise at room temperature. The resulting mixture was stirred for an additional 3 h at 15°C. The reaction was quenched by the addition of NaiSiCh (17g) and NaHSOs (3 g) at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, AQ-C18 silica gel; mobile phase, MeCN in Water (0.1% HC1), 10% to 35% gradient in 16 min; detector, ELSD. This resulted in 6-(3- (dimethylamino)propoxy)undecanedioic acid hydrochloride salt (2.9 g, 30.35%) as a light yellow oil.
[0311] Synthesis of 1,1 l-bis(pentadecan-8-yl) 6-[3 -(dimethyl lamino)propoxy]undecanedioate
Figure imgf000121_0002
[0312] Into a 100 mL round-bottom flask were added 6-(3- (Dimethylamino)propoxy)undecanedioic acid hydrochloride salt (850 mg, 2.402 mmol, 1 equiv), pentadecan-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) at room temperature. To the above mixture was added EDCI (1.4 g, 7.303 mmol, 3.04 equiv) in portions at room temperature. The resulting mixture was stirred for an additional 16 h at room temperature. The resulting mixture was diluted with water (50 mL). The resulting mixture was extracted with heptane (3x20 mL). The combined organic layers were washed with sat. Na2CCh (2x20 mL), Me0H/H20 (4: 1, 5x20 mL), H2O (3x20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% TFA), 35% to 70% gradient in 10 min; detector, Ms. The product fraction was concentrated under vacuum to removed ACN and sat. Na2CCh (30 mL) added. The resulting mixture was extracted with heptane (3 x20 mL). The combined organic layers were washed with MeOH/FbO (4: 1, 5x20 mL), H2O (3x20 mL) and brine (20 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 1,1 l-bis(pentadecan-8-yl) 6-[3- (dimethyl lamino)propoxy]undecanedioate (680 mg, HPLC: 95.1%, yield: 34.24%) as a light yellow oil. LCMS: (ES, m/z): 738.7 [M+H]+; 'H NMR: (400 MHz, Chloroforms/) 34.892- 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).
Example 13. Synthesis of di(heptadecan-9-yl) 4-(3- (dimethylamino)propoxy)heptanedioate (L-13)
Figure imgf000122_0001
[0313] Synthesis of nona-l,8-dien-5-ol
Figure imgf000123_0001
64.01 %
[0314] To a stirred solution of Mg (32.81 g, 1349.910 mmol, 5.0 equiv) and I2 (200 mg, 0.788 mmol) in THF (100 mL) was added 4-bromo-l -butene (91.12 g, 674.955 mmol, 2.5 equiv) dropwise at room temperature under nitrogen atmosphere. The mixture was stirred for
1 h at 55°C under nitrogen atmosphere. To the above mixture was added ethyl formate (20 g, 269.982 mmol, 1 equiv) dropwise at 0°C. The resulting mixture was stirred for an additional
2 h at room temperature. The reaction was quenched with sat. NH4Q (aq.) (400 mL, 20 V) at 0°C. The resulting mixture was extracted with EtOAc (2 x 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, eluted with PE / EA (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent PE / EA =92/8). A sample was analyzed by TLC(PE / EA = 10: 1), to afford nona-l,8-dien-5-ol (26.0 g, 64.01%) as a light yellow oil.
[0315] Synthesis of N,N-dimethyl-3-(nona-l,8-dien-5-yloxy)propan-l-amine
Figure imgf000123_0002
[0316] To a stirred solution of nona-l,8-dien-5-ol (26 g, 185.415 mmol, 1 equiv) in toluene (500 mL, 20 V) was added NaH (22.25 g, 556.245 mmol, 3.0 equiv, 60%) in portions at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 8 h at 80°C under nitrogen atmosphere. To the above mixture was added (3 -chloropropyl) dimethylamine hydrochloride (58.68 g, 371.428 mmol, 2.0 equiv) at 80°C. The resulting mixture was stirred for an additional 16 h at 80°C. The reaction was quenched with sat. NH4Q (aq.) (500 mL, 20 V) at room temperature. The resulting mixture was extracted with EtOAc (2 x 250 mL, 20 V). 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, eluted with CH2CI2 / MeOH (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent CH2CI2 / MeOH =90/10). Took sample for TLC analysis. (CH2CI2 / MeOH = 10:1), to afford N,N-dimethyl-3-(nona-l,8-dien-5- yloxy)propan-l -amine (35 g, 74.96%) as a light yellow oil.
[0317] Synthesis of 4-(3-(dimethylamino)propoxy)heptanedioic acid
Figure imgf000124_0001
[0318] To a stirred solution of N,N-dimethyl-3-(nona-l,8-dien-5-yloxy)propan-l-amine (12 g, 53.244 mmol, 1 equiv) in AcOH (240 mL, 20 V) was added KMnO4 (33.66 g, 212.976 mmol, 4.0 equiv) dissolved in water (1.2 L, 100 V) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with solid NaiSiCh (33.66 g, 212.976 mmol, 4.0 equiv) at room temperature and stirred for 30 min at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by slurring with DMF (IL, 80 V). The crude product was used in the next step directly without further purification.
[0319] Synthesis of di(heptadecan-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate
Figure imgf000124_0002
[0320] 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 equiv) and 9-heptadecanol (14.72 g, 57.402 mmol, 2.50 equiv) was added EDCI (13.20 g, 68.883 mmol, 3.00 equiv) at room temperature. The resulting mixture was stirred for 16 h at room temperature. 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 x 300 mL, 60 V). The combined organic layers were washed with brine (300 mL, 30 V), 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 90: 10, and collected product eluent DCM/MeOH=95/5). for a sample was analyzed by TLC (DCM : MeOH = 10: 1), to afford di(heptadecan-9-yl) 4-(3-(dimethylamino)propoxy)heptanedioate (601.1 mg, 3.55%) as a yellow oil. LCMS: (ES, m/z): 738.7 [M+H]+; XH NMR (400 MHz, Chloroform-d) 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).
Example 14. Synthesis of (3-{[l,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2- yl]oxy}propyl)dimethylamine (L-14)
Figure imgf000125_0001
[0321] Synthesis of 2,2-dimethyl-l,3-dioxan-5-ol
Figure imgf000126_0001
[0322] Into a 500 mL round-bottom flask was place 2,2-dimethyl-l,3-dioxan-5-one (15 g, 115.258 mmol, 1 equiv) and THF (150 mL) at room temperature under N2 atmosphere. To this was added LiAlH4 (4.37 g, 115.258 mmol, 1.0 equiv) at 0°C. The mixture was stirring for 1 hour at 0°C. The reaction was quenched by the addition of water (4.5 mL), 4.5 mL (wt% 15% NaOH) and 13.5 mL water at 0°C. The resulting solution was diluted with 300 mL of THF and to this was added Na2SO4 (30 g). The mixture was warmed to room temperature and stirred for 15 minutes. The resulting mixture was filtered; the filter cake was washed with EA (2x100 mL). The filtrate was concentrated under reduced pressure to afford 2,2-dimethyl-l,3- dioxan-5-ol (12 g, 78.78%) as a colorless oil.
[0323] Synthesis of {3-[(2,2-dimethyl-l,3-dioxan-5-yl)oxy]propyl}dimethylamine
Figure imgf000126_0002
[0324] Into a 100 mL round-bottom flask was placed 2,2-dimethyl-l,3-dioxan-5-ol (5 g, 37.833 mmol, 1 equiv) and DMF (100 mL) at room temperature under N2 atmosphere. To this was added NaH (2.27 g, 94.582 mmol, 2.5 equiv) at 0°C. The mixture was stirring for 1 hour at room temperature. To this was added (3 -chloropropyl) dimethylamine (5.52 g, 45.400 mmol, 1.2 equiv., in 10 mL DMF) at room temperature. The mixture was stirred for 16 hours at 50°C. The reaction was quenched by the addition of NH4Q (sat. 50 mL) at 5°C. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, and eluted with washed (MeOH/DCM=l/l) to afford {3-[(2,2- dimethyl- 1,3 -di oxan-5-yl)oxy]propyl} dimethylamine (2.1 g, 25.54%) as a yellow solid.
[0325] Synthesis of 2-[3-(dimethylamino)propoxy]propane-l,3-diol hydrochloride salt
Figure imgf000127_0001
[0326] Into a 40 mL vial was place {3-[(2,2-dimethyl-l,3-dioxan-5-yl) oxy]propyl] dimethylamine (1.0 g, 4.602 mmol, 1 equiv) and DCM (20 mL) at room temperature under N2 atmosphere. To this was added HCl(gas)in 1,4-di oxane (4M, 5 mL) at 0°C. The mixture was stirring for 1 hour at 0°C. The resulting mixture was concentrated under vacuum to afford 2-[3-(dimethylamino)propoxy]propane-l,3-diol hydrochloride salt (780 mg, crude) as a yellow oil. The crude product was used in the next step directly without further purification.
Figure imgf000127_0002
[0328] Into a 250 mL round-bottom flask was place 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) at room temperature under N2 atmosphere. To this was added 4-nitrophenyl carb onochlori date (4.32 g, 21.445 mmol, 1.1 equiv., in 20 mL THF) at 0°C. The mixture was stirring for 2 hours at 70°C. The resulting mixture was diluted with THF (100 mL). The resulting mixture was filtered; the filter cake was washed with THF (2x3 OmL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with HepZEA (volume ratio) (gradient from 100:0 to 90: 10, and collected product eluent Hep/EA=97/3). A sample was analyzed by TLC(Hep: EA = 10: 1) to afford heptadecan-9-yl 4-nitrophenyl carbonate (3.5 g, 41.52%) as a light yellow oil.
[0329] Synthesis of (3-{[l,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2- yl]oxy}propyl)dimethylamine
Figure imgf000128_0001
[0330] Into a 250 mL round-bottom flask was place 2-[3 - (dimethylamino)propoxy]propane-l,3-diol hydrochloride salt (0.79 g, 3.736 mmol, 0.45 equiv), DMAP (0.23 g, 1.868 mmol, 0.5 equiv), TEA (1.51 g, 14.944 mmol, 4 equiv) and DMF (16 mL) at room temperature under N2 atmosphere. To this was added heptadecan-9-yl 4-nitrophenyl carbonate (3.5 g, 8.302 mmol, 1 equiv. in 30 mL DMF) at room temperature. The mixture was stirring for 16 hours at 80°C. The resulting mixture was diluted with EA (300 mL) and washed with 5% wt Citric acid (1x50 mL), NaHCO3 (1x50 mL) and washed with water (2x150 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with DCM/MeOH (volume ratio) (gradient from 100:0 to 90: 10, and collected product eluent DCM/MeOH=97/3). A sample was analyzed by TLC . (DCM: MeOH = 10: 1 0.2), to afford (3-{[l,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2- yl]oxy}propyl)dimethylamine (800 mg) as a yellow oil. The products were dissolved in n- heptane (100 mL). The n-heptane phase was washed with MeOH/EEO (4: 1) (2 x 10 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford (3-{[l,3-bis({[(heptadecan-9-yloxy)carbonyl]oxy})propan-2- yl]oxy}propyl)dimethylamine (584.1 mg, 9.07%) as a yellow oil. LCMS: (ES, m/z): 742.18 [M+l]+; 'H N R (400 MHz, Chloroforms/) 3 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).
Example 15. Synthesis of l,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propyljnonanedioate (L-15)
Figure imgf000129_0001
[0331] Synthesis of methyl 3-(2,6-dioxocyclohexyl)propanoate
Figure imgf000129_0002
step 1
[0332] Into a 250 mL round-bottom flask was placed 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). The reaction mixture was stirred at 80°C for 12 h. The reaction was then quenched by the addition of 200 mL of water/ice. The pH value of the solution was adjusted to 6 with HC1 (1 mol/L). The resulting solution was extracted with 3x200 mL of ethyl acetate and the organic layers combined. The resulting mixture was washed with 200 mL of NaCl. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in methyl 3-(2,6- dioxocyclohexyljpropanoate (35 g, crude) as a yellow oil.
[0333] Synthesis of 5-oxononanedioic acid
Figure imgf000130_0001
[0334] Into a 250 mL round-bottom flask was placed methyl 3-(2,6- dioxocyclohexyl)propanoate (35 g, 176.573 mmol, 1 equiv) and HC1 (IM) (70 mL). The reaction mixture was stirred at 110°C for 12 h. The resulting mixture was concentrated under vacuum. The residue was dissolved in 200 mL of MTBE and stirred for 1 hr. The solids were collected by filtration. This resulted in 5-oxononanedioic acid (15 g, 42.01%) as a brown solid.
Figure imgf000130_0002
[0336] Into a 1.0 L round-bottom flask was placed 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). The reaction mixture was stirred at 20°C for 3 h. The reaction mixture was poured into 500 mL ice water. The resulting mixture was extracted with EA (3 x 500 mL). The combined organic layers were washed with water (3 x 100 mL) and NaCl (100 mL, aq), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford pentadecan-8-ol (10 g, crude) as a white solid.
[0337] Synthesis of l,9-bis(pentadecan-8-yl) 5 -oxononanedi oate
Figure imgf000131_0001
[0338] Into a 250 mL round-bottom flask was placed 5 -oxononanedioic acid (5.0 g, 24.727 mmol, 1 equiv), pentadecan-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). 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 the addition of Citric acid (5% aq) (60 mL) at 5°C and washed with 2x30 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, and eluted with Hep/EA (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent Hep/EA=95/5). A sample was analyzed by TLC (Hep: EA = 10: 1 0.2). The resulting mixture was concentrated under reduced pressure to afford l,9-bis(pentadecan-8-yl) 5-oxononanedioate (10.2 g, 66.21%) as a light yellow oil. [0339] Synthesis of l,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]-5- hydroxynonanedioate
Figure imgf000131_0002
[0340] Into a 250 mL round-bottom flask, was place l,9-bis(pentadecan-8-yl) 5- oxononanedioate (5.5 g, 8.828 mmol, 1 equiv) and THF (44 mL) at room temperature under N2 atmosphere. To this was added [3-(chloromagnesio)propyl]dimethylamine (88.28 mL, 88.280 mmol, 10 equiv) dropwise at -60°C. The mixture was stirring for 2 hours at -60°C. The reaction was quenched by the addition of NH4Q (aq) (80 mL) at 5°C and washed with 2x40 mL of water. The organic phase was dried over anhydrous Na2SO4. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with DCM/MeOH (volume ratio), (gradient from 100:0 to 70:30, and collected product eluent DCM/MeOH=82/18). A sample was analyzed by TLC. (DCM:MeOH = 2: 1 0.1). The resulting mixture was concentrated under reduced pressure to afford l,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.7 g, 43.07%) as light yellow oil.
[0341] Synthesis of l,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propylidene]nonanedioate
Figure imgf000132_0001
[0342] Into a 40 mL vial, was place l,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propyl]-5-hydroxynonanedioate (2.5 g, 3.520 mmol, 1 equiv) and DCM (5 mL) at room temperature under N2 atmosphere. To this was added EtsSiH (4.09 g, 35.200 mmol, 10 equiv) and BF3 Et2O (5.00 g, 35.200 mmol, 10 equiv) at room temperature. The mixture was stirring for 2 hours at 45°C. The resulting mixture was diluted with DCM (100 mL). The reaction was quenched by the addition of NaHCCh (aq) (25 mL) at 5°C and washed with 2x25 mL of water. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluted with DCM/MeOH (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent DCM/MeOH=94/6). Took sample for TLC analysis. (DCM: MeOH = 10: 1 0.15). The resulting mixture was concentrated under reduced pressure to afford l,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propylidene]nonanedioate (1.6 g, 62.51%) as light yellow oil. [0343] Synthesis of l,9-bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]nonanedioate
(L-15)
Figure imgf000133_0001
[0344] Into a 100 mL vial was place l,9-bis(pentadecan-8-yl) 5-[3- (dimethylamino)propylidene]nonanedioate (1.6 g, 2.312 mmol, 1 equiv) and EtOH (32 mL). To this was added Pd/C (0.49 g, 0.462 mmol, 0.2 equiv, 10%) at room temperature. The mixture was stirring for 2 hours at room temperature under H2 (30 psi) atmosphere. The resulting mixture was filtered; the filter cake was washed with EA (2x32 mL). 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 90: 10, and collected product eluent DCM/MeOH=95/5). A 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, 100 V). Then n-heptane phase was washed with MeOH/H2O (4: 1) (2 x 32 mL, 10 V), MeCN/H2O (4: 1) (2 x 32 mL, 10 V), water (32 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford 1,9- bis(pentadecan-8-yl) 5-[3-(dimethylamino)propyl]nonanedioate (1.0640 g, 66.31%) as light yellow oil. LCMS: (ES, m/z): 694.9 [M+H]+. XH NMR (400 MHz, Chloroform-d) S 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). Example 16. Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5- tris(dodecanoyloxy)pentan-2-yl dodecanoate (L-16)
Figure imgf000134_0001
[0345] Into a 40 mL vial were added 3-[3-(dimethylamino)propoxy]pentane-l,2,4,5-tetrol (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) at room temperature. The resulting mixture was stirred for 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 x 150mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: C LCN, A: water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The fraction was removed C LCN under reduce pressure and was basified to pH 8 with saturated NaHCOs (aq.). The aqueous layer was extracted with n-Heptane (2x100 mL) and dried over anhydrous Na2SO4; After filtration, the filtrate was concentrated under reduced pressure. This resulted in 3-[3- (dimethylamino)propoxy]-l,4,5-tris(dodecanoyloxy)pentan-2-yl dodecanoate (0.6852 g, 20.24%) as a yellow oil. LCMS: (ES, m/z): 967 [M+l]+. 'H NMR (300 MHz, Chloroform-d) 5 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). Example 17. Synthesis of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl 5- [3-(dimethylamino)propyl] nonanedioate (L-17)
Figure imgf000135_0001
[0346] Synthesis of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid
Figure imgf000136_0001
[0347] A solution of 5-oxononanedioic 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 pentadecan-8-ol (2.94 g, 12.858 mmol, 0.65 equiv) at 25°C under nitrogen atmosphere followed by the addition of EDCI (4.17 g, 21.760 mmol, 1.1 equiv) in portions at 25°C. The resulting mixture was stirred for 18h at 25°C under nitrogen atmosphere. The mixture was acidified to pH 5 with 0.05 M HC1. The resulting mixture was washed with 1x50 mL of 0.05 M HC1 and 1x100 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 160 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (2: 1) (gradient from 10: 1 to 2: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 1 : 1), combined qualified products. This afforded 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (4 g, 49.01%) as a light yellow oil. LCMS: (ES, m/z): 413.2 [M±H]+. 'H NMR: (400 MHz, Chloroform-d) S 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).
[0348] Synthesis of 2-(bromomethyl)-octahydro-lH-indene
Figure imgf000136_0002
[0349] A solution of octahydro- lH-inden-2-ylmethanol (16 g, 103.726 mmol, 1 equiv) in DCM (300 mL) was treated with triphenylphosphine (40.81 g, 155.589 mmol, 1.5 equiv) at 0°C under nitrogen atmosphere followed by the addition of carbon tetrabromide (51.60 g, 155.589 mmol, 1.5 equiv) in portions at 0°C. The resulting mixture was stirred for 18 h at 25°C under nitrogen atmosphere. 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 160 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (100: 1) (gradient from 1:0 to 30: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 20: 1), combined qualified products. This resulted in 2-(bromomethyl)- octahydro-lH-indene (22 g, 97.67%) as a colorless oil. 'H NMR: (400 MHz, Chloroform-d) S 3.480-3.378 (m, 2H), 2.663-2.368 (m, 1H), 2.072-1.691 (m, 4H), 1.644-0.887 (m, 10H). [0350] Synthesis of 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-lH-inden- 2-yl)propyl]-octahydro-lH-indene
Figure imgf000137_0001
[0351] A mixture of NaH (5.80 g, 144.898 mmol, 2.3 equiv, 60%) in DMSO (200 mL) was stirred for 1 h at 25°C under nitrogen atmosphere. To the above mixture was added toluenesulfonylmethyl isocyanide (TosMIC) (12.3 g, 62.999 mmol, 1.00 equiv) and TBAI (2327 mg, 6.300 mmol, 0.1 equiv) in portions at 25°C. The resulting mixture was stirred for additional 3 h at 25°C. To the above mixture was added 2-(bromomethyl)-octahydro-lH- indene (21.89 g, 100.798 mmol, 1.6 equiv) in DMSO (100 mL) dropwise over 20 min at 25°C. The resulting mixture was stirred for additional 18 h at 25°C. The reaction was quenched with sat. NH4Q (aq.) at 25°C. The resulting mixture was extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (3x300 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 500 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (20: 1) (gradient from 50:0 to 10: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 10: 1), combined qualified products. This resulted in 2-[2-isocyano-2-(4- methylbenzenesulfonyl)-3-(octahydro-lH-inden-2-yl)propyl]-octahydro-lH-indene (9 g, 30.54%) as a yellow oil. LCMS: (ES, m/z): 468.4 [M±H]+. 'H NMR: (400 MHz, Chloroform- d) S 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).
[0352] Synthesi s of 1 , 3 -bi s(octahy dro- 1 H-inden-2-yl)propan-2-one
Figure imgf000138_0001
[0353] A solution of 2-[2-isocyano-2-(4-methylbenzenesulfonyl)-3-(octahydro-lH-inden- 2-yl)propyl]-octahydro-lH-indene (9 g, 19.243 mmol, 1 equiv) in DCM (60 mL) was stirred at 25°C. To the above mixture was added HC1 (5 mL, 4M in MeOH) dropwise at 25°C. The resulting mixture was stirred for additional 3 h at 25°C. Brine (100 mL) was added in. The resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with Brine (2 x 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 300 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (30: 1) (gradient from 50:0 to 10: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 10: 1), combined qualified products. The residue was purified by silica gel column chromatography, eluted with PE / EA (30: 1) to afford l,3-bis(octahydro-lH-inden-2-yl)propan-2-one (5 g, 85.90%) as a light yellow solid. LCMS-: (ES, m/z): 303.4 [M±H]+. 'H NMR: (400 MHz, Chloroform-d) S 2.668-2.308 (m, 6H), 2.039-1.690 (m, 8H), 1.633-0.949 (m, 20H).
[0354] Synthesis of l,3-bis(octahydro-lH-inden-2-yl)propan-2-ol
Figure imgf000139_0001
Step 5
[0355] A solution of l,3-bis(octahydro-lH-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 followed by the addition of methanol (50 mL) dropwise at 0°C. The resulting mixture was stirred for 3 h at 25°C. The reaction was quenched by the addition of sat. NH4Q (aq.) (20mL) at 0°C. The resulting mixture was extracted with EA (2 x 100 mL). The combined organic layers were washed with Brine (2 x 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (30: 1) (gradient from 50:0 to 20: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 20: 1), combined qualified products. This resulted in l,3-bis(octahydro-lH-inden-2-yl)propan-2-ol (4.6 g, 91.39%) as a colorless oil. LCMS: (ES, m/z): 287.3 [M-18±H]+. 'H NMR: (400 MHz, Chloroform-d) S 3.698-3.574 (m, 1H), 2.394-2.044 (m, 2H), 1.970-1.701 (m, 8H), 1.605-
1.090 (m, 25H).
[0356] Synthesis of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl
5 di t
Figure imgf000139_0002
[0357] A solution of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (6 g, 14.542 mmol, 1 equiv) in DCM (120 mL) was treated with l,3-bis(octahydro-lH-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) at 25°C under nitrogen atmosphere followed by the addition of EDCI (3.64 g, 18.913 mmol, 1.3 equiv) in portions at 25°C. The resulting mixture was stirred for 18 h at 45°C under N2 atmosphere. The resulting mixture was diluted with DCM (120 mL). The combined organic layers were washed with 0.05M HC1 (1x100 mL) and Brine (1x200 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 300 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with PE / EA (5: 1) (gradient from 20: 1 to 4: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 3: 1), combined qualified products. This resulted in l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl 5-oxononanedioate (3.7 g, 36.40%) as a light yellow oil. LCMS: (ES, m/z): 721.5 [M+Na]+. 'H NMR: (400 MHz, Chloroform-d) S 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).
[0358] Synthesis of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate
Figure imgf000140_0001
[0359] To a stirred solution of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl 5-oxononanedioate (3.5 g, 5.006 mmol, 1 equiv) in tetrahydrofuran (50 mL) was added [3-(chloromagnesio)propyl]dimethylamine (75.10 mL, 75.090 mmol, 15 equiv) dropwise at -60°C under nitrogen atmosphere. The resulting mixture was stirred for 2 h at - 60°C under nitrogen atmosphere. The reaction was quenched by the addition of sat. NH4Q (aq.) (50 mL) at -60°C. The mixture was acidified to pH 6 with 0.05M HC1. The resulting mixture was extracted with CH2CI2 (2 x 100 mL). The combined organic layers were washed with Brine (1x200 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with CH2CI2 / MeOH (20: 1) (gradient from 50: 1 to 10: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (CH2CI2 / MeOH = 10: 1), combined qualified products. This resulted in l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl 5-[3- (dimethylamino)propyl]-5-hydroxynonanedioate (2.5 g, 63.51%) as a light yellow oil.
LCMS: (ES, m/z): 786.7 [M±l]+. XH NMR: (400 MHz, Chloroform-d) S 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).
[0360] Synthesis of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl (5Z)-5-[3-(dimethylamino)propylidene]nonanedioate
Figure imgf000141_0001
[0361] To a stirred solution of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2.4 g, 3.052 mmol, 1 equiv) and triethylsilane (3.55 g, 30.520 mmol, 10 equiv) in DCM (50 mL) was added BF3.Et2O (4.33 g, 30.520 mmol, 10 equiv) dropwise at 0°C under nitrogen atmosphere. The resulting mixture was stirred for 2 h at 40°C under nitrogen atmosphere. The combined organic layers were washed with NaHCCh (2x50 mL), Brine (1x50 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. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 100 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using CombiFlash to purify the product. Eluted with CH2CI2 / MeOH (20: 1) (gradient from 50: 1 to 10: 1, collected every 200 ± 10 mL). Took sample for TLC analysis (CH2CI2 / MeOH = 10: 1), combined qualified products. This resulted in 1 -[ 1 ,3 - bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl (5Z)-5-[3- (dimethylamino)propylidene]nonanedioate (1.5 g, 63.97%) as a light yellow oil. LCMS: (ES, m/z): 768.7 [M+H]+. 'H NMR: (400 MHz, Chloroform-d) S 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).
[0362] Synthesis of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9-pentadecan-8-yl 5 - [3 -(dimethylamino)propyl]nonanedioate (L- 17)
Figure imgf000142_0001
[0363] To a stirred solution of l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl (5Z)-5-[3-(dimethylamino)propylidene]nonanedioate (1.3 g, 1.692 mmol, 1 equiv) in EtOH (40 mL) were added Pd/C (0.26 g, 2.443 mmol, 1.44 equiv) at 25°C under nitrogen atmosphere. The flask was evacuated and flushed three times with nitrogen, followed by flushing with hydrogen. The mixture was stirred 2 h at room temperature under an atmosphere of hydrogen. The resulting mixture was filtered, the filter cake was washed with EA (2x100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in Heptane (150 mL). The resulting mixture was washed with 2x100 mL of ACN/H2O (5: 1, 50 mL), 1x100 mL of ACN (50 mL). The heptane phase was concentrated under reduced pressure. This resulted in l-[l,3-bis(octahydro-lH-inden-2-yl)propan-2-yl] 9- pentadecan-8-yl 5-[3-(dimethylamino)propyl]nonanedioate (0.5205 g, 38.46%) as a light yellow oil. LCMS: (ES, m/z): 770.7 [M+H]+. 'H NMR: (400 MHz, Chloroform-d) S 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).
Example 18. Synthesis of l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9- pentadecan-8-yl 5-[3-(dimethylamino) propyljnonanedioate (L-18)
Figure imgf000144_0001
[0364] Synthesis of 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid
Figure imgf000145_0001
[0365] Into a 500 mL round-bottom flask were added 5-oxononanedioic acid (11 g, 54.400 mmol, 1 equiv), DCM (200 mL), pentadecan-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) at room temperature. The resulting mixture was stirred overnight at room temperature under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, and eluted with PE / EA (10: 1) to afford 5,9-dioxo-9-(pentadecan-8-yloxy)nonanoic acid (10 g, yield: 44.55%) as a yellow oil. [0366] Synthesis of methyl 2-(l,3-dihydroinden-2-ylidene)acetate
Figure imgf000145_0002
[0367] Into a 2000 mL 3 -necked round-bottom flask were added methyl 2- (dimethoxyphosphoryl)acetate (103.35 g, 567.485 mmol, 2.5 equiv) and THF (900 mL) at room temperature. To the above mixture was added NaH (22.70 g, 567.485 mmol, 2.5 equiv, 60%) in portions at 0°C. The resulting mixture was stirred for additional 30 min at 0 °C. To the above mixture was added 2-indanone (30 g, 226.994 mmol, 1 equiv, in 200 mL of THF) dropwise at 0°C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched by the addition of sat. NH4Q (aq.) (300 mL) at 0°C. The resulting mixture was extracted with Heptane (3 x 200 mL). The combined organic layers were washed with aqueous sat. Na2CCh (2x50 mL), water (2x50 mL), MeOH / H2O (4: 1, 4x50 mL), water (2x50 mL) and brine (1x50 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 afford methyl 2-(l ,3- dihydroinden-2-ylidene)acetate (25.6 g, yield: 59.68%) as an orange oil. [0368] Synthesis of methyl 2-(octahydro-lH-inden-2-yl)acetate
Figure imgf000146_0001
[0369] To a solution of methyl 2-(l,3-dihydroinden-2-ylidene)acetate (25 g, 132.819 mmol, 1 equiv) in 750 mL AcOH was added Pd/C (10%, 12.5 g) in a 1 L pressure tank reactor. The mixture was hydrogenated at 110°C under 40 atm. of hydrogen pressure for 24 h. The resulting mixture was cold 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 x 200 mL). The combined organic layers were washed with water (2x500 mL), sat. NaHCCh (2x300 mL), MeOH (4x200 mL), and brine (1x300 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in methyl 2-(octahydro-lH-inden-2- yl)acetate (21 g, yield: 80.55%) as a light yellow solid.
[0370] Synthesis of 2-(octahydro-lH-inden-2-yl)ethanol
Figure imgf000146_0002
[0371] Into a 500 mL 4-necked round-bottom flask were added methyl 2-(octahydro-lH- inden-2-yl)acetate (20 g, 101.890 mmol, 1 equiv) and THF (200 mL) at room temperature. To the above mixture was added LiAlHi (40 mL, 80.000 mmol, 0.79 equiv) dropwise at 0°C.
The resulting mixture was stirred overnight at room temperature. The reaction was quenched by the addition of water (3 mL) at 0°C. The resulting mixture was added dropwise with aqueous NaOH (3 mL, 15 % w/w) and water (9 mL). After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in heptane (500 mL). The resulting mixture was washed with 2x200 mL of water, 3x200 mL of water/MeOH (1:4), 2x200 mL of aqueous citric acid (5% w/w), 2x200 mL of sat. NaHCCh 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 afford 2-(octahydro-lH-inden-2-yl)ethanol (14.3 g, yield: 83.40%) as a light yellow oil. [0372] Synthesis of afford 2-(2-bromoethyl)-octahydro-lH-indene
H°
Figure imgf000147_0001
[0373] Into a 500 mL 3-necked round-bottom flask were added 2-(octahydro-lH-inden-2- yl)ethanol (14 g, 83.195 mmol, 1 equiv) and DCM (280 mL) at room temperature. To the above mixture was added PPhs (32.73 g, 124.792 mmol, 1.5 equiv) in portions at room temperature. The resulting mixture was stirred for an additional 10 min at room temperature. To the above mixture was added CBn (41.38 g, 124.792 mmol, 1.5 equiv, in 200 mL of DCM) dropwise 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 out; the filter cake was washed with heptane (2x50 mL). The filtrate was washed with 2x200 mL of water, 3x200 mL of water/MeOH (1 :4), 2x200 mL of aqueous citric acid (5% w/w), 2x200 mL of sat. NaHCCh 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, eluted with heptane to afford 2-(2-bromoethyl)-octahydro-lH-indene (15.1 g, yield: 77.96%) as a light yellow oil.
[0374] Synthesis of 2-[3-isocyano-3-(4-methylbenzenesulfonyl)-5-(octahydro-lH-inden- 2-yl)pentyl] -octa hydro- 1 H-indene
Figure imgf000147_0002
[0375] Into a 250 mL 3-necked round-bottom flask were added DMSO (30 mL) and NaH (1.13 g, 28.252 mmol, 2.31 equiv, 60%) at room temperature. The resulting mixture was stirred for an additional 1 h at room temperature. To the above mixture was added TosMIC (2.39 g, 12.241 mmol, 1 equiv) in portions over 10 min at room temperature. The resulting mixture was stirred for an additional 1 h at room temperature. To the above mixture was added TBAI (0.45 g, 1.224 mmol, 0.1 equiv) and 2-(2-bromoethyl)-octahydro-l H-indene (6.00 g, 25.706 mmol, 2.1 equiv, 99.3%) dropwise over 1 h at 20 °C. The resulting mixture was stirred overnight at room temperature. The resulting mixture was diluted with sat. NH4Q (120 mL). The resulting mixture was extracted with hexane (3 x 60 mL). The combined organic layers were washed with water (2x150 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 PE / EA (95:5) to afford 2-[3-isocyano-3-(4- methylbenzenesulfonyl)-5-(octahydro-lH-inden-2-yl)pentyl]-octa hydro- IH-indene (4.8 g, yield: 49.62%) as a light yellow oil.
[0376] Synthesis of l,5-bis(octahydro-lH-inden-2-yl)pentan-3-one
Figure imgf000148_0001
[0377] Into a 250 mL 3-necked round-bottom flask were added 2-[3-isocyano-3-(4- methylbenzenesulfonyl)-5-(octahydro-lH-inden-2-yl)pentyl]-octahydro-lH-indene (4 g, 7.592 mmol, 1 equiv, 94.1%) and HC1 (gas) in 1,4-dioxane at 0 °C. The resulting mixture was stirred for an additional 3 h at room temperature. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with hexane (3 x 30 mL). The combined organic layers were washed with sat. NaHCOs (2x50 mL), water (2x50 mL) and brine (1x50 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 PE / EA (95:5) to afford l,5-bis(octahydro-lH-inden-2-yl)pentan-3-one (2.8 g, yield: 95.13%) as a light yellow oil.
[0378] Synthesis of l,5-bis(octahydro-lH-inden-2-yl)pentan-3-ol
Figure imgf000148_0002
[0379] Into a 100 mL 3-necked round-bottom flask were added l,5-bis(octahydro-lH- inden-2-yl)pentan-3-one (2.5 g, 7.351 mmol, 1 equiv, 97.2%) and MeOH (25 mL) at 0 °C. To the above mixture was added NaBH4 (0.28 g, 7.351 mmol, 1 equiv) in portions over 2 min at 0 °C. The resulting mixture was stirred for an additional 2 h at 0 °C. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with hexane (3 x 30 mL). The combined organic layers were washed with sat. NaHCOs (2x50 mL), water (2x50 mL) and brine (1x50 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 PE / EA (95:5) to afford l,5-bis(octahydro-lH-inden-2- yl)pentan-3-ol (2.3 g, yield: 91.72%) as an off-white solid.
[0380] Synthesis of l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5 -oxononanedi oate
Figure imgf000149_0001
[0381] Into a 100 mL 3-necked round-bottom flask were added l,5-bis(octahydro-lH- inden-2-yl)pentan-3-ol (1.8 g, 5.277 mmol, 1 equiv, 97.5%) 5,9-dioxo-9-(pentadecan-8- yloxy)nonanoic acid (2.61 g, 6.332 mmol, 1.2 equiv), DCM (36 mL), DIEA (1.36 g, 10.554 mmol, 2 equiv) and DMAP (128.94 mg, 1.055 mmol, 0.2 equiv) at room temperature. To the above mixture was added EDCI (1.52 g, 7.915 mmol, 1.5 equiv) in portions 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 resulting mixture was diluted with water (30 mL). The resulting mixture was extracted with heptane (3 x 20 mL). The combined organic layers were washed with sat. Na2CCh (30 mL), MeOH/ELO (4: 1, 2x50 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, and eluted with PE / EA (9: 1) to afford l-[l,5-bis(octahydro-lH- inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5-oxononanedioate (3.6 g, yield: 93.25%) as a light yellow oil.
[0382] Synthesis of l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate
Figure imgf000150_0001
[0383] Into a 250 mL 3-necked round-bottom flask were added l-[l,5-bis(octahydro-lH- inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5-oxononanedioate (3 g, 4.101 mmol, 1 equiv, 99.4%) and THF (30 mL) at room temperature. To the above mixture was added [3- (chloromagnesio)propyl]dimethylamine (41.01 mL, 41.010 mmol, 10 equiv) dropwise at - 50°C. The resulting mixture was stirred for an additional 2 h at -40 °C. The reaction was quenched by the addition of sat. NELCl (aq.) (20 mL) at -30°C. The mixture was allowed to warm to room temperature. The resulting mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine (1x20 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 CH2CI2 / MeOH (13:87) to afford l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5-[3- (dimethylamino)propyl]-5-hydroxynonanedioate (2.1 g, yield: 59.11%) as a light yellow oil. [0384] Synthesis of l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate
Figure imgf000151_0001
[0385] Into a 50 mL round-bottom flask were added l-[l,5-bis(octahydro-lH-inden-2- yl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (2 g, 2.309 mmol, 1 equiv, 94%), and DCM (20 mL) at room temperature. To the above mixture was added EtsSiH (2.68 g, 23.090 mmol, 10 equiv) and BF3.Et2O (3.28 g, 23.090 mmol, 10 equiv) dropwise at room temperature. The resulting mixture was stirred for an additional 2 h at 40°C. The resulting mixture was diluted with water (20 mL). The resulting mixture was extracted with CH2CI2 (3 x 20 mL). The combined organic layers were washed with brine (1 x 20 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 CH2CI2 / MeOH (13:87) to afford l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9- pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (1.28 g, yield: 49.05%) as a light yellow oil.
[0386] Synthesis of l-[l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl] 9-pentadecan-8-yl 5 - [3 -(dimethyl amino) propyl ] nonanedi oate
Figure imgf000152_0001
[0387] Into a 50 mL round-bottom flask were added l-[l,5-bis(octahydro-lH-inden-2- yl)pentan-3-yl] 9-pentadecan-8-yl (4Z)-5-[3-(dimethylamino)propyl]non-4-enedioate (650 mg, 0.789 mmol, 1 equiv, 96.7%) and EtOH (13 mL) at room temperature. To the above mixture was added Pd/C (150 mg) at room temperature. The mixture was hydrogenated at room temperature under 30 psi of hydrogen pressure for 3h. After filtration, the filtrate was concentrated under reduced pressure. The residue was diluted with heptane (80 mL). The solid was filtered out. The filtrate was washed with MeOH / H2O (4: 1, 2x50 mL), water (50 mL) and brine (1x50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in l-[l,5-bis(octahydro-lH-inden-2- yl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-(dimethylamino) propyl]nonanedioate (504.9 mg, yield: 45.01%) as a colorless oil. LCMS: (ES, m/z): 798.7 [M+H]+. 'H NMR: (400 MHz, Chloroform-t/, ppm) S 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).
Example 19. l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-
(dimethylamino)propyl]nonanedioate (L-19)
Figure imgf000153_0001
[0389] Into a 500 mL 4-necked round-bottom flask were added DMSO (120 mL) and
NaH (1.74 g, 72.329 mmol, 2.4 equiv) at room temperature. To the above mixture was added TosMIC (5.88 g, 30.137 mmol, 1 equiv) in portions over 10 min at room temperature. The resulting mixture was stirred for additional 0.5 h at room temperature. To the above mixture was added TBAI (1.11 g, 3.014 mmol, 0.1 equiv) in portions at room temperature. The resulting mixture was stirred for additional 15 min at room temperature. To the above mixture was added l-(2-bromoethyl)-4-methylbenzene (6 g, 30.137 mmol, 1 equiv) dropwise over 15 min at room temperature. The resulting mixture was stirred for additional 18 h at room temperature. The reaction was quenched with 300 mL of sat. NH4Q (aq.) at 0 °C. The resulting mixture was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with water (2 x 200 mL) and brine (1 x 200 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 PE / EA (4: 1) to afford l-[3-isocyano-3-(4- methylbenzenesulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene (5 g, 38.44%) as a light yellow oil.
[0390] Synthesis of l,5-bis(4-methylphenyl)pentan-3-one
Figure imgf000154_0001
[0391] Into a 250 mL round-bottom flask were added l-[3-isocyano-3-(4- methylbenzenesulfonyl)-5-(4-methylphenyl)pentyl]-4-methylbenzene (5 g, 11.585 mmol, 1 equiv) and HC1 (g) in MeOH (100 mL, 4 M) at room temperature. The resulting mixture was stirred for 18 hr at room temperature. LCMS showed the reaction was completed. The resulting mixture was concentrated under vacuum. The resulting mixture was diluted with 60 mL of sodium carbonate (5.0%, aq.). The resulting mixture was extracted with EA (2 x 100 mL). The combined organic layers were washed with water (2 x 100 mL) and brine (1 x 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 PE / EA (4: 1) to afford l,5-bis(4-methylphenyl)pentan-3-one (2.5 g, 81.01%) as a light yellow oil. [0392] Synthesis of l,5-bis(4-methylphenyl)pentan-3-ol
Figure imgf000155_0001
[0393] Into a 250-mL 3-necked round bottom flask, was placedl,5-bis(4- methylphenyl)pentan-3-one (2.5 g, 9.385 mmol, 1 equiv) MeOH (4 mL) and THF (8 mL) at 0°C. This was followed by the addition of NaBH4 (0.99 g, 26.041 mmol, 1.5 equiv), in portions at 0 degrees. The resulting solution was stirred at 0°C for 10 min. LCMS showed the reaction was completed. The resulting mixture was quenched by the addition of citric acid (5%, 50 mL) at 0°C. The resulting mixture was extracted with EA (2 x 100 mL). The combined organic layer was washed with water (1 x 200 mL), brine (1 x 200 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 EA: Heptane (1 : 8) to afford l,5-bis(4-methylphenyl)pentan-3-ol (2.3 g, 91.31%) .
[0394] Synthesis of l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5- oxononanedioate
Figure imgf000155_0002
[0395] Into a 250 mL 3-necked round bottom flask was added 1 , 5-bis(4- methylphenyl)pentan-3-ol (2.3 g, 8.569 mmol, 1 equiv), 5,9-dioxo-9-(pentadecan-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) at room temperature. The mixture was stirred for 18 h at room temperature. LCMS showed the reaction was completed. The resulting solution was diluted with 250 mL of DCM. The resulting solution was washed with 1 x 200 mL of citric acid (5%, aq.), 2 x 200 mL of water, and 1 x 200 mL of brine, dried with anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with Hep/EA (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent Hep/EA=95/5) to afford 1- [l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-oxononanedioate (5.1 g, 89.77%) as a colorless oil.
[0396] Synthesis of l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-[3- (dimethylamino)propyl]-5-hydroxynonanedioate
Figure imgf000156_0001
[0397] Into a 250 mL round-bottom flask, was placel-[l,5-bis(4-methylphenyl)pentan-3- yl] 9-pentadecan-8-yl 5-oxononanedioate (3.5 g, 5.279 mmol, 1 equiv) and tetrahydrofuran (10.5 mL) at room temperature under N2 atmosphere. The mixture was cooled to -60°C. To this was added [3-(chloromagnesio)propyl]dimethylamine (52.79 mL, 52.790 mmol, 10 equiv) with dropwise at -60°C . The mixture was stirring for 2 hours at -60°C. LCMS showed the reaction was completed. The reaction was quenched by the addition of 80 mL of NH4Q (sat.) under 0°C. The resulting mixture was extracted with EA (2 x 100 mL). The combined organic layers were washed with water (2 x 100 mL) and brine (1 x 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 collected product eluent DCM/MeOH=82/18) to afford l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-[3- (dimethylamino)propyl]-5-hydroxynonanedioate (1.3 g, 32.83%) as colorless oil.
[0398] Synthesis of inseparable l-(l,5-di-p-tolylpentan-3-yl) 9-(pentadecan-8-yl) (Z)-5- (3-(dimethylamino)propylidene)nonanedioate, l-(l,5-di-p-tolylpentan-3-yl) 9-(pentadecan-8- yl) (E)-5-(3-(dimethylamino)propyl)non-4-enedioate, and 9-(l,5-di-p-tolylpentan-3-yl) 1- (pentadecan-8-yl) (E)-5-(3-(dimethylamino)propyl)non-4-enedioate
Figure imgf000157_0001
[0399] Into a 50 mL round-bottom flask, was place l-[l,5-bis(4-methylphenyl)pentan-3- yl] 9-pentadecan-8-yl 5-[3-(dimethylamino)propyl]-5-hydroxynonanedioate (1.3 g, 1.733 mmol, 1 equiv) and DCM (0.55 mL, 8.665 mmol, 5 equiv) at room temperature under N2 atmosphere. To this was added BF3 Et20 (2.46 g, 17.330 mmol, 10 equiv) and EtsSiH (2.02 g, 17.330 mmol, 10 equiv) at room temperature. The mixture was stirring for 2 hours at 45°C. LCMS showed the reaction was completed. The reaction was quenched by the addition of 25 mL of NaHCOs (sat.) at 0°C. The resulting mixture was extracted with DCM (2 x 50 mL). The combined organic layers were washed with water (2 x 100 mL) and brine (1 x 100 mL), dried over anhydrous Na2SO4. The residue was purified by silica gel column chromatography, eluted with DCM/MeOH (volume ratio), (gradient from 100:0 to 90: 10, and collected product eluent DCM/MeOH=94/6) to afford an inseparable mixture of product isomers (1.1 g, 86.82%) as colorless oil.
[0400] Synthesis of l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-[3- (dimethylamino)propyl]nonanedioate
Figure imgf000158_0001
[0401] Into a 100 mL round-bottom flask, was placed (1.1 g, 1.505 mmol, 1 equiv), and EtOH (22 mL). To this was added Pd/C (0.22 g, 10%) at room temperature. The mixture was stirring for 4 hours at room temperature under Eb (30 psi) atmosphere. LCMS showed the reaction was completed. The resulting mixture was filtered; the filter cake was washed with EA (2 x 30 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in n-heptane (110 mL, 100 V). Then n-heptane phase was washed with 2 x 30 mL of MeOH/H2O (4: 1), 2 x 30 mL of MeCN/EEO (4: 1), 1 x 30 mL of water, dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to afford l-[l,5-bis(4-methylphenyl)pentan-3-yl] 9-pentadecan-8-yl 5-[3-
(dimethylamino)propyl]nonanedioate (539.5 mg, 48.84%) as colorless oil. LCMS:(ES, m/z): 733.6 [M+H]+. XH NMR (400 MHz, Chloroforms/, ppm) 3 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).
Example 20. Synthesis of 3-(3-(diethylamino)propoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (L-20)
Figure imgf000159_0001
[0402] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methanol
Figure imgf000159_0002
[0403] Into a 2 L 3-necked round-bottom flask were added xylitol (40 g, 262.905 mmol, 1 equiv), MeOH (400 mL, 10 V), 2,2-dimethoxypropane (600 mL, 15 v) and TsOH.ELO (10 g, 52.581 mmol, 0.2 equiv) at room temperature. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. To the above mixture was added K2CO3 (7.263 g, 0.2 eq) in portions at room temperature. The resulting mixture was stirred for additional 30 min at room temperature. The resulting mixture was filtered, the filtrate was concentrated under reduced pressure. To the mixture was added 150 g of silica gel (type: ZCX-2, 100-200 mesh, 1.5 w./w.). This mixture was then concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. 700 g silica gel (type: ZCX-2, 100- 200 mesh, 9.0 w./w.) was charged to the column, followed by the prepared dry silica gel which absorbed the reaction mixture of the last step. Using combi-flash to purify the product. Eluted with n-heptane/EA (Gradient from 100:0 to 90: 10). Took sample for TLC analysis (EA:n-heptane=l :4) and combined qualified products. To afford bis(2,2-dimethyl-l,3- dioxolan-4-yl)methanol (36.1 g, Yield 59.12%) as a light yellow oil. [0404] Synthesis of bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl methanesulfonate
Figure imgf000160_0001
[0405] A solution of bis(2,2-dimethyl-l,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 nitrogen atmosphere. Followed by the addition of MsCI (19.08 g, 166.613 mmol, 1.5 equiv) dropwise at 0 °C. The resulting mixture was stirred for overnight at room temperature. The reaction was quenched by the addition of NH4Q saturated solution at room temperature. The resulting mixture was extracted with DCM (3 x 300 mL). The combined organic layers were washed with brine (2x300 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl methanesulfonate (35.8 g crude) as a brown solid.
[0406] Synthesis of 3-(bis(2,2-dimethyl-l,3-dioxolan-4-yl)methoxy)-N,N-diethylpropan- 1 -amine
Figure imgf000160_0002
[0407] Into a 1 L 3-necked round-bottom flask were added 3-(diethylamino)propan-l-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 nitrogen atmosphere. To the above mixture was added bis(2,2-dimethyl-l,3-dioxolan-4-yl)methyl methanesulfonate (10 g, crude) in portions at 0°C. The resulting mixture was stirred for 18 h at room temperature under nitrogen atmosphere. The reaction was quenched with sat. NH4Q (aq.) at 0°C. The resulting mixture was extracted with EtOAc (2x300 mL). The combined organic layers were concentrated under reduced pressure. To the mixture was added 20 g of silica gel (type: ZCX-2, 100-200 mesh, 1.5 w./w.). This mixture was then concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. 200 g silica gel (type: ZCX-2, 100-200 mesh, 9.0 w./w.) was charged to the column, followed by the prepared dry silica gel which absorbed the reaction mixture of the last step. Using combi-flash to purify the product. Eluted with DCM/MeOH (Gradient from 100:0 to 90: 10). Took sample for TLC analysis (DCM/MeOH (10: 1)) and combined qualified products. To afford 3-(bis(2,2-dimethyl-l,3- dioxolan-4-yl)methoxy)-N,N-diethylpropan-l -amine (1.9 g) as a light yellow oil
[0408] Synthesis of 3-(3-(diethylamino)propoxy)pentane-l,2,4,5-tetraol
Figure imgf000161_0001
[0409] Into a lOOmL round-bottom flask were added 3-(bis(2,2-dimethyl-l,3-dioxolan-4- yl)methoxy)-N,N-diethylpropan-l -amine (1.9 g, 5.500 mmol, 1 equiv) and HC1 (6 M) (20 mL) at room temperature. The resulting mixture was stirred for 3 h at 60°C under nitrogen atmosphere. The resulting mixture was concentrated under vacuum. The crude product (1.8 g) was used in the next step directly without further purification.
[0410] Synthesis of 3-(3-(diethylamino)propoxy)pentane-l,2,4,5-tetrayl tetraki s(decanoate)
Figure imgf000161_0002
[0411] Into a 250 mL round-bottom flask were added 3-(3- (diethylamino)propoxy)pentane-l,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) at room temperature. The resulting mixture was stirred for overnight at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in water (200 mL). The resulting mixture was extracted with heptane (3x200 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, C LCN and Water (0.05% TFA), 50% C LCN to 95 % gradient in 15 min; detector, UV 200 nm. The product eluent was collected and concentrated. The resulting mixture was diluted with n-heptane (300 mL) and basified to pH 8-9 with saturated Na2CCh (3 %). The organic layer was washed with brine (2x100 mL) and H2O/MeOH=l/5
(1x100 mL). The organic layers were dried over anhydrous Na2SO4 and concentrated. This resulted in 3-(3-(diethylamino)propoxy)pentane-l,2,4,5-tetrayl tetrakis(decanoate) (526.8 mg, Yield 8.80 %) as a light yellow oil. LCMS: (ES, m/z): 883 [M+l]+. ’H NMR (300 MHz, CDCh,/2pm) 5 5.484 (t, J= 5.4 Hz, 1H), 5.347-5.311 (m, 1H), 5.205-5.171 (m,lH), 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).
Example 21. Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(octahydro-lH- inden-2-yl)propanoyl]oxy})pentan-2-yl 3-(octahydro-lH-inden-2-yl)propanoate
Figure imgf000162_0001
oxylic acid
Figure imgf000163_0001
[0413] To a solution of 2,3-dihydro-lH-indene-2-carboxylic acid (25 g, 154.142 mmol, 1 equiv) in 500 mL AcOH was added Pd/C (25 g) in a pressure tank. The mixture was hydrogenated at 120°C under 30 psi of hydrogen pressure for 24 h. 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 x 250 mL). The combined organic layers were washed with brine (lx 250 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in octahydro-lH-indene-2-carboxylic acid (22 g, 75.08%) as a brown oil.
[0414] Synthesis of octahydro- lH-inden-2-ylmethanol
Figure imgf000163_0002
[0415] To a stirred mixture of octahydro-lH-indene-2-carboxylic acid (22 g, 115.730 mmol, 1 equiv, 88.5%) in THF was added LiAlHi (6.59 g, 173.595 mmol, 1.5 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 4 h at room temperature. 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 x 150 mL). The combined organic layers were washed with brine (1x200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in octahydro- lH-inden-2-ylmethanol (16 g, 79.68%) as a yellow oil. [0416] Synthesis of octahydro- lH-indene-2-carbaldehy de
Figure imgf000163_0003
[0417] Into a 250 mL 3-necked round-bottom flask were added octahydro- lH-inden-2- ylmethanol (15 g, 97.243 mmol, 1 equiv) and DCM (150 mL) at room temperature. To the above mixture was added Dess-Martin (45.37 g, 106.967 mmol, 1.1 equiv) in portions at 0°
C. The resulting mixture was stirred for additional 2 h at room temperature. The reaction was quenched by the addition of Na2S2Ch (aq.) (100 mL) at room temperature. The resulting mixture was extracted with CH2CI2 (2 x 200 mL). The combined organic layers were washed with NaHCOs (aq) (3x200 mL), dried over anhydrous MgSCU. After filtration, the filtrate was concentrated under reduced pressure. This resulted in octahydro- lH-indene-2-carbaldehy de (5.95 g, 26.69%) as a yellow oil.
[0418] Synthesis of methyl (2E)-3-(octahydro-lH-inden-2-yl)prop-2-enoate
Figure imgf000164_0001
[0419] Into a 250 mL 3-necked round-bottom flask were added octahydro- lH-indene-2- carbaldehyde (5.7 g, 37.442 mmol, 1 equiv), 2-MeTHF (60 mL) and methyl 2-(triphenyl- lambda5-phosphanylidene)acetate (15.02 g, 44.930 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for 2 h at room temperature. Combined work up with previous 200 mg reaction. The resulting mixture was concentrated under reduced pressure. The mixture was dissolved in DCM (100 mL) and 24 g of silica gel (type: ZCX-2, 100-200 mesh, 4 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35 °C. Charged 200 g of silica gel (type: ZCX-2, 100-200 mesh, 20 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using combi-flash to purify the product. Eluted with PE / EA (gradient from 100:0 to 90:10, collected every 200 ± 10 mL). Took sample for TLC analysis (EA:PE = 5: 1), combined qualified products. This resulted in methyl (2E)-3 -(octahydro- 1 H-inden-2-yl)prop- 2-enoate (5.51 g, 63.00%) as a colorless oil.
[0420] Synthesis of methyl 3 -(octahydro- lH-inden-2-yl)propanoate
Figure imgf000164_0002
[0421] To a solution of methyl (2E)-3 -(octahydro- lH-inden-2-yl)prop-2-enoate (5 g, 24.004 mmol, 1 equiv) in 50 mL MeOH was added Pd/C (2.5 g) in a pressure tank. The mixture was hydrogenated at room temperature under 30 psi of hydrogen pressure for 12 h. Combined work up with previous 500 mg reaction. Filtered through a Celite pad and the filtrate was concentrated under reduced pressure. The resulting mixture was filtered, the filter cake was washed with MeOH (2x20 mL). The filtrate was concentrated under reduced pressure. This resulted in methyl 3 -(octahydro- lH-inden-2-yl)propanoate (5.1 g, 72.19%) as a colorless oil.
[0422] Synthesis of 3-(octahydro-lH-inden-2-yl)propanoic acid
Figure imgf000165_0001
[0423] Into a 250 mL 3-necked round-bottom flask were added methyl 3 -(octahydro- 1H- inden-2-yl)propanoate (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) at room temperature. The resulting mixture was stirred for 2 h at room temperature. The organic solvent was removed under reduced pressure. The resulting mixture was extracted with EtOAc (2 x 50mL) and Collected water phase. The water phase was acidified to pH= 2 with HC1 (6M). The resulting mixture was extracted with EtOAc (2 x lOOmL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 3 -(octahydro- lH-inden-2- yl)propanoic acid (3.941 g, 74.10%) as a white solid.
[0424] Synthesis of 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(octahydro-lH-inden-2- yl)propanoyl] oxy } )pentan-2-yl 3 -(octahydro- 1 H-inden-2-yl)propanoate
Figure imgf000166_0001
[0425] Into a 80 mL vial were added 3-(octahydro-lH-inden-2-yl)propanoic acid (0.85 g, 4.330 mmol, 1 equiv), ACN (8.5 mL), 3-[3-(dimethylamino)propoxy]pentane-l,2,4,5-tetrol (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) at room temperature. The resulting mixture was stirred for additional 3 h at room temperature. The reaction was quenched by the addition of water (10 mL) at room temperature. The resulting mixture was extracted with Heptane (3x150 mL). The collected organic phase was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase HP-flash chromatography with the following conditions: column, XSelect CSH Prep C18 5um; mobile phase, B: C LCN, A: water (0.1% TFA), 50% to 95% gradient in 15 min; Flow: 50 mL/min: detector, ELSD. The resulting mixture was concentrated under reduced pressure and basified to pH 8 with saturated Na2CCh (aq.). The aqueous layer was extracted with heptane (3x100 mL). The resulting mixture was concentrated under reduced pressure. This resulted in 3-[3-(dimethylamino)propoxy]-l,4,5-tris({[3-(octahydro-lH-inden- 2-yl)propanoyl]oxy})pentan-2-yl 3 -(octahydro- lH-inden-2-yl)propanoate (0.5272 g, 12.90%) as a yellow oil. LCMS: (ES, m/z): 950.9 [M+l]+. ’H NMR (300 MHz, Chloroform-d) 5 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). Example 22. Synthesis of 5-(dimethylamino)-l-((2-heptylnonanoyl)oxy)pentane-2,3-diyl bis(decanoate) (L-22)
Figure imgf000167_0001
[0426] Synthesis of 2-(5-(hydroxymethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)acetaldehyde
Figure imgf000167_0002
30 min
[0427] Into a 500 mL 3-necked round-bottom flask were added 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) at room temperature. To the above mixture was added 2,2-dimethoxypropane (18.64 g, 178.930 mmol, 1.2 equiv) dropwise at 0 °C. The resulting mixture was stirred for additional 30 min at 0 °C. The mixture was basified to pH 8 with saturated NaHCOs (aq.). The resulting mixture was concentrated under reduced pressure to remove the acetone. The resulting mixture was diluted with water (200 mL). The resulting mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 2-(5-(hydroxymethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)acetaldehyde (23 g, crude,
GCMS purity: 78.2%) as a light yellow oil.
[0428] Synthesis of (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-l,3-dioxolan-4- yl)methanol
Figure imgf000168_0001
[0429] Into a 250 mL 3-necked round-bottom flask were added 2-((4S,5S)-5- (hydroxymethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)acetaldehyde (6 g, 34.444 mmol, 1 equiv), THF (30 mL), MeOH (60 mL) and AcOH (2.6 g, 43.296 mmol, 1.26 equiv) at room temperature. To the above mixture was added dimethylamine (2M in THF, 22.4 mL, 44.800 mmol, 1.30 equiv) dropwise at room temperature. The resulting mixture was stirred for additional 30 min at room temperature. To the above mixture was added NaBHsCN (3.0 g, 47.740 mmol, 1.39 equiv) in portions over 10 min at room temperature. The resulting mixture was stirred for additional 4 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% NH3.H2O), 5 % hold for 3 min, 5% to 55% gradient in 12 min; detector, UV 220 nm. This resulted in (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)methanol (3.2 g, 45.70%) as a light yellow oil.
[0430] Synthesis of (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl
Figure imgf000168_0002
[0431] Into a 250 mL 3-necked round-bottom flask were added ((4S,5S)-5-(2-
(dimethylamino)ethyl)-2,2-dime thyl-l,3-dioxolan-4-yl)methanol (3.2 g, 15.742 mmol, 1 equiv), ACN (64 mL), 2-heptylnonanoic acid (4.04 g, 15.742 mmol, 1 equiv) and DMAP (0.58 g, 4.723 mmol, 0.3 equiv) at room temperature. To the above mixture was added EDCI (4.53 g, 23.613 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred over night at room temperature. The resulting mixture was diluted with water (150 mL). The resulting mixture was extracted with heptane (3 x 80 mL). The combined organic layers were washed with aqueous. Na2CCh (2x80 mL), water (100 mL) and brine (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 CH2CI2 / MeOH (98:2) to afford (5-(2-(dimethylamino)ethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl 2- heptylnonanoate (4.7 g, 54.96%) as a light yellow oil.
[0432] Synthesis of 5-(dimethylamino)-2,3-dihydroxypentyl 2-heptylnonanoate
Figure imgf000169_0001
[0433] Into a 100 mL 3-necked round-bottom flask were added ((4S,5S)-5-(2- (dimethylamino)ethyl)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl 2-heptylnonanoate (1.0 g, 2.264 mmol, 1 equiv) and DCM (20 mL) at -10 °C. To the above mixture was added a mixture of TFA (5 mL, 67.315 mmol, 29.73 equiv) and H2O (0.5 mL, 27.755 mmol, 12.26 equiv) dropwise at -10 °C. The resulting mixture was stirred for additional 1 h maintained the temperature between-10 °C and -5 °C. The resulting mixture was diluted with water (100 mL). The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with sta. Na2CCh (2x50 mL), water (2x50 mL) and brine (1x50 mL), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. This resulted in 5-(dimethylamino)-2,3-dihydroxypentyl 2-heptylnonanoate (920 mg, purity: 72.6 %, yield: 73.45%) as a light yellow oil. The crude product was used in the next step directly without further purification. [0434] Synthesis of 5-(dimethylamino)-l-((2-heptylnonanoyl)oxy)pentane-2,3-diyl bis(decanoate)
Figure imgf000170_0001
[0435] Into a 100 mL round-bottom flask were added 5-(dimethylamino)-2,3- dihydroxypentyl 2-heptylnonanoate (900 mg, 1.627 mmol, 1 equiv, 72.6%), capric acid (560.51 mg, 3.254 mmol, 2 equiv), ACN (18 mL) and DMAP (59.63 mg, 0.488 mmol, 0.3 equiv) at room temperature. To the above mixture was added EDCI (779.67 mg, 4.067 mmol, 2.5 equiv) in portions at room temperature. The resulting mixture was stirred for additional 18 h at room temperature. The resulting mixture was diluted with water (60 mL). The resulting mixture was extracted with Heptane (2 x 80 mL). The combined organic layers were washed with aqueous sat. Na2CCh (2x50 mL), water (2x50 mL), MeOH/H2O (4: 1, 4x50 mL), water (2x50 mL) and brine (1x50 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 CH2CI2 / MeOH (98:2) to afford 5-(dimethylamino)-l- ((2-heptylnonanoyl)oxy)pentane-2,3-diyl bis(decanoate) (531.3 mg, 43.73%) as a light yellow oil. LCMS: (ES, m/z): 710.7 [M+H]+; 'H NMR: (400 MHz, Chloroforms/) 3 5.222- 5.143(m, 2H), 4.346 (dd, 1H, J= 12.0, 3.2 Hz), 4.100 (dd, 1H, J= 12.0, 3.2 Hz), 2.349-2.264 (m, 13H), 1.783-1.766 (m, 2H), 1.613-1.540 (m, 6H), 1.709-1.461-1.394 (m, 2H), 1.264- 1.251 (m, 44H), 0.893-0.861 (m, 12H).
Example 23. 2-(decanoyloxy)-6-(dimethylamino)-l-[(2-heptylnonanoyl)oxy]hexan-3-yl decanoate (L-23)
Figure imgf000171_0001
[0436] Synthesis of l-[2,2-dimethyl-l,3-dioxolan-4-yl]pent-4-en-l-ol
Figure imgf000171_0002
[0437] To a mixture of 2,2-dimethyl-l,3-dioxolane-4-carbaldehyde (5.5 g, 42.261 mmol, 1 equiv) in THF (110 mL) was added ZnCh (11.12 mL, 21.131 mmol, 0.5 equiv) and bromo(but-3-en-l-yl)magnesium (105.65 mL, 105.653 mmol, 2.5 equiv) at -30 °C. The mixture was stirred at -30 °C for 6 h. The reaction was quenched by the addition of water (20 mL) at 0 °C. The resulting mixture was diluted with EA (220 mL). The resulting mixture was washed with 2 x 110 mL of water, brine (110 mL). The resulting solution was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (55 mL) and 27.5 g of silica gel (type: ZCX-2, 100-200 mesh, 5.00 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 110 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using combi-flash to purify the product. Eluted with PE / EA (100: 1) (gradient from 100:0 to 90: 10, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 95:5), combined qualified products. This resulted in l-[2,2-dimethyl-l,3- dioxolan-4-yl]pent-4-en-l-ol (3.5 g, 44.47%) as a light yellow oil.
[0438] Synthesis of hept-6-ene-l,2,3-triol
Figure imgf000172_0001
[0439] To a mixture of l-[2,2-dimethyl-l,3-dioxolan-4-yl]pent-4-en-l-ol (3.3 g, 17.718 mmol, 1 equiv) in MeOH (33 mL) was added HC1 (6 M) (0.13 g, 3.544 mmol, 0.2 equiv) . The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by trituration with MTBE (33 mL) to afford hept-6-ene-l,2,3-triol (1.2 g, 46.33%) as a white solid.
[0440] Synthesis of 2,3-dihydroxyhept-6-en-l-yl 2-heptylnonanoate
Figure imgf000172_0002
s ep
[0441] To a mixture of hept-6-ene-l,2,3-triol (1.1 g, 7.525 mmol, 1 equiv), 2- heptylnonanoic acid (1.83 g, 7.149 mmol, 0.95 equiv) and DMAP (0.92 g, 7.525 mmol, 1 equiv) in DCM (22 mL) was added EDCI (1.44 g, 7.525 mmol, 1 equiv). The reaction mixture was stirred at 20 °C for 6 h. The resulting mixture was diluted with DCM (110 mL). The resulting mixture was washed with 3 x 33 mL of water, brine (33 mL). The resulting solution was dried over anhydrous Na2SO4. After filtration, the resulting mixture was concentrated under reduced pressure. The residue was dissolved in DCM (22 mL) and 5.5 g of silica gel (type: ZCX-2, 100-200 mesh, 5.00 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 22 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using combi-flash to purify the product. Eluted with PE / EA (80:20) (gradient from 100:0 to 70:30, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 5: 1), combined qualified products. This resulted in
2,3-dihydroxyhept-6-en-l-yl 2-heptylnonanoate (1.6 g, 55.29%) as a light-yellow oil.
[0442] Synthesis of 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]hept-6-en-3-yl decanoate
Figure imgf000173_0001
[0443] A solution of 2,3-dihydroxyhept-6-en-l-yl 2-heptylnonanoate (1.5 g, 3.900 mmol,
1 equiv) in DCM (120 mL) was added capric acid (1.48 g, 8.580 mmol, 2.2 equiv) and
DMAP (0.48 g, 3.900 mmol, 1 equiv) at 20°C under nitrogen atmosphere followed by the addition of EDCI (1.87 g, 9.750 mmol, 2.5 equiv) in portions at 20°C. The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was diluted with DCM (300 mL). The resulting mixture was washed with 3 x 100 mL of water, brine (100 mL). The resulting solution was dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (15 mL) and 7.5 g of silica gel (type: ZCX-2, 100-200 mesh, 5.00 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 30 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using combi-flash to purify the product. Eluted with n-Heptane / EA (95:5) (gradient from 100:0 to 90: 10, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 20: 1), combined qualified products. This resulted in 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]hept-6-en-3-yl decanoate (2.1 g, 77.69%) as a light yellow oil.
[0444] Synthesis of 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]-6-oxohexan-3-yl decanoate
Figure imgf000173_0002
[0445] A mixture of 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]hept-6-en-3-yl decanoate (2.0 g, 2.886 mmol, 1 equiv) was in THF (80 mL) and H2O (40 mL) was added K2OSO4.2H2O (127.58 mg, 0.346 mmol, 0.12 equiv). The reaction mixture was stirred at 20 °C for 10 min, NalCb (3.09 g, 14.430 mmol, 5.0 equiv) and 2,6-lutidine (1.55 g, 14.430 mmol, 5.0 equiv) was added at 20 °C. The reaction mixture was stirred at 20 °C for 18 h. The resulting mixture was diluted with EA (200 mL). The resulting mixture was washed with 3 x 60 mL of water, brine (60 mL). The resulting solution was dried over anhydrous Na2SO4. The resulting mixture 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, 5.00 w./w.) was added. Concentrated to no fraction under vacuum while maintaining the temperature below 35°C. Charged 40 g of silica gel (type: ZCX-2, 100-200 mesh, 20.00 w/w.) to the column, followed by the last step prepared dry silica gel which absorbed the reaction mixture. Using combi- flash to purify the product. Eluted with n-Heptane / EA (90: 10) (gradient from 100:0 to 90: 10, collected every 200 ± 10 mL). Took sample for TLC analysis (PE / EA = 10: 1), combined qualified products. This resulted in 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]-6- oxohexan-3-yl decanoate (1.4 g, 69.80%) as a light yellow oil.
[0446] Synthesis of 2-(decanoyloxy)-6-(dimethylamino)-l-[(2- heptylnonanoyl)oxy]hexan-3-yl decanoate
Figure imgf000174_0001
[0447] To a solution of 2-(decanoyloxy)-l-[(2-heptylnonanoyl)oxy]-6-oxohexan-3-yl decanoate (1.4 g, 2.014 mmol, 1 equiv) and dimethylamine (0.45 mL, 10.070 mmol, 5 equiv) in THF (28 mL) was added NaBH(OAc)3 (1.28 g, 6.042 mmol, 3 equiv). The reaction mixture was stirred at 20 °C for 12 h. The resulting mixture was diluted with EA (140 mL). The resulting mixture was washed with 3x28 mL of water, brine (28 mL), dried over anhydrous Na2SO4. After filtration. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in water (0.05% CF3COOH), 30% to 90% gradient in 26 min; detector, ELSD. The collect liquid was added 28 mL NaHCOs (2M, aq). The resulting mixture was extracted with n-Heptane (3 x 70 mL). The combined organic layers were washed with 3 x 28 mL of MeOH/H2O(4/l), water (28 mL). The resulting mixture was concentrated under reduced pressure to afford 2- (decanoyloxy)-6-(dimethylamino)-l-[(2-heptylnonanoyl)oxy]hexan-3-yl decanoate (0.5497 g, 37.69%) as a colorless oil. LCMS:(ES, m/z): 724.6 [M+H]+. 'H NMR: (400 MHz, Chloroform-d,/2pm) 3 5.223-5.115 (m, 2H), 4.348 (dd, J= 2.8 Hz, 4.0 Hz, 1H), 4.118 (dd, J = 7.2 Hz, 12.0 Hz, 1H), 2.381-2.182 (m, 13H), 1.698-1.553 (m, 8H), 1.546-1.383 (m, 4H), 1.337-1.220 (m, 44H), 0.928-0.856 (m, 12H).
Example 24. Synthesis of l-(l,5-bis(octahydro-lH-inden-2-yl)pentan-3-yl) 7- (pentadecan-8-yl) 4-(3-(dimethylamino)propyl)heptanedioate (L-24)
Figure imgf000175_0001
[0448] L-24 is synthesized in a manner similar to L-18, substituting 4-oxoheptanedioic acid instead of 5-oxononanedioic acid. Example 25. Synthesis of 3-(3-(dimethylamino)propoxy)-l,5-bis((3-(octahydro-lH- inden-2-yl)propanoyl)oxy)pentane-2,4-diyl bis(decanoate) (L-25)
Figure imgf000176_0001
[0449] L-25 is synthesized in a manner similar to L-21, but instead the xylitol core is acylated first with 3-(octahydro-lH-inden-2-yl)propanoic acid at primary then the secondary alcohols are acylated with decanoyl chloride to provide the final product.
Example 26. Synthesis of l-(3-(dimethylamino)propanamido)-4-((2- heptylnonanoyl)oxy)butane-2,3-diyl bis(3-(octahydro-lH-inden-2-yl)propanoate) (L-26)
Figure imgf000176_0002
[0450] L-26 is synthesized in a manner similar to L-5 and L-23. Example 27. Preparation and analysis of lipid nanoparticle formulations with varying ionizable lipids
[0451] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above and detailed in Fig. 1 A.
[0452] Wild type female BALB/c mice (approximately 7-8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 5 mL/kg body weight. The DNA-LNPs were administered at 1 mg/kg based on the weight of the DNA payload. Four hours after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. IB. The DNA-LNP formulation with ionizable lipid L-3 produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC-0315 (FIG. IB). The DNA-LNP formulation with ionizable lipid L-2 produced measurable but somewhat lower EPO expression levels, as did the exemplary ionizable lipid MC3 (FIG. IB). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 1C. The DNA-LNP formulations with ionizable lipids L-2 and L-3 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid MC3, which were higher than the IL-6 levels generated by formulations with the exemplary ionizable lipid ALC-0315 (FIG. 1C).
Example 28. Preparation and analysis of further LNP formulations with varying additional ionizable lipids
[0453] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above. The LNP formulations using DSPC as the phospholipid are detailed in Fig. 2A. The LNP formulations using DOPE as the phospholipid are detailed in Fig. 2B.
[0454] Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Seven days after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 2C. When formulated with DSPC, the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315, MC3, and LP01, and comparable to the exemplary ionizable lipids SM102 and ARCT (FIG. 2C). When formulated with DOPE, the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipids MC3, LP01, and SM102, and comparable to the exemplary ionizable lipids ALC-0315 and ARCT (FIG. 2C). When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-9 produced somewhat lower EPO expression levels than L-15, comparable to several of the exemplary ionizable lipids (FIG. 2C). When formulated with DSPC or DOPE, the DNA-LNPs with ionizable lipid L-5 produced substantially lower EPO expression levels, below the levels of the exemplary ionizable lipids (FIG. 2C). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 2D. When formulated with DSPC, the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid ALC-0315, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2D). When formulated with DOPE, the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of IL-6 that were generally similar compared to all the exemplary ionizable lipids (FIG. 2D). Four hours after dosing, blood was collected via a retro-orbital bleed and serum levels of multiple cytokines determined as presented in FIG. 2E and FIG. 2F. When formulated with DSPC, the DNA- LNPs with ionizable lipids L-5, L-15, and L-9 generated similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipid ALC-0315, which were typically lower than the cytokine levels generated by formulations with the exemplary ionizable lipids MC3, LP01, SMI 02, and ARCT (FIG. 2E). When formulated with DOPE, the DNA-LNPs with ionizable lipids L-5, L-15, and L-9 generated serum levels of cytokines that were generally similar compared to all the exemplary ionizable lipids (FIG. 2F).
Example 29: Preparation and analysis of further LNP formulations with varying additional ionizable lipids
[0455] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above. The LNP formulations using DSPC as the phospholipid are detailed in Fig. 3 A. The LNP formulations using DOPE as the phospholipid are detailed in Fig. 3B.
[0456] Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs were administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Seven days after dosing, blood was collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 3C. When formulated with DSPC, the DNA-LNP with ionizable lipid L-12 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC- 0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C). When formulated with DSPC, the DNA-LNPs with ionizable lipids L-13 and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipid ssOP, and comparable to the exemplary ionizable lipid ALC-0315 (FIG. 3C). When formulated with DOPE, the DNA- LNPs with ionizable lipids L-12, L-13, and L-14 produced robust EPO expression levels, higher than the exemplary ionizable lipids ALC-0315 and ssOP, and comparable to the exemplary ionizable lipid A9 (FIG. 3C). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 3D. When formulated with DSPC, the DNA-LNP with ionizable lipid L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D). When formulated with DSPC, the DNA-LNPs with ionizable lipids L-12 and L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipid A9, which were lower than the IL-6 levels generated by formulations with the exemplary ionizable lipids ALC-0315 and ssOP (FIG. 3D). When formulated with DOPE, the DNA-LNPs with ionizable lipids L- 12 and L-14 resulted in substantially lower IL-6 serum levels compared to the other formulations tested (FIG. 3D). When formulated with DOPE, the DNA-LNP with ionizable lipid L-13 generated similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP (FIG. 3D). Four hours after dosing, blood was collected via a retro-orbital bleed and serum levels of multiple cytokines determined as presented in FIG. 3E and FIG. 3F. When formulated with DSPC, the DNA-LNPs with ionizable lipids L-12, L-13, and L-14 produced generally similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP, although for several cytokines L-14 produced the lowest levels and ssOP produced the highest levels (FIG. 3E). When formulated with DOPE, the DNA-LNPs with ionizable lipids L-12, L-13, and L-14 produced generally similar, or lower, serum levels of cytokines compared to the exemplary ionizable lipids ALC-0315, A9, and ssOP, although for several cytokines L-14 produced the lowest levels (FIG. 3F).
Example 30: Preparation and analysis of further LNP formulations with varying additional ionizable lipids
[0457] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above. The LNP formulations using DSPC as the phospholipid are detailed in Fig. 4A.
[0458] Wild type female BALB/c mice (approximately 8 weeks old) were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs are administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Three days after dosing, blood is collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 4B. When formulated with DSPC, the DNA-LNP with ionizable lipid L-15 produced robust EPO expression levels, higher than the exemplary ionizable lipid ALC-0315 (FIG. 4B). When formulated with DSPC, the DNA-LNPs with ionizable lipids L-9 and L-ll produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC- 0315 (FIG. 4B). When formulated with DSPC, the DNA-LNPs with ionizable lipids L-16 and L-10 produced measurable EPO expression levels, somewhat lower than the exemplary ionizable lipid ALC-0315 (FIG. 4B). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 4C. When formulated with DSPC, the DNA-LNPs with ionizable lipids L-15, L-9, L-16, L-10, and L- 11 produced generally similar serum levels of IL-6 compared to the exemplary ionizable lipid ALC-0315, with L-16 resulting in the highest IL-6 levels (FIG. 4C).
Example 31: Preparation and analysis of further LNP formulations with varying additional ionizable lipids
[0459] DNA payloads were formulated into lipid nanoparticles (LNPs) comprising an ionizable lipid, as described above. The LNP formulations using DSPC as the phospholipid are detailed in Fig. 5A.
[0460] Wild type adult BALB/c mice were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The DNA-LNPs are administered at 1 or 0.3 mg/kg based on the weight of the DNA payload. Three days after dosing, blood is collected via a retro-orbital bleed and EPO levels in serum determined as presented in FIG. 5B. When formulated with DSPC, the DNA-LNP with ionizable lipid L-21 produced robust EPO expression levels, comparable to the exemplary ionizable lipid ALC-0315 (FIG. 5B). When formulated with DSPC, the DNA-LNPs with ionizable lipids L-17 and L-20 produced robust EPO expression levels, comparable to the exemplary ionizable lipids ARCT and CL1 and higher than the exemplary lipid ALC-0315 (FIG. 5B). When formulated with DSPC, the DNA-LNPs with ionizable lipid L-19 produced robust EPO expression levels, higher than all three exemplary ionizable lipids (FIG. 5B). Four hours after dosing, blood was collected via a retro-orbital bleed and IL-6 levels in serum determined as presented in FIG. 5C. When formulated with DSPC, the DNA-LNPs with ionizable lipids L-17, L-19, and L-21 produced generally similar serum levels of IL-6 compared to the exemplary ionizable lipids ALC-0315, ARCT, and CL1 (FIG. 5C).
Example 32: Preparation and analysis of LNPs comprising varying ionizable lipids coformulated with both DNA and mRNA
[0461] DNA and mRNA payloads were co-formulated into lipid nanoparticles (LNPs) comprising ionizable lipids of the present disclosure, using a 1 :3 (w/w) ratio of DNA:mRNA, as described above. The LNP formulations using DOPE as the phospholipid are detailed in Fig. 6A.
[0462] Wild type adult BALB/c mice were dosed once by a single i.v. bolus injection into the tail vein at 10 mL/kg body weight. The LNPs are administered at 0.5 mg/kg based on the weight of the DNA payload (e.g., 1.5 mg/kg based on the weight of the mRNA payload). Twenty-one days after dosing, blood was collected via a retro-orbital bleed and human FIX levels in plasma determined as presented in FIG. 6B. The LNPs with ionizable lipid L-15 produced FIX expression levels about 2-fold higher compared to exemplary ionizable lipid CL1 (FIG. 6B), while the LNPs with ionizable lipids L-17 and L-18 produced FIX expression levels about 3- to 4-fold higher compared to the exemplary ionizable lipid CL1 (FIG. 6B). Four hours after dosing, blood was collected via a retro-orbital bleed and the serum levels of a panel of cytokines were determined The LNPs with ionizable lipid L-15 elicited generally similar serum levels of cytokines as the exemplary ionizable lipid CL1 (FIG. 6C - 6K), while the LNPs with ionizable lipids L-17 and L-18 elicited approximately 2-fold lower serum levels of IL-6 compared to the exemplary ionizable lipid CL1 (FIG. 6C - 6K).
Equivalents and incorporation by reference
[0463] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
[0464] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
[0465] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
[0466] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.
[0467] All references, issued patents and patent applications mentioned or cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:
1. An ionizable lipid compound of formula (I):
(Z-L-Y)-Wn-(X-R)(n-i)
(I) wherein:
Z is an ionizable head group;
L is an optionally substituted (Ci-Ci2)alkylene;
Y is a linking group;
Wn is a linear alkyl core of n carbon atoms, wherein n is 3 to 6;
X is an optional linking group; and each R is independently a lipid tail.
2. The compound of claim 1, wherein n is 4 to 6.
3. The compound of claim 2, wherein n is 4.
4. The compound of claim 2, wherein n is 5.
5. The compound of claim 2, wherein n is 6.
6. The compound of claim 1, wherein n is 3.
7. The compound of any one of claims 1 to 6, wherein Wn is selected from:
Figure imgf000183_0001
Figure imgf000184_0001
wherein:
* depicts the point of attachment to Y; each ** depicts a point of attachment to X;
G1 is H, or a group cyclically linked with Y that together with the carbon atom of Wn to which they are attached provide a heterocycle; and
G2 is H or -CH2OH.
8. The compound of any one of claims 1 to 6, wherein Y is selected from — O — , — C(R10)2— — OC(O)— , — C(O)O— , — OC(O)O— , — OC(O)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O— , and — NR10C(O)S — , wherein R10 is selected from H and C1-6 alkyl.
9. The compound of claim 8, wherein Y is selected from — O — , — OC(O) — , and — OC(O)NR10— .
10. The compound of claim 8, wherein Y is — CH2 — .
11. The compound of claim 7, wherein G1 is a group that is cyclically linked with Y and together with the carbon atom of Wn to which they are attached provides a heterocycle.
12. The compound of any one of claims 1 to 11, wherein L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene.
13. The compound of claim 12, wherein L is -(CH2)2- The compound of claim 12, wherein L is -(CH2)3- The compound of any one of claims 1 to 14, wherein Z comprises a tertiary amino group. The compound of claim 15, wherein Z is -NRUR12, wherein R11 and R12 are each independently alkyl or substituted alkyl. The compound of claim 16, wherein R11 and R12 are each Ci-6 alkyl. The compound of claim 17, wherein R11 and R12 are each C1-3 alkyl. The compound of claim 18, wherein R11 and R12 are each methyl. The compound of claim 18, wherein R11 and R12 are each ethyl. The compound of any one of claims 1 to 20, wherein each X is independently selected from — (CH2)SOC(O)— , — (CH2)SC(O)O— , — (CH2)SOC(O)O— ,— (CH2)SOC(O)NR10— , — (CH2)SO— , — (CH2)SSC(O)NR10— , — (CH2)SC(O)NR10— , — (CH2)sNR10C(O)— , — (CH2)sS— , — (CH2)sNR10— , — (CH2)sNR10C(O)O— , and — (CH2)SNR10C(O)S — , wherein R10 is selected from H and Ci-6 alkyl and s is 0-6. The compound of 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)NR10— , — SC(O)NR10— , — C(O)NR10— , — NR10C(O)— , — S— , —NR10—, — NR10C(O)O— , and — NR10C(O)S — , wherein R10 is selected from H and Ci-6 alkyl. The compound of claim 22, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— . The compound of claim 23, wherein each — X-R is — OC(O)R. The compound of any one of claims 1 to 24, wherein each R is independently an aliphatic hydrocarbon group that is straight chain or branched, saturated or unsaturated and/or optionally comprises a cyclic group. The compound of any one of claims 1 to 25, wherein each R is a linear hydrocarbon group optionally comprising one or more cyclic groups. The compound of any one of claims 1 to 26, wherein each R is selected from a C5-C2o alkyl, C5-C2o alkenyl, and a C5-C2o alkynyl. The compound of claim 27, wherein each R is selected from a C6-C12 alkyl, and Ce- C12 alkenyl. The compound of claim 26, wherein at least one R is a linear hydrocarbon group comprising a cyclic group. The compound of claim 29, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted. The compound of any one of claims 1 to 25, wherein at least one R is a branched hydrocarbon group optionally comprising a cyclic group. The compound of claim 31, wherein each R is a branched hydrocarbon group. The compound of claim 32, wherein the branched hydrocarbon group comprises 8-20 carbon atoms. The compound of any one of claims 31 to 33, wherein the branched hydrocarbon group is saturated. The compound of any one of claims 31 to 33, wherein the branched hydrocarbon group is unsaturated. The compound of any one of claims 31 to 35, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl. The compound of claim 31, wherein at least one R is a branched hydrocarbon group comprising a cyclic group. The compound of claim 37, wherein the cyclic group is a monocyclic or bicyclic group selected from cycloalkyl, aryl, heterocycle, and heteroaryl, wherein any of the monocyclic or bicyclic groups are optionally substituted. The compound of claim 1, wherein the compound is of formula (IIA):
Figure imgf000186_0001
(IIA). The compound of claim 39, wherein Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — , wherein R10 is selected from H and Ci-6 alkyl. The compound of claim 40, wherein Y is — O — . The compound of claim 40, wherein Y is — OC(O) — , The compound of claim 40, wherein Y is — OC(O)NR10 — . The compound of any one of claims 39 to 43, wherein L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene. The compound of claim 44, wherein L is -(CH2)2- The compound of claim 44, wherein L is -(CFb)?-. The compound of claim 44, wherein L is -(CH2)4- The compound of any one of claims 39 to 47, wherein Z is -NRnR12, wherein R11 and
R12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl. The compound of claim 48, wherein R11 and R12 are each C1-3 alkyl. The compound of claim 49, wherein R11 and R12 are each methyl. The compound of claim 49, wherein R11 and R12 are each ethyl. The compound of any one of claims 39 to 51, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— . The compound of any one of claims 39 to 52, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl. The compound of any one of claims 39 to 52, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group. The compound of claim 54, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
56. The compound of claim 39, wherein the compound is of formula (IIIA):
Figure imgf000188_0001
wherein:
R11 and R12 are each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , — OC(O) — , and — OC(O)NR10 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and - (CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10.
57. The compound of claim 1, wherein the compound is of formula (IIB):
Figure imgf000188_0002
58. The compound of claim 57, wherein Y is selected from — O — , — OC(O) — , — OC(O)NR10— , — NR10C(O)— , — NR10C(O)O— , and — NR10C(O)S— , wherein R10 is selected from H and C1-6 alkyl.
59. The compound of claim 58, wherein Y is selected from — NHC(O) — , —
NHC(O)O— , and — NHC(O)S— .
60. The compound of any one of claims 57 to 59, wherein L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene.
61. The compound of claim 60, wherein L is -(CH2)2-
62. The compound of claim 60, wherein L is -(CFb)?-.
63. The compound of claim 60, wherein L is -(CH2)4-
64. The compound of any one of claims 57 to 59, wherein Z is -NRnR12, wherein R11 and
R12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl.
65. The compound of claim 64, wherein R11 and R12 are each C1-3 alkyl.
66. The compound of claim 64, wherein R11 and R12 are each methyl.
67. The compound of any one of claims 57 to 66, wherein each X is independently selected from — OC(O)— , — C(O)O— , and — OC(O)O— .
68. The compound of any one of claims 57 to 67, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
69. The compound of any one of claims 57 to 67, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic groups.
70. The compound of claim 69, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
71. The compound of claim 57, wherein the compound is of formula (IIIB):
Figure imgf000189_0001
wherein: R11 and R12 are each independently selected from C1-3 alkyl or C 1-4 heteroalkyl; q is 1 to 4;
Y is selected from — NHC(O) — , — NHC(O)O — , and — NHC(O)S — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and - (CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and t and u are each independently 1-10. The compound of claim 1, wherein the compound is of formula (IIC):
Figure imgf000190_0001
(IIC). The compound of claim 72, wherein Y is selected from — O — , — OC(O) — , — OC(O)NR10 — , and — C(R10)2 — , wherein R10 is selected from H and Ci-6 alkyl. The compound of claim 73, wherein Y is — O — . The compound of claim 73, wherein Y is — C(R10)2 — . The compound of any one of claims 72 to 75, wherein L is (C2-Ce)alkylene or substituted (C2-Ce)alkylene. The compound of any one of claims 76, wherein L is -(CH2)2- The compound of any one of claims 76, wherein L is -(CFb)?-. The compound of any one of claims 76, wherein L is -(CH2)4- The compound of any one of claims 72 to 79, wherein Z is -NRnR12, wherein R11 and
R12 are each independently Ci-6 alkyl or substituted Ci-6 alkyl. The compound of claim 80, wherein R11 and R12 are each selected from C1-3 alkyl and Ci-4 heteroalkyl. The compound of claim 81, wherein R11 and R12 are each methyl. The compound of any one of claims 72 to 82, wherein each X is independently selected from — (CH2)SOC(O)— , — (CH2)SC(O)O— , — (CH2)SOC(O)O— , wherein s is 0-6.
84. The compound of any one of claims 72 to 82, wherein each s is 0.
85. The compound of any one of claims 72 to 82, wherein each s is 1.
86. The compound of any one of claims 72 to 82, wherein each s is 3.
87. The compound of any one of claims 72 to 86, wherein each R is selected from C5-C20 alkyl, C5-C20 alkenyl, and a C5-C20 alkynyl.
88. The compound of any one of claims 72 to 86, wherein at least one R is a branched hydrocarbon group comprising 8-20 carbon atoms optionally further comprising one or more cyclic group.
89. The compound of claim 88, wherein R is -CH(R7)2, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl.
90. The compound of claim 72, wherein the compound is of formula (IIIC):
Figure imgf000191_0001
wherein:
R11 and R12 is each independently selected from C1-3 alkyl and Ci-4 heteroalkyl; q is 1 to 4;
Y is selected from — O — , and — C(R10)2 — ; each s is independently 0, 1 or 2;
W is — O — , and — C(R10)2 — ; and each R is independently selected from C5-C20 alkyl, C5-C20 alkenyl, -CH(R7)2, and - (CH2)tJ(CH2)u, wherein each R7 is independently C5-C12 alkyl, or C5-C12 alkenyl, J is a cyclic group, and each of t and u are 1-10.
91. The compound of any one of claims 1 to 90, wherein each R is independently
Figure imgf000192_0001
CyA and CyB is each independently a bond or an optionally substituted, saturated, partially unsaturated, or aromatic cyclic group selected from 5- to 12-membered monocyclyl, bicyclyl, bridged polycyclyl, and spirocyclyl;
Rx and Ry is each independently a bond, or an optionally substituted, straight or branched, saturated or partially unsaturated, C1-C20 aliphatic group; and r, p, and q is each independently an integer from 0 to 20.
92. The compound of claim 91, wherein at least one R comprises a moiety selected from
Figure imgf000192_0002
Figure imgf000193_0001
to X, or the point of attachment to a linear or branched hydrocarbon chain of R. A lipid nanoparticle comprising an ionizable lipid compound according to any one of claims 1 to 92. The lipid nanoparticle of claim 93, further comprising a neutral lipid and a lipid capable of reducing aggregation. The lipid nanoparticle of claim 94, wherein the neutral lipid comprises a phospholipid. The lipid nanoparticle of claim 94 or 95, wherein the neutral lipid comprises cholesterol. The lipid nanoparticle of claim 96, comprising: a) a nucleic acid, b) an ionizable lipid, c) a phospholipid, d) cholesterol, and e) a lipid capable of reducing aggregation. The lipid nanoparticle of claim 97, wherein the nucleic acid comprises DNA. The lipid nanoparticle of claim 98, wherein the nucleic acid comprises RNA. The lipid nanoparticle of claim 98, wherein the nucleic acid comprises DNA and
RNA. The lipid nanoparticle of claim 100, wherein the RNA is selected from mRNA, gRNA, and siRNA. The lipid nanoparticle of any one of claims 97 to 101, wherein the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), and a phosphatidylglycerol (PG), and derivatives thereof. The lipid nanoparticle of claim 102, wherein the phospholipid is a phosphatidylethanolamine (PE). The lipid nanoparticle of claim 103, wherein the phospholipid is a phosphatidylcholine (PC). The lipid nanoparticle of any one of claims 97 to 104, wherein the phospholipid comprises hydrocarbon chains each independently having 12-24 carbons. The lipid nanoparticle of claim 105, wherein the phospholipid comprises hydrocarbon chains each independently having 16-20 carbons. The lipid nanoparticle of claim 105 or 106, wherein the hydrocarbon chains are saturated. The lipid nanoparticle of claim 105 or 106, wherein the hydrocarbon chains are unsaturated and/or further comprise a carbocyclyl. The lipid nanoparticle of claim 108, wherein the hydrocarbon chains each independently comprise 1-4 double bonds. The lipid nanoparticle of any one of claims 94 to 109, wherein the phospholipid comprises two different hydrocarbon chains. The lipid nanoparticle of claim 103, wherein the phospholipid comprises 1,2-dioleyl- sn-glycero-3-phosphoethanolamine (DOPE). The lipid nanoparticle of claim 103, wherein the phospholipid comprises l-stearoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). The lipid nanoparticle of claim 104, wherein the phospholipid comprises 1,2- dipalmitoleoyl-sn-glycero-3-phosphocholine (A9A9-Cis PC). The lipid nanoparticle of claim 106, wherein the lipid nanoparticle comprises 1,2- di stearoyl -sn-gly cero-3 -phosphocholine (D SPC) . The lipid nanoparticle of claim 104, wherein the lipid nanoparticle comprises 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC). The lipid nanoparticle of any one of claims 103 to 115 wherein the lipid capable of reducing aggregation is a PEG-lipid. The lipid nanoparticle of claim 116, wherein the PEG-lipid is 1,2-dimyristoyl-rac- gly cero-3 -methoxypolyethylene glycol-2000 (PEG-DMG[2K]) or PEG-1, 2- distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (PEG-DSG[2K]). The lipid nanoparticle of any one of claims 94 to 117, further comprising a targeting ligand. The lipid nanoparticle of claim 118, wherein the targeting ligand comprises GalNAc. The lipid nanoparticle of claim 118 or 119, wherein the targeting ligand is linked to the lipid capable of reducing aggregation. The lipid nanoparticle of claim 120, wherein the lipid capable of reducing aggregation is PEG- l,2-distearoyl-rac-glycero-3-methylpoly oxy ethylene 2000 (PEG-DSG[2K]). The lipid nanoparticle of any one of claims 94 to 121, wherein the N/P ratio (ratio of moles of the amine groups of cationic lipids to those of the phosphate ones of DNA) is from 5 to 30. The lipid nanoparticle of claim 122, wherein the N/P ratio is 7. The lipid nanoparticle of claim 122, wherein the N/P ratio is 14. The lipid nanoparticle of claim 122, wherein the N/P ratio is 28. The lipid nanoparticle of any one of claims 94 to 125, comprising: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 6 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. The lipid nanoparticle of any one of claims 94 to 125, comprising: a) an ionizable lipid at 40 to 60 mol % of the total lipid present; b) a phospholipid at 10 to 20 mol % of the total lipid present; c) cholesterol at 35 to 45 mol % of the total lipid present; and d) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. The lipid nanoparticle of any one of claims 94 to 125, comprising: e) an ionizable lipid at 40 to 49 mol % of the total lipid present; f) a phospholipid at 10 to 20 mol % of the total lipid present; g) cholesterol at 35 to 45 mol % of the total lipid present; and h) a lipid capable of reducing aggregation at 1.5 to 2.5 mol % of the total lipid present. A pharmaceutical composition comprising a lipid nanoparticle of any one of claims 94 to 128 and a pharmaceutically acceptable excipient, carrier, or diluent. A method for delivering a nucleic acid into a cell, the method comprising contacting the cell with a lipid nanoparticle of any one of claims 94 to 128. The method according to claim 130, wherein the cell is in vitro. The method according to claim 130, wherein the cell is in vivo. A method for delivering a nucleic acid for in vivo production of target protein, the method comprising: administering systemically to a subject in need thereof a pharmaceutical composition of claim 129, wherein the nucleic acid encodes a target protein and is encapsulated within the lipid nanoparticles, and the administering of the pharmaceutical composition results in the prolonged stable expression of the target protein.
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