Nothing Special   »   [go: up one dir, main page]

WO2011090965A1 - Novel cationic lipids for oligonucleotide delivery - Google Patents

Novel cationic lipids for oligonucleotide delivery Download PDF

Info

Publication number
WO2011090965A1
WO2011090965A1 PCT/US2011/021619 US2011021619W WO2011090965A1 WO 2011090965 A1 WO2011090965 A1 WO 2011090965A1 US 2011021619 W US2011021619 W US 2011021619W WO 2011090965 A1 WO2011090965 A1 WO 2011090965A1
Authority
WO
WIPO (PCT)
Prior art keywords
dien
octadeca
yloxy
bis
compound
Prior art date
Application number
PCT/US2011/021619
Other languages
French (fr)
Inventor
Daniel Zewge
Francis Gosselin
Original Assignee
Merck Sharp & Dohme Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Sharp & Dohme Corp. filed Critical Merck Sharp & Dohme Corp.
Priority to EP11735061A priority Critical patent/EP2525781A1/en
Priority to US13/574,315 priority patent/US9670487B2/en
Publication of WO2011090965A1 publication Critical patent/WO2011090965A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C07C217/28—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 one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C07C217/40—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 one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the same carbon atom of the carbon skeleton, e.g. amino-ketals, ortho esters
    • 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
    • C07C217/52—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups or amino groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • the present invention relates to novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides, to facilitate the cellular uptake and endosomal escape, and to knockdown target mRNA both in vitro and in vivo.
  • Cationic lipids and the use of cationic lipids in lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, have been previously disclosed.
  • WO 2009/127060 See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602. Lipid nanoparticles and use of lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, has been previously disclosed. (See US patent applications: US 2006/0240554 and US 2008/0020058). Oligonucleotides (including siRNA and miRNA) and the synthesis of oligonucleotides has been previously disclosed. (See US patent applications: US 2006/0240554 and US 2008/0020058).
  • cationic lipids such as DLinDMA have been employed for siRNA delivery to liver but suffer from extended tissue half lives. It is an object of the instant invention to provide a cationic lipid scaffold that is susceptible to hydrolytic instability that may translate into reduced liver residence times and reduced hepatocellular toxicity.
  • the present invention employs acetals and ketals to provide a low pH sensitive chemical handle for degradation.
  • the instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that is susceptible to hydrolytic instability that may translate into reduced liver residence times and reduced hepatocellular toxicity.
  • the present invention employs acetals and ketals to provide a low pH sensitive chemical handle for degradation.
  • FIGURE 1 Lipid (Compound 2) pH dependent hydrolytic stability.
  • FIGURE 2 Lipid (Compound 2) pH dependent hydrolytic stability within LNP assembly.
  • FIGURE 3 Lipid (Compound 31) efficacy in mouse.
  • FIGURE 4 Lipid (Compound 31) efficacy in rat.
  • FIGURE 5 Absolute liver levels of cationic lipid (Compound 31) in rat at 48 hours.
  • FIGURE 6 Lipid (Compound 34) efficacy in NHP.
  • the various aspects and embodiments of the invention are directed to the utility of novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miRNA, to any target gene.
  • novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miRNA, to any target gene.
  • the cationic lipids of the instant invention are useful components in a lipid nanoparticle for the delivery of oligonucleotides, specifically siRNA and miRNA.
  • n 0, 1 or 2;
  • R 1 and R 2 are independently selected from H, (C 1 -C6)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';
  • R 3 is selected from H and (C 1 -C 6 )alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R 3 can be taken together with Rl to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';
  • R 4 is selected from H, (C 1 -C 6 )alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R';
  • R 5 is selected from H and (C 1 -C6)alkyl; or R 5 can be taken together with R 1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';
  • R' is independently selected from halogen, R", OR", SR", CN, C0 2 R" and
  • R" is selected from H and (C 1 -C 6 )alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;
  • L 1 is a C 4 -C 22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R';
  • L 2 is a C 4 -C 22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R';
  • the invention features a compound having Formula A, wherein:
  • L 1 and L 2 are ; and all other variables are as defined in the first embodiment;
  • the cationic lipids are illustrated by the following abbreviations:
  • n 0, 1 or 2;
  • R 1 and R 2 are independently selected from H and (C 1 -C 4 )alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
  • R 3 is selected from H and (C 1 -C 4 )alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R 3 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R 3 can be taken together with R 4 to form cyclopropyl or cyclobutyl;
  • R 4 is selected from H and (C 1 -C 4 )alkyl, said alkyl is optionally substituted with one or more substituents selected from R';
  • R 5 is selected from H and (C 1 -C 4 )alkyl, or R 5 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
  • R' is independently selected from halogen, R" and OR";
  • R" is selected from H and (C 1 -C 4 )alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;
  • L 1 is a C 4 -C 22 alkyl or a C 4 -C 22 alkenyl
  • L 2 is a C 4 -C 22 alkenyl
  • the cationic lipids are illustrated by the following abbreviations:
  • n 0, 1 or 2;
  • R 1 and R 2 are independently selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from R', or
  • R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R 1 ;
  • R 3 is selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from R', or R 3 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R 3 can be taken together with R 4 to form cyclopropyl;
  • R 4 is selected from H, methyl and ethyl, said methyl and ethyl are optionally substituted with one or more substituents selected from R';
  • R 5 is selected from H, methyl and ethyl, or R 5 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
  • R' is independently selected from OH and R";
  • R" is selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from halogen and OH;
  • L 1 is a C 4 -C 22 alkyl or a C 4 -C 22 alkenyl; and L 2 is a C 4 -C 22 alkenyl;
  • Specific cationic lipids are:
  • the cationic lipids disclosed are useful in the preparation of lipid nanoparticles.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of oligonucleotides.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA and miRNA.
  • the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA.
  • the cationic lipids of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1 1 19-1 190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention.
  • the cationic lipids disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
  • substituents and substitution patterns on the cationic lipids of the instant invention can be selected by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
  • Si atoms can be incorporated into the cationic lipids of the instant invention by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials.
  • the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
  • the present invention is meant to include all suitable isotopic variations of the compounds of Formula A.
  • different isotopic forms of hydrogen (H) include protium (lH) and deuterium (2 H ).
  • Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
  • Isotopically-enriched compounds within Formula A can be prepared without undue
  • alkyl means a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • alkenyl means a straight chain, cyclic or branched unsaturated aliphatic hydrocarbon having the specified number of carbon atoms including but not limited to diene, triene and tetraene unsaturated aliphatic hydrocarbons.
  • heterocyclyl or “heterocycle” means an aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O. N and S, and includes bicyclic groups.
  • “Heterocyclyl” or “heterocycle” therefore includes, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazo
  • quinazolinyl quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1 ,4-dioxanyl,
  • polyamine means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.
  • halogen means Br, Cl, F and I.
  • n 0.
  • n 1
  • n 2
  • R 1 and R 2 are independently selected from H and (C 1 -C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
  • R 1 and R 2 are independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R 1 and R 2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R 1 .
  • R 1 and R 2 are independently selected from H, methyl, ethyl and propyl.
  • R 1 and R 2 are independently selected from H and methyl.
  • R 1 and R 2 are both methyl.
  • R 3 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R'. or R 3 can be taken together with R 1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
  • R 3 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R 1 , or R 3 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R 3 can be taken together with R 4 to form cyclopropyl or cyclobutyl.
  • R 3 is selected from H, methyl, ethyl and propyl.
  • R 3 is selected from H, methyl and ethyl. In an embodiment of Formula A, R 3 is methyl.
  • R 3 is H.
  • R 4 is selected from H, methyl, ethyl and propyl.
  • R 4 is selected from H and methyl.
  • R 4 is methyl
  • R 4 is H.
  • R 5 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R 5 can be taken together with R 1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
  • R 5 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R 5 can be taken together with R 1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R'.
  • R 5 is selected from H, methyl, ethyl and propyl.
  • R 5 is selected from H and methyl.
  • R 5 is methyl
  • R 5 is H.
  • R' is OH and R".
  • R' is R".
  • R" is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more OH.
  • R" is selected from H, methyl and ethyl wherein said methyl and ethyl are optionally substituted with one or more OH.
  • L 1 is selected from C 4 -C 22 alkyl and C 4 -C 22 alkenyl, which are optionally substituted with halogen and OH.
  • L 1 is selected from C 4 -C 22 alkyl and C 4 -C 22 alkenyl.
  • L 1 is selected from C 6 -C 18 alkyl and C 6 -C 18 alkenyl.
  • L 2 is a C 4 -C 24 alkenyl, which is optionally substituted with halogen and OH.
  • L 2 is a C 4 -C 24 alkenyl.
  • L 2 is C 18 alkenyl.
  • L 2 is
  • heterocyclyl is pyrolidine, piperidine, morpholine, imidazole or piperazine.
  • "monocyclic heterocycle” is pyrolidine, piperidine, morpholine, imidazole or piperazine.
  • monocyclic heterocycle is pyrolidine or piperidine.
  • polyamine is putrescine, cadaverine, spermidine or spermine.
  • alkyl is a straight chain saturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • alkenyl is a straight chain unsaturated aliphatic hydrocarbon having the specified number of carbon atoms.
  • the free form of cationic lipids of Formula A is the free form of cationic lipids of Formula A, as well as the pharmaceutically acceptable salts and stereoisomers thereof.
  • Some of the isolated specific cationic lipids exemplified herein are the protonated salts of amine cationic lipids.
  • the term "free form" refers to the amine cationic lipids in non-salt form.
  • the encompassed pharmaceutically acceptable salts not only include the isolated salts exemplified for the specific cationic lipids described herein, but also all the typical pharmaceutically acceptable salts of the free form of cationic lipids of Formula A.
  • the free form of the specific salt cationic lipids described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium
  • the free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention.
  • the pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods.
  • the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
  • pharmaceutically acceptable salts of the cationic lipids of this invention include the conventional non-toxic salts of the cationic lipids of this invention as formed by reacting a basic instant cationic lipids with an inorganic or organic acid.
  • conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic (
  • salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, ⁇ , ⁇ 1 - dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.
  • the cationic lipids of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.
  • Primary amine containing acetals/ketals are prepared starting with the phthalamide containing ethyl acetal, conversion of this to the dilipid acetal/ketal and deprotection of the phthalamide protecting group.
  • acetals/ketals could be prepared directly from the aldehyde/ketone by direct acetal/ketal formation. Deprotection generates the secondary amine cationic lipids. Reductive amination gives tertiary amine cationic lipids.
  • Geminally di-substituted cationic lipids are prepared by protecting the starting aminoalchol as the phthalamide and then oxidizing the alcohol to the ketone. Acetal/ketal formation was followed by deprotection with hydrazine.
  • Cyclic ketals were prepared by first protecting the free amine of the diethyl ketal followed by ketalization with the lipid alcohol. Deprotection of the amine gives the free secondary amine. Reducive amination provides the tertiary amine catinic lipids. GENERAL SCHEME 5
  • General scheme 6 is an extension of General Scheme 1 wherein the alkyl bromide is homologated.
  • General Scheme 7 is an extension of General Scheme 6 wherein the alkylating agent is a phthalamide protected primary amine. Deprotection of this amine with hydrazine reveals cationic lipids of type xx.
  • General scheme 8 is an extension of General Scheme 4 wherein the intermediate aldehyde/ketone ix is accessed via the Weinreb amide xxi.
  • the Weinreb amide is prepared from ester xxi.
  • General scheme 9 outlines the preparation of mixed acetals of type xxv.
  • the dimethyl acetal is first converted to a mixed Iipid/methyl acetal using TMSOTf/lutidine followed by addition of lipid alcohol.
  • the final mixed acetal is prepared by converting the intermediate mixed acetal xxiii to mixed lipid acetal xxiv employing similar conditions.
  • General Scheme 10 is analogous to General Scheme 9 wherein the starting material is a phthalamide protected amine acetal. Iterative mixed acetal formation followed by deprotection of the amine generates final compounds of type xxviii. "
  • Compound 4 is S-Octyl CLinDMA which is generically described in US 7,514,099.
  • Compound 10 can be prepared according to General Scheme 3 above.
  • Compound 63 is DLinDMA as described in J. Controlled Release, 2005, 107, 276-287, US 2006/0083780 Al, and US 2006/0008910 Al .
  • Compound 64 is DLin C2DMA as described in Nature Biotechnology, 2010, 28, 172-176, WO 2010/042877 Al , WO 2010/048536 A2, WO 2010/088537 A2, and WO 2009/127060 Al .
  • lipid nanoparticle compositions of the instant invention are useful for the delivery of oligonucleotides, specifically siRNA and miRNA:
  • the Lipid Nano-Particles are prepared by an impinging jet process.
  • the particles are formed by mixing lipids dissolved in alcohol with siRNA dissolved in a citrate buffer.
  • the mixing ratio of lipids to siRNA are targeted at 45-55% lipid and 65-45% siRNA.
  • the lipid solution contains a novel cationic lipid of the instant invention, a helper lipid
  • the ratio of the lipids has a mole percent range of 25-98 for the cationic lipid with a target of 35-65, the helper lipid has a mole percent range from 0-75 with a target of 30-50, the PEG lipid has a mole percent range from 1-15 with a target of 1-6, and the DSPC has a mole precent range of 0-15 with a target of 0-12.
  • the siRNA solution contains one or more siRNA sequences at a concentration range from 0.3 to 1 .0 mg mL with a target of 0.3 -0.9 mg/mL in a sodium citrate buffered salt solution with pH in the range of 3.5-5.
  • the two liquids are heated to a temperature in the range of 15-40°C, targeting 30-40°C, and then mixed in an impinging jet mixer instantly forming the LNP.
  • the teelD has a range from 0.25 to 1.0 mm and a total flow rate from 10 -600 mL/min.
  • the combination of flow rate and tubing ID has effect of controlling the particle size of the LNPs between 30 and 200 nm.
  • the solution is then mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol but targeting 1 :2 vol: vol.
  • This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C.
  • the mixed LNPs are held from 30 minutes to 2 hrs prior to an anion exchange filtration step.
  • the temperature during incubating is in the range of 15-40°C, targeting 30-40°C.
  • After incubating the solution is filtered through a 0.8 um filter containing an anion exchange separation step. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min.
  • the LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the citrate buffer is exchanged for the final buffer solution such as phosphate buffered saline.
  • the ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range from 30 -500 KD.
  • the membrane format can be hollow fiber or flat sheet cassette.
  • the TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer; final buffer wastes.
  • the TFF process is a multiple step process with an initial concentration to a siRNA
  • the LNPs solution is diafiltered against the final buffer for 10 -20 volumes to remove the alcohol and perform buffer exchange. The material is then concentrated an additional 1-3 fold. The final steps of the LNP process are to sterile filter the concentrated LNP solution and vial the product.
  • siRNA duplex concentrations are determined by Strong Anion-Exchange High-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector.
  • the LNPs otherwise referred to as RNAi Delivery Vehicles (RDVs)
  • RDVs RNAi Delivery Vehicles
  • SAX separation using a Dionex BioLC DNAPac PA 200 (4 ⁇ 250 mm) column with UV detection at 254 nm.
  • Mobile phase is composed of A: 25 mM NaCl0 4> 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaCl0 4 , 10 mM Tris, 20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1 ml/min.
  • the si NA amount is determined by comparing to the siRNA standard curve.
  • Fluorescence reagent SYBR Gold is employed for RNA quantitation to monitor the encapsulation rate of RDVs.
  • RDVs with or without Triton X- 100 are used to determine the free siRNA and total siRNA amount.
  • the assay is performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples are excited at 485 run and fluorescence emission was measured at 530 nm. The siRNA amount is determined by comparing to the siRNA standard curve.
  • Encapsulation rate ( 1 - free siRN A/total siRNA) * 100%
  • RDVs containing 1 ⁇ g siRNA are diluted to a final volume of 3 ml with 1 x xBS.
  • the particle size and polydispersity of the samples is measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instruments Corporation,
  • the scattered intensity is measured with He-Ne laser at 25°C with a scattering angle of 90°.
  • RDVs containing 1 ⁇ g siRNA are diluted to a final volume of 2 ml with 1 mM Tris buffer (pH 7.4). Electrophoretic mobility of samples is determined using ZetaPALS instrument (Brookhaven Instruments Corporation. Holtsville, NY) with electrode and He-Ne laser as a light source. The Smoluchowski limit is assumed in the calculation of zeta potentials.
  • lipid concentrations are determined by Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation. Milford MA) with a Corona charged aerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, MA).
  • Individual lipids in RDVs are analyzed using an Agilent Zorbax SB- Cl 8 (50 x 4.6 mm, 1.8 ⁇ particle size) column with CAD at 60 °C.
  • the mobile phase is composed of A: 0.1% TFA in H 2 0 and B: 0.1% TFA in IPA.
  • the gradient changes from 60% mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min:
  • Oligonucleotide synthesis is well known in the art. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US
  • the siR A targets the mRNA transcript for the firefly (Photinus pyralis) luciferase gene (Accession # Ml 5077).
  • the primary sequence and chemical modification pattern of the luciferase siRNA is displayed above.
  • the in vivo luciferase model employs a transgenic mouse in which the firefly luciferase coding sequence is present in all cells.
  • ROSA26- LoxP-Stop-lx>xP-Luc (LSL-Luc) transgenic mice licensed from the Dana Farber Cancer Institute are induced to express the Luciferase gene by first removing the LSL sequence with a recombinant Ad-Cre virus (Vector Biolabs). Due to the organo-tropic nature of the virus, expression is limited to the liver when delivered via tail vein injection. Luciferase expression levels in liver are quantitated by measuring light output, using an IVIS imager (Xenogen) following administration of the luciferin substrate (Caliper Life Sciences). Pre-dose luminescence levels are measured prior to administration of the RDVs.
  • Luciferin in PBS 15mg/mL is intraperitoneally (IP) injected in a volume of 150 uL. After a four minute incubation period mice are anesthetized with isoflurane and placed in the IVIS imager. The RDVs (containing siRNA) in PBS vehicle were tail vein injected n a volume of 0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA. PBS vehicle alone was dosed as a control. Three hours post dose, mice were bled retro-orbitally to obtain plasma for cytokine analysis. Mice were imaged 48 hours post dose using the method described above.
  • Table 1 Mouse In Vivo efficacy data. Average % Inhibition of Bioluminescence by LNPs prepared from Compound 2 compared against Compound 4 at 0.3 mg Kg -1 .
  • LNPs utilizing compounds in the nominal compositions described above were evaluated for in vivo efficacy and increases in alanine amino transferase and aspartate amino transferase in Sprague-Dawley (Crl:CD(SD) female rats (Charles River Labs).
  • the siRNA targets the mRNA transcript for the ApoB gene (Accession # NM 019287).
  • the primary sequence and chemical modification pattern of the ApoB siRNA is displayed above.
  • the RDVs (containing siRNA) in PBS vehicle were tail vein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately 3 ml/min. Five rats were used in each dosing group. After LNP administration, rats are placed in cages with normal diet and water present.
  • liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing a rat ApoB commercial probe set (Applied Biosystems Cat #
  • RNA samples were analyzed for RNA expression in Eukaryotic cells.
  • the PCR reaction was performed on an ABI 7500 instrument with a 96- well Fast Block.
  • the ApoB mRNA level is normalized to the housekeeping PPIB (NM 01 1149) mRNA.
  • PPIB mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio of ApoB mRNA/ PPIB mRNA. All mRNA data is expressed relative to the PBS control dose.
  • Serum ALT and AST analysis were performed on the Siemens Advia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanine aminotransferase (Cat# 03039631) and aspartate aminotransferase (Cat# 03039631) reagents. Similar efficacy and better tolerability was observed in rats dosed with Compound 31 containing RDV than with the RDV containing the DLinKC2DMA cationic lipid 64 ( Figure 4).
  • Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w of water using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 ⁇ aliquot of each tissue homogenate was mixed with 300 L of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 ⁇ L ⁇ of each supernatant was then transferred to separate wells of a 96- well plate and 10 ⁇ samples were directly analyzed by LC S-MS.
  • OPS Diagnostics 1600 strokes/min, 5min.
  • a 50 ⁇ aliquot of each tissue homogenate was mixed with 300 L of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 ⁇ L ⁇ of each supernatant was then transferred to separate wells
  • Absolute quantification versus standards prepared and extracted from rat liver homogenate was performed using an Aria LX-2 HPLC system (Thermo Scientific) coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 ⁇ sample was injected onto a BDS Hypersil C8 HPLC column (Thermo, 50 x 2mm, 3 ⁇ ) at ambient temperature ( Figure 5).
  • LNPs utilizing compound 34 in the nominal compositions described above were evaluated for in vivo efficacy in male or female Macaca mulatta (rhesus) monkeys.
  • the siRNA targets the mRNA transcript for the ApoB gene (Accession # XM 001097404).
  • the primary sequence and chemical modification pattern of the ApoB siRNA is displayed above.
  • the RDVs (containing siRNA) in PBS vehicle were administered by intravenous injection in the saphenous vein at an injection rate of 20 mL/minute to a dose level of 1 mg/kilogram siRNA.
  • the injection volumes were from 1.9 to 2.1 mL/kilogram and monkeys ranged in weight from 2.5 to 4.5 kilograms.
  • the RDV or PBS control were administered to three monkeys.
  • Incubation mixtures (600 ⁇ xL total volume) containing 10 ⁇ test compound, 50% (v/v) methanol, 50% (v/v) various pH buffers (pH 5.0, 20mM sodium acetate buffer; pH 6.0: 20mM sodium acetate buffer; and pH 7.5: 20 mM sodium hydrogen phosphate buffer).
  • the reaction mixture was incubated at 37°C with gentle shaking.
  • a 50 ⁇ aliquot was removed at time: 0, 30min, lhr, 4hr and 24hr, and transferred to a collection tube containing 200 ⁇ reaction stopping buffer (95% H 2 0/5% methanol/1 OmM ammonium formate/0. l%formic acid with 500 nM internal standard).
  • Absolute quantification was performed using a Perkin Elmer HPLC system coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 ⁇ L of sample was injected onto a Phenomenex Luna C 18(2) HPLC column (5 ⁇ , 50 x 2mm) with the temperature set to 50°C (column heater. Restek, Model PTC050). A dual eluent system was used: 95% H 2 0/5% methanol/ 1 OmM ammonium formate/0. l%formic acid (A) and 40% methanol/60% n-propanol/10mM ammonium formate/0. l%formic acid (B). The flow rate was 0.4 mL/min and the gradient was as follows: hold at 100% A for 1 min, ramp to 0% A at 2.5 min, hold at 0% A for 2 min, then hold at 100% A for 1.5 min. Total run time was 7.0 min.
  • the API 4000 instrument source parameters were CAD: 4, CUR: 15, GS1 : 65, GS2: 35, IS: 4000, TEM: 600, DP: 80, CE: 29, CXP: 10, EP: 10, and the ion-transition condition was 602.7/336.3.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a catiomc lipid scaffold that is susceptible to hydrolytic instability that may translate into reduced liver residence times and reduced hepatocellular toxicity. The present invention employs acetals and ketals to provide a low pH sensitive chemical handle for degradation.

Description

TITLE OF THE INVENTION
NOVEL CATIONIC LIPIDS FOR OLIGONUCLEOTIDE DELIVERY
BACKGROUND OF THE INVENTION
The present invention relates to novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides, to facilitate the cellular uptake and endosomal escape, and to knockdown target mRNA both in vitro and in vivo.
Cationic lipids and the use of cationic lipids in lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, have been previously disclosed. (See US patent applications: US 2006/0008910, US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US 2009/0285881 and PCT patent applications: WO 2009/086558, WO 2010/042877, WO 2010/048536, WO 2010/088537, and
WO 2009/127060). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602. Lipid nanoparticles and use of lipid nanoparticles for the delivery of oligonucleotides, in particular siRNA and miRNA, has been previously disclosed. (See US patent applications: US 2006/0240554 and US 2008/0020058). Oligonucleotides (including siRNA and miRNA) and the synthesis of oligonucleotides has been previously disclosed. (See US patent applications: US 2006/0240554 and US 2008/0020058).
Traditional cationic lipids such as DLinDMA have been employed for siRNA delivery to liver but suffer from extended tissue half lives. It is an object of the instant invention to provide a cationic lipid scaffold that is susceptible to hydrolytic instability that may translate into reduced liver residence times and reduced hepatocellular toxicity. The present invention employs acetals and ketals to provide a low pH sensitive chemical handle for degradation.
SUMMARY OF THE INVENTION
The instant invention provides for novel cationic lipids that can be used in combination with other lipid components such as cholesterol and PEG-lipids to form lipid nanoparticles with oligonucleotides. It is an object of the instant invention to provide a cationic lipid scaffold that is susceptible to hydrolytic instability that may translate into reduced liver residence times and reduced hepatocellular toxicity. The present invention employs acetals and ketals to provide a low pH sensitive chemical handle for degradation.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 : Lipid (Compound 2) pH dependent hydrolytic stability. FIGURE 2: Lipid (Compound 2) pH dependent hydrolytic stability within LNP assembly.
FIGURE 3: Lipid (Compound 31) efficacy in mouse.
FIGURE 4: Lipid (Compound 31) efficacy in rat.
FIGURE 5. Absolute liver levels of cationic lipid (Compound 31) in rat at 48 hours.
FIGURE 6: Lipid (Compound 34) efficacy in NHP.
The various aspects and embodiments of the invention are directed to the utility of novel cationic lipids useful in lipid nanoparticles to deliver oligonucleotides, in particular, siRNA and miRNA, to any target gene. (See US patent applications: US 2006/0008910, US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US
2009/0285881 and PCT patent applications: WO 2009/086558, WO 2010/042877,
WO 2010/048536, WO 2010/088537, and WO 2009/127060). See also Semple S. C. et al., Rational design of cationic lipids for siRNA delivery, Nature Biotechnology, published online 17 January 2010; doi:10.1038/nbt.l602.
The cationic lipids of the instant invention are useful components in a lipid nanoparticle for the delivery of oligonucleotides, specifically siRNA and miRNA.
In a first embodiment of this invention, the cationic lipids are illustrated by the
Formula A:
Figure imgf000003_0001
wherein:
n is 0, 1 or 2;
R1 and R2 are independently selected from H, (C1-C6)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';
R3 is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R3 can be taken together with Rl to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'; R4 is selected from H, (C1-C6)alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R';
R5 is selected from H and (C1-C6)alkyl; or R5 can be taken together with R1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R';
R' is independently selected from halogen, R", OR", SR", CN, C02R" and
CON(R")2;
R" is selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;
L1 is a C4-C22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R'; and
L2 is a C4-C22 alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R';
or any pharmaceutically acceptable salt or stereoisomer thereof.
In a second embodiment, the invention features a compound having Formula A, wherein:
L1 and L2 are
Figure imgf000004_0001
; and all other variables are as defined in the first embodiment;
or any pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment of this invention, the cationic lipids are illustrated by the
Formula A:
Figure imgf000004_0002
wherein:
n is 0, 1 or 2;
R1 and R2 are independently selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
R3 is selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R4 to form cyclopropyl or cyclobutyl; R4 is selected from H and (C1-C4)alkyl, said alkyl is optionally substituted with one or more substituents selected from R';
R5 is selected from H and (C1-C4)alkyl, or R5 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
R' is independently selected from halogen, R" and OR";
R" is selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;
L1 is a C4-C22 alkyl or a C4-C22 alkenyl; and
L2 is a C4-C22 alkenyl;
or any pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment of this invention, the cationic lipids are illustrated by the
Formula A:
Figure imgf000005_0001
wherein:
n is 0, 1 or 2;
R1 and R2 are independently selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from R', or
R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R1;
R3 is selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from R', or R3 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R4 to form cyclopropyl;
R4 is selected from H, methyl and ethyl, said methyl and ethyl are optionally substituted with one or more substituents selected from R';
R5 is selected from H, methyl and ethyl, or R5 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
R' is independently selected from OH and R";
R" is selected from H, methyl and ethyl, wherein said methyl and ethyl are optionally substituted with one or more substituents selected from halogen and OH;
L1 is a C4-C22 alkyl or a C4-C22 alkenyl; and L2 is a C4-C22 alkenyl;
or any pharmaceutically acceptable salt or stereoisomer thereof.
Specific cationic lipids are:
N,N-dimethyl-2,2-bis[(9Z, 122)-octadeca-9, 12-dien- 1 -yloxy]ethanamine (Compound 2);
N,N-diniethyl-2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan- 1 -amine (Compound 3);
2.2- bis[(9Z,12Z)-octadeca-9,12-dien-l -yloxy]ethanamine (Compound 6);
(2R)-2- { bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } pyrrolidine (Compound 8);
(2R)-2- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } - 1 -methylpyrrolidine (Compound 9);
2-[(2R)-2- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } pyrrolidin- 1 -yl]ethanol
(Compound 10);
(2S)-l,l -bis[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine (Compound 11);
2-methyl- 1 , 1 -bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -y loxy]propan-2-amine (Compound 15);
4,4-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]piperidine (Compound 18);
3 ,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjpyrrolidine (Compound 19);
N,N-dimethyl-3,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan-l -amine (Compound 20):
1- {3,3-bis[(9Z,12Z)-octadeca-9,12-dien-l -yloxyjpropyl} pyrrolidine (Compound 21);
3.3- bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan- 1 -amine (Compound 22);
N,N-dimethyl-2,2-bis[(9Z)-octadec-9-en-l -yloxyjethanamine (Compound 23);
l-{bis[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}cyclopropanamine (Compound 28);
N.N-dimethyl-2-[(9Z, 12Z)-octadeca-9,l 2-dien- 1 -yloxy]-2-(octyloxy)ethanamine (Compound
3D;
2- (decyloxy)-N,N-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethanamine (Compound 32);
2-[(8Z)-dodec-8-en- 1 -yloxy]-N^-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien-l - yloxyjethanamine (Compound 33);
1 - {2-[(8Z)-dodec-8-en- 1 -yloxy]-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethyl } pyrrolidine (Compound 34);
l-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-(octyloxy)ethyl}pynOlidine (Compound 35); [(2S)- 1 - {2-(decyloxy)-2- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl }pyrrolidin-2-yl]methanol (Compound 36);
1 - {2-(decyloxy)-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethyl}piperidin-4-ol (Compoundl 37); N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyJ-3-(octyloxy)propa mine
(Compound 38);
2-(heptyloxy)-N,N-dimethyl-2-[(9Z,12Z)-octade namine (Compound
39) ;
N,N-dimethyl-2-(nonyloxy)-2-[(9Z,2Z)-octadeca-9,12-dien-l-yloxyjethanamine (Compound
40) ; 1 - { 3-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-3-(octyloxy)propyl } pyrrolidine (Compound 41); 2-(liexyloxy)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]ethanamine (Compound 44);
2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(octyloxy)ethanamine (Compound 45);
2-(decyloxy)-2-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]ethanamine (Compouind 46);
2-(decyloxy)-2-[(9Z)-octadec-9-en-l -yloxy]ethanamine (Compound 47):
2- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(tetradecyloxy)ethanamine (Compound 48);
[(2R)- 1 -{2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl } pyrrolidin-2-yI]methanol
(Compound 49);
[(2S)- 1 - {2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl} pyrrolidin-2-yl]inethanol
(Compound 50);
(3i¾,55)-5-{bis[(9Z,12Z)-octadeca-9!12-dien-1-yloxy]methyl}pyrrolidin-3-ol (Compound 53); (2S)-2-amino-3,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan- 1 -ol (Compound 54);
4- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjmethyl } piperidine (Compound 55);
3- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } piperidine (Compound 56);
3-amino-l,l-bis[(9Z,12Z)-octadeca-9,12-dien-l -yloxy]propan-2-ol (Compound 61); and l-methyl-4,4-bis[(9Z,12Z)-octadeca-9,l 2-dien-l -yloxy]piperidine (Compound 62)
or any pharmaceutically acceptable salt or stereoisomer thereof.
In another embodiment, the cationic lipids disclosed are useful in the preparation of lipid nanoparticles.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of oligonucleotides.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA and miRNA.
In another embodiment, the cationic lipids disclosed are useful components in a lipid nanoparticle for the delivery of siRNA.
The cationic lipids of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1 1 19-1 190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, being included in the present invention. In addition, the cationic lipids disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
It is understood that substituents and substitution patterns on the cationic lipids of the instant invention can be selected by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
It is understood that one or more Si atoms can be incorporated into the cationic lipids of the instant invention by one of ordinary skill in the art to provide cationic lipids that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials.
In the compounds of Formula A, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds of Formula A. For example, different isotopic forms of hydrogen (H) include protium (lH) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
Isotopically-enriched compounds within Formula A can be prepared without undue
experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Scheme and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
As used herein, "alkyl" means a straight chain, cyclic or branched saturated aliphatic hydrocarbon having the specified number of carbon atoms.
As used herein, "alkenyl" means a straight chain, cyclic or branched unsaturated aliphatic hydrocarbon having the specified number of carbon atoms including but not limited to diene, triene and tetraene unsaturated aliphatic hydrocarbons.
Examples of a cyclic "alkyl" or "alkenyl are:
Figure imgf000008_0001
As used herein, "heterocyclyl" or "heterocycle" means an aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O. N and S, and includes bicyclic groups. "Heterocyclyl" or "heterocycle" therefore includes, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl. quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1 ,4-dioxanyl,
hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl> dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofiiranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl,
dihydrothiadiazolyk dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof all of which are optionally substituted with one or more substituents selected from R'.
As used herein, "polyamine" means compounds having two or more amino groups. Examples include putrescine, cadaverine, spermidine, and spermine.
As used herein, "halogen" means Br, Cl, F and I.
In an embodiment of Formula A, n is 0.
In an embodiment of Formula A, n is 1.
In an embodiment of Formula A, n is 2.
In an embodiment of Formula A, R1 and R2 are independently selected from H and (C1-C6)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
In an embodiment of Formula A, R1 and R2 are independently selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R1.
In an embodiment of Formula A, R1 and R2 are independently selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, R1 and R2 are independently selected from H and methyl.
In an embodiment of Formula A, R1 and R2 are both methyl.
R3 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R'. or R3 can be taken together with R1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
R3 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R1, or R3 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R4 to form cyclopropyl or cyclobutyl.
In an embodiment of Formula A, R3 is selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, R3 is selected from H, methyl and ethyl. In an embodiment of Formula A, R3 is methyl.
In an embodiment of Formula A, R3 is H.
In an embodiment of Formula A, R4 is selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, R4 is selected from H and methyl.
In an embodiment of Formula A, R4 is methyl.
In an embodiment of Formula A, R4 is H.
In an embodiment of Formula A, R5 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R5 can be taken together with R1 to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R'.
In an embodiment of Formula A, R5 is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more substituents selected from R', or R5 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R'.
In an embodiment of Formula A, R5 is selected from H, methyl, ethyl and propyl.
In an embodiment of Formula A, R5 is selected from H and methyl.
In an embodiment of Formula A, R5 is methyl.
In an embodiment of Formula A, R5 is H.
In an embodiment of Formula A, R' is OH and R".
In an embodiment of Formula A, R' is R".
In an embodiment of Formula A, R" is selected from H, methyl, ethyl and propyl, wherein said methyl, ethyl and propyl are optionally substituted with one or more OH.
In an embodiment of Formula A, R" is selected from H, methyl and ethyl wherein said methyl and ethyl are optionally substituted with one or more OH. In an embodiment of Formula A, L1 is selected from C4-C22 alkyl and C4-C22 alkenyl, which are optionally substituted with halogen and OH.
In an embodiment of Formula A, L1 is selected from C4-C22 alkyl and C4-C22 alkenyl.
In an embodiment of Formula A, L1 is selected from C6-C18 alkyl and C6-C18 alkenyl.
In an embodiment of Formula A, L2 is a C4-C24 alkenyl, which is optionally substituted with halogen and OH.
In an embodiment of Formula A, L2 is a C4-C24 alkenyl.
In an embodiment of Formula A, L2 is C18 alkenyl.
In an embodiment of Formula A, L2 is
Figure imgf000011_0001
Figure imgf000011_0002
In an embodiment of Formula A, "heterocyclyl" is pyrolidine, piperidine, morpholine, imidazole or piperazine.
In an embodiment of Formula A, "monocyclic heterocycle" is pyrolidine, piperidine, morpholine, imidazole or piperazine.
In an embodiment of Formula A, "monocyclic heterocycle" is pyrolidine or piperidine.
In an embodiment of Formula A, "polyamine" is putrescine, cadaverine, spermidine or spermine.
In an embodiment, "alkyl" is a straight chain saturated aliphatic hydrocarbon having the specified number of carbon atoms.
In an embodiment, "alkenyl" is a straight chain unsaturated aliphatic hydrocarbon having the specified number of carbon atoms.
Included in the instant invention is the free form of cationic lipids of Formula A, as well as the pharmaceutically acceptable salts and stereoisomers thereof. Some of the isolated specific cationic lipids exemplified herein are the protonated salts of amine cationic lipids. The term "free form" refers to the amine cationic lipids in non-salt form. The encompassed pharmaceutically acceptable salts not only include the isolated salts exemplified for the specific cationic lipids described herein, but also all the typical pharmaceutically acceptable salts of the free form of cationic lipids of Formula A. The free form of the specific salt cationic lipids described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium
bicarbonate. The free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention.
The pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
Thus, pharmaceutically acceptable salts of the cationic lipids of this invention include the conventional non-toxic salts of the cationic lipids of this invention as formed by reacting a basic instant cationic lipids with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic (TFA) and the like.
When the cationic lipids of the present invention are acidic, suitable
"pharmaceutically acceptable salts" refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, Ν,Ν1- dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like. The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et l.,
"Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19.
It will also be noted that the cationic lipids of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.
EXAMPLES
Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof. The reagents utilized in synthesizing the cationic lipids are either commercially available or are readily prepared by one of ordinary skill in the art.
Synthesis of the novel cationic lipids is a linear process starting with acetal/ketal formation followed by amine displacement of the alkyl bromide. NERAL SCHEME 1
Figure imgf000013_0002
Primary amine containing acetals/ketals are prepared starting with the phthalamide containing ethyl acetal, conversion of this to the dilipid acetal/ketal and deprotection of the phthalamide protecting group.
GEN RAL SCHEME 2
Figure imgf000013_0001
In some examples, acetals/ketals could be prepared directly from the aldehyde/ketone by direct acetal/ketal formation. Deprotection generates the secondary amine cationic lipids. Reductive amination gives tertiary amine cationic lipids.
Figure imgf000014_0001
Geminally di-substituted cationic lipids are prepared by protecting the starting aminoalchol as the phthalamide and then oxidizing the alcohol to the ketone. Acetal/ketal formation was followed by deprotection with hydrazine.
Figure imgf000014_0002
Cyclic ketals were prepared by first protecting the free amine of the diethyl ketal followed by ketalization with the lipid alcohol. Deprotection of the amine gives the free secondary amine. Reducive amination provides the tertiary amine catinic lipids. GENERAL SCHEME 5
Figure imgf000015_0001
General scheme 6 is an extension of General Scheme 1 wherein the alkyl bromide is homologated.
Figure imgf000015_0002
General Scheme 7 is an extension of General Scheme 6 wherein the alkylating agent is a phthalamide protected primary amine. Deprotection of this amine with hydrazine reveals cationic lipids of type xx.
Figure imgf000015_0003
General scheme 8 is an extension of General Scheme 4 wherein the intermediate aldehyde/ketone ix is accessed via the Weinreb amide xxi. The Weinreb amide is prepared from ester xxi.
Figure imgf000016_0001
General scheme 9 outlines the preparation of mixed acetals of type xxv. The dimethyl acetal is first converted to a mixed Iipid/methyl acetal using TMSOTf/lutidine followed by addition of lipid alcohol. The final mixed acetal is prepared by converting the intermediate mixed acetal xxiii to mixed lipid acetal xxiv employing similar conditions.
Finally, the bromide is displaced with an amine to provide the final lipids.
Figure imgf000016_0002
General Scheme 10 is analogous to General Scheme 9 wherein the starting material is a phthalamide protected amine acetal. Iterative mixed acetal formation followed by deprotection of the amine generates final compounds of type xxviii. "
Figure imgf000017_0001
General Scheme 1 1 outlines preparation of lipids of type xxxii from
commercially available alcohols xxix. Oxidation of the alcohol affords aldehyde or ketones of type xxx. Acetal/ketal formation followed by Boc deprotection provides the final compounds.
Figure imgf000017_0002
General Scheme 12 outlines the preparation of amino alcohol cationic lipids of type xxvi. Commercial epoxide xxxi ii is opened with azide to give azido alcohol xxxiv. Lipid acetal formation followed by reduction of the azide delivers final compounds.
Figure imgf000017_0003
General Scheme 13 outlines the preparation of ketals of type xxxviii from the corresponding ketones xxxvii using acid and lipid alcohol.
Figure imgf000018_0001
SCHEMES
( 62.9Z)- 18- { 2-bromo- 1 -\ (9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxylethoxy ) octadeca-6.9-diene (Compound \)
Figure imgf000018_0002
To a stirred mixture of bromoacetaldehyde dimethylacetal (l .Og, 5.92 mmol) and linoleyl alcohol (3.15g, 1 1.83 mmol) was added camphor sulfonic acid (0.07g, 0.296 mmol) in one portion. The mixture heated to 70 °C for several hours. The reaction mixture was loaded directly onto a silica column and purified by flash chromatography to give product (2.5g) in 66% yield. 1H NMR (400 MHz, CDC13) δ 5.34 (4H, m), 4.63 (1H, t, J=5.5), 3.60 (2H, m), 3.47 (2H, m), 3.35 (2H, J=5.5), 2.76 (4H, t, J=6.5), 2.03 (8H, q, J=6.3), 1.58 (4H, br m), 1.4-1.22 (32H, br m), 0.88 (6H, t, J=6.6) ppm.
N,N-dimethyl-2.2-bis[(9Z,12Z)-octadeca-9.12-dien-l-yloxylethanamine (Compound 2)
Figure imgf000018_0003
A solution of the bromide (1, 3.5g, 5.49 mmol) in 0.89g/mL dimethylamine in DMF (0.278 mL, 5.49 mmol) was heated to 150 °C under microwave irradiation for 30 minutes. The reaction was partitioned between water/ethyl acetate. The organics were washed with brine, dried over sodium sulfate, filtered and evaporated in vacuo to generate pure product (2, 2.6g) in 79% yield. 1H NMR (400 MHz, CDC13) 6 5.33 (8H, m), 4.56 (1H, t, J =5.5), 3.57 (2H q, J =7.5), 3.47 (2H q, J -7.5), 2.44 (2H d, J =5.4), 2.27 (6H s), 1.56 (4H br m), 1.4-1.22 (32H, br m), 0.88 (6H, t, J=6.6) ppm.
N,N-dimethyl-2.2-bis[i9Z.12Z)-octadeca-9.12-dien-1-yloxy]propan-1 -amine (Compound 3)
Figure imgf000019_0001
Compound 3 can be prepared according to General Scheme 1 above.
1.0 g, 33% yield. C41H77N02: HRMS (ESI positive) M+H, theory m/z 616.6027, measured m/z 616.6038 amu. 1H NMR (400 MHz, CDC13) 6 5.35, (m, 8H), 3.40 (t, 4H, J = 6.83 Hz), 2.77 (t, 4H, J= 6.59 Hz), 2.42 (s, 3H), 2.29 (s, 6H), 2.05 (q, 8H, J = 6.84 Hz), 1.51 (m, 2H), 1.30 (m, 36H), 0.89 (t, 6H, J = 6.83 Hz) ppm.
Compound 4 is S-Octyl CLinDMA which is generically described in US 7,514,099.
Figure imgf000019_0002
Compound 4
2- (2.2-bis f(9Z.12ZV octadeca-9.12-dien- 1 -yloxylethyl } - 1 H-isoindole- 13(2H)-dione
(Compound 5~)
Figure imgf000019_0003
To a mixture of phthalimidoacetaldehyde diethyl acetal (3.5 g, 13.3 mmol) and cis, cis - 9, 12-octadecadien-1-ol (10.6 g, 39.9 mmol) was added pyridinium p-toluenesulfonate (0.167 g, 0.665 mmol) and heated to 105 °C. After 48 h, the reaction was diluted in 300 ml of dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0% ethyl acetate/ hexane → 20% ethyl acetate/ hexane) to give the title compound (7.6 g). MS 726.6 (M+Na). 2.2-bisf ( 9Z.12Z)-octadeca-9.12-dien- 1 -yloxyjethanamine (Compound 6)
Figure imgf000020_0001
To solution of 2- {2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl } - 1 H- isoindole-l ,3(2H)-dione in benzene (40 ml) was added 6.3 ml of methylhydrazine and heated to 70 °C. After 5 h, the reaction was filtrated and the solution was diluted in 200 ml of dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (1% MeOH/
dichloromethane→ 15% MeOH/ dichloromethane) to give the title compound (5.3 g). HRMS 574.5558 (M+l). 1H NMR δ (ppm)(CHCl 3-d): 5.40-5.29 (8 H, m), 4.39 (1 H, t, J = 5.13 Hz), 3.62 (2 H, dt, J = 9.36, 6.65 Hz), 3.45 (2 H, dt, J = 9.36, 6.67 Hz), 2.77 (6 H, t, J = 5.85 Hz), 2.05 (8 H, q, J = 7.05 Hz), 1.62-1.54 (6 H, m), 1.50-1.14 (32 H, m), 0.89 (6 H, t, J = 6.74 Hz).
(2R)-2- 1 bisf (9Z.12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl ) pyrrolidine (Compound 8)
Figure imgf000020_0002
To a mixture of (_¾)-N-Boc-prolinal (4.9 g, 24.6 mmol) and cis, cis - 9, 12- octadecadien-1-ol (16.4 g, 61.5 mmol) was added pyridinium p-toluenesulfonate (0.309 g, 1.23 mmol) and heated to 105 °C. After 72 h, the reaction was diluted in 300 ml of THF and cooled to 0 °C. Gaseous HCl was bubbled into the THF solution for 3 minutes at 0 °C. The reaction was stirred from 0 °C to ambient temperature for 5 h. The solvent was evaporated and reaction was redissolved by 300 ml dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel
chromatography (1% MeOH/ dichloromethane→ 12% MeOH/ dichloromethane) to give the title compound (8.2 g). MS 614.9(M+1). HRMS 614.5873 (M+l). 1H NMR δ (ppm)(CHCl 3-d): 5.39-5.30 (8 H, m), 4.25 (1 H, d, J = 6.83 Hz), 3.68-3.58 (2 H, m), 3.48 (2 H, p, J = 7.71 Hz), 3.23 (1 H, q, J = 7.14 Hz), 3.02-2.95 (1 H, m), 2.90-2.84 (1 H, m), 2.77 (4 H, t, J = 6.82 Hz), 2.16-1.96 (8 H, m), 1.89-1.80 (1 H, m), 1.80-1.68 (2 H, m), 1.62-1.52 (5 H, m), 1.45-1.20 (32 H, m), 0.99-0.76 (6 H, m).
(2R)-2-{bis[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy1methvn-1-methylpyrrolidme (Comp
Figure imgf000021_0001
To 8 ml THF solution of (2^)-2-{bis[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]methyl} pyrrolidine (Compound 8) (2.15 g, 3.50 mmol) was added 37% formaldehyde aqueous solution (2.61 ml, 35 mmol), acetic acid (0.401 ml, 7.00 mmol) and 8 M borane- pyridine complex (1.09 ml, 8.75 mmol) and stirred at ambient temperature. After 4 h, the reaction was diluted in 100 ml of dichloromethane. The organic was washed by 50 ml of saturated sodium bicarbonate solution twice and brine (50 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0% MeOH/ dichloromethane→ 12% MeOH/ dichloromethane) to give the title compound (1.05 g). MS 628.9(M+1). HRMS 628.6039 (M+l). 1H NMR δ (ppm)(CHCl 3-d): 5.40-5.31 (8 H, m), 4.29 (1 H, d, J = 6.40 Hz), 3.65 (1 H, dt, J = 9.22, .67 Hz), 3.58-3.46 (4 H, m), 3.05 (1 H, t, J = 7.41 Hz), 2.77 (4 H, t, J = 6.71 Hz), 2.45 (4 H, ), 2.28-2.20 (1 H, m), 2.07-2.01 (8 H, m), 1.87 (1 H, t, J = 9.78 Hz), 1.80-1.64 (2H, m), 1.63-1.52 (4 H, m), 1.45-1.20 (32 H, m), 0.89 (6 H, t, J = 6.58 Hz).
2- 2RV2-{bisr(9Z.12Z -octadeca-9.12-dien-1-yloxy]methvnpyri lidin-1-yl]ethanol
(Compound 10)
Figure imgf000021_0002
Compound 10 can be prepared according to General Scheme 3 above.
MS 658.8 (M+l). HRMS 658.6156 (M+l). 1H NMR δ (ppm)(CHCl 3-d): 5.41-5.29 (8 H, m), 4.25 (1 H, d, J = 6.26 Hz), 3.67-3.53 (4 H, m), 3.52-3.43 (2 H, m), 3.18-3.05 (2 H, m), 2.95- 2.70 (5 H, m), 2.59 (1 H, dt, J = 12.59, 3.75 Hz), 2.36 (1 H, q, J = 8.37 Hz), 2.05 (8 H, q, J = 7.11 Hz), 1.87-1.71 (4 H, m), 1.64-1.45 (4 H, m), 1.42-1.22 (32 H, m), 0.89 (6 H, t, J = 6.73 Hz).
(2S^-l.l-bis[('9Z, 12Z -octadeca-9.12-dien-1-yloxy1propan-2-amine (Compound 1 1)
Figure imgf000022_0003
Compound 11 can be prepared according to General Scheme 3 above.
HRMS 588.5731 (M+l). 1H NMR 5 (ppm)(CHCl 3-d): 5.39-5.30 (8 H, m), 4.08 (1 H, d, J = 5.87 Hz), 3.64 (2 H, ddt, J = 22.87, 9.24, 6.57 Hz), 3.45 (2 H, dt, J = 9.23, 6.72 Hz), 2.97 (1 H, t, J = 6.30 Hz), 2.77 (4 H, t, J = 6.49 Hz), 2.05 (8 H, q, J = 6.91 Hz), 1.65-1.5 (4 H, m), 1.45 - 1.20 (32 H, m), 1.07 (3 H, d, J = 6.51 Hz), 0.89 (6 H, t, J = 6.76 Hz).
2-( 1 -hvdroxy-2-methylpropan-2-yl)- 1 H-isoindole- ,3(2H)-dione (Compound 12)
Figure imgf000022_0001
To 12 ml D F solution of 2-amino-2-methylpropan-1-ol (1.70 g, 19.1 mmol) was added phthalic anhydride (3.11 g, 21.0 mmol) and triethyl amine (5.85 ml, 42 mmol). The mixture was microwaved at 145 °C for 2 h. . After evaporation of solvent, the residue was piuified by silica gel chromatography (0% eOH/ dichloromethane→ 5% MeOH/
dichloromethane) to give the title compound (1.9 g). MS 220.3 (M+l).
2-( 1.3-dioxo- 1.3-dihvdiO-2H-isoindol-2-yl)-2-methylpropanal (Compound 13)
Figure imgf000022_0002
To 150 ml dichloromethane solution of 2-(l-hydroxy-2-methylpropan-2-yl)-lH- isoindoIe-1,3(2 )-dione (Compound 12) (6.2 g, 28.3 mmol) was added Dess-Martin periodinane (14.4 g, 33.9 mmol) and stirred at ambient temperature for 16 h. The reaction was diluted in 200 ml of dichloromethane. The organic was washed by 100 ml 1 M sodium thiosulfate aqueous solution twice, saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0 % MeOH/ dichloromethane→ 5% MeOH/ dichloromethane) to give the title compound (5.6 g). MS 218.3 (M+l). 2- 12-methyl- 1.1 -bisf(9Z.12Z)-octadeca-9.12-dien- 1 -yloxylpropan-2-yl ) - 1 H-isoindole- 1 (2H>- dione (Compound 14)
Figure imgf000023_0001
To a mixture of 2-(1,3-dioxo-13-dihydro-2H-isoindol-2-yl)-2-methylpropanal (4.78 g, 22.0 mmol) and cis, cis - 9, 12-octadecadien-1-ol (17.6 g. 66.0 mmol) was added pyridinium p-toluenesulfonate (0.553 g, 0.220 mmol) and heated to 120 °C. After 48 h, the reaction was diluted in 300 ml of dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0% ethyl acetate/ hexane→ 20% ethyl acetate/ hexane) to give the title compound (9.1 g). MS 754.9 (M+Na).
2-methyl-l,l -bisf(9Z, 12Z)-octadeca-9.12-dien- 1 -yloxylpropan-2-amine (Compound 15)
Figure imgf000023_0002
To a 140 ml benzene solution of 2-{2-methy]-l,l -bis[(9Z,12Z -octadeca-9,12- dien-1-yloxy]propan-2-yl}-lH-isoindole-1,3(2H)-dione (Compound 14) (7.0 g, 9.56 mmol) was added methyl hydrazine (3.52 g, 76 mmol) and heated to 70 °C. After 5 h, the reaction was filtrated and the solution was diluted with 200 ml of dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0.5 % MeOH/ dichloromethane→ 10% MeOH dichloromethane) to give the title compound (3.5 g). MS 602.8 (M+l). HRMS 602.5877 (M+l). 1H NMR δ (ppm)(CHCh-d): 5.41-5.30 (8 H, m), 4.00 (1 H, s), 3.78 (2 H, dt, J = 9.04, 6.44 Hz), 3.47 (2 H, dt, J = 9.08, 6.78 Hz), 2.77 (4 H, t, J = 6.73 Hz), 2.05 (8 H, q, J = 7.00 Hz), 1.63-1.45 (4H, m), 1.42-1.20 (32 H, m), 1.06 (6 H, s), 0.89 (6 H, t, J = 6.67 Hz).
9H-fluoren-9-ylmethyl 4.4-diethoxypiperidine-1-carboxylate (Compound 16)
Figure imgf000024_0002
To 40 ml ethanol solution of l-benzyl-4,4-diethoxypiperidine (3.3 g, 12.5 mmol) was added 0.4 g palladium hydroxide on carbon (20 vvt %). The reaction was hydrogenated under 45 psi hydrogen. After 16 h, the reaction was filtrated and evaporated of solvent. The residue was stirred with l-{[(9H-fluoren-9-ylmethoxy)carbonyl]oxy}pynOlidine- 2,5-dione (5.14 g, 15.2 mmol) and sodium bicarbonate (5.33 g, 63.5 mmol) in 180 ml of 10 : 1 dioxane / water solvent. After 16 h, the reaction was diluted with 400 ml of dichloromethane. The organic was washed by 300 ml of saturated sodium bicarbonate solution twice and brine (300 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0% ethyl acetate/ hexane→ 60% ethyl acetate/ hexane) to give the title compound (4.8 g). MS 418.4 (M+Na).
9H-fluoren-9-ylmethyl 4.4-bis((9Z.12ZVoctadeca-9, 12-dien- 1 -ylox vjpiperidine- 1 -carboxylate (Compound 17)
Figure imgf000024_0001
To 120 ml toluene solution of 9H-fluoren-9-ylmethyl 4,4-diethoxypiperidine-1- carboxylate (4.8 g, 12.1 mmol) and cis, cis - 9, 12-octadecadien-1-ol (9.70 g, 36.4 mmol) was added pyridinium p-toluenesulfonate (0.305 g. 1.21 mmol) and heated to 100 °C. After 48 h, the reaction was diluted in 300 ml of dichloromethane. The organic was washed by 100 ml of saturated sodium bicarbonate solution twice and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0% ethyl acetate/ hexane→ 20% ethyl acetate/ hexane) to give the title compound (7.5 g). 1H NMR δ (ppm)(CHCl » -d): 7.76 (2 H, d, J = 7.60 Hz), 7.58 (2 H, d, J = 7.53 Hz), 7.40 (2 H, t, J = 7.49 Hz), 7.32 (2 H, d, J = 7.56 Hz), 5.42-5.31 (8 H, m), 4.41 (2 H, d, J = 6.92 Hz), 4.25 (1 H, t, J = 6.93 Hz), 3.48 (4 H, s, b), 3.39 (4 H, t, J = 6.78 Hz), 2.78 (4 H, t, J = 6.86 Hz), 2.05 (8 H, d, J =» 8.02 Hz), 1.72 (4 H, s, b), 1.60-1.50 (4 H, m), 1.42-1.20 (32 H, m), 0.89 (6 H, t, J = 6.61 Hz).
4.4-bisf(9Z,12Z)-octadeca-9.12-dien-1-yloxy1piperidine ( Compound 18)
Figure imgf000025_0001
To 150 ml dichloromethane solution of 9H-fluoren-9-ylmethyl 4,4- bis[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]piperidine-1-carboxylate (Compound 17) (7.3 g, 8.73 mmol) was added piperidine (7.43 g, 87 mmol). After 16 h at ambient temperature, the reaction was filtrated and the solution was diluted with 300 ml of dichloromethane. The organic was washed by 150 ml of saturated sodium bicarbonate solution twice and brine (150 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (0 % MeOH/ dichloromethane→ 12% MeOH/ dichloromethane) to give the title compound (3.9 g). MS 614.7 (M+l). HRMS 614.5863 (M+l). 1H NMR δ (ppm)(CHCl j-d): 5.40-5.31 (8 H, m), 3.38 (4 H, t, J = 6.76 Hz), 2.84 (4 H, t, J - 5.27 Hz), 2.77 (4 H, t, J = 6.81 Hz), 2.05 (8 H, q, J = 7.03 Hz), 1.71 (4 H, s, b), 1.56-1.49 (4 H, m), 1.42-1.2 (32 H, m), 0.89 (6 H, t, J = 6.74 Hz).
3.3-bis|Y9Z.12Z)-octadeca-9, 12-dien- 1 -yloxy-|pyiTolidine (Compound 19)
Figure imgf000025_0002
Compound 19 can be prepared according to General Scheme 3 above. MS
600.8 (M+l). HRMS 600.5711 (M+l). 1H NMR 6 (ppm)(CHC -d): 5.41-5.31 (8 H, m), 3.41 (4 H, t, J = 6.80 Hz), 3.10 (2 H, t, J = 7.30 Hz), 2.98 (2 H, s), 2.77 (4 H, t, J = 6.82 Hz), 2.05 (8 H, q, J = 7.04 Hz), 1.95 (2 H, t, J = 7.30 Hz), 1.55 (4 H, t, J = 7.01 Hz), 1.51-1.20 (32 H, m), 0.89 (6 H, t, J = 6.76 Hz).
N V-dimethyl-3,3-bis[( 9Z.12Z)-octadeca-9, 12-dien- 1 -yloxy^propan- 1 -amine (Compound 20)
Figure imgf000025_0003
Compound 20 was prepared according to General Scheme 6 above and with chemistry as described for Compound 2. 1H NMR 6 (ppm) (CHC -d): 5.41-5.30 (8 H, m), 4.54 (1 H, t, J = 5.7 Hz), 3.60-3.55 (2 H, m), 3.43-3.39 (2 H, m), 2.79-2.76 (2 H,m), 2.34-2.31 (2 H, m), 2.22 (6 H, s), 2.07-2.02 (8 H, m), 1.80-1.76 (2 H, m), 1.58-1.53 (4H, m), 1.39-1.25 (32 H, m), 0.91-0.87 (6 H, m).
1 -(3.3-bisf( 9Z.12Z)-octadeca-9.12-dien- 1 -yloxy]propyl ) yrrolidine (Compound 21 )
Figure imgf000026_0001
Compound 21 was prepared according to General Scheme 6 above and with chemistry as described for Compound 2. HRMS (M+l) calc'd: 642.61 1 1, found: 642.6169.
3.3-bis[(9Z, 12Z)-octadeca-9.12-dien- 1 -yloxy)propan- 1 -amine (Compound 22)
Figure imgf000026_0002
Compound 22 was prepared according to General Scheme 7 above and with chemistry as described for compounds 2 and 6. HRMS (M+l) calc'd: 588.5641 , found:
588.5712.
N,N-dimethyl-2,2-bisf(9Z)-octadec-9-en-l -yloxy]ethanamine (Compound 23)
Figure imgf000026_0003
Compound 23 was prepared according to General Scheme 1 with chemistry as described for compound 2. HRMS (M+l) calc'd: 606.611 1 , found: 606.6184. rt-butyl l-(1.3-dioxo-1.3-dihvdiO-2H-isoindol-2-yl)cvclopropanecarboxylate (Compound 24)
Figure imgf000026_0004
To 45 mL DMF solution of /erf-butyl 1 -aminocyclopropanecarboxylate (1.8 g, 1 1.5 mmol) was added phthalic anhydride (2.04 g, 13.7 mmol) and diisopropylethylamine (10.0 mL, 57.2 mmol). After heating at 160 °C for 16 h, the reaction was diluted by 150 ml of dichloromethane. The organic was washed by IX 100 mL of saturated NaHCOs solution, water and brine, respectively. The organic was dried over a2S04, filtrated and concentrated. The residue was purified by silica gel chromatography (0% EtOAC / hexane→ 50% EtOAc / hexane) give title compound (2.80 g). MS 310.2 (M+Na). l-(13-dioxo-13-dihvdiO-2H-isoindol-2-ylVN-methoxy-N-methylcvclopropanecarboxamide
(Compound 25)
Figure imgf000027_0001
To a 45 mL dichloromethane solution of ter/-butyl l -(1,3-dioxo-l ,3-dihydro-
2H-isoindol-2-yl)cyclopropanecarboxylate (2.8 g, 9.75 mmol) was added 5 mL of TFA. After 16 h at ambient temperature, the reaction was concentrated. The residue was retaken by 150 ml of dichloromethane and IX 100 mL of IN HCl solution, water and brine. Evaporation of solvent gave l-(l ,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)cyclopropanecarboxylic acid (2.2 g). MS 254.1 (M+Na). l-(1,3-dioxo-1,3-dihydro-2//-isoindol-2-yl)cyclopropanecarboxylic acid (2.2 g, 9.52 mmol) was dissolved in 50 mL of dichloromethane/DMF 4:1 solvent. To this, N,0-dimethylhydroxylamine hydrochloride (1.39 g, 14.3 mmol) was added in followed by EDC (4.01 g, 20.9 mmol), HOBt (1.60 g, 10.5 mmol) and diisopropylethylamine (6.65 mL, 38.1 mmol). After 16 h at ambient temperature, the reaction was concentrated and the residue was retaken by 150 mL of dichloromethane, washed by IX 100 mL of saturated NaHCC solution, water and brine. The organic was dried over Na2S04, filtrated and concentrated. The residue was purified by silica gel chromatography (0% EtOAC / hexane→ 65% EtOAc / hexane) give title compound (2.45 g). MS 275.2 (M+l). l-n.3-dioxo-1,3-dihvdro-2H-isoindol-2-vncvclopropanecarbaldehvde (Compound 26)
Figure imgf000027_0002
To a 100 mL THF solution of l-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N- methoxy-N-methylcyclopropanecarboxamide (2.45 g, 8.93 mmol) at -78 °C was added 1 M DiBAL solution in Tolune (19.65 mL). After 3 h at -78 °C, the reaction was quenched with 30 mL of saturated NH4C1 solution. The solution was concentrated and the residue was retaken by 200 mL of EtOAc. The organic was washed by IX 100 mL of 0.5 N Rochelle's salt, saturated NaHCC>3 solution, water and brine. The organic was dried over NajSO.*, filtrated and concentrated. The residue was purified by silica gel chromatography (5% EtOAC / hexane— > 75% EtOAc / hexane) to give title compound (1.32 g). MS 216.1 (M+l).
2-(\-t bis[(9Z.12ZVoctadeca-9.12-dien- 1 -yloxylmethyl ) cvclopropylV 1 H-isoindole- 13(2H)- dione (Compound 27)
Figure imgf000028_0001
To a 10 mL Toluene solution of l-(1,3-dioxo-1,3-dihydro-2H-isoindol-2- yl)cyclopropanecarbaldehyde (1.3 g, 6.04 mmol) and (9Z,12Z)-octadeca-9,l 2-dien- l-ol (5.68 mL, 18.1 mmol) was added camphorsulfonic acid (0.14 g, 0.604 mmol). The reaction was heated at 1 1 °C for 36 hours. The reaction was purified directly by silica gel chromatography (0% EtOAC / hexane→ 16% EtOAc / hexane) to give title compound (2.5 g). MS 730.9 (M+l).
1 - (bis[(9Z.12ZVoctadeca 9.12 dien 1 -yloxy]methyl} cvclopropanamine (Compound 28)
Figure imgf000028_0002
(27)
To 100 mL solution of 2-(l-{bis[(9Z,12Z)-octadeca-9,12-dien-l - yloxy]methyl}cyclopropyl)-lH-isoindole-l ,3(2H)-dione (2.4 g, 3.29 mmol) was added methylhydrazine (1.73 mL, 32.9 mmol). The reaction was refluxed for 10 hours. The solvent was evaporated and the residue was retaken by 200 mL of hexane and filtrated. The solution was washed by 2 x 100 mL of NaHC03 solution, water and brine, respectively. The organic was dried over Na2S04, filtrated and concentrated. The residue was purified by reverse-phase HPLC (70%- 100% CH3CN/water) to give the title compound (1.45 g). MS 600.9 (M+l). HRMS 600.5719 (M+l). 1H NMR 6 (ppm) (CHCh-d): 5.41-5.30 (8 H, m), 4.07 (1 H, s), 3.64- 3.60 (2 H, m), 3.48-3.43 (2 H, m), 2.79-2.76 (4 H,m), 2.07-2.02 (8 H, m), 1.61-1.55 (4 H, m), 1.39-1.26 (32 H, m), 0.91-0.87 (6 H, m), 0.62-0.55 (4H, m).
(6Z9Z 18-(2-bromo-l -methoxyethoxy octadeca-6.9-diene (Compound 29)
Figure imgf000029_0001
To 200 ml dichloroethane solution of 2-bromo-l,l-dimethoxyethane (5.0 g, 29.0 mmol) under an ice bath was added 2,6-Iutidine (11.33 mL, 98 mmol) and TMSOTf (11.8 mL, 65.1 mmol). The reaction stirred under the ice bath for one hour. After which, (9Z,12Z)- octadeca-9,12-dien-1-ol (27.8 mL. 89 mmol) was added in and the reaction was stirred from 0 °C to 20 °C for 1 hours. The reaction was diluted with 300 mL dichloromethane and washed by 200 mL of NaHC03 solution, water, brine, respectively. The organic was dried over Na2SC>4, filtrated and purified by silica gel chromatography (0 % ethyl acetate/ hexane→ 10% ethyl acetate / hexane) to give (6Z,9Z)-18-(2-bromo-1-methoxyethoxy)octadeca-6,9-diene (Compound 29) (1 1.3 g).
( 6Z.9ZV 18-f 2-bromo- 1 -(octyloxy)ethoxy]octadeca-6<9-diene (Compound 30)
Figure imgf000029_0002
To 50 ml dichloromethane solution of (6Z,9Z)-18-(2-bromo-1- methoxyethoxy)octadeca-6,9-diene (Compound 29) (4.0 g, 9.91 mmol) under an ice bath was added collidine (4.32 mL, 32.7 mmol) and TMSOTf (4.12 mL, 22.8 mmol). The reaction stirred under the ice bath for 3 hours. After which, 1-octanol (7.81 mL, 49.6 mmol) was added in and the reaction was stirred from 0 °C to 20 °C for 16 hours. The reaction was diluted with 200 mL dichloromethane and washed by 100 mL of NaHCOj solution, water, brine. The organic was dried over Na2S04, filtrated and purified by silica gel chromatography (0% dichloromethane/ hexane→ 50% dichloromethane/ hexane) to give title compound (3.6 g). MS 523.6 (M+Na).
N.N-dimethyl-2-[(9Z.12Z)-octadeca-9, 12-dien-l -yloxy -2-(octyloxy)ethanamine (Compound m
Figure imgf000029_0003
To (6Z,9Z)-18-[2-bromo-1-(octyloxy)ethoxy]octadeca-6,9-diene (Compound 30) (1.76 g, 3.51 mmol) in a microwave tube was added 40% dimethylamine (4.44 mL) aqueous solution. The reaction was heated to 160 °C for 30 minutes under microwave. After which, the reaction was diluted with 150 mL of Hexane, and washed by 2 x 100 mL of NaHCOj solution, water and brine, respectively. The organic was dried over Na2S04 and filtrated. Evaporation of solvent to dryness gave the title compound (1.60 g). MS 466.4 (M+l). HRMS 466.4627 (M+l). 1H NMR δ (ppm)(CHC -d): 5.39-5.31 (4 H, m), 4.58 (1 H, t, J = 5.3 Hz), 3.61-3.56 (2 H, m), 3.48-3.43 (2 H, m), 2.77 (2 H, J - 6.7 Hz), 2.46 (2 H, d, J = 5.3 Hz), 2.29 (6 H, s), 2.07-2.02 (4 H, m), 1.60-1.55 (4 H, m), 1.37-1.27 (26 H, m), 0.90-0.86 (6 H, m).
2-( decyloxyVN.N-dimethyl-2-f (9Z, 12Z)-octadeca-9.12-dien- 1 -yloxy]ethanamine (Compound 32}
Figure imgf000030_0001
Compound 32 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 494.4859; found: 494.4922.
2-[(8Z)-dodec-8-en- 1 -yloxyl-N.N-dimethyl-2-r(9Z.12Z)-octadeca-9.12-dien- 1 - yloxy]ethanamine (Compound 33)
Figure imgf000030_0002
Compound 33 was prepared according to General Scheme 9 as described for Compound 31. 1H NMR 6 (ppm) (CHCh-d): 5.39-5.32 (6 H, m), 4.58 (1 H, t, J = 5.3 Hz), 3.60-3.56 (2 H, m), 3.48-3.43 (2 H, m), 2.77 (2 H, t, J = 6.6 Hz), 2.46 (2 H, d, J = 5.4 Hz), 2.29 (6 H, s), 2.06-1.99 (8 H, m), 1.60-1.55 (6 H, m), 1.39-1.29 (24 H, m), 0.92-0.87 (6 H, m). HRMS (M+l) calc'd: 520.5015; found: 520.5073.
1 - ( 2-f (8Z)-dodec-8-en- 1 -yloxy]-2-f (9Z.12Z)-octadeca-9.12-dien- 1 -yloxylethyl pyrrolidine (Compound 34)
Figure imgf000030_0003
Compound 34 was prepared according to General Scheme 9 as described for
Compound 31. HRMS (M+l) calc'd: 546.5172; found: 546.5235.
1 - ( 2-f (9Z.12Z)-octadeca-9.12-dien- 1 -yloxy1-2-(octyloxy)ethyl ) pyrrolidine (Compound 35)
Figure imgf000030_0004
Compound 35 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 492.4702; found: 492.4788. rf2^-1-(2-(decyloxyV2-f(9Z, 12Z>-octadeca-9.12-dien-1-yloxy)ethyl}pyrrolidin-2-yl)methanol (Compound 36)
Figure imgf000031_0001
Compound 36 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 550.5121 ; found: 550.521 1.
Figure imgf000031_0002
(
Compound 37 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 550.5121 ; found: 550.5208.
N.N-dimethyl-3- IY92.12Z)-octadeca-9, 12-dien- 1 -yloxy-)-3-(octyloxy)pi pan- 1 -amine
(Compound 38)
Figure imgf000031_0003
Compound 38 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 480.4702; found: 480.4763.
2-(heptyloxyVN.N-dimethyl-2-f (92.12Z)-octadeca-9.12-dien- 1 -yloxylethanamine (Compound 39)
Figure imgf000031_0004
Compound 39 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l ) calc'd: 452.4389; found: 452.446.
N.N-dimethyl-2-(nonyloxy)-2-f (9Z.12Z)-octadeca-9.12-dien- 1 -yloxylethanamine (Compound 40)
Figure imgf000032_0004
Compound 40 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l ) calc'd: 480.4702; found: 480.4772.
1 - ( 3-[(9ZJ 2ZVoctadeca-9, 12-dien- 1 -yloxy]-3-( octyloxy'lpropyl ) pyrrolidine (Compound 41 )
Figure imgf000032_0001
Compound 41 was prepared according to General Scheme 9 as described for Compound 31. HRMS (M+l) calc'd: 506.4859; found: 506.4929.
2- 2-ethoxy-2-f(9Z.12ZVoctadeca-9.12-dien- 1 -yloxylethyl ) - /H-isoindole- 1.3(2HVdione (Compound 42)
Figure imgf000032_0002
To 50 ml dichloromethane solution of 2-(2,2-diethoxyethyl)-/H-isoindole- 1,3(2H)-dione (6.8 g, 25.8 mmol) under an ice bath was added 2,6-lutidine (9.89 mL, 85 mmol) and TMSOTf (10.3 mL, 56.8 mmol). The reaction stirred under the ice bath for one hour. After which, (9Z,12Z)-octadeca-9,l 2-dien- l-ol (24.3 mL, 77 mmol) was added in and the reaction was stirred from 0 °C to 20 °C for 16 hours. The reaction was diluted with 200 mL dichloromethane and washed by 100 mL of NaHC03 solution, water, brine. The organic was dried over Na2S04, filtrated and purified by silica gel chromatography (0 % ethyl acetate/ hexane→ 18% ethyl acetate / hexane) to give title compound (12.1 g). MS 506.5 (M+Na).
2-(2-(hexyloxyV2-f(9Z.12ZVoctadeca-9.12-dien-1-yloxy1ethyll-lH-isoindole-1.3(2H)-dione (Compound 43)
Figure imgf000032_0003
( )
To 40 ml dichloroethane solution of 2-{2-ethoxy-2-[(9Z,12Z)-octadeca-9,12- dien-1-yloxy]ethyl}-7H-isoindole-1,3(2H)-dione (compound 42) (4.10 g, 8.48 mmol) under ice bath was added 2,6-lutidine (3.25 mL, 28.0 mmol) and TMSOTf (3.37 mL, 18.7 minol). The reaction stirred under the ice bath for one hour. After which, 1-hexanol (5.04 mL, 42.4 mmol) was added in and the reaction was stirred from 0 °C to 20 °C for 16 hours. The reaction was diluted with 200 mL dichloromethane and washed by 100 mL of NaHC03 solution, water, brine, respectively. The organic was dried over Na2SC>4, filtrated and purified by silica gel chromatography (0 % ethyl acetate/ hexane→ 20% ethyl acetate / hexane) to give title compound (Compound 43) (2.8 g). MS 562.7 (M+Na).
2-(hexyloxy)-2-|Y9Z, 12Z)-octadeca-9.12-dien- 1 -yloxy]ethanamme (Compound 44)
Figure imgf000033_0001
To 30 ml ethanol solution of 2-{2-(hexyloxy)-2-[(9Z,12Z)-octadeca-9,12-dien- l-yloxy]ethyl}-lH-isoindole-1,3(2H)-dione (compound 43) (2.75 g, 5.09 mmol was added 1.61 mL of methylhydrazine. The reaction was refluxed for 5 hours. The solvent was evaporated and the residue was retaken by 200 mL of hexane and filtrated. The solution was washed by 2 x 100 mL of NaHC03 solution, water and brine, respectively. The organic was dried over Na2S04 and filtrated. Evaporation of solvent to dryness gave the title compound (2.01 g). MS 410.6 (M+l). HRMS 410.3980 (M+l). 'H NMR 6 (ppm)(CHCl3-d): 5.41-5.30 (4 H, m), 4.39 (1 H, t, J = 5.2 Hz), 3.65-3.60 (2 H, m), 3.48-3.43 (2 H, m), 2.78-2.76 (4 H, m), 2.07-2.00 (4 H, m), 1.61-1.55 (4 H, m), 1.35-1.29 (22 H, m), 0.90-0.87 (6 H, m).
2-f (9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(octyloxy)ethanamine (Compound 45)
H
Figure imgf000033_0002
Compound 45 was prepared according to General Scheme 10 as described for Compound 44. HRMS (M+l) calc'd: 438.4233; found: 438.4309.
2-(decyloxy)-2-f (9Z.12Z)-octadeca-9.12-dien- 1 -yloxylethanamine (Compouind 46)
H
Figure imgf000033_0003
Compound 46 was prepared according to General Scheme 10 as described for Compound 44. HRMS (M+l) calc'd: 466.4546; found: 466.4609. 2-(decyloxy)-2-|Y9Z)-octadec-9-en-l -yloxyjethanamine (Compound 47)
Figure imgf000034_0001
Compound 47 was prepared according to General Scheme 10 as described for Compound 44. HRMS (M+l) calc'd: 468.4702; found: 468.4763.
2-|Y9Z, 12Z)-octadeca-9¾ 12-dien- 1 -vIoxy-|-2-(tetradecyloxy)ethanamine (Compound 48)
Figure imgf000034_0002
( )
Compound 48 was prepared according to General Scheme 10 as described for Compound 44. HRMS (M+l) calc'd: 522.5172; found: 522.5259.
\(2R)- 1 - (2.2-bisf (9Z, 12Z)-octadeca-9.12-dien- 1 -yloxy1ethvU yiTolidin-2-yl1methanol (Compound 49)
Figure imgf000034_0003
Compound 49 was prepared according to General Scheme 1 as described for Compound 2. HRMS (M+l) calc'd: 658.6060; found: 658.6128. f(2S)-l -i2.2-bis ('9Z, 12Z)-octadeca-9, 12-dien-l -yloxylethvnpyffolidin-2-yllmethanol (Compound 50)
Figure imgf000034_0004
Compound 50 was prepared according to General Scheme 1 as described for
Compound 2. HRMS (M+l) calc'd: 658.6060; found: 658.6144.
/err-butyl (2SARY2- ( bisf (9Z.12Z)-octadeca-9.12-dien- 1 -yloxylmethyl ) -4-hvdroxypyrrolidine- 1-carboxylate (Compound 52)
Figure imgf000035_0002
To 100 mL dichloromethane solution of ter/-butyl (2S',4/?)-4-hydroxy-2- (hydroxymethyl)pyrrolidine-1-carboxylate (5.86 g, 27 mmol) was added Dess-Martin periodinane (10.9 g, 25.6 mmol). After 5 h at ambient temperature, the reaction was diluted with another 200 mL dichloromethane. The dichloromethane solution was washed by IX 100 mL of 0.5 N Na2S203 solution, saturated NaHC03 solution, water and brine. The organic was concentrated and the residue was purified by silica gel chromatography (0% EtOAC / hexane -→ 70% EtOAc / hexane) to give tert-butyl (2S',4R)-2-formyl-4-hydroxypyrrolidine-1- carboxylate (3.5 g).
To tert-butyl (2S,4.?)-2-foi-myl-4-hydroxypyiTolidine-1-carboxylate (3.4 g, 15.8 mmol) in (9Z,12Z)-octadeca-9,12-dien-1-ol (16.8 g, 63.2 mmol) was added pyridinium -toluene- sulfonate (0.40 g, 1.58 mmol). After 16 h at 100 °C, the reaction was purified directly by silica gel chromatography (0% EtOAC / hexane→ 35% EtOAc / hexane) to give title compound (1.9 g). ). MS 752.8 (M+Na). f 3 R.5S)-5-i bisf(9Z.12ZVoctadeca-9.12-dien- 1 -yloxy)methyl ) pyrrolidin-3-ol (Compound 53)
Figure imgf000035_0001
To 150 mL THF solution of te/7-butyl (2S,4/?)-2-{bis[(9Z,12Z)-octadeca-9,12- dien-1-yIoxy]methyl}-4-hydroxypynolidine-1-carboxylate (1.78 g, 2.44 mmol) was bubbled gas HCl for 3 minutes at 0 °C. After that, the reaction was stirred from 0 °C to ambient temperature for 2 hours. The solvent was evaporated and the residue was retaken by 200 mL of dichloromethane and washed by 2 x 100 mL of NaHC0 solution, water and brine, respectively. The organic layer was concentrated and purified by reverse-phase HPLC (80%- 100% CH3CN/water) to give the title compound (0.32 g). MS 630.9 (M+l). HRMS 630.5817 (M+l). 1H NMR δ (ppm) (CHC -d): 5.42-5.30 (8 H, m), 4.42-4.40 (1 H, m), 4.26-4.24 (1 H, m), 3.66-3.59 (2 H, m), 3.55-3.43 (3 H, m), 3.13-3.09 (1 H, m), 2.88-2.85 (1 H, m), 2.79-2.75 (4 H, m), 2.08-2.02 (8 H, m), 1.85-1.78 (4 H, m), 1.62-1.52 (4 H, m), 1.39-1.26 (32 H, m), 0.91-0.87 (6 H, m).
(2.SV2-amino-3.3-bisf ( 9Z.12Z)-octadeca-9.12-dien- 1 -yloxy]propan- 1 -ol ( Compound 54)
Figure imgf000036_0001
Compound 54 was prepared according to General Scheme 1 1 as described for Compound 53. HRMS (M+l) calc'd: 604.5590; found: 604.5675.
4- ( bis[(9Z.12Z)-octadeca-9.12-dien- 1 -yloxyjmethyl ) piperidine (Compound 55)
Figure imgf000036_0002
Compound 55 was prepared according to General Scheme 1 1 as described for Compound 53. HRMS (M+l) calc'd: 628.5954; found: 628.6042.
3- 1 bis[(9Z.12Z)-octadeca-9.12-dien- 1 -yloxy]methyl } piperidine (Compound 56)
Figure imgf000036_0003
Compound 56 was prepared according to General Scheme 11 as described for Compound 53. HRMS (M+l) calc'd: 628.5954; found: 628.6017.
3-azido- 1.1 -bisf (9Z, 12Z)-octadeca-9.12-dien- 1 -yloxy]propan-2-ol (Compound 60)
Figure imgf000036_0004
To the solution of 2-(diethoxymethyl)oxirane (4.79 mL, 30.8 mmol) in 80 mL of EtOH/pH 7.0 phosphate buffer 1 : 1 was added sodium azide (4.0 g, 61.6 mmol). The reaction was heated to 55 °C for 16 hours. The reaction was diluted with 300 mL of dichlormethane and washed by 2 x 100 mL of NaHCOj solution, water and brine, respectively. The organic was concentrated and purified by silica gel chromatography (0 % MeOH / CH2C12 → 5 % MeOH/' CH2C12) to give 3-azido-l,l-diethoxypropan-2-ol (compound 59) (5.5 g).
To 3-azido-l,l-diethoxypropan-2-ol (compound 59) (2.80 g, 14.8 mmol) was added (9Z,12Z)-octadeca-9,12-dien-1-ol (15.1 mL, 48.8 mmol), followed by pPTS ( 0.37 g, 1.48 mmol). The reaction was heated to 80 °C for 1 hours. After which, the reaction was purified by reverse phase HPLC (70 % 0.1 % TFA-water/ CHjCN→ 100% CHjCN) to give title compound (2.53 g). MS 652.8 (M+Na).
3-amino- 1.1 -bis[(9Z.12Z)-octadeca-9> 12-dien- 1 -yloxy)propan-2-ol (Compound 61 )
Figure imgf000037_0001
(61)
To 50 ml THF solution of 3-azido- 1,1 -bis[(9Z,12Z)-octadeca-9,l 2-dien- 1- yloxy]propan-2-ol (compound 60) (2.56 g, 4.06 mmol) at 0 °C was added triphenylphosphine (3.52 g, 13.41 mmol). The reaction was stirred from 0 °C to 20 °C for 5 hours. After which, water (3 mL) was added into the reaction. The reaction was stirred at 55 °C for 16 h. The solvent was evaporated and the residue was retaken by 200 mL of hexane and filtrated. The hexane solution was washed by 2 x 100 mL of NaHC03 solution, water and brine,
respectively. The organic was dried over Na2S04 and filtrated. Evaporation of solvent to dryness gave the title compound (2.1 g). MS 604.8 (M+l). HRMS 604.5669 (M+l). 1H NMR δ (ppm)(CHC -d): 5.40-5.30 (8 H, m), 4.37 (1 H, d, J = 5.9 Hz), 3.71-3.66 (2 H, m), 3.57-3.47 (3 H, m), 2.93-2.89 (1 H, m), 2.81-2.75 (4 H, m), 2.08-2.02 (8 H, m), 1.62-1.56 (4 H, m), 1.39- 1.27 (34 H, m), 0.91-0.87 (6 H, m).
1 -methyl-4.4-bis[( 9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]piperidine (Compound 62)
Figure imgf000037_0002
Combined ketone (l .Og. 8.84mmol), linoleyl alcohol (5.18g, 19.44mmol) and tosic acid (1.85g, 9.72mmol) and refluxed with a Dean-Stark trap for 3 hours. LCMS shows desired product but substantial unreacted alcohol. Appoximately 500mg of ketone added and additional ~500mg of TsOH added and the reaction was refluxed for ~ 3 hours. Toluene removed in vacuo and loaded directly onto Torrent silica column. 100 DCM --> 85 DCM / 15 MeOH. Isolated fractions stripped down and then washed with aqueous NaOH and extracted into hexanes. Dried with Na2S04 and solvent removed to afford light yellow oil. MS 628.8 (M+l). 1H NMR δ (ppmXCHCl j-d): 5.37 (8H, m), 3.38 (4H, m), 2.78 (4H, m), 2.39 (4H, bs), 2.22 (3H, s), 2.02 (4H, m), 1.79 (4H, m), 1.53 (4H, m), 1.30 (36H, m), 0.86 (6H, m).
Compound 63 is DLinDMA as described in J. Controlled Release, 2005, 107, 276-287, US 2006/0083780 Al, and US 2006/0008910 Al .
Figure imgf000038_0001
Compound 64 is DLin C2DMA as described in Nature Biotechnology, 2010, 28, 172-176, WO 2010/042877 Al , WO 2010/048536 A2, WO 2010/088537 A2, and WO 2009/127060 Al .
Figure imgf000038_0002
UTILITY
LNP COMPOSITIONS
The following lipid nanoparticle compositions (LNPs) of the instant invention are useful for the delivery of oligonucleotides, specifically siRNA and miRNA:
Cationic Lipid / Cholesterol / PEG-DMG 56.6/38/5.4;
Cationic Lipid / Cholesterol / PEG-DMG 60/38/2;
Cationic Lipid/ Cholesterol / PEG-DMG 67.3/29/3.7;
Cationic Lipid / Cholesterol / PEG-DMG 49.3/47/3.7;
Cationic Lipid / Cholesterol / PEG-DMG 50.3/44.3/5.4;
Cationic Lipid / Cholesterol / PEG-C-DMA / DSPC 40/48/2/10; and
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 40/48/2/10.
LNP process description:
The Lipid Nano-Particles (LNP) are prepared by an impinging jet process. The particles are formed by mixing lipids dissolved in alcohol with siRNA dissolved in a citrate buffer. The mixing ratio of lipids to siRNA are targeted at 45-55% lipid and 65-45% siRNA.
The lipid solution contains a novel cationic lipid of the instant invention, a helper lipid
(cholesterol) , PEG (e.g. PEG-C-DMA, PEG-DMG) lipid, and DSPC at a concentration of 5- 15 mg mL with a target of 9-12 mg/mL in an alcohol (for example ethanol). The ratio of the lipids has a mole percent range of 25-98 for the cationic lipid with a target of 35-65, the helper lipid has a mole percent range from 0-75 with a target of 30-50, the PEG lipid has a mole percent range from 1-15 with a target of 1-6, and the DSPC has a mole precent range of 0-15 with a target of 0-12. The siRNA solution contains one or more siRNA sequences at a concentration range from 0.3 to 1 .0 mg mL with a target of 0.3 -0.9 mg/mL in a sodium citrate buffered salt solution with pH in the range of 3.5-5. The two liquids are heated to a temperature in the range of 15-40°C, targeting 30-40°C, and then mixed in an impinging jet mixer instantly forming the LNP. The teelD has a range from 0.25 to 1.0 mm and a total flow rate from 10 -600 mL/min. The combination of flow rate and tubing ID has effect of controlling the particle size of the LNPs between 30 and 200 nm. The solution is then mixed with a buffered solution at a higher pH with a mixing ratio in the range of 1 : 1 to 1 :3 vol: vol but targeting 1 :2 vol: vol. This buffered solution is at a temperature in the range of 15-40°C, targeting 30-40°C. The mixed LNPs are held from 30 minutes to 2 hrs prior to an anion exchange filtration step. The temperature during incubating is in the range of 15-40°C, targeting 30-40°C. After incubating the solution is filtered through a 0.8 um filter containing an anion exchange separation step. This process uses tubing IDs ranging from 1 mm ID to 5 mm ID and a flow rate from 10 to 2000 mL/min. The LNPs are concentrated and diafiltered via an ultrafiltration process where the alcohol is removed and the citrate buffer is exchanged for the final buffer solution such as phosphate buffered saline. The ultrafiltration process uses a tangential flow filtration format (TFF). This process uses a membrane nominal molecular weight cutoff range from 30 -500 KD. The membrane format can be hollow fiber or flat sheet cassette. The TFF processes with the proper molecular weight cutoff retains the LNP in the retentate and the filtrate or permeate contains the alcohol; citrate buffer; final buffer wastes. The TFF process is a multiple step process with an initial concentration to a siRNA
concentration of 1 -3 mg/mL. Following concentration, the LNPs solution is diafiltered against the final buffer for 10 -20 volumes to remove the alcohol and perform buffer exchange. The material is then concentrated an additional 1-3 fold. The final steps of the LNP process are to sterile filter the concentrated LNP solution and vial the product.
Analytical Procedure:
1) siRNA concentration
The siRNA duplex concentrations are determined by Strong Anion-Exchange High-Performance Liquid Chromatography (SAX-HPLC) using Waters 2695 Alliance system (Water Corporation, Milford MA) with a 2996 PDA detector. The LNPs, otherwise referred to as RNAi Delivery Vehicles (RDVs), are treated with 0.5% Triton X-100 to free total siRNA and analyzed by SAX separation using a Dionex BioLC DNAPac PA 200 (4 χ 250 mm) column with UV detection at 254 nm. Mobile phase is composed of A: 25 mM NaCl04> 10 mM Tris, 20% EtOH, pH 7.0 and B: 250 mM NaCl04, 10 mM Tris, 20% EtOH, pH 7.0 with liner gradient from 0-15 min and flow rate of 1 ml/min. The si NA amount is determined by comparing to the siRNA standard curve.
2) Encapsulation rate
Fluorescence reagent SYBR Gold is employed for RNA quantitation to monitor the encapsulation rate of RDVs. RDVs with or without Triton X- 100 are used to determine the free siRNA and total siRNA amount. The assay is performed using a SpectraMax M5e microplate spectrophotometer from Molecular Devices (Sunnyvale, CA). Samples are excited at 485 run and fluorescence emission was measured at 530 nm. The siRNA amount is determined by comparing to the siRNA standard curve.
Encapsulation rate = ( 1 - free siRN A/total siRNA) * 100%
3) Particle size and polvdispersitv
RDVs containing 1 μg siRNA are diluted to a final volume of 3 ml with 1 x xBS. The particle size and polydispersity of the samples is measured by a dynamic light scattering method using ZetaPALS instrument (Brookhaven Instruments Corporation,
Holtsville, NY). The scattered intensity is measured with He-Ne laser at 25°C with a scattering angle of 90°.
4) Zeta Potential analysis
RDVs containing 1 μg siRNA are diluted to a final volume of 2 ml with 1 mM Tris buffer (pH 7.4). Electrophoretic mobility of samples is determined using ZetaPALS instrument (Brookhaven Instruments Corporation. Holtsville, NY) with electrode and He-Ne laser as a light source. The Smoluchowski limit is assumed in the calculation of zeta potentials.
5) Lipid analysis
Individual lipid concentrations are determined by Reverse Phase High- Performance Liquid Chromatography (RP-HPLC) using Waters 2695 Alliance system (Water Corporation. Milford MA) with a Corona charged aerosol detector (CAD) (ESA Biosciences, Inc, Chelmsford, MA). Individual lipids in RDVs are analyzed using an Agilent Zorbax SB- Cl 8 (50 x 4.6 mm, 1.8 μιη particle size) column with CAD at 60 °C. The mobile phase is composed of A: 0.1% TFA in H20 and B: 0.1% TFA in IPA. The gradient changes from 60% mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B at 1.00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min:
40% mobile phase A and 60% mobile phase B from 5.00 min to 25% mobile phase A and 75% mobile phase B at 10.00 min; 25% mobile phase A and 75% mobile phase B from 10.00 min to 5% mobile phase A and 95% mobile phase B at 15.00 min; and 5% mobile phase A and 95% mobile phase B from 15.00 to 60% mobile phase A and 40% mobile phase B at 20.00 min with flow rate of 1 ml/min. The individual lipid concentration is determined by comparing to the standard curve with all the lipid components in the RDVs with a quadratic curve fit. The molar percentage of each lipid is calculated based on its molecular weight. Utilizing the above described LNP process, specific LNPs with the following ratios were identified:
Nominal composition:
Cationic Lipid / Cholesterol / PEG-DMG 60/38/2
Cationic Lipid / Cholesterol / PEG-DMG 67.3/29/3.7.
Luc siRNA
5'-iB-At/AAGGCUAC/GAAGAGAi ATT-iB 3' (SEQ.ID.NO.:l)
3'-WUAUUCCGAUACUUCUCUAVS' (SEQ.ID.NO.:2)
AUGC - Ribose
iB - Inverted deoxy abasic
UC - 2' Fluoro
AGT - 2' Deoxy
AGU - 2' OCH3
Nominal composition
Cationic Lipid /Cholesterol/PEG-DMG 60/38/2
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 40/48/2/10
Cationic Lipid / Cholesterol / PEG-DMG / DSPC 58/30/2/10
ApoB siRNA
5'-iB-CUUUAACAAUUCCUGA 4UTsT-iB-3' (SEQ ID NO.:3) 3'-UsUG L4,4UC/GUUAAGGACUsUsUsA-5' (SEQ ID NO.:4)
AUGC - Ribose
iB - Inverted deoxy abasic
UC - 2' Fluoro
AGT - 2' Deoxy
AGU - 2' OCHj
UsA - phophorothioate linkage
Oligonucleotide synthesis is well known in the art. (See US patent applications: US 2006/0083780, US 2006/0240554, US 2008/0020058, US 2009/0263407 and US
2009/0285881 and PCT patent applications: WO 2009/086558, WO2009/ 127060,
WO2009/132131 , WO2010/042877, WO2010/054384, WO2010/054401 , WO2010/054405 and WO2010/054406). The Luc siRNA incorporated in the LNPs disclosed and utilized in the Examples were synthesized via standard solid phase procedures. EXAMPLE 1
In Vivo Evaluation of Efficacy and Toxicity
LNPs utilizing the compounds of the instant invention, in the nominal compositions described immediately above, were evaluated for in vivo efficacy and induction of inflammatory cytokines in a luciferase mouse model. The siR A targets the mRNA transcript for the firefly (Photinus pyralis) luciferase gene (Accession # Ml 5077). The primary sequence and chemical modification pattern of the luciferase siRNA is displayed above. The in vivo luciferase model employs a transgenic mouse in which the firefly luciferase coding sequence is present in all cells. ROSA26- LoxP-Stop-lx>xP-Luc (LSL-Luc) transgenic mice licensed from the Dana Farber Cancer Institute are induced to express the Luciferase gene by first removing the LSL sequence with a recombinant Ad-Cre virus (Vector Biolabs). Due to the organo-tropic nature of the virus, expression is limited to the liver when delivered via tail vein injection. Luciferase expression levels in liver are quantitated by measuring light output, using an IVIS imager (Xenogen) following administration of the luciferin substrate (Caliper Life Sciences). Pre-dose luminescence levels are measured prior to administration of the RDVs. Luciferin in PBS (15mg/mL) is intraperitoneally (IP) injected in a volume of 150 uL. After a four minute incubation period mice are anesthetized with isoflurane and placed in the IVIS imager. The RDVs (containing siRNA) in PBS vehicle were tail vein injected n a volume of 0.2 mL. Final dose levels ranged from 0.3 to 3 mg/kg siRNA. PBS vehicle alone was dosed as a control. Three hours post dose, mice were bled retro-orbitally to obtain plasma for cytokine analysis. Mice were imaged 48 hours post dose using the method described above. Changes in luciferin light output directly correlate with luciferase mRNA levels and represent an indirect measure of luciferase siRNA activity. In vivo efficacy results are expressed as % inhibition of luminescence relative to pre-dose luminescence levels. Plasma cytokine levels were determined using the SearchLight multiplexed cytokine chemoluminescent array
(Pierce/Thermo). Systemic administration of the luciferase siRNA RDVs decreased luciferase expression in a dose dependant manner. Greater efficacy was observed in mice dosed with Compound 2 containing RDVs than with the RDV containing the octyl-CLinDMA cationic lipid, Compound 4, (Table 1). Similar efficacy was observed in mice dosed with Compound 31 containing RDVs than with RDV containing DLinDMA cationic lipid, Compound 63
(Figure 3).
Table 1 : Mouse In Vivo efficacy data. Average % Inhibition of Bioluminescence by LNPs prepared from Compound 2 compared against Compound 4 at 0.3 mg Kg-1.
Figure imgf000043_0001
Rat In Vivo Evaluation of Efficacy and Toxicity
LNPs utilizing compounds in the nominal compositions described above, were evaluated for in vivo efficacy and increases in alanine amino transferase and aspartate amino transferase in Sprague-Dawley (Crl:CD(SD) female rats (Charles River Labs). The siRNA targets the mRNA transcript for the ApoB gene (Accession # NM 019287). The primary sequence and chemical modification pattern of the ApoB siRNA is displayed above. The RDVs (containing siRNA) in PBS vehicle were tail vein injected in a volume of 1 to 1.5 mL. Infusion rate is approximately 3 ml/min. Five rats were used in each dosing group. After LNP administration, rats are placed in cages with normal diet and water present. Six hours post dose, food is removed from the cages. Animal necropsy is performed 24 hours after LNP dosing. Rats are anesthetized under isoflurane for 5 minutes, then maintained under anesthesia by placing them in nose cones continuing the delivery of isoflurane until ex-sanguination is completed. Blood is collected from the vena cava using a 23 guage butterfly venipuncture set and aliquoted to serum separator vacutainers for serum chemistry analysis. Punches of the excised caudate liver lobe are taken and placed in RNALater (Ambion) for mRNA analysis. Preserved liver tissue was homogenized and total RNA isolated using a Qiagen bead mill and the Qiagen miRNA-Easy RNA isolation kit following the manufacturer's instructions. Liver ApoB mRNA levels were determined by quantitative RT-PCR. Message was amplified from purified RNA utilizing a rat ApoB commercial probe set (Applied Biosystems Cat #
RN01499054_ml). The PCR reaction was performed on an ABI 7500 instrument with a 96- well Fast Block. The ApoB mRNA level is normalized to the housekeeping PPIB (NM 01 1149) mRNA. PPIB mRNA levels were determined by RT-PCR using a commercial probe set (Applied Biosytems Cat. No. Mm00478295_ml). Results are expressed as a ratio of ApoB mRNA/ PPIB mRNA. All mRNA data is expressed relative to the PBS control dose. Serum ALT and AST analysis were performed on the Siemens Advia 1800 Clinical Chemistry Analyzer utilizing the Siemens alanine aminotransferase (Cat# 03039631) and aspartate aminotransferase (Cat# 03039631) reagents. Similar efficacy and better tolerability was observed in rats dosed with Compound 31 containing RDV than with the RDV containing the DLinKC2DMA cationic lipid 64 (Figure 4).
Determination of Cationic Lipid Levels in Rat Liver
Liver tissue was weighed into 20-ml vials and homogenized in 9 v/w of water using a GenoGrinder 2000 (OPS Diagnostics, 1600 strokes/min, 5min). A 50 ί aliquot of each tissue homogenate was mixed with 300 L of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 μL· of each supernatant was then transferred to separate wells of a 96- well plate and 10 μΐ samples were directly analyzed by LC S-MS.
Standards were prepared by spiking known amounts of a methanol stock solution of Compound 1 or OCD into untreated rat liver homogenate (9 vol water/weight liver). Aliquots (50 μί) each standard/liver homogenate was mixed with 300 μΐ, of extraction/protein precipitating solvent (50/50 acetonitrile/methanol containing 500 nM internal standard) and the plate was centrifuged to sediment precipitated protein. A volume of 200 μΐ, of each supernatant was transferred to separate wells of a 96-well plate and 10 μΐ of each standard was directly analyzed by LC/MS-MS.
Absolute quantification versus standards prepared and extracted from rat liver homogenate was performed using an Aria LX-2 HPLC system (Thermo Scientific) coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 μί sample was injected onto a BDS Hypersil C8 HPLC column (Thermo, 50 x 2mm, 3 μηι) at ambient temperature (Figure 5).
Mobile Phase A: 95% H20/5% methanol/ 10 mM ammonium
formate/0. l%formic acid Mobile Phase B: 40% methanol/60% n-propanol/10 mM ammonium formate/0. l%formic acid The flow rate was 0.5 mL/min and gradient elution profile was as follows: hold at 80% A for 0.25 min, linear ramp to 100% B over 1.6 min, hold at 100% B for 2.5 min, then return and hold at 80% A for 1.75 min. Total run time was 5.8 min. API 4000 source parameters were CAD: 4, CUR: 15, GS1 : 65, GS2: 35, IS: 4000, TEM: 550, CXP: 15, DP: 60, EP: 10.
Rhesus Monkey In Vivo Evaluation of Efficacy
LNPs utilizing compound 34 in the nominal compositions described above, were evaluated for in vivo efficacy in male or female Macaca mulatta (rhesus) monkeys. The siRNA targets the mRNA transcript for the ApoB gene (Accession # XM 001097404). The primary sequence and chemical modification pattern of the ApoB siRNA is displayed above. The RDVs (containing siRNA) in PBS vehicle were administered by intravenous injection in the saphenous vein at an injection rate of 20 mL/minute to a dose level of 1 mg/kilogram siRNA. The injection volumes were from 1.9 to 2.1 mL/kilogram and monkeys ranged in weight from 2.5 to 4.5 kilograms. The RDV or PBS control were administered to three monkeys. At multiple days post dose, 1 mL blood samples were drawn from the femoral artery for serum chemistry analysis. Monkeys were fasted overnight prior to blood draws. As a measure of efficacy, LDL-C was monitored as a downstream surrogate marker of ApoB mRNA reduction. At 2-12 days post systemic administration of RDVs containing compound 34, serum levels of LDL-C were reduced to less than 30% of pre-dose levels (figure 6).
In Vitro Evaluation of Hydrolytic Stability
Stability of acetal/ketal di-linoleyl cationic lipids were evaluated under three pH conditions (5.0, 6.0, and 7.5).
A: Incubation procedures:
Incubation mixtures (600 \xL total volume) containing 10 μΜ test compound, 50% (v/v) methanol, 50% (v/v) various pH buffers (pH 5.0, 20mM sodium acetate buffer; pH 6.0: 20mM sodium acetate buffer; and pH 7.5: 20 mM sodium hydrogen phosphate buffer). The reaction mixture was incubated at 37°C with gentle shaking. A 50 μί aliquot was removed at time: 0, 30min, lhr, 4hr and 24hr, and transferred to a collection tube containing 200 μί reaction stopping buffer (95% H20/5% methanol/1 OmM ammonium formate/0. l%formic acid with 500 nM internal standard). After a brief vortex and a 10 min centrifugation at lOOOg at 4'C, 100 μL· of the supernatants were transferred to a 96-well plate, followed a 10 fold dilution with reaction stopping buffer. ΙΟμί samples were directly injected and analyzed by LC/MS- MS.
B. LC-MS/MS analysis:
Absolute quantification was performed using a Perkin Elmer HPLC system coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems). For each run, a total of 10 μL of sample was injected onto a Phenomenex Luna C 18(2) HPLC column (5μη , 50 x 2mm) with the temperature set to 50°C (column heater. Restek, Model PTC050). A dual eluent system was used: 95% H20/5% methanol/ 1 OmM ammonium formate/0. l%formic acid (A) and 40% methanol/60% n-propanol/10mM ammonium formate/0. l%formic acid (B). The flow rate was 0.4 mL/min and the gradient was as follows: hold at 100% A for 1 min, ramp to 0% A at 2.5 min, hold at 0% A for 2 min, then hold at 100% A for 1.5 min. Total run time was 7.0 min.
The API 4000 instrument source parameters were CAD: 4, CUR: 15, GS1 : 65, GS2: 35, IS: 4000, TEM: 600, DP: 80, CE: 29, CXP: 10, EP: 10, and the ion-transition condition was 602.7/336.3.

Claims

WHAT IS CLAIMED IS:
1. A cationic lipid of Formula A:
Figure imgf000047_0001
wherein: n is 0, 1 or 2;
R1 and R2 are independently selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R1 and R2 can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R'; R3 is selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R', or R3 can be taken together with R4 to form cyclopropyl or cyclobutyl; R4 is selected from H and (C1-C4)alkyl, said alkyl is optionally substituted with one or more substituents selected from R'; R5 is selected from H and (C1-C4)alkyl. or R5 can be taken together with R1 to form a monocyclic heterocycle which is optionally substituted with one or more substituents selected from R';
R' is independently selected from halogen, R" and OR";
R" is selected from H and (C1-C4)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH;
L1 is a C4-C22 alkyl or a C4-C22 alkenyl; and
L2 is a C4-C22 alkenyl; or any pharmaceutically acceptable salt or stereoisomer thereof.
2. A cationic lipid of Formula A according to Claim 1 , wherein:
L1 and L-2 are
Figure imgf000048_0001
; and all other variables are as defined in Claim 1 ; or any pharmaceutically acceptable salt or stereoisomer thereof.
3. A cationic lipid according to Claim 1 which is selected from:
N,N-dimethyl-2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethanamine;
N^V-dimethyl-2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjpropan- 1 -amine;
2,2-bis[(9Z.12Z)-octadeca-9, 12-dien- 1 -yloxy]ethanamine;
(2R)-2-{bis[(9Z,l 2Z)-octadeca-9, 12-dien- 1 -yloxy]methyl}pyrrolidine;
(2R)-2- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } - 1 -methylpyrrolidine;
2-[(2R)-2- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl }pyrrolidin- 1 -yljethanol;
(2S)- 1 ,1 -bis[(9Z,l 2Z)-octadeca-9, 12-dien- 1 -yloxy]propan-2-amine;
2-methyl- 1 , 1 -bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan-2-amine;
4,4-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjpiperidine; and
3,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy pyrrolidine;
N,N-dimethyl-3 ,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjpropan- 1 -amine;
1 - { 3 ,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propyl } pyrrolidine;
3 ,3-bis [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjpropan- 1 -amine;
N,N-dimethyl-2,2-bis[(9Z)-octadec-9-en- 1 -yloxy]ethanamine;
1 - {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } cyclopropanamine;
N,N-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(octyloxy)ethanamine;
2-(decyloxy)-NiN-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethanamine;
2-[(8Z)-dodec-8-en- 1 -yloxy]-N,N-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 - yloxyjethanamine;
1 - {2-[(8Z)-dodec-8-en- 1 -yloxy]-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl } pyrrolidine; 1 - {2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(octyloxy)ethyl } pyrrolidine;
[(2S)- 1 - {2-(decyloxy)-2- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethyl } pyrrolidin-2- yljmethanol;
1 - {2-(decyloxy)-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl} piperidin-4-ol;
N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-3-(octyloxy)propan- 1 -amine;
2-(heptyloxy)-N^V-dimethyl-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethanamine; N,N-dimethyl-2-(nonyloxy)-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]ethanamine;
1- {3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propyl}pyrrolidine;
2- (hexyloxy)-2-[(9Z, 12Z)-octadeca-9.12-dien- 1 -yloxyjethanamine;
2-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]-2-(octyloxy)ethanamine;
2-(decyloxy)-2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxyjethanamine;
2-(decyloxy)-2-[(9Z)-octadec-9-en- 1 -yloxy]ethanamine;
2- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-(tetradecyloxy)ethanamine;
[(2R)-\ - {2,2-bis[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]ethyl}pyrrolidin-2-yl]methanol;
[(2S)- 1 - {2,2-bis[(9Z.12Z)-octadeca-9, 12-dien- 1 -yloxy]ethyl } pyrrolidin-2-yl]methanol ;
(3R,5S)-5- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } pyrrolidin-3-ol ;
(2S)-2-amino-3,3-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan- 1 -ol;
4- { bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } piperidine;
3- {bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]methyl } piperidine;
3-amino- 1 , 1 -bis[(9Z,l 2Z)-octadeca-9.12-dien-l -yloxy]propan-2-ol; and
1 -methyl-4,4-bis[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]piperidine or any pharmaceutically acceptable salt or stereoisomer thereof.
4. The use of a cationic lipid according to Claim 1 for the preparation of lipid nanoparticles.
5. The use of a cationic lipid according to Claim 1 as a component in a lipid nanoparticle for the delivery of oligonucleotides.
6. The use according to Claim 5 wherein the oligonucleotides are siRNA or miRNA.
7. The use according to Claim 5 wherein the oligonucleotides are siRNA.
PCT/US2011/021619 2010-01-22 2011-01-19 Novel cationic lipids for oligonucleotide delivery WO2011090965A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP11735061A EP2525781A1 (en) 2010-01-22 2011-01-19 Novel cationic lipids for oligonucleotide delivery
US13/574,315 US9670487B2 (en) 2010-01-22 2011-01-19 Cationic lipids for oligonucleotide delivery

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US29737710P 2010-01-22 2010-01-22
US61/297,377 2010-01-22
US32590810P 2010-04-20 2010-04-20
US61/325,908 2010-04-20

Publications (1)

Publication Number Publication Date
WO2011090965A1 true WO2011090965A1 (en) 2011-07-28

Family

ID=44307168

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/021619 WO2011090965A1 (en) 2010-01-22 2011-01-19 Novel cationic lipids for oligonucleotide delivery

Country Status (3)

Country Link
US (4) US20130116419A1 (en)
EP (2) EP2525781A1 (en)
WO (1) WO2011090965A1 (en)

Cited By (109)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130053572A1 (en) * 2010-01-22 2013-02-28 Steven L. Colletti Novel Cationic Lipids for Oligonucleotide Delivery
WO2013059496A1 (en) * 2011-10-18 2013-04-25 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
CN103906504A (en) * 2011-11-04 2014-07-02 日东电工株式会社 Method of producing lipid nanoparticles for drug delivery
WO2014152540A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2015006747A2 (en) 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use.
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
WO2015110957A2 (en) 2014-01-21 2015-07-30 De Beer Joel Hybridosomes, compositions comprising the same, processes for their production and uses thereof
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2016065349A2 (en) 2014-10-24 2016-04-28 University Of Maryland, Baltimore Short non-coding protein regulatory rnas (sprrnas) and methods of use
US9579338B2 (en) 2011-11-04 2017-02-28 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
WO2017070613A1 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Human cytomegalovirus vaccine
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US10106490B2 (en) 2014-06-25 2018-10-23 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
WO2018232006A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding coagulation factor viii
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
EP3395797A4 (en) * 2015-12-25 2019-09-04 Kyowa Hakko Kirin Co., Ltd. Compounds as cationic lipids
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019217964A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
US10501513B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
WO2020033791A1 (en) 2018-08-09 2020-02-13 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
WO2020061284A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. Peg lipids and uses thereof
WO2020061295A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. High-purity peg lipids and uses thereof
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021026310A1 (en) 2019-08-06 2021-02-11 L.E.A.F. Holdings Group Llc Processes of preparing polyglutamated antifolates and uses of their compositions
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021061815A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
WO2021061707A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein b (apob) gene expression
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
WO2021183720A1 (en) 2020-03-11 2021-09-16 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022032154A2 (en) 2020-08-06 2022-02-10 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2022200575A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
WO2022223556A1 (en) 2021-04-20 2022-10-27 Anjarium Biosciences Ag Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof
WO2022232286A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2022233880A1 (en) 2021-05-03 2022-11-10 Curevac Ag Improved nucleic acid sequence for cell type specific expression
WO2022261394A1 (en) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
WO2023014974A1 (en) 2021-08-06 2023-02-09 University Of Iowa Research Foundation Double stranded mrna vaccines
US11590229B2 (en) 2011-12-07 2023-02-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081526A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2023086465A1 (en) 2021-11-12 2023-05-19 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2023135273A2 (en) 2022-01-14 2023-07-20 Anjarium Biosciences Ag Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023154818A1 (en) 2022-02-09 2023-08-17 Modernatx, Inc. Mucosal administration methods and formulations
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2023239756A1 (en) 2022-06-07 2023-12-14 Generation Bio Co. Lipid nanoparticle compositions and uses thereof
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
EP4342460A1 (en) 2022-09-21 2024-03-27 NovoArc GmbH Lipid nanoparticle with nucleic acid cargo
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
WO2024134199A1 (en) 2022-12-22 2024-06-27 Mina Therapeutics Limited Chemically modified sarna compositions and methods of use
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
US12129223B2 (en) 2021-12-16 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2024223728A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024223724A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
US12133923B2 (en) 2022-12-08 2024-11-05 Recode Therapeutics, Inc. Lipid nanoparticle compositions and uses thereof

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10550385B2 (en) * 2012-12-20 2020-02-04 Sirna Therapeutics, Inc. Post-synthetic orthogonal amidation plus metal catalyzed azide-alkyne cycloaddition click chemistry on siRNA
CN114423869A (en) 2019-07-19 2022-04-29 旗舰先锋创新Vi有限责任公司 Recombinase compositions and methods of use
IL298363A (en) 2020-05-20 2023-01-01 Flagship Pioneering Innovations Vi Llc Immunogenic compositions and uses thereof
JP2023526422A (en) 2020-05-20 2023-06-21 フラッグシップ パイオニアリング イノベーションズ シックス，エルエルシー Coronavirus antigen compositions and their uses
CN116018405A (en) 2020-05-29 2023-04-25 旗舰先锋创新Vi有限责任公司 TREM compositions and related methods
CA3182026A1 (en) 2020-05-29 2021-12-02 Flagship Pioneering Innovations Vi, Llc. Trem compositions and methods relating thereto
TW202218669A (en) 2020-09-03 2022-05-16 美商旗艦先鋒創新有限責任公司 Immunogenic compositions and uses thereof
EP4267732A1 (en) 2020-12-23 2023-11-01 Flagship Pioneering Innovations VI, LLC Compositions of modified trems and uses thereof
EP4313109A1 (en) 2021-03-31 2024-02-07 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
EP4377457A1 (en) 2021-07-26 2024-06-05 Flagship Pioneering Innovations VI, LLC Trem compositions and uses thereof
WO2023023055A1 (en) 2021-08-16 2023-02-23 Renagade Therapeutics Management Inc. Compositions and methods for optimizing tropism of delivery systems for rna
CN118043466A (en) 2021-09-03 2024-05-14 葛兰素史克生物有限公司 Substitution of nucleotide bases in self-amplified mRNA
JP2024534697A (en) 2021-09-14 2024-09-20 レナゲード セラピューティクス マネージメント インコーポレイテッド Acyclic lipids and methods of use thereof
EP4402123A1 (en) 2021-09-14 2024-07-24 Renagade Therapeutics Management Inc. Cyclic lipids and methods of use thereof
JP2024534428A (en) 2021-09-17 2024-09-20 フラッグシップ パイオニアリング イノベーションズ シックス，エルエルシー Compositions and methods for producing circular polyribonucleotides
AU2022370530A1 (en) 2021-10-18 2024-05-02 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
EP4426832A1 (en) 2021-11-03 2024-09-11 The J. David Gladstone Institutes, A Testamentary Trust Established under The Will of J. David Gladstone Precise genome editing using retrons
EP4436983A1 (en) 2021-11-24 2024-10-02 Flagship Pioneering Innovations VI, LLC Varicella-zoster virus immunogen compositions and their uses
EP4436984A1 (en) 2021-11-24 2024-10-02 Flagship Pioneering Innovations VI, LLC Coronavirus immunogen compositions and their uses
KR20240122785A (en) 2021-11-24 2024-08-13 플래그쉽 파이어니어링 이노베이션스 브이아이, 엘엘씨 Immunogenic composition and use thereof
AU2022413687A1 (en) 2021-12-17 2024-06-27 Flagship Pioneering Innovations Vi, Llc Methods for enrichment of circular rna under denaturing conditions
AU2022417517A1 (en) 2021-12-22 2024-06-27 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
EP4452928A1 (en) 2021-12-23 2024-10-30 Renagade Therapeutics Management Inc. Constrained lipids and methods of use thereof
WO2023122789A1 (en) 2021-12-23 2023-06-29 Flagship Pioneering Innovations Vi, Llc Circular polyribonucleotides encoding antifusogenic polypeptides
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
AU2023239151A1 (en) 2022-03-25 2024-10-03 Sail Biomedicines, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
AU2023251104A1 (en) 2022-04-07 2024-10-17 Renagade Therapeutics Management Inc. Cyclic lipids and lipid nanoparticles (lnp) for the delivery of nucleic acids or peptides for use in vaccinating against infectious agents
WO2023196634A2 (en) 2022-04-08 2023-10-12 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2023220083A1 (en) 2022-05-09 2023-11-16 Flagship Pioneering Innovations Vi, Llc Trem compositions and methods of use for treating proliferative disorders
WO2023220729A2 (en) 2022-05-13 2023-11-16 Flagship Pioneering Innovations Vii, Llc Double stranded dna compositions and related methods
WO2023242817A2 (en) 2022-06-18 2023-12-21 Glaxosmithkline Biologicals Sa Recombinant rna molecules comprising untranslated regions or segments encoding spike protein from the omicron strain of severe acute respiratory coronavirus-2
WO2023250112A1 (en) 2022-06-22 2023-12-28 Flagship Pioneering Innovations Vi, Llc Compositions of modified trems and uses thereof
WO2024020346A2 (en) 2022-07-18 2024-01-25 Renagade Therapeutics Management Inc. Gene editing components, systems, and methods of use
WO2024030856A2 (en) 2022-08-01 2024-02-08 Flagship Pioneering Innovations Vii, Llc Immunomodulatory proteins and related methods
WO2024035952A1 (en) 2022-08-12 2024-02-15 Remix Therapeutics Inc. Methods and compositions for modulating splicing at alternative splice sites
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2024049979A2 (en) 2022-08-31 2024-03-07 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same
US20240174732A1 (en) 2022-10-05 2024-05-30 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additional polypeptides and their use in treating cancer
WO2024097664A1 (en) 2022-10-31 2024-05-10 Flagship Pioneering Innovations Vi, Llc Compositions and methods for purifying polyribonucleotides
WO2024102799A1 (en) 2022-11-08 2024-05-16 Flagship Pioneering Innovations Vi, Llc Compositions and methods for producing circular polyribonucleotides
WO2024129988A1 (en) 2022-12-14 2024-06-20 Flagship Pioneering Innovations Vii, Llc Compositions and methods for delivery of therapeutic agents to bone
WO2024133160A1 (en) 2022-12-19 2024-06-27 Glaxosmithkline Biologicals Sa Hepatitis b compositions
WO2024151583A2 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations Vii, Llc Vaccines and related methods
WO2024151673A2 (en) 2023-01-09 2024-07-18 President And Fellows Of Harvard College Recombinant nucleic acid molecules and their use in wound healing
US20240269251A1 (en) 2023-01-09 2024-08-15 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
WO2024151685A1 (en) 2023-01-09 2024-07-18 Beth Israel Deaconess Medical Center, Inc. Recombinant nucleic acid molecules and their use in wound healing
US20240269263A1 (en) 2023-02-06 2024-08-15 Flagship Pioneering Innovations Vii, Llc Immunomodulatory compositions and related methods
WO2024173307A2 (en) 2023-02-13 2024-08-22 Flagship Pioneering Innovation Vii, Llc Cleavable linker-containing ionizable lipids and lipid carriers for therapeutic compositions
WO2024173828A1 (en) 2023-02-17 2024-08-22 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified uracil
US20240293582A1 (en) 2023-02-17 2024-09-05 Flagship Pioneering Innovations Vii, Llc Dna compositions comprising modified cytosine
WO2024192422A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Immunogenic compositions and uses thereof
WO2024192291A1 (en) 2023-03-15 2024-09-19 Renagade Therapeutics Management Inc. Delivery of gene editing systems and methods of use thereof
WO2024192277A2 (en) 2023-03-15 2024-09-19 Renagade Therapeutics Management Inc. Lipid nanoparticles comprising coding rna molecules for use in gene editing and as vaccines and therapeutic agents
WO2024192420A1 (en) 2023-03-15 2024-09-19 Flagship Pioneering Innovations Vi, Llc Compositions comprising polyribonucleotides and uses thereof
WO2024216191A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Modified trems, compositions, and related methods thereof
WO2024216128A1 (en) 2023-04-12 2024-10-17 Flagship Pioneering Innovations Vi, Llc Trems for use in correction of missense mutations
WO2024220746A2 (en) 2023-04-21 2024-10-24 Flagship Pioneering Innovations Vii, Llc Rnai agents targeting fatty acid synthase and related methods

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4124641A (en) * 1975-12-12 1978-11-07 Canadian D. A. Stuart Oil Co. Limited Oxyethylated acetals
US20090023673A1 (en) * 2006-10-03 2009-01-22 Muthiah Manoharan Lipid containing formulations
US20100015218A1 (en) * 2007-02-16 2010-01-21 Vasant Jadhav Compositions and methods for potentiated activity of biologically active molecules

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2569664C (en) 2004-06-07 2013-07-16 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
CA2569645C (en) 2004-06-07 2014-10-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
AU2006336384B2 (en) 2005-02-14 2010-12-16 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US8114636B2 (en) 2006-02-10 2012-02-14 Life Technologies Corporation Labeling and detection of nucleic acids
CN104193815A (en) * 2006-09-08 2014-12-10 Ambrx公司 Modified human plasma polypeptide or Fc scaffolds and their uses
CA3044134A1 (en) 2008-01-02 2009-07-09 Arbutus Biopharma Corporation Improved compositions and methods for the delivery of nucleic acids
US20090263407A1 (en) 2008-04-16 2009-10-22 Abbott Laboratories Cationic Lipids and Uses Thereof
AU2009298802A1 (en) 2008-09-23 2010-04-08 Alnylam Pharmaceuticals, Inc. Chemical modifications of monomers and oligonucleotides with cycloaddition
EP2743265B1 (en) 2008-10-09 2017-03-15 Arbutus Biopharma Corporation Improved amino lipids and methods for the delivery of nucleic acids
WO2010048536A2 (en) 2008-10-23 2010-04-29 Alnylam Pharmaceuticals, Inc. Processes for preparing lipids
CA2751342C (en) 2009-01-29 2019-05-07 Alnylam Pharmaceuticals, Inc. Lipid formulations comprising cationic lipid and a targeting lipid comprising n-acetyl galactosamine for delivery of nucleic acid
US8569256B2 (en) * 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
US20130116419A1 (en) * 2010-01-22 2013-05-09 Daniel Zewge Post-synthetic chemical modification of rna at the 2'-position of the ribose ring via "click" chemistry
US20110250641A1 (en) * 2010-04-08 2011-10-13 Matthew Jacob Powell Zwitterionic acid-labile surfactants and methods of use

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4124641A (en) * 1975-12-12 1978-11-07 Canadian D. A. Stuart Oil Co. Limited Oxyethylated acetals
US20090023673A1 (en) * 2006-10-03 2009-01-22 Muthiah Manoharan Lipid containing formulations
US20100015218A1 (en) * 2007-02-16 2010-01-21 Vasant Jadhav Compositions and methods for potentiated activity of biologically active molecules

Cited By (149)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9670487B2 (en) * 2010-01-22 2017-06-06 Sirna Therapeutics, Inc. Cationic lipids for oligonucleotide delivery
US20130053572A1 (en) * 2010-01-22 2013-02-28 Steven L. Colletti Novel Cationic Lipids for Oligonucleotide Delivery
WO2013059496A1 (en) * 2011-10-18 2013-04-25 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
US11059807B2 (en) 2011-10-18 2021-07-13 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
US9549983B2 (en) 2011-10-18 2017-01-24 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
US10144725B2 (en) 2011-10-18 2018-12-04 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
US10155945B2 (en) 2011-11-04 2018-12-18 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
US9579338B2 (en) 2011-11-04 2017-02-28 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
CN103906504A (en) * 2011-11-04 2014-07-02 日东电工株式会社 Method of producing lipid nanoparticles for drug delivery
TWI615156B (en) * 2011-11-04 2018-02-21 日東電工股份有限公司 Single use system for sterilely producing lipid-nucleic acid particles
US11633480B2 (en) 2011-12-07 2023-04-25 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11633479B2 (en) 2011-12-07 2023-04-25 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11679158B2 (en) 2011-12-07 2023-06-20 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11612657B2 (en) 2011-12-07 2023-03-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
US11590229B2 (en) 2011-12-07 2023-02-28 Alnylam Pharmaceuticals, Inc. Biodegradable lipids for the delivery of active agents
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US11564998B2 (en) 2012-04-02 2023-01-31 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
US10772975B2 (en) 2012-04-02 2020-09-15 Modernatx, Inc. Modified Polynucleotides for the production of biologics and proteins associated with human disease
US10501513B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US10703789B2 (en) 2012-04-02 2020-07-07 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US10493167B2 (en) 2012-04-02 2019-12-03 Modernatx, Inc. In vivo production of proteins
US10463751B2 (en) 2012-04-02 2019-11-05 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
CN104411338A (en) * 2012-04-02 2015-03-11 现代治疗公司 Modified polynucleotides for the production of biologics and proteins associated with human disease
US10577403B2 (en) 2012-04-02 2020-03-03 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US10583203B2 (en) 2012-04-02 2020-03-10 Modernatx, Inc. In vivo production of proteins
EP4074834A1 (en) 2012-11-26 2022-10-19 ModernaTX, Inc. Terminally modified rna
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2014152540A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015006747A2 (en) 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use.
EP3971287A1 (en) 2013-07-11 2022-03-23 ModernaTX, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2015075557A2 (en) 2013-11-22 2015-05-28 Mina Alpha Limited C/ebp alpha compositions and methods of use
EP3985118A1 (en) 2013-11-22 2022-04-20 MiNA Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
EP3594348A1 (en) 2013-11-22 2020-01-15 Mina Therapeutics Limited C/ebp alpha short activating rna compositions and methods of use
WO2015110957A2 (en) 2014-01-21 2015-07-30 De Beer Joel Hybridosomes, compositions comprising the same, processes for their production and uses thereof
EP3791863A1 (en) 2014-01-21 2021-03-17 Anjarium Biosciences AG Process for the production of hybridosomes
US11944706B2 (en) 2014-01-21 2024-04-02 Anjarium Biosciences Ag Hybridosomes, compositions comprising the same, processes for their production and uses thereof
US10106490B2 (en) 2014-06-25 2018-10-23 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2016065349A2 (en) 2014-10-24 2016-04-28 University Of Maryland, Baltimore Short non-coding protein regulatory rnas (sprrnas) and methods of use
US10221127B2 (en) 2015-06-29 2019-03-05 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11168051B2 (en) 2015-06-29 2021-11-09 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017070613A1 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Human cytomegalovirus vaccine
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
EP3395797A4 (en) * 2015-12-25 2019-09-04 Kyowa Hakko Kirin Co., Ltd. Compounds as cationic lipids
US10525138B2 (en) 2015-12-25 2020-01-07 Kyowa Hakko Kirin Co., Ltd. Compound as cationic lipid
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
WO2018232006A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Polynucleotides encoding coagulation factor viii
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4219715A2 (en) 2017-09-08 2023-08-02 MiNA Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4233880A2 (en) 2017-09-08 2023-08-30 MiNA Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019048631A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Hnf4a sarna compositions and methods of use
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019197845A1 (en) 2018-04-12 2019-10-17 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
EP4242307A2 (en) 2018-04-12 2023-09-13 MiNA Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2019217964A1 (en) 2018-05-11 2019-11-14 Lupagen, Inc. Systems and methods for closed loop, real-time modifications of patient cells
WO2020033791A1 (en) 2018-08-09 2020-02-13 Verseau Therapeutics, Inc. Oligonucleotide compositions for targeting ccr2 and csf1r and uses thereof
WO2020061284A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. Peg lipids and uses thereof
WO2020061295A1 (en) 2018-09-19 2020-03-26 Modernatx, Inc. High-purity peg lipids and uses thereof
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2020208361A1 (en) 2019-04-12 2020-10-15 Mina Therapeutics Limited Sirt1-sarna compositions and methods of use
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021026310A1 (en) 2019-08-06 2021-02-11 L.E.A.F. Holdings Group Llc Processes of preparing polyglutamated antifolates and uses of their compositions
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021061815A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. COMPOSITIONS AND METHODS FOR MODULATING HEPATOCYTE NUCLEAR FACTOR 4-ALPHA (HNF4α) GENE EXPRESSION
WO2021061707A1 (en) 2019-09-23 2021-04-01 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein b (apob) gene expression
DE202021004123U1 (en) 2020-02-04 2022-10-26 Curevac Ag Coronavirus Vaccine
EP4147717A1 (en) 2020-02-04 2023-03-15 CureVac SE Coronavirus vaccine
DE202021003575U1 (en) 2020-02-04 2022-01-17 Curevac Ag Coronavirus Vaccine
DE202021004130U1 (en) 2020-02-04 2022-10-26 Curevac Ag Coronavirus Vaccine
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
DE112021000012T5 (en) 2020-02-04 2021-11-18 Curevac Ag Coronavirus vaccine
WO2021183720A1 (en) 2020-03-11 2021-09-16 Omega Therapeutics, Inc. Compositions and methods for modulating forkhead box p3 (foxp3) gene expression
WO2021195214A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing factor ix therapeutics
WO2021195218A1 (en) 2020-03-24 2021-09-30 Generation Bio Co. Non-viral dna vectors and uses thereof for expressing gaucher therapeutics
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2022023284A1 (en) 2020-07-27 2022-02-03 Anjarium Biosciences Ag Compositions of dna molecules, methods of making therefor, and methods of use thereof
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022032154A2 (en) 2020-08-06 2022-02-10 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
WO2022122872A1 (en) 2020-12-09 2022-06-16 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
WO2022200575A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200810A1 (en) 2021-03-26 2022-09-29 Mina Therapeutics Limited Tmem173 sarna compositions and methods of use
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
WO2022223556A1 (en) 2021-04-20 2022-10-27 Anjarium Biosciences Ag Compositions of dna molecules encoding amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase, methods of making thereof, and methods of use thereof
WO2022232289A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing therapeutic antibodies and uses thereof
WO2022232286A1 (en) 2021-04-27 2022-11-03 Generation Bio Co. Non-viral dna vectors expressing anti-coronavirus antibodies and uses thereof
WO2022233880A1 (en) 2021-05-03 2022-11-10 Curevac Ag Improved nucleic acid sequence for cell type specific expression
WO2022261394A1 (en) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Rna polymerase iii promoters and methods of use
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
WO2023014974A1 (en) 2021-08-06 2023-02-09 University Of Iowa Research Foundation Double stranded mrna vaccines
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023081526A1 (en) 2021-11-08 2023-05-11 Orna Therapeutics, Inc. Lipid nanoparticle compositions for delivering circular polynucleotides
WO2023086465A1 (en) 2021-11-12 2023-05-19 Modernatx, Inc. Compositions for the delivery of payload molecules to airway epithelium
WO2023099884A1 (en) 2021-12-01 2023-06-08 Mina Therapeutics Limited Pax6 sarna compositions and methods of use
WO2023104964A1 (en) 2021-12-09 2023-06-15 Ucl Business Ltd Therapeutics for the treatment of neurodegenerative disorders
US12129223B2 (en) 2021-12-16 2024-10-29 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2023135273A2 (en) 2022-01-14 2023-07-20 Anjarium Biosciences Ag Compositions of dna molecules encoding factor viii, methods of making thereof, and methods of use thereof
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023154818A1 (en) 2022-02-09 2023-08-17 Modernatx, Inc. Mucosal administration methods and formulations
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023170435A1 (en) 2022-03-07 2023-09-14 Mina Therapeutics Limited Il10 sarna compositions and methods of use
WO2023177655A1 (en) 2022-03-14 2023-09-21 Generation Bio Co. Heterologous prime boost vaccine compositions and methods of use
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2023239756A1 (en) 2022-06-07 2023-12-14 Generation Bio Co. Lipid nanoparticle compositions and uses thereof
WO2024033901A1 (en) 2022-08-12 2024-02-15 LifeEDIT Therapeutics, Inc. Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2024040222A1 (en) 2022-08-19 2024-02-22 Generation Bio Co. Cleavable closed-ended dna (cedna) and methods of use thereof
EP4342460A1 (en) 2022-09-21 2024-03-27 NovoArc GmbH Lipid nanoparticle with nucleic acid cargo
WO2024062001A1 (en) 2022-09-21 2024-03-28 Novoarc Gmbh Lipid nanoparticle with nucleic acid cargo
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
DE202023106198U1 (en) 2022-10-28 2024-03-21 CureVac SE Nucleic acid-based vaccine
WO2024102730A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and nanoparticle compositions for delivering polynucleotides
WO2024102677A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Circular rna compositions
WO2024102762A1 (en) 2022-11-08 2024-05-16 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024119051A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Novel polyglycerol-conjugated lipids and lipid nanoparticle compositions comprising the same
WO2024119074A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticle compositions for cell targeting
WO2024119039A2 (en) 2022-12-01 2024-06-06 Generation Bio Co. Stealth lipid nanoparticles and uses thereof
WO2024119103A1 (en) 2022-12-01 2024-06-06 Generation Bio Co. Lipid nanoparticles comprising nucleic acids and lipid-anchored polymers
US12133923B2 (en) 2022-12-08 2024-11-05 Recode Therapeutics, Inc. Lipid nanoparticle compositions and uses thereof
WO2024134199A1 (en) 2022-12-22 2024-06-27 Mina Therapeutics Limited Chemically modified sarna compositions and methods of use
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024205657A2 (en) 2023-03-29 2024-10-03 Orna Therapeutics, Inc. Lipids and lipid nanoparticle compositions for delivering polynucleotides
WO2024223728A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024223724A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation

Also Published As

Publication number Publication date
US20130116419A1 (en) 2013-05-09
EP2526113B1 (en) 2016-08-10
US20150211008A1 (en) 2015-07-30
US20130053572A1 (en) 2013-02-28
US9441228B2 (en) 2016-09-13
EP2526113A1 (en) 2012-11-28
US9670487B2 (en) 2017-06-06
US20160348109A1 (en) 2016-12-01
EP2526113A4 (en) 2013-11-27
EP2525781A1 (en) 2012-11-28

Similar Documents

Publication Publication Date Title
US9670487B2 (en) Cationic lipids for oligonucleotide delivery
US10337014B2 (en) Low molecular weight cyclic amine containing cationic lipids for oligonucleotide delivery
US9981907B2 (en) Low molecular weight cationic lipids for oligonucleotide delivery
EP2467357B1 (en) Novel cationic lipids with various head groups for oligonucleotide delivery
EP2621480B1 (en) Low molecular weight cationic lipids for oligonucleotide delivery
ES2888231T3 (en) Low molecular weight cationic lipids for oligonucleotide delivery
EP2575767B1 (en) Novel low molecular weight cationic lipids for oligonucleotide delivery
DK2830594T3 (en) DIETHER-BASED BIOLOGICALLY DEGRADABLE CATIONIC LIPIDS FOR siRNA RELEASE
US9044512B2 (en) Amino alcohol cationic lipids for oligonucleotide delivery
WO2013016058A1 (en) Novel bis-nitrogen containing cationic lipids for oligonucleotide delivery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11735061

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2011735061

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 13574315

Country of ref document: US