EP4724418A1 - Tricine and citric acid-based cationic lipids - Google Patents

Tricine and citric acid-based cationic lipids

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Publication number
EP4724418A1
EP4724418A1 EP24731386.9A EP24731386A EP4724418A1 EP 4724418 A1 EP4724418 A1 EP 4724418A1 EP 24731386 A EP24731386 A EP 24731386A EP 4724418 A1 EP4724418 A1 EP 4724418A1
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EP
European Patent Office
Prior art keywords
independently
alkyl
compound
mol
pharmaceutically acceptable
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP24731386.9A
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German (de)
French (fr)
Inventor
Ramesh Dasari
Hongfeng Deng
Frank Derosa
Saswata KARMAKAR
Shrirang KARVE
Neha KAUSHAL
Peter Justin Mikochik
Apiwat WANGWEERAWONG
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Sanofi SA
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Sanofi SA
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Filing date
Publication date
Application filed by Sanofi SA filed Critical Sanofi SA
Publication of EP4724418A1 publication Critical patent/EP4724418A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • 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
    • C07D295/14—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero 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
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl

Definitions

  • the present invention provides, among other things, a novel class of tricine and citric acid-based cationic lipid compounds for improved in vivo delivery of therapeutic agents, such as nucleic acids. It is contemplated that the compounds provided herein can be capable of highly effective in vivo delivery while maintaining a favorable toxicity profile. It is contemplated that the compounds provided herein can be capable of highly effective intranasal delivery. Indeed, Examples 43 and 45 shows that lipid nanoparticles comprising compounds of the present invention (e.g. compound 64 or a compound in Table 6, or a pharmaceutically acceptable salt thereof) are particularly effective at intranasal delivery.
  • compounds of the present invention e.g. compound 64 or a compound in Table 6, or a pharmaceutically acceptable salt thereof
  • the invention features a cationic lipid having a structure according to Formula (A2): or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkyl, C 4 -C 30 alkenyl, C 4 -C 30 alkynyl, or C 4 -C 30 heteroalkyl; A is a substructure selected from: , , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L 1 is a carbonyl, ester, or amide; L 2 is C 2 -C 10 alkylene or a C 2 -C 10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R 4 is C1-C10 alkylene; and wherein A is only substructure (a4)
  • the invention features a cationic lipid having a structure according to Formula (A1): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; A is a substructure selected from: , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L 1 is a carbonyl, ester, or amide; L 2 is C 2 -C 10 alkylene or a C 2 -C 10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH 3 , and CH 3 ; R 4 is C1-C10 alkylene; and wherein A is only substructure (a4) when
  • the invention features a cationic lipid having a structure according to Formula (A): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkyl, C 4 -C 30 alkenyl, C 4 -C 30 alkynyl, or C 4 -C 30 heteroalkyl; A is a substructure selected from: , , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L 1 is a carbonyl, ester, or amide; L 2 is C 2 -C 10 alkylene or a C 2 -C 10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH 3 , and CH 3 ; R 4 is C2-C10 alkylene; and wherein A is only substructure selected from: , B
  • X is O and/or B is independently an ionizable nitrogen-containing group.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4- C 30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently a C 4 -C 30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C6 alkyl.
  • each of R 1 , R 2 , and R 3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L 3 is OC(O), CO 2 , or (O)CO; o is an integer of 2-5; and R 5 is C 4 -C 24 alkyl; or L 3 is OC(O), CO 2 , or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • B is independently , , , , , , [014]
  • a compound has a structure according to Formula (I): , or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , and R 3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group. [015] In embodiments, B is independently an ionizable nitrogen containing group.
  • m is 2 and/or n is 2 or 3.
  • B is independently , , , , [020] In embodiments, B is independently , , , , [021] In embodiments, .
  • a compound is selected from the group consisting of: Compounds (1), (2), (3), (4), (6), (9), (17), (24), (26), (27), (33), and (35).
  • a compound is Compound (3).
  • a compound has a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , and R 3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. [032] In embodiments, R 1 , R 2 , and R 3 is C 4 -C 30 heteroalkyl. [033] In embodiments, m is 2 and/or n is 2 or 3.
  • B is independently , , , , [035] In embodiments, B is independently , , , , [036] In embodiments, B is independently , , , , , , , . [037] In embodiments, B is independently , , , , [038] In embodiments, B is . [039] In embodiments, a compound is Compound (8) or (39). [040] In embodiments, a compound has a structure according to Formula (V1): or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, 4, 5, 6, or 7; and B is independently an ionizable nitrogen-containing group.
  • V1 Formula (V1): or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, 4, 5, 6, or 7; and B is independently an ionizable nitrogen-containing group.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • L 3 is OC(O), CO 2 , or (O)CO; o is an integer of 6-12; and R 5 is C 1 -C 6 alkyl.
  • B is independently , , , , , .
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (112), (113), (114), (115), (116), (117), (126), (127), (128), and (129).
  • a compound has a structure according to Formula (V): , or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • L 3 is OC(O), CO 2 , or (O)CO; o is an integer of 6-12; and R 5 is C 1 -C 6 alkyl [ , .
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (114), and (116).
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), and (40).
  • a compound has a structure according to Formula (VI): , or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , and R 3 is independently C 6 -C 30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group.
  • B is independently , , , , , . [063] In embodiments, B is independently , , , , [064] In embodiments, . [065] In embodiments, a compound is Compound (21).
  • a compound has a formula according to Formula (VII), , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkyl, C 4 -C 30 alkenyl, C 4 -C 30 alkynyl, or C 4 -C 30 heteroalkyl; each m is an integer of 2-10; each L 4 is a carbonyl, ester, or amide; Z is –(CH2)q1–N–(CH2)q2–, wherein q1 and q2 are independently integers of 2-10; or Z is C6H3-Z 1 , wherein Z 1 is a carbonyl, ester, or amide that is covalently attached to the –CH2(CH2)qB moiety; q is an integer of 1-9; and B is independently an ionizable nitrogen-containing group.
  • X is independently O or NH
  • X is O; each L 4 is –C(O)O–; m is an integer of 2, 3, or 4; and/or q is 1.
  • Z is –(CH 2 ) q1 –N–(CH 2 ) q2 –, and q1 and q2 are each 2.
  • Z is . . [074] In embodiments, . [075] In embodiments, a compound is Compound (16) or (31).
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of intranasal delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of intranasal delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
  • a compound is selected from the group of Compounds 41-64 and 90, or a pharmaceutically acceptable salt thereof:
  • a compound is selected from the group of Compounds 91-111, or a pharmaceutically acceptable salt thereof:
  • a compound is selected from the group of Compounds 112-117 and
  • a compound is selected from the group of Compounds 118-125, or a pharmaceutically acceptable salt thereof:
  • a composition comprises an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is any cationic lipid described herein.
  • a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is any cationic lipid described herein.
  • a composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • a nucleic acid is an mRNA encoding a peptide or polypeptide.
  • an mRNA that encodes a peptide or polypeptide for use in vaccine encodes an antigen.
  • an antigen is from an infectious agent.
  • a composition is formulated for a route of administration that is oral, rectal, vaginal, transmucosal, pulmonary, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal.
  • parenteral delivery including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal.
  • pulmonary delivery is intratracheal or inhaled.
  • a route of administration is intranasal.
  • a route of administration is intramuscular.
  • a route of administration is pulmonary.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X 1A is independently O or NR 1A ; R 1A is H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X 2A and X 2B is O and the other is a covalent bond; one of X 3A and X 3B is O
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1;
  • X 1A is independently O or NR 1A ;
  • R 1A is H or C 1 -C 6 alkyl;
  • X 1B is a covalent bond, C(O), CH 2 CO 2 , or CH 2 C(O); one of X 2A and X 2B is O and the other is a covalent bond;
  • one of X 3A and X 3B is O and the other is a covalent bond;
  • one of X 4A and X 4B is O and the other is a covalent bond;
  • R 1 is independently L 1 -B 1 , C6-C30 alkyl
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B1): wherein L 1 is straight-chain C5 alkylene; and R 2 , R 3 , and R 4 are as defined for Formula (B).
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B1): wherein L 1 is as defined for Formula (B); and R 2 , R 3 , and R 4 are straight-chain C 8 alkylene.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B1): , wherein L 1 is as defined for Formula (B); and R 2 , R 3 , and R 4 are straight-chain C8 alkylene.
  • a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B1): , wherein L 1 is as defined for Formula (B); and R 2 , R 3 , and R 4 are straight-chain C8 alkylene.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B1): , wherein L 1 is as defined for Formula (B); and R 2 , R 3 , and R 4 are straight-chain C 8 alkylene.
  • the present invention also provides compounds of formula (B1) as described herein. Said compounds may be incorporated into any of the compositions disclosed herein and may be used in any of the methods described herein.
  • a composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
  • a nucleic acid is an mRNA encoding a peptide or polypeptide.
  • a composition comprises an mRNA that encodes a peptide or polypeptide for use in vaccine.
  • an mRNA encodes an antigen.
  • an antigen is from an infectious agent.
  • a compound is selected from the group consisting of Compounds (65)- (89), or a pharmaceutically acceptable salt thereof:
  • a compound is selected from the group consisting of Compounds (118)-(125), or a pharmaceutically acceptable salt thereof:
  • delivery is intranasal.
  • delivery is intramuscular.
  • delivery is pulmonary.
  • FIG. 1 depicts % survival of BALB/c mice inoculated 1) intranasally (IN) with Lipid
  • Nanoparticles encapsulating CA09 HA mRNA (these LNPs comprised either Compound 64 or the lipid OF-02); 2) intramuscularly (IM) with LNPs encapsulating CA09 HA mRNA (these LNPs comprised OF-02 only); or 3) a phosphate-buffered saline (PBS) buffer control administered intranasally (IN).
  • LNPs comprising Compound 64 administered intranasally in a lethal mouse influenza challenge model.
  • FIG. 2 depicts % body weight change of BALB/c mice inoculated 1) intranasally (IN) with Lipid Nanoparticles (LNPs) encapsulating CA09 HA mRNA (these LNPs comprised either Compound 64 or the lipid OF-02); 2) intramuscularly (IM) with LNPs encapsulating CA09 HA mRNA (these LNPs comprised OF-02 only); or 3) a phosphate-buffered saline (PBS) buffer control administered intranasal ly (IN).
  • LNPs Lipid Nanoparticles
  • IM intramuscularly
  • PBS phosphate-buffered saline
  • FIG. 3 and FIG. 4 depict data obtained from various studies comparing intranasal administration of Compound 64 (a compound of the invention) with intramuscular administration of known compound OF-02 which are described in more detail in Example 47 herein.
  • amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally occurring amino acid.
  • an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid.
  • Standard amino acid refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids, including carboxy- and/or aminoterminal amino acids in peptides can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond.
  • Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).
  • chemical entities e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.
  • amino acid is used interchangeably with "amino acid residue/' and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a bovine, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a bovine, a primate, and/
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery”).
  • patient's circulation system e.g., serum
  • expression refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme).
  • intact protein e.g., enzyme
  • post-translational modification e.g., enzyme
  • a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • Helper lipid refers to any neutral or zwitterionic lipid material including cholesterol. Without wishing to be held to a particular theory, helper lipids may add stability, rigidity, and/or fluidity within lipid bilayers/nanoparticles.
  • improve, increase, or reduce As used herein, the terms “improve,” “increase,” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.
  • in vivo refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is "pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • Liposome refers to any lamellar, multilamellar, or solid nanoparticle vesicle.
  • a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s).
  • a liposome suitable for the present invention contains a cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEG-modified lipid(s).
  • messenger RNA As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. The term “modified mRNA” related to mRNA comprising at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
  • mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.
  • an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguagua
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • “nucleic acid” encompasses ribonucleic acids (RNA), including but not limited to any one or more of interference RNAs (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA).
  • RNAi interference RNAs
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • aRNA antisense RNA
  • mRNA messenger RNA
  • mmRNA modified messenger RNA
  • IncRNA micro-RNA
  • miRNA multimeric coding nucleic acid
  • PCNA
  • nucleic acid encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and complementary DNA (cDNA). In some embodiments, "nucleic acid” encompasses both RNA and DNA.
  • DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro-RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73
  • patient refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes.
  • Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans).
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
  • a patient is a human.
  • a human includes pre- and post-natal forms.
  • compositions that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (CI. 4 alkyl ) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate.
  • Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
  • Systemic distribution or delivery As used herein, the terms “systemic distribution” or “systemic delivery,” or grammatical equivalents thereof, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of "local distribution or delivery.”
  • Subject As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term "subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Target tissues refers to any tissue that is affected by a disease to be treated.
  • target tissues include those tissues that display disease-associated pathology, symptom, or feature.
  • therapeutically effective amount As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • Aliphatic refers to C1-C50 hydrocarbons and includes both saturated and unsaturated hydrocarbons.
  • An aliphatic may be linear, branched, or cyclic.
  • C1-C20 aliphatics can include C1-C20 alkyls (e.g., linear or branched C1-C20 saturated alkyls), C2-C20 alkenyls (e.g., linear or branched C4-C20 dienyls, linear or branched C6-C20 trienyls, and the like), and C2-C20 alkynyls (e.g., linear or branched C2-C20 alkynyls).
  • C1-C20 aliphatics can include C3-C20 cyclic aliphatics (e.g., C3-C20 cycloalkyls, C4-C20 cycloalkenyls, or C8-C20 cycloalkynyls).
  • the aliphatic may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide.
  • An aliphatic group is unsubstituted or substituted with one or more substituent groups as described herein.
  • an aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR”, -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', - OCO2R", -NH2, -NHR", -N(R") 2 , -SR" or-SO2R”, wherein each instance of R" independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • substituents e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents
  • R" independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms.
  • alkyl means acyclic linear and branched hydrocarbon groups, e.g. "C1-C30 alkyl” refers to alkyl groups having 1-30 carbons.
  • An alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, isohexyl, etc.
  • lower alkyl means an alkyl group straight chain or branched alkyl having 1 to 6 carbon atoms.
  • Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure.
  • An alkyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • an alkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -CO2H, - CO2R", -CN, -OH, -OR", -OCOR', -OCO 2 R", -NH 2 , -NHR", -N(R") 2 , -SR" or-SO 2 R", wherein each instance of R" independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • substituents e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents
  • R" independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkyl group is substituted with a-OH group and may also be referred to herein as a "hydroxyalkyl" group, where the prefix denotes the -OH group and "alkyl" is as described herein.
  • alkyl also refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms (“C1-C50 alkyl”). In some embodiments, an alkyl group has 1 to 40 carbon atoms (“C1-C40 alkyl”). In some embodiments, an alkyl group has 1 to 30 carbon atoms (“C1-C30 alkyl”). In some embodiments, an alkyl group has 1 to 20 carbon atoms (“C1-C20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-C10 alkyl”).
  • an alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("Ci-Cg alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-C7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("Ci-Cg alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“Ci- C 5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl").
  • an alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”).
  • Ci-Cg alkyl groups include, without limitation, methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), iso-butyl (C 4 ), n-pentyl (C 5 ), 3- pentanyl (C 5 ), amyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butanyl (C 5 ), tertiary amyl (C 5 ), and n-hexyl (Cg).
  • alkyl groups include n-heptyl (C 7 ), n-octyl (Cg) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C1-C50 alkyl. In certain embodiments, the alkyl group is a substituted C1-C50 alkyl.
  • Affixing the suffix "-ene" to a group indicates the group is a divalent moiety, e.g., arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.
  • Alkylene represents a saturated divalent straight or branched chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene and the like.
  • alkenylene represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain
  • alkynylene herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur in any stable point along the chain.
  • an alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide.
  • an alkylene, alkenylene, or alkynylene may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, -CO 2 H, -CO 2 R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO 2 R’’, -NH 2 , -NHR’’, -N(R’’) 2 , -SR’’ or - SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl).
  • R’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 al
  • R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is unsubstituted C 1 -C 3 alkyl. In certain embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, an alkylene, alkenylene, or alkynylene does not include any heteroatoms.
  • alkenyl means any linear or branched hydrocarbon chains having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, e.g. “C2-C30 alkenyl” refers to an alkenyl group having 2-30 carbons.
  • an alkenyl group includes prop-2-enyl, but-2-enyl, but-3-enyl, 2- methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like.
  • the alkenyl comprises 1, 2, or 3 carbon-carbon double bond.
  • the alkenyl comprises a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated.
  • An alkenyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • an alkenyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, - CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or-SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C 1 -C 3 alkyl).
  • R’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C 1 -C 3 alkyl).
  • R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C 1 - C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R’’ independently is unsubstituted C 1 -C 3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • an alkenyl group is substituted with a–OH group and may also be referred to herein as a “hydroxyalkenyl” group, where the prefix denotes the –OH group and “alkenyl” is as described herein.
  • alkenyl also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C 2 -C 50 alkenyl”). In some embodiments, an alkenyl group has 2 to 40 carbon atoms (“C2-C40 alkenyl”).
  • an alkenyl group has 2 to 30 carbon atoms (“C2-C30 alkenyl”). In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C2-C20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2- C 10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C 2 -C 9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C 2 -C 8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C 2 -C 7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2 -C 3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C 2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • Examples of C 2 -C 4 alkenyl groups include, without limitation, ethenyl (C 2 ), 1- propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like.
  • Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.
  • each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents.
  • the alkenyl group is an unsubstituted C 2 -C 50 alkenyl.
  • the alkenyl group is a substituted C 2 - C50 alkenyl.
  • alkynyl means any hydrocarbon chain of either linear or branched configuration, having one or more carbon-carbon triple bonds occurring in any stable point along the chain, e.g., “C2-C30 alkynyl”, refers to an alkynyl group having 2-30 carbons. Examples of an alkynyl group include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3- methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. In embodiments, an alkynyl comprises one carbon-carbon triple bond.
  • An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein.
  • an alkynyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, - CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or-SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C 1 -C 3 alkyl).
  • R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C 1 - C 20 alkyl, C 1 -C 15 alkyl, C 1 -C 10 alkyl, or C 1 -C 3 alkyl). In embodiments, R’’ independently is unsubstituted C 1 -C 3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).
  • alkynyl also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C 2 -C 50 alkynyl”).
  • An alkynyl group that has one or more triple bonds and one or more double bonds is also referred to as an “ene-yne”.
  • an alkynyl group has 2 to 40 carbon atoms (“C2-C40 alkynyl”).
  • an alkynyl group has 2 to 30 carbon atoms (“C2-C30 alkynyl”). In some embodiments, an alkynyl group has 2 to 20 carbon atoms (“C2-C20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C 2 -C 9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C 2 -C 8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C 2 -C 7 alkynyl”).
  • an alkynyl group has 2 to 6 carbon atoms (“C 2 -C 6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon--carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • Examples of C 2 -C 4 alkynyl groups include, without limitation, ethynyl (C 2 ), 1- propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
  • Examples of C 2 -C 6 alkenyl groups include the aforementioned C2-C4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.
  • each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents.
  • the alkynyl group is an unsubstituted C 2 -C 50 alkynyl. In certain embodiments, the alkynyl group is a substituted C 2 -C 50 alkynyl.
  • Aryl refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein said ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic and wherein each ring in the system contains 4 to 7 ring members.
  • an aryl group has 6 ring carbon atoms (“C6 aryl,” e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C 10 aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C 14 aryl,” e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • aryls include phenyl, naphthyl, and anthracene.
  • aryl also refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”).
  • an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).
  • an aryl group has 14 ring carbon atoms (“C 14 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
  • the aryl group is an unsubstituted C6-C14 aryl.
  • the aryl group is a substituted C 6 -C 14 aryl.
  • Arylene The term “arylene” as used herein refers to an aryl group that is divalent (that is, having two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene).
  • Carbocyclyl As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of a non- aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3 -C 8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3 -C 7 carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3 -C 6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-C6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-C6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-C10 carbocyclyl”).
  • Exemplary C3-C6 carbocyclyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C4), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
  • Exemplary C3-C8 carbocyclyl groups include, without limitation, the aforementioned C3-C6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like.
  • Exemplary C 3 -C 10 carbocyclyl groups include, without limitation, the aforementioned C 3 -C 8 carbocyclyl groups as well as cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents.
  • the carbocyclyl group is an unsubstituted C 3 -C 10 carbocyclyl.
  • the carbocyclyl group is a substituted C 3 -C 10 carbocyclyl.
  • “carbocyclyl” or “carbocyclic” is referred to as a “cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”).
  • a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3-C8 cycloalkyl”).
  • a cycloalkyl group has 3 to 6 ring carbon atoms (“C 3-C6, cycloalkyl”).
  • a cycloalkyl group has 4 to 6 ring carbon atoms (“C 4 -C 6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5 -C 6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C 5 -C 10 cycloalkyl”). Examples of C 5 -C 6 cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ).
  • C3-C6 cycloalkyl groups include the aforementioned C5-C6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4).
  • C3-C8 cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8).
  • each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
  • the cycloalkyl group is an unsubstituted C 3 -C 10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-C10 cycloalkyl.
  • Halogen As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine.
  • Heteroalkyl The term “heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 4 to 50 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P.
  • a heteroalkyl group has 4 to 40 carbon atoms (“C4-C40 heteroalkyl”). In some embodiments, a heteroalkyl group has 6 to 30 carbon atoms (“C6-C30 heteroalkyl”). In some embodiments, a heteroalkyl group has 4 to 20 carbon atoms (“C4-C20 heteroalkyl”). Heteroalkyls include tertiary amines, secondary amines, ethers, esters, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides.
  • a heteroalkyl group may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members.
  • heteroalkyls include polyethers, such as methoxymethyl and ethoxyethyl.
  • a heteroalkyl comprises an alkyl group as described herein (e.g., a C4-C50 alkyl or C6-C30 alkyl) which may be unsubstituted or substituted (e.g., comprising a hydroxy group, an oxo group, or an ionizable nitrogen group as described herein), and/or comprises 1 or 2 heteroatoms selected from N and O.
  • a heteroalkyl comprises an oxo substitutent group (e.g., a heteroalkyl comprises an ester group where the oxo is adjacent to an oxygen atom).
  • Heteroalkylene The term “heteroalkylene,” as used herein, represents a divalent form of a heteroalkyl group as described herein.
  • Heteroaryl The term “heteroaryl,” as used herein, is fully unsaturated heteroatom- containing ring wherein at least one ring atom is a heteroatom such as, but not limited to, nitrogen and oxygen.
  • heteroaryl also refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-14 membered heteroaryl”).
  • heteroaryl groups that contain one or more nitrogen atoms
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system.
  • Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heteroaryl").
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heteroaryl").
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl").
  • the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl”) with one or more substituents.
  • the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.
  • Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6- membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7- membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6, 6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
  • heterocyclyl refers to a radical of a 3- to 14- membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-14 membered heterocyclyl").
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)). and can be saturated or can contain one or more carbon-carbon double or triple bonds.
  • Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • each instance of heterocyclyl is independently unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents.
  • the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heterocyclyl").
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heterocyclyl").
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl").
  • the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • the 5-6 membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
  • Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5- membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione.
  • Exemplary 5- membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5- membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
  • Exemplary 6 -membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1, 8- naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, lH-benzo[e][l,4-
  • Heterocycloalkyl is a non-aromatic ring wherein at least one atom is a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon.
  • the heterocycloalkyl group can be substituted or unsubstituted.
  • alkyl, alkenyl, alkynyl, acyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., "substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, "substituted” or 'unsubstituted” heteroalkynyl, "substituted” or “unsubstituted” carbocyclyl, "substituted” or “unsubstituted” heterocyclyl, "substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group.
  • substituted or unsubstituted
  • substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • substituted is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound.
  • the present invention contemplates any and all such combinations in order to arrive at a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • each instance of R aa is, independently, selected from C1-C50 alkyl, C 2 -C 5 o alkenyl, C 2 -C 5 o alkynyl, C 3 -Cio carbocyclyl, 3-14 membered heterocyclyl, Cg-Ci4 aryl, and 5-14 membered heteroaryl, or two R aa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;
  • halo or halogen refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).
  • a “counterion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality.
  • Exemplary counterions include halide ions (e.g., F-, Cl-, Br-, I-), NO 3 -, ClO 4 -, OH-, H 2 PO 4 -, HSO 4 -, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).
  • carboxylate ions e.g., acetate, ethanoate
  • Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms.
  • the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • Nitrogen protecting groups such as carbamate groups include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l-
  • Nitrogen protecting groups such as sulfonamide groups include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy- 4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchrornan-6-sulfonamide (Pmc), me
  • Ts p-toluenesulfonamide
  • Mtr 2,
  • nitrogen protecting groups include, but are not limited to, phenothiazinyl-(lO)- acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N' -phenylaminothioacyl derivative, N- benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N- phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1, 1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5-triazacyclohexan
  • the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group).
  • Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
  • L 1 is a carbonyl
  • L 1 is an amide. In embodiments, L 1 is -NHC(O)-. In embodiments, L 1 is -C(O)NH-
  • L 2 is C2-C10 alkylene. In embodiments, L 2 is unsubstituted C2-C10 alkylene. In embodiments, L 2 is substituted C2-C10 alkylene.
  • L 2 is C2-C10 alkenylene. In embodiments, L 2 is unsubstituted C2-C10 alkenylene. In embodiments, L 2 is substituted C2-C10 alkenylene.
  • Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3.
  • X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group.
  • each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkyl, C 4 -C 30 alkenyl, or C 4 - C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted. In embodiments, each of R 1 , R 2 , and R 3 is substituted (e.g., comprising 1-3 substituents as described herein).
  • each of R 1 , R 2 , and R 3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C 1 -C 6 alkyl.
  • B is independently , , , , [0220] In embodiments, B is independently , , , , [0221] In embodiments, B is independently , , , , , ,
  • B is independently H
  • B is independently Me
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently [0230] In embodiments, B is independently
  • B is independently
  • B is independently
  • B is independently [0234] In embodiments, B is independently
  • B is independently
  • B is independently [0239] In embodiments, B is independently [0240] In embodiments, B is independently
  • B is independently
  • B is independently
  • the invention features a cationic lipid having a structure according to
  • X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl;
  • A is a substructure selected from:
  • B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10;
  • L 1 is a carbonyl, ester, or amide
  • L 2 is C2-C10 alkylene or a C2-C10 alkenylene
  • Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3;
  • R 4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R 1 , R 2 , and R 3 is independently a C 4 -
  • C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • A is only substructure (a4) when each of R 1 , R 2 , and R 3 is independently
  • X is independently O.
  • X is independently NH.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 heteroalkyl (e.g., Cg-Cso heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted Cg-Cso heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 heteroalkyl (e.g., substituted Cg-Cso heteroalkyl).
  • B is independently an ionizable nitrogen-containing group.
  • B is independently a permanently charged nitrogen group.
  • n is an integer of 2-10.
  • n is an integer of 2-10.
  • L 1 is a carbonyl
  • L 1 is an ester. In embodiments, L 1 is -OC(O)-. In embodiments, L 1 is - (O)CO-.
  • L 1 is an amide. In embodiments, L 1 is -NHC(O)-. In embodiments, L 1 is -C(O)NH-
  • L 2 is C2-C10 alkylene. In embodiments, L 2 is unsubstituted C2-C10 alkylene. In embodiments, L 2 is substituted C2-C10 alkylene.
  • L 2 is C2-C10 alkenylene. In embodiments, L 2 is unsubstituted C2-C10 alkenylene. In embodiments, L 2 is substituted C2-C10 alkenylene.
  • Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3. [0265] In embodiments, R 4 is unsubstituted C1-C10 alkylene. Inembodiments, R 4 is substituted
  • X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C 4 - C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted. In embodiments, each of R 1 , R 2 , and R 3 is substituted (e.g., comprising 1-3 substituents as described herein).
  • each of R 1 , R 2 , and R 3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • B is independently H [0270] In embodiments, B is independently [0271] In embodiments, B is independently , , , ,
  • B is independently , , ,
  • B is independently H
  • B is independently
  • B is independently
  • B is independently
  • B is independently [0278] In embodiments, B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • the invention features a cationic lipid having a structure according to
  • X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl;
  • A is a substructure selected from: B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10;
  • L 1 is a carbonyl, ester, or amide
  • L 2 is C2-C10 alkylene or a C2-C10 alkenylene
  • Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3;
  • R 4 is C2-C10 alkylene.
  • A is only substructure (a4) when each of R 1 , R 2 , and R 3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or
  • X is independently O.
  • X is independently NH.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 heteroalkyl (e.g., Cg-Cso heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted Cg-Cso heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 heteroalkyl (e.g., substituted Cg-Cso heteroalkyl).
  • B is independently an ionizable nitrogen-containing group.
  • B is independently a permanently charged nitrogen group.
  • n is an integer of 2-10.
  • n is an integer of 2-10.
  • L 1 is a carbonyl
  • L 1 is an ester. In embodiments, L 1 is -OC(O)-. In embodiments, L 1 is - (O)CO-.
  • L 1 is an amide. In embodiments, L 1 is -NHC(O)-. In embodiments, L 1 is -C(O)NH-
  • L 2 is C2-C10 alkylene. In embodiments, L 2 is unsubstituted C2-C10 alkylene. In embodiments, L 2 is substituted C2-C10 alkylene.
  • L 2 is C2-C10 alkenylene. In embodiments, L 2 is unsubstituted C2-C10 alkenylene. In embodiments, L 2 is substituted C2-C10 alkenylene.
  • Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3.
  • R 4 is unsubstituted C2-C10 alkylene. In embodiments, R 4 is substituted C2-C10 alkylene.
  • X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C 4 - C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is unsubstituted. In embodiments, each of R 1 , R 2 , and R 3 is substituted (e.g., comprising 1-3 substituents as described herein).
  • each of R 1 , R 2 , and R 3 is independently a C4-C30 heteroalkyl comprising
  • . M ,.R 5 a disulfide bond or having a structure that is ° L , wherein L is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl; or L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • B is independently H , , ,
  • B is independently , , ,
  • B is independently , , ,
  • B is independently H
  • B is independently
  • B is independently
  • B is independently
  • B is independently [0328] In embodiments, B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently
  • B is independently [0339] In embodiments, B is independently . [0340] In embodiments, B is independently . [0341] In embodiments, B is independently . [0342] In embodiments, B is independently .
  • Formula (I) [0343] In embodiments, a compound has a structure according to Formula (I): or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , R 3 , B, m, and n is independently according to any combination of embodiments described herein.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl, C 6 -C 30 alkenyl, C 6 -C 30 alkynyl, or C 4 -C 30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C6-C30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C6-C30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently C6-C30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C 6 -C 30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C 6 -C 30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C6-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C6-C30 alkynyl. [0348] In embodiments, each of R 1 , R 2 , and R 3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C 4 -C 30 heteroalkyl (e.g., unsubstituted C 6 -C 30 heteroalkyl).
  • each of R 1 , R 2 , and R 3 is independently substituted C 4 -C 30 heteroalkyl (e.g., substituted C 6 -C 30 heteroalkyl).
  • B is independently an ionizable nitrogen containing group.
  • B is independently a permanently charged nitrogen containing group.
  • m is 2 and/or n is 2 or 3.
  • n is 2.
  • m is 2 and n is 3.
  • B is independently , , , , , [0355] In embodiments, B is independently , , , , , ,
  • B is independently ,
  • a compound is selected from the group consisting of: Compounds (1), (2), (3), (4), (6), (9), (17), (24), (26), (27), (33), and (35).
  • Formula (III) [0362]
  • a compound has a structure according to Formula (III): , or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , R 3 , R 4 , and B is independently according to any combination of embodiments described herein.
  • each of R 1 , R 2 , and R 3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; R 4 is C2-C10 alkylene; and B is independently an ionizable nitrogen-containing group.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C 6 -C 30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C6-C30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently C6-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C6-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C 6 -C 30 alkenyl. [0366] In embodiments, each of R 1 , R 2 , and R 3 is independently C6-C30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C 6 -C 30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C 6 -C 30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl).
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl).
  • each of R 1 , R 2 , and R 3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl).
  • R 4 is unsubstituted C 2 -C 10 alkylene.
  • R 4 is substituted C2-C10 alkylene.
  • R 4 is -CH2CH2-.
  • B is independently > > [0372] In embodiments, B is independently [0373] In embodiments, B is independently , , , ,
  • a compound is Compound (3).
  • Formula (IV) [0377] In embodiments, a compound has a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , R 3 , m, n, and B is independently according to any combination of embodiments described herein.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl, C 6 -C 30 alkenyl, C 6 -C 30 alkynyl, or C 4 -C 30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. [0379] In embodiments, each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C 6 -C 30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C 6 -C 30 alkyl.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C6-C30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C6-C30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently C6-C30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C 6 -C 30 alkynyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C 6 -C 30 alkynyl. [0382] In embodiments, each of R 1 , R 2 , and R 3 is independently C 4 -C 30 heteroalkyl (e.g., C 6 -C 30 heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl).
  • each of R 1 , R 2 , and R 3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl).
  • R 1 , R 2 , and R 3 is C 4 -C 30 heteroalkyl.
  • m is 2 and/or n is 2 or 3.
  • m is 2 and n is 2.
  • m is 2 and n is 3.
  • B is independently , , , , , [0388] In embodiments, B is independently , , , , [0389] In embodiments, B is independently , , , , , , ,
  • B is independently , , ,
  • a compound is Compound (8) or (39).
  • a compound has a structure according to Formula (VI): or a pharmaceutically acceptable salt thereof, wherein each of R 5 , L 3 , n, o, and B is independently according to any combination of embodiments described herein.
  • n is an integer of 2, 3, 4, 5, 6, or 7;
  • B is independently an ionizable nitrogen-containing group.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is Ci-Cg alkyl.
  • B is independently H , , , ,
  • B is independently , , ,
  • B is independently , , ,
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (112), (113), (114), (115), (116), (117), (126), (127), (128), and (129).
  • a compound has a structure according to Formula (V): or a pharmaceutically acceptable salt thereof, wherein each of R 5 , L 3 , n, o, and B is independently according to any combination of embodiments described herein.
  • n is an integer of 2, 3, or 4;
  • B is independently an ionizable nitrogen-containing group.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R 5 is C4-C24 alkyl.
  • L 3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R 5 is C1-C5 alkyl.
  • B is independently H , , , , ,
  • B is independently
  • B is independently [0411] In embodiments, B is .
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (114), and (116).
  • a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), and (40).
  • a compound has a structure according to Formula (VI): or a pharmaceutically acceptable salt thereof, wherein each of R 1 , R 2 , R 3 , n, and B is independently according to any combination of embodiments described herein.
  • each of R 1 , R 2 , and R 3 is independently C 6 -C 30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group.
  • B is independently , , , [0419] In embodiments, B is independently , , , [0420] In embodiments, B is .
  • a compound is Compound (21).
  • Formula (VII) [0422]
  • a cationic lipid has a formula according to Formula (VII), , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R 1 , R 2 , and R 3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; each m is an integer of 2-10; each L 4 is a carbonyl, ester, or amide; Z is –(CH 2 ) q1 –N–(CH 2 ) q2 –, wherein q1 and q2 are independently integers of 2-10; or Z is C 6 H 3 -Z 1 , wherein Z 1 is a carbonyl, ester, or amide that is covalently attached to the –CH 2 (CH 2 ) q B moiety; q is an integer of
  • each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkyl. [0424] In embodiments, each of R 1 , R 2 , and R 3 is independently C4-C30 alkenyl. In embodiments, each of R 1 , R 2 , and R 3 is independently unsubstituted C 4 -C 30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C 4 -C 30 alkenyl.
  • each of R 1 , R 2 , and R 3 is independently C 4 -C 30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C4-C30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently substituted C4-C30 alkynyl.
  • each of R 1 , R 2 , and R 3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl).
  • each of R 1 , R 2 , and R 3 is independently unsubstituted C 4 -C 30 heteroalkyl (e.g., unsubstituted C 6 -C 30 heteroalkyl). In embodiments, each of R 1 , R 2 , and R 3 is independently substituted C 4 -C 30 heteroalkyl (e.g., substituted C 6 -C 30 heteroalkyl).
  • L 4 is a carbonyl.
  • L 4 is an ester.
  • L 4 is –OC(O)–.
  • L 4 is – (O)CO–.
  • L 4 is an amide.
  • L 4 is –NHC(O)–. In embodiments, L 4 is -C(O)NH–.
  • X is O; each L 4 is –C(O)O–; m is an integer of 2, 3, or 4; and/or q is 1.
  • Z is –(CH2)q1–N–(CH2)q2–, and q1 and q2 are each 2.
  • B is independently , , , [0436] In embodiments, B is independently , , , , ,
  • a compound is Compound (16) or (31).
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of intranasal delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of intranasal delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
  • a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
  • Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition with the nasal mucosa, nasal turbinates or sinus cavity.
  • the pharmaceutical compositions for administration may be applied in a single administration or in multiple administrations. For example, one dose can be placed in each nostril during administration.
  • bi-dose delivery can be used with the compositions according to the invention.
  • Bi-dose devices contain two sub-doses of a single dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time.
  • a mono-dose device may be used for administering the compositions according to the invention.
  • the composition can be given in one, two, three, four, or more doses, so that the subject is given a first dose (which can be a bi-dose or mono-dose, as described above), and then a second dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days, or 4 ,5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years apart.
  • Exemplary devices for intranasal administration of the compositions according to the invention are spray devices.
  • Suitable commercially available nasal spray devices include AccusprayTM (Becton Dickinson). Nebulizers produce a very fine spray (such as a mist) which can be easily inhaled and are also contemplated herein.
  • Exemplary spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art.
  • the invention provides in a further aspect a pharmaceutical kit comprising an intranasal administration device as described herein containing a formulation according to the invention.
  • compositions according to the invention may be administered in other forms e.g. as a powder.
  • a cationic lipid is selected from the group of Compounds 41-64 and
  • a cationic lipid is selected from the group of Compounds 91-111 or a pharmaceutically acceptable salt thereof:
  • a cationic lipid is selected from the group of Compounds 112-117 and 126-129, or a pharmaceutically acceptable salt thereof:
  • a cationic lipid is selected from the group of Compounds 118-125 or a pharmaceutically acceptable salt thereof:
  • the invention features a cationic lipid according to Formula (B). or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X 1A is independently O or NR 1A ; R 1A is H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X 2A and X 2B is O and the other is a covalent bond; one of X 3A and X 3B is O and the other is a covalent bond; one of X 4A and X 4B is O and the other is a covalent bond; R 1 is independently L 1 -B 1 , C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R 2 is independently L 2 -B 2 , C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R 2 is independently L 2
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition
  • a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B) as described herein, or a pharmaceutically acceptable salt thereof.
  • the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B) as described herein, or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (B) is selected from the group consisting of Compounds (65)-(89), or a pharmaceutically acceptable salt thereof:
  • a compound of Formula (B) is selected from the group consisting of Compounds (65), (66), (67), (70), (71), (74), (75), (77), (81), (84), (87), (88), and (89).
  • a compound of Formula (B) is selected from the group consisting of Compounds (68), (69), (72), (73), (76), (78), (79), (80), (82), (83), (85), and (86). [0456] In embodiments, a compound of Formula (B) excludes Compounds (68), (69), (72), (73),
  • R2, R3, and R 4 are straight-chain Cg alkylene.
  • Li is straight-chain C 5 alkylene
  • R2, R3, and R 4 are as defined for Formula (B) [0459]
  • a compound of Formula (B) or (Bl) is selected from the group consisting of Compounds (118)-(125), or a pharmaceutically acceptable salt thereof:
  • Exemplary compounds of the invention include those cationic lipids described in Table 1.
  • a cationic lipid is Compound (1) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (2) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (3) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (4) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (6) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (8) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (9) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (11) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (12) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (13) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (14) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (16) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (17) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (21) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (22) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (24) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (25) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (26) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (27) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (28) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (29) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (30) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (31) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (32) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (33) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (34) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (35) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (36) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (38) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (39) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (40) or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds of the invention include those cationic lipids described in Table
  • a cationic lipid is Compound (41) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (42) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (43) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (44) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (45) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (46) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (47) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (48) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (49) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (50) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (51) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (52) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (53) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (54) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (55) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (56) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (57) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (58) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (59) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (60) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (61) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (62) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (63) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (64) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (90) or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds of the invention include those cationic lipids described in Table
  • a cationic lipid is Compound (65) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (66) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (67) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (68) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (69) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (70) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (71) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (72) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (73) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (74) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (75) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (76) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (77) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (78) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (79) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (80) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (81) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (82) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (83) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (84) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (85) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (86) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (87) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (88) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (89) or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds of the invention include those cationic lipids described in Table 9.
  • a cationic lipid is Compound (91) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (92) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (93) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (94) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (95) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (96) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (97) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (98) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (99) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (100) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (101) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (102) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (103) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (104) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (105) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (106) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (107) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (108) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (109) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (110) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (111) or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds of the invention include those cationic lipids described in Table
  • a cationic lipid is Compound (112) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (113) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (114) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (115) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (116) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (117) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (126) or a pharmaceutically acceptable salt thereof.
  • a cationic lipid is Compound (127) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (128) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (129) or a pharmaceutically acceptable salt thereof.
  • Exemplary compounds of the invention include those cationic lipids described in Table
  • a cationic lipid is Compound (118) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (119) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (120) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (121) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (122) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (123) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (124) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (125) or a pharmaceutically acceptable salt thereof.
  • compositions comprising any compound of the invention include compositions comprising the cationic lipid of any one of the preceding embodiments, one or more non-cationic lipids, one or more cholesterol-based lipids, and/or one or more PEG- modified lipids.
  • this composition is a lipid nanoparticle.
  • the one or more cationic lipid(s) constitute(s) about 30 mol %-60 mol % of the lipid nanoparticle.
  • the one or more non-cationic lipid(s) constitute(s) 10 mol%-50 mol% of the lipid nanoparticle.
  • the one or more PEG-modified lipid(s) constitute(s) 1 mol%-10 mol% of the lipid nanoparticle.
  • the cholesterol-based lipid constitutes 10 mol%- 50 mol% of the lipid nanoparticle.
  • the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 50%.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 55%.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 60%.
  • the lipid nanoparticles have an encapsulation percentage for mRNA of at least 65%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 70%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 75%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 80%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 85%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 90%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 95%.
  • composition of any one of the proceeding embodiments is for use in therapy.
  • the composition of any one of the proceeding embodiments is for use in a method of treating or preventing a disease amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
  • the composition is administered intravenously, intrathecally or intramuscularly, or by pulmonary delivery, optionally through nebulization or use of an inhaler (e.g., a metered dose inhaler, a dry powder inhaler, or a soft mist inhaler).
  • an inhaler e.g., a metered dose inhaler, a dry powder inhaler, or a soft mist inhaler.
  • Mucosal routes of administration such as oral and intransal administration can elicit immune responses at both the local and distal mucosal sites. Such routes of administration can also give rise to systemic immune responses.
  • routes of administration such as intransal when administering the compositions of the present invention particularly with a view to generating protective immunity.
  • the methods and compositions of the invention may induce mucosal immunity in a subject.
  • the methods and compositions of the invention may induce mucosal immunity at distal mucosal sites.
  • the methods and compositions of the invention may generate protective mucosal immunity.
  • protection mucosal immunity is meant immunity or an immune response against an infectious agent which is exhibited by a subject, whereby said immunity or immune response prevents or ameliorates an infection or reduces one or more symptoms thereof.
  • the compositions and methods described herein may elicit an antibody response in a subject.
  • said antibody may be IgA.
  • said antibody may be IgG.
  • compositions and methods described herein may enable a subject to produce an CD4 memory T cell population.
  • the compositions and methods described herein may induce a CD4 memory T cell population in a subject, optionally wherein said population is induced in the mucosa.
  • compositions useful for the delivery of nucleic acids can be used to prepare compositions useful for the delivery of nucleic acids.
  • Nucleic acids according to the present invention may be synthesized according to any known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, mutated T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7, mutated T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7, mutated T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
  • a desired amino acid sequence e.g., an enzyme sequence
  • Optimization algorithms may then be used for selection of suitable codons.
  • the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency
  • mRNA according to the present invention may be synthesized as unmodified or modified mRNA.
  • Modified mRNA includes nucleotide modifications in the RNA.
  • a modified mRNA according to the invention can thus include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications.
  • mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g., 1-methyl-adenine, 2-methyl-adenine, 2- methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio- cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl- guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl
  • the compounds of the invention as described herein, as well as pharmaceutical and liposomal compositions comprising such lipids can be used in formulations to facilitate the delivery of encapsulated materials (e.g., one or more polynucleotides such as mRNA) to, and subsequent transfection of one or more target cells.
  • encapsulated materials e.g., one or more polynucleotides such as mRNA
  • cationic lipids described herein are characterized as resulting in one or more of receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and 187ncapsulel87ytosis, fusogenicity, endosomal or lysosomal disruption and/or releasable properties that afford such compounds advantages relative other similarly classified lipids.
  • a nucleic acid e.g., mRNA encoding a protein (e.g., a full length, fragment or portion of a protein) as described herein may be delivered via a delivery vehicle comprising a compound of the invention as described herein.
  • delivery vehicle As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle,” or grammatical equivalents thereof, are used interchangeably.
  • the present invention provides a composition (e.g., a pharmaceutical composition) comprising a compound described herein and one or more polynucleotides.
  • a composition e.g., a pharmaceutical composition
  • a composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells.
  • Such methods generally comprise the step of contacting the one or more target cells with the cationic lipids and/or pharmaceutical compositions disclosed herein (e.g., a liposomal formulation comprising a compound described herein encapsulating one or more polynucleotides) such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides).
  • the terms "transfect” or “transfection” refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell (e.g., into a target cell).
  • the introduced polynucleotide may be stably or transiently maintained in the target cell.
  • transfection efficiency refers to the relative amount of such encapsulated material (e.g., polynucleotides) taken up by, introduced into, and/or expressed by the target cell which is subject to transfection. In practice, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection.
  • the compounds and pharmaceutical compositions described herein demonstrate high transfection efficiencies thereby improving the likelihood that appropriate dosages of the encapsulated materials (e.g., one or more polynucleotides) will be delivered to the site of pathology and subsequently expressed, while at the same time minimizing potential systemic adverse effects or toxicity associated with the compound or their encapsulated contents.
  • the encapsulated materials e.g., one or more polynucleotides
  • the production of the product (e.g., a polypeptide or protein) encoded by such polynucleotide may be stimulated and the capability of such target cells to express the polynucleotide and produce, for example, a polypeptide or protein of interest is enhanced.
  • transfection of a target cell by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.
  • delivery vehicles described herein may be prepared to preferentially distribute to other target tissues, cells or organs, such as the heart, lungs, kidneys, spleen.
  • the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to the target cells and tissues.
  • polynucleotides e.g., mRNA
  • encapsulated in one or more of the compounds or pharmaceutical and liposomal compositions described herein can be delivered to and/or transfect targeted cells or tissues.
  • thel89ncapsuleated polynucleotides are capable of being expressed and functional polypeptide products produced (and in some instances excreted) by the target cell, thereby conferring a beneficial property to, for example the target cells or tissues.
  • Such encapsulated polynucleotides may encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.
  • a composition is a suitable delivery vehicle.
  • a composition is a liposomal delivery vehicle, e.g., a lipid nanoparticle.
  • liposomal delivery vehicle and “liposomal composition” are used interchangeably.
  • Enriching liposomal compositions with one or more of the cationic lipids disclosed herein may be used as a means of improving (e.g., reducing) the toxicity or otherwise conferring one or more desired properties to such enriched liposomal composition (e.g., improved delivery of the encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of a liposomal composition).
  • the compounds of the invention as described herein may be used as a component of a liposomal composition to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).
  • encapsulated materials e.g., one or more therapeutic agents
  • target cells e.g., by permeating or fusing with the lipid membranes of such target cells.
  • liposomal delivery vehicles e.g., lipid nanoparticles
  • lipid nanoparticles are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).
  • Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
  • compositions e.g., liposomal compositions
  • are loaded with or otherwise encapsulate materials such as for example, one or more biologically-active polynucleotides (e.g., mRNA).
  • a composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein, encapsulated within a liposome.
  • a liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol- based lipids and one or more PEG-modified lipids, and wherein at least one cationic lipid is a compound of the invention as described herein.
  • a composition comprises an mRNA encoding for a protein (e.g., any protein described herein).
  • a composition comprises an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein.
  • a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a compound described herein.
  • a nucleic acid is an mRNA encoding a peptide or protein.
  • an mRNA encodes a peptide or protein for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • an mRNA encodes a peptide or protein for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein).
  • OTC ornithine transcarbamylase
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net positive charge e.g., a lipid nanoparticle
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net negative charge e.g., a net negative charge
  • a liposomal delivery vehicle e.g., a lipid nanoparticle
  • a net neutral charge e.g., a lipid nanoparticle
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more compounds of the invention as described herein.
  • the amount of a compound of the invention as described herein in a composition can be described as a percentage ("wt%") of the combined dry weight of all lipids of a composition (e.g., the combined dry weight of all lipids present in a liposomal composition).
  • a compound of the invention as described herein is present in an amount that is about 0.5 wt% to about 30 wt% (e.g., about 0.5 wt% to about 20 wt%) of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition).
  • a compound of the invention as described herein is present in an amount that is about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition).
  • a compound of the invention as described herein is present in an amount that is about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in a composition such as a liposomal delivery vehicle.
  • the amount of a compound of the invention as described herein is present in an amount that is at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
  • a composition e.g., a liposomal composition
  • the amount of a compound of the invention as described herein is present in an amount that is no more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
  • a composition e.g., a liposomal composition
  • a composition e.g., a liposomal delivery vehicle such as a lipid nanoparticle
  • a delivery vehicle comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of a compound described herein.
  • a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of a compound described herein.
  • the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
  • the amount of a compound of the invention as described herein in a composition also can be described as a percentage ("mol%") of the combined molar amounts of total lipids of a composition (e.g., the combined molar amounts of all lipids present in a liposomal delivery vehicle).
  • a compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%) of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • a compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol %, or about 45 mol% to about 60 mol% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
  • a compound of the invention as described herein is present in an amount that is about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol% or about 5 mol% to about 25 mol% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle
  • a compound of the invention as described herein can comprise from about 0.1 mol% to about 50 mol%, or from 0.5 mol% to about 50 mol%, or from about 1 mol% to about 50 mol%, or from about 5 mol% to about 50 mol%, or from about 10 mol% to about 50 mol%, or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol%, of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).
  • a composition e.g., a liposomal delivery vehicle
  • a compound of the invention as described herein can comprise greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol% of the total amount of lipids in the lipid nanoparticle.
  • a compound as described can comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol %, less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).
  • a composition e.g., a liposomal delivery vehicle
  • the amount of a compound of the invention as described herein is present in an amount that is at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined molar amounts of total lipids in a composition (e.g., a liposomal composition).
  • a composition e.g., a liposomal composition
  • the amount of a compound of the invention as described herein is present in an amount that is no more than about 5 mol%, about 10 mol%, about 15 mol%, about
  • composition e.g., a liposomal composition
  • the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
  • a composition of the invention (e.g., a liposomal composition) comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol- based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the invention as described herein.
  • a composition suitable for practicing the invention has four lipid components comprising a compound of the invention as described herein as the cationic lipid component, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid.
  • the non-cationic lipid may be DOPE or DEPE.
  • the cholesterol-based lipid may be cholesterol.
  • the PEG-modified lipid may be DMG-PEG2K.
  • pharmaceutical (e.g., liposomal) compositions comprise one or more of a PEG-modified lipid, a non-cationic lipid and a cholesterol lipid.
  • such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids.
  • such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids and one or more cholesterol lipids.
  • a composition e.g., lipid nanoparticle
  • a nucleic acid e.g., mRNA encoding a peptide or protein
  • lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.
  • a composition e.g., lipid nanoparticle
  • a nucleic acid e.g., mRNA encoding a peptide or protein
  • lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein)
  • a nucleic acid e.g., mRNA encoding a peptide or protein
  • lipids selected from the group consisting of a cationic lipid, a noncationic lipid, and a PEGylated lipid
  • further comprises a cholesterol-based lipid e.g., lipid nanoparticle
  • such a composition has four lipid components comprising a compound of the invention as described herein as the cationic lipid component, a non-cationic lipid (e.g., DOPE), a cholesterol- based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K).
  • a non-cationic lipid e.g., DOPE
  • a cholesterol- based lipid e.g., cholesterol
  • PEG-modified lipid e.g., DMG-PEG2K
  • a lipid nanoparticle that encapsulates a nucleic acid comprises one or more compounds of the invention as described herein, as well as one or more lipids selected from the group consisting of a cationic lipid, a noncationic lipid, a PEGylated lipid, and a cholesterol-based lipid.
  • the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
  • lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art.
  • suitable methods include methods described in International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety.
  • Process A relates to a conventional method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles.
  • an ethanol lipid solution and an aqueous buffered solution of mRNA are prepared separately.
  • a solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) is prepared by dissolving lipids in ethanol.
  • the mRNA solution is prepared by dissolving the mRNA in citrate buffer, resulting in mRNA at a concentration of 0.0833mg/ml in citrate buffer with a pH of 4.5. Then, these two solutions are mixed using a pump system. In some instances, the two solutions are mixed using a gear pump system. In certain embodiments, the two solutions are mixing using a T junction (or "Y" junction). The mixture is then purified by diafiltration with a TFF process. The resultant formulation is concentrated and stored at 2-8 °C until further use.
  • a second exemplary process for lipid nanoparticle formulation is Process B of WO 2018/089801 (see, e.g., Example 2 and Figure 2 of WO 2018/089801).
  • Process B (“B”) refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA.
  • mRNA messenger RNA
  • a range of different conditions, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations, may be employed in Process B.
  • lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process.
  • the resultant formulation mixture comprises empty lipid nanoparticles in citrate buffer containing alcohol.
  • the formulation is then subjected to a TFF purification process wherein buffer exchange occurs.
  • the resulting suspension of preformed empty lipid nanoparticles is then mixed with mRNA using a pump system.
  • heating the solution post-mixing can result in a higher percentage of lipid nanoparticles containing mRNA and a higher total yield of mRNA.
  • a composition may comprise one or more additional cationic lipids.
  • liposomes may comprise one or more additional cationic lipids.
  • cationic lipid refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.
  • Suitable additional cationic lipids for use in the compositions include the cationic lipids as described in the literature.
  • compositions may also comprise one or more helper lipids.
  • helper lipids include non-cationic lipids.
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl -phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dim
  • a noncationic or helper lipid suitable for practicing the invention is dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • DEPE l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • the percentage of non-cationic lipid in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%.
  • the percentage of non-cationic lipid in a liposome is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.
  • a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition.
  • the percentage of non-cationic lipid in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total noncationic lipids in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%.
  • the percentage of non-cationic lipid in a liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.
  • the percentage total non-cationic lipids in a liposome may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.
  • a composition (e.g., a liposomal composition) comprises one or more cholesterol-based lipids.
  • a suitable cholesterol-based lipid for practicing the invention is cholesterol.
  • Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE), which has the following structure,
  • a cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.
  • a cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%.
  • the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.
  • a composition (e.g., a liposomal composition) comprises one or more further PEGylated lipids.
  • a suitable PEG-modified or PEGylated lipid for practicing the invention is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).
  • PEG-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1- [succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of compounds of the invention as described herein and, in some embodiments, other lipids together which comprise the liposome.
  • particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C M or Cig).
  • Contemplated further PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of Cg-Czo length.
  • a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235- 237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
  • PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, or about 3% to about 5% of the total lipid present in the composition (e.g., a liposomal composition).
  • compositions e.g., to construct liposomal compositions
  • encapsulated materials e.g., one or more therapeutic polynucleotides
  • target cells e.g., by permeating or fusing with the lipid membranes of such target cells
  • a mRNA encodes a polypeptide.
  • a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein.
  • OTC ornithine transcarbamylase
  • the route of delivery used in the methods of the invention allows for non-invasive, selfadministration of the compounds of the invention.
  • the methods involve intratracheal or pulmonary administration by aerosolization, nebulization, or instillation of a compositions comprising mRNA encoding a therapeutic protein in a suitable transfection or lipid carrier vehicles as described above.
  • the protein is encapsulated with a liposome.
  • the liposome comprises a lipid, which is a compound of the invention.
  • administration of a compound of the invention includes administration of a composition comprising a compound of the invention.
  • the utility of the compounds of the invention and methods of the invention extend beyond production of therapeutic protein in lung cells and tissues of the lung and can be used to delivery to non-lung target cells and/or tissues. They are useful in the management and treatment of a large number of diseases, and in particular peripheral diseases which result from both secreted and non-secreted protein and/or enzyme deficiencies (e.g., one or more lysosomal storage disorders).
  • the compounds of the invention, used in the methods of the invention result in the distribution of the mRNA encapsulated nanoparticles and production of the encoded protein in the liver, spleen, heart, and/or other non- lung cells.
  • both the local cells of the lung and the peripheral non-lung cells can serve as biological reservoirs or depots capable of producing and/or secreting a translation product encoded by one or more polynucleotides.
  • the present invention is not limited to the treatment of lung diseases or conditions, but rather can be used as a non-invasive means of facilitating the delivery of polynucleotides, or the production of enzymes and proteins encoded thereby, in peripheral organs, tissues and cells (e.g., hepatocytes) which would otherwise be achieved only by systemic administration.
  • Exemplary peripheral non- lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
  • the protein product may be detectable in the peripheral target tissues at a concentration (e.g., a therapeutic concentration) of at least 0.025-1.5 pg/ml (e.g., at least 0.050 pg/ml, at least 0.075 pg/ml, at least 0.1 pg/ml, at least 0.2 pg/ml, at least 0.3 pg/ml, at least 0.4 pg/ml, at least 0.5 pg/ml, at least 0.6 pg/ml, at least 0.7 pg/ml, at least 0.8 pg/ml, at least 0.9 pg/ml, at least 1.0 pg/ml, at least 1.1 pg/ml, at least 1.2 pg/ml, at least 1.3 pg/ml, at least 1.4 pg/ml, or at least 1.5 pg/ml), for at least about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10,
  • nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of an aerosol mist produced by a liquid nebulizer or the use of a dry powder apparatus such as that described in U.S. patent 5,780,014, incorporated herein by reference.
  • the compounds of the invention may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or upon administration to the subject. Such compounds may be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject.
  • suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject.
  • such devices facilitate the administration of a predetermined mass, volume or dose of the compositions (e.g., about 0.5 mg/kg of mRNA per dose) to the subject.
  • a predetermined mass, volume or dose of the compositions e.g., about 0.5 mg/kg of mRNA per dose
  • the compounds of the invention are administered to a subject using a metered dose inhaler containing a suspension or solution comprising the compound and a suitable propellant.
  • the compounds of the invention may be formulated as a particulate powder (e.g., respirable dry particles) intended for inhalation.
  • compositions of the invention formulated as respirable particles are appropriately sized such that they may be respirable by the subject or delivered using a suitable device (e.g., a mean D50 or D90 particle size less than about 500pm, 400pm, 300pm, 250pm, 200pm, 150pm, 100pm, 75pm, 50pm, 25pm, 20pm, 15pm, 12.5pm, 10pm, 5pm, 2.5pm or smaller).
  • a suitable device e.g., a mean D50 or D90 particle size less than about 500pm, 400pm, 300pm, 250pm, 200pm, 150pm, 100pm, 75pm, 50pm, 25pm, 20pm, 15pm, 12.5pm, 10pm, 5pm, 2.5pm or smaller.
  • the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).
  • the compounds of the invention are administered to a subject such that a concentration of at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, at least 5.0 mg/kg, at least 6.0 mg/kg, at least 7.0 mg/kg, at least 8.0 mg/kg, at least 9.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, or at least 100 mg/kg body weight is administered in
  • the compounds of the invention are administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg mRNA is administered in one or more doses.
  • DIPEA N,N-Diisopropylethylamine
  • reaction mixture was quenched by cold saturated NaHCO3 upto pH 7, and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO 2 : 0-20 % ethyl acetate in hexane), to give the desired 6-hydroxyhexyl pentanoate [C-3] (6.2 g, 36 %, Yield) as a light yellow oil.
  • JC-TL1-16D-E5-9 (Compound (29)) tricarboxylate [D-5] (0.7 g, 0.00078 mol) and 3-(dimethylamino)propanoic acid [D-6] (0.36 g, 0.00312 mol) in dichloromethane (20 mL) was cooled to 0 o C, EDC.HCl (0.59 g, 0.00312 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.09 g, 0.00078 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (10 mL) was added to the reaction mixture and extracted with DCM (3x25 mL).
  • reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL) and dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [F-3] (5.1 g, 32.23%) as yellow liquid.
  • Reaction mass was diluted with DCM (25.0 mL), washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain desired product TL1- 14D-E8-4 (0.30 g, 25%) as yellow colour liquid.
  • reaction mass was quenched with DM water (100 ml), extracted with DCM (2x 50.0 mL), dried over sodium sulphate and concentrated. Crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [TL1-16D-E6-8] 0.250 g, (32.25%) as yellow colour liquid mass.
  • reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water 01 L, dichloromethane layer was separated and dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain [H-3] 5.2 g, (29.42%) as yellow colour liquid mass.
  • TL1-16D-E8-6 (Compound (54)) [0627] To a stirred solution tris(8-(heptanoyloxy)octyl) 2-hydroxypropane-1,2,3-tricarboxylate [H-5] (0.820 g, 0.00897 mol) and N,Ndimethylpropionic acid [Int-6] (0.420 g, 0.00359 mol) in dichloromethane (50 ml ), was added EDCI (0.690 g, 0.00359 mol,) followed by addition of DMAP (0.109g, 0.000897 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed).
  • reaction mass was washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 20 % ethyl acetate in n-heptane to obtain desired product [TL1-16D-E8-6] 0.250 g, (27.77%) as yellow colour liquid mass.
  • reaction mass was allowed to stirred at RT for 16 h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane. Obtained pure wt 7.3 g of I-3, (30.12%) as yellow colour liquid mass.
  • Reaction mass was stirred for 05 minute then added DMAP (4.17 g, 0.034 mol) followed by nonanoyl chloride (6.0 g, 0.034 mol) drop wise. Resulting reaction mass was allowed to stirred at RT for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get [J-3] (5.2 g, 26.55%) as yellow colour liquid mass.
  • reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water 20 ml, dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [TL1-12D-E8-2] (Compound ( (0.300 g, 25%) as yellow colour liquid.
  • reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate and concentrated under reduced pressure. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [M-3] (17.0 g, 60.71%) as yellow colour liquid.
  • reaction mixture was washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 30% ethyl acetate in n-heptane to get desired product [TL1-14D-E5-7-2i] 1.2 g, (44.44%) as yellow colour liquid mass.
  • Reaction mixture was stirred for 16 hr at r.t. under nitrogen atmosphere. Reaction progress monitored by TLC / ELSD, after consumption of starting material reaction mass diluted with water and dichloromethane. Organic layer was separated, dried over sodium sulphate. Solvent was distilled out under reduced pressure. Crude was purified with column chromatography using 15-20% ethyl acetate, to get compound O-5 (0.9 g; yield: 83.42 %) as a light yellow colour less liquid.
  • Reaction mixture was allowed to stirred at RT for 18 h. Progress of reaction was monitored by ELSD / TLC. Reaction mixture was diluted with cold water (500.0 mL) and extracted with dichloromethane (3x 100 ml). Combined organic layer was washed with fresh water (2x 100 mL) and brine solution (2x 100.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude was purified with column chromatography using 10% Ethyl acetate in Hexanes, to give 5-hydroxypentyl 7-methyloctanoate [Int-3] (12 g, 49.1 mmol) as colourless clear liquid.
  • Reaction mixture was allowed to stirred at 25°C for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with cold water (100.0 mL) and extracted with dichloromethane (3x 100 mL). Combined organic layer was washed with fresh water (2x 50 mL) and brine solution (2x 50.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure.
  • TL1-12D-011 [0744] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2- ⁇ [3-( ⁇ 2-[(tert- butyldimethylsilyl)oxy]ethyl ⁇ (methyl)amino)propanoyl]oxy ⁇ propane-1,2,3-tricarboxylate [U-12] (0.4 g, 373 ⁇ mol) in tetrahydrofuran (10 mL, 123 mmol), hydrogen pyridine fluoride (202 ⁇ L, 2.24 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [V-3] (16 g, 69.5 mmol) as light yellow colour liquid.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [V-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [W-3] (16 g, 69.5 mmol) as light yellow colour liquid.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [W-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • reaction mass was diluted with 25.0 mL of DCM and washed with water (25.0 mL). Organic layer was dried over anhy sodium sulphate and concentrated. Crude was purified over silica using 3% MeOH in DCM to obtain pure product [TL1-12D-028] (Compound (50) (120 mg, 125 ⁇ mol) as brown colour mass.
  • Trifluoroacetic acid (4.98 mL, 65.1 mmol) was added into a solution of 1,2,3-tris[5- (octanoyloxy)pentyl] 2-[(3- ⁇ [(tert-butoxy)carbonyl](methyl)amino ⁇ propanoyl)oxy]propane-1,2,3- tricarboxylate [X-10] (3 g, 2.96 mmol) in dichloromethane (40 mL, 625 mmol) at 0 o C, allow to stirred for until reaction completion. The progress of reaction was monitored by TLC. After completion the reaction, reaction mixture was concentrated under reduced pressure to remove excess trifluoroacetic acid.
  • reaction mass was cooled, filter by sintered glass funnel.
  • the filtrate was concentrated under reduced pressure and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired 1,2,3-tris[5- (octanoyloxy)pentyl] 2- ⁇ [3-( ⁇ 3-[(tert- butyldimethylsilyl)oxy]propyl ⁇ (methyl)amino)propanoyl]oxy ⁇ propane-1,2,3-tricarboxylate [X-12] (1.0 g, 56 % yield) as pale yellow liquid.
  • Reaction mass cooled to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with ethyl acetate (200 ml). Organic layer was washed with brine solution (500.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure, get crude. Crude was purified with column chromatography using 5-10% Ethyl acetate in Hexanes, to give compound Y-3 (16 g, 69.5 mmol) as light yellow colour liquid.
  • Reaction mixture was stirred for 16 hr at RT under inert atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (100.0 mL) and dichloromethane (2x 50.0 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure. Crude was purified with flash chromatography using 10-15% ethyl acetate in hexane to give compound Y-5 (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • reaction mass was stirred over night at RT. Reaction progress was monitored by ELSD. Reaction mixture was diluted with water (50.0 mL) and extracted with DCM (50 ml). Organic layer was washed with brine solution (500.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure to get compound Y-7 (0.2 g, 50.5 ⁇ mol) as off white semi solid crude. Which was proceeded to next step without further purification.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [Z-3] (16 g, 69.5 mmol) as light yellow colour liquid.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [Z-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • Reaction mass was stirred for 36 h at RT. Reaction progress was monitored by TLC and ELSD. Reaction mass was diluted with DCM (100.0 mL) and washed with water (2x 50.0 mL). Organic layer was separated and dried with sodium sulphate and distil out under reduced pressure.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [AA-3] (16 g, 69.5 mmol) as light yellow colour liquid.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [AA-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • Reaction mass was stirred for 36 h at RT. Reaction progress was monitored by TLC and ELSD. Reaction mass was diluted with DCM (100.0 mL) and washed with water (2x 50.0 mL). Organic layer was separated and dried with sodium sulphate and distil out under reduced pressure.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [BB-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure.
  • Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [CC-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid.
  • Reaction mass was diluted with DCM (25.0 mL) and washed with DM water (50 ml) and dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10 % ethyl acetate in n-heptane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(7-bromoheptanoyl)oxy]propane-1,2,3-tricarboxylate [Int-7] (0.4 g, 392 ⁇ mol) as yellow gummy mass.
  • Example 31 Synthesis of Compound (41): Synthetic Scheme EE-1 Procedure [0833] To a 100 mL RBF containing the aliphatic alcohol (2.24 g, 14.2 mmol) was added citric acid (816 mg, 4.25 mmol) and TsOH (269 mg, 1.42 mmol). The solids were then washed down with toluene (10 mL) and heated at 110 o C overnight equipped with Dean-Stark apparatus. The remaining residue was slightly diluted with hexanes and loaded onto a large silica gel column. Purification using 0% to 20% EtOAc in hexanes afforded the product EE-3A as clear light-yellow oil (2.20 g, 85% yield).
  • Example 38 Synthesis of Compound (34): Synthetic Scheme LL 5-hydroxypentyl decanoate (MM-3) [0852] To a solution of pentane-1,5-diol (MM-1) (2.5 mL, 24.00 mmol) in tetrahydrofuran (40 mL) was added decanoyl chloride (2) (2.5 mL, 12.00 mmol) at 0 o C. To that was slowly added triethylamine (1.67 mL, 12.00 mmol) over 10 minutes, then reaction mixture was warmed to room temperature and stirred for 16 h. MS analysis indicated completion of the reaction. The reaction mixture suspension was diluted with dichloromethane washed with sat.
  • the reaction mixture was diluted with dichloromethane washed with sat. NaHCO3 solution, water and brine. The organic layer was dried over Na2SO4 and concentrated. The crude residue was purified to get the desired mono-product (3.83 g, 58%).
  • the reaction mixture was stirred at room temperature for 16h.
  • the reaction was diluted with water (100 mL), its organic layer was separated, and aqueous layer was extracted with dichloromethane 2 ⁇ 100 mL. Combined organic layer was washed with brine solution (100 mL), dried over sodium sulphate, and concentrated under reduced pressure.
  • the resulting crude was purified over silica using 5-10% ethyl acetate in n-heptane as eluent system to give product hexyl 7-hydroxyheptanoate [OO-3] (2.1 g, Yield – 29.62%) as a colourless liquid.
  • reaction mass was stirred for 15 min at room temperature and Hexyl 7-hydroxyheptanoate [OO-3] (16.8 g, 3.5 eq., 72.9 mmol) was added in to reaction mixture.
  • Reaction mixture was stirred for 48 h at room temperature.
  • the resulting reaction mixture was quenched with water (500 mL), organic layer was separated, and aqueous layer was extracted with dichloromethane 2 ⁇ 100 mL.
  • the resulting organic layer was combined, dried over Na2SO4 and concentrated under reduced pressure.
  • the resulting crude material was purified by silica using 10-20% ethyl acetate in n-heptane as eluent system.
  • reaction mass was diluted with ethyl acetate, filtered through celite and washed with ethyl acetate to get crude product.
  • the crude was purified by flash column chromatography (SiO 2 : 0-30% Ethyl acetate in Hexanes), to give the desired [PP-7] (2.0 g, 18.7% yield) as yellow liquid.
  • TL1-12D-024 (Compound 100): [0875] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [PP-7] (140 mg, 159 ⁇ mol) in tetrahydrofuran (5 mL, 61.4 mmol) was added 1- methylpiperazine [PP-8] (15.9 mg, 159 ⁇ mol). Reaction mass was stirred at RT for 48 h.

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Abstract

The present invention provides, in part, tricine and citric acid lipids, or a pharmaceutically acceptable salt thereof. Compounds provided herein include Compounds of Formula (A) and can be useful for delivery and expression of mRNA and encoded protein, e.g., as a component of liposomal delivery vehicle, and accordingly can be useful for treating various diseases, disorders and conditions, such as those associated with deficiency of one or more proteins.

Description

TRICINE AND CITRIC ACID-BASED CATIONIC LIPIDS RELATED APPLICATIONS This application claims priority to European application no. EP23305930.2 filed on 12th June 2023, European application no. EP23306900.4 filed on 2nd November 2023, and European application no. EP23306902.0 filed on 2nd November 2023, the entire disclosures of which are hereby incorporated by reference. BACKGROUND [001] Delivery of nucleic acids has been explored extensively as a potential therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for treatment of various diseases, including for those associated with deficiency of one or more proteins. SUMMARY OF THE INVENTION [002] The present invention provides, among other things, a novel class of tricine and citric acid-based cationic lipid compounds for improved in vivo delivery of therapeutic agents, such as nucleic acids. It is contemplated that the compounds provided herein can be capable of highly effective in vivo delivery while maintaining a favorable toxicity profile. It is contemplated that the compounds provided herein can be capable of highly effective intranasal delivery. Indeed, Examples 43 and 45 shows that lipid nanoparticles comprising compounds of the present invention (e.g. compound 64 or a compound in Table 6, or a pharmaceutically acceptable salt thereof) are particularly effective at intranasal delivery. [003] In one aspect, the invention features a cationic lipid having a structure according to Formula (A2): or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; A is a substructure selected from: , , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [004] In one aspect, the invention features a cationic lipid having a structure according to Formula (A1): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; A is a substructure selected from: , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. [005] In one aspect, the invention features a cationic lipid having a structure according to Formula (A): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; A is a substructure selected from: , , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C2-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. [006] In embodiments, X is O and/or B is independently an ionizable nitrogen-containing group. [007] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4- C30 alkynyl. [008] In embodiments of Formula (A2), each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [009] In embodiments, each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. , [013] In embodiments, B is independently , , , , , [014] In embodiments, a compound has a structure according to Formula (I): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group. [015] In embodiments, B is independently an ionizable nitrogen containing group. [016] In embodiments, m is 2 and/or n is 2 or 3. [017] In embodiments, B is independently , , , , [020] In embodiments, B is independently , , , , [021] In embodiments, . [022] In embodiments, a compound is selected from the group consisting of: Compounds (1), (2), (3), (4), (6), (9), (17), (24), (26), (27), (33), and (35). [023] In embodiments, a compound has a structure according to Formula (III): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; R4 is C2-C10 alkylene; and B is independently an ionizable nitrogen-containing group. [024] In embodiments, R4 is –CH2CH2–. [025] In embodiments, B is independently , , , , [026] In embodiments, B is independently , , , , , . [030] In embodiments, a compound is Compound (3). [031] In embodiments, a compound has a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. [032] In embodiments, R1, R2, and R3 is C4-C30 heteroalkyl. [033] In embodiments, m is 2 and/or n is 2 or 3. [034] In embodiments, B is independently , , , , [035] In embodiments, B is independently , , , , [036] In embodiments, B is independently , , , , , , , . [037] In embodiments, B is independently , , , , [038] In embodiments, B is . [039] In embodiments, a compound is Compound (8) or (39). [040] In embodiments, a compound has a structure according to Formula (V1): or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, 4, 5, 6, or 7; and B is independently an ionizable nitrogen-containing group. [041] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl. [042] In embodiments of Formula (A1), Formula (A), and Formula (V1), L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. In embodiments of Formula (A2) and Formula (V1), L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [043] In embodiments, B is independently , , , , , . [048] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (112), (113), (114), (115), (116), (117), (126), (127), (128), and (129). [049] In embodiments, a compound has a structure according to Formula (V): , or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. [050] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl. [051] In embodiments of Formula (A1), Formula (A), and Formula (V), L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. In embodiments of Formula (A2) and Formula (V), L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl [ , . [057] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (114), and (116). [058] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), and (40). [059] In embodiments, a compound has a structure according to Formula (VI): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. [060] In embodiments, B is independently , , , , , . [063] In embodiments, B is independently , , , , [064] In embodiments, . [065] In embodiments, a compound is Compound (21). [066] In embodiments, a compound has a formula according to Formula (VII), , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; each m is an integer of 2-10; each L4 is a carbonyl, ester, or amide; Z is –(CH2)q1–N–(CH2)q2–, wherein q1 and q2 are independently integers of 2-10; or Z is C6H3-Z1, wherein Z1 is a carbonyl, ester, or amide that is covalently attached to the –CH2(CH2)qB moiety; q is an integer of 1-9; and B is independently an ionizable nitrogen-containing group. [067] In embodiments, X is O; each L4 is –C(O)O–; m is an integer of 2, 3, or 4; and/or q is 1. [068] In embodiments, Z is –(CH2)q1–N–(CH2)q2–, and q1 and q2 are each 2. [069] In embodiments, Z is . . [074] In embodiments, . [075] In embodiments, a compound is Compound (16) or (31). [076] In one aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof. [077] In one aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof. [078] In one aspect, the invention features a method of intranasal delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof. [079] In one aspect, the invention features a method of intranasal delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (A1) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof. [080] In another aspect, a compound is selected from the group of Compounds 41-64 and 90, or a pharmaceutically acceptable salt thereof: [081] In another aspect, a compound is selected from the group of Compounds 91-111, or a pharmaceutically acceptable salt thereof:
[082] In another aspect, a compound is selected from the group of Compounds 112-117 and
126-129, or a pharmaceutically acceptable salt thereof:
[083] In another aspect, a compound is selected from the group of Compounds 118-125, or a pharmaceutically acceptable salt thereof:
[084] In embodiments, a composition comprises an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is any cationic lipid described herein.
[085] In embodiments, a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is any cationic lipid described herein. [086] In embodiments, a composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
[087] In embodiments, a nucleic acid is an mRNA encoding a peptide or polypeptide.
[088] In embodiments, an mRNA that encodes a peptide or polypeptide for use in vaccine. [089] In embodiments, an mRNA encodes an antigen.
[090] In embodiments, an antigen is from an infectious agent.
[091] In embodiments, a composition is formulated for a route of administration that is oral, rectal, vaginal, transmucosal, pulmonary, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. [092] In embodiments, pulmonary delivery is intratracheal or inhaled. [093] In embodiments, a route of administration is intranasal. [094] In embodiments, a route of administration is intramuscular. [095] In embodiments, a route of administration is pulmonary. [096] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. [097] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. [098] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B1): wherein L1 is straight-chain C5 alkylene; and R2, R3, and R4 are as defined for Formula (B). [099] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B1): wherein L1 is as defined for Formula (B); and R2, R3 , and R4 are straight-chain C8 alkylene. [0100] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B1): , wherein L1 is as defined for Formula (B); and R2, R3, and R4 are straight-chain C8 alkylene. [0101] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B1): , wherein L1 is as defined for Formula (B); and R2, R3 , and R4 are straight-chain C8 alkylene. [0102] In embodiments the present invention also provides compounds of formula (B1) as described herein. Said compounds may be incorporated into any of the compositions disclosed herein and may be used in any of the methods described herein. [0103] In embodiments, a composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. [0104] In embodiments, a nucleic acid is an mRNA encoding a peptide or polypeptide. [0105] In embodiments, a composition comprises an mRNA that encodes a peptide or polypeptide for use in vaccine. [0106] In embodiments, an mRNA encodes an antigen. [0107] In embodiments, an antigen is from an infectious agent. [0108] In embodiments, a compound is selected from the group consisting of Compounds (65)- (89), or a pharmaceutically acceptable salt thereof:
[0109] In embodiments, a compound is selected from the group consisting of Compounds (118)-(125), or a pharmaceutically acceptable salt thereof:
[0110] In embodiments, delivery is intranasal.
[0111] In embodiments, delivery is intramuscular.
[0112] In embodiments, delivery is pulmonary. BRIEF DESCRIPTION OF DRAWINGS
[0113] FIG. 1 depicts % survival of BALB/c mice inoculated 1) intranasally (IN) with Lipid
Nanoparticles (LNPs) encapsulating CA09 HA mRNA (these LNPs comprised either Compound 64 or the lipid OF-02); 2) intramuscularly (IM) with LNPs encapsulating CA09 HA mRNA (these LNPs comprised OF-02 only); or 3) a phosphate-buffered saline (PBS) buffer control administered intranasally (IN). These results demonstrate high efficacy of LNPs comprising Compound 64 administered intranasally in a lethal mouse influenza challenge model.
[0114] FIG. 2 depicts % body weight change of BALB/c mice inoculated 1) intranasally (IN) with Lipid Nanoparticles (LNPs) encapsulating CA09 HA mRNA (these LNPs comprised either Compound 64 or the lipid OF-02); 2) intramuscularly (IM) with LNPs encapsulating CA09 HA mRNA (these LNPs comprised OF-02 only); or 3) a phosphate-buffered saline (PBS) buffer control administered intranasal ly (IN). These results demonstrate high efficacy of LNPs comprising Compound 64 administered intranasally in a lethal mouse influenza challenge model. The data presented in the graph of FIG.2 extends to 14 days post-challenge
[0115] FIG. 3 and FIG. 4 depict data obtained from various studies comparing intranasal administration of Compound 64 (a compound of the invention) with intramuscular administration of known compound OF-02 which are described in more detail in Example 47 herein.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[0116] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
[0117] Amino acid: As used herein, the term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or aminoterminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue/' and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
[0118] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a bovine, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
[0119] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0120] Biologically active: As used herein, the term "biologically active" refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
[0121] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").
[0122] Expression: As used herein, "expression" of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms "expression" and "production," and grammatical equivalents thereof, are used interchangeably.
[0123] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
[0124] Half-life: As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
[0125] Helper lipid: The term "helper lipid" as used herein refers to any neutral or zwitterionic lipid material including cholesterol. Without wishing to be held to a particular theory, helper lipids may add stability, rigidity, and/or fluidity within lipid bilayers/nanoparticles.
[0126] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
[0127] In Vitro: As used herein, the term "in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.
[0128] In Vivo: As used herein, the term "in vivo” refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
[0129] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
[0130] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). In some embodiments, a liposome suitable for the present invention contains a cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEG-modified lipid(s).
[0131] messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. The term "modified mRNA" related to mRNA comprising at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2' -fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-/V-phosphoramidite linkages).
[0132] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acids (RNA), including but not limited to any one or more of interference RNAs (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), micro-RNA (miRNA) multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, DNA may be in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In embodiments, RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), micro-RNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satellite RNA, or derivatives of these groups. In some embodiments, a nucleic acid is a mRNA encoding a protein such as an enzyme.
[0133] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.
[0134] Pharmaceutically acceptable: The term "pharmaceutically acceptable," as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0135] Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI.4 alkyl )4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
[0136] Systemic distribution or delivery: As used herein, the terms "systemic distribution" or "systemic delivery," or grammatical equivalents thereof, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of "local distribution or delivery." [0137] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
[0138] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0139] Target tissues: As used herein, the term "target tissues" refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.
[0140] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
[0141] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
Chemical definitions [0142] Acyl: As used herein, the term "acyl" refers to RZ-(C=O)-, wherein Rz is, for example, any alkyl, alkenyl, alkynyl, heteroalkyl or heteroalkylene.
[0143] Aliphatic: As used herein, the term aliphatic refers to C1-C50 hydrocarbons and includes both saturated and unsaturated hydrocarbons. An aliphatic may be linear, branched, or cyclic. For example, C1-C20 aliphatics can include C1-C20 alkyls (e.g., linear or branched C1-C20 saturated alkyls), C2-C20 alkenyls (e.g., linear or branched C4-C20 dienyls, linear or branched C6-C20 trienyls, and the like), and C2-C20 alkynyls (e.g., linear or branched C2-C20 alkynyls). C1-C20 aliphatics can include C3-C20 cyclic aliphatics (e.g., C3-C20 cycloalkyls, C4-C20 cycloalkenyls, or C8-C20 cycloalkynyls). In certain embodiments, the aliphatic may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide. An aliphatic group is unsubstituted or substituted with one or more substituent groups as described herein. For example, an aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR”, -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', - OCO2R", -NH2, -NHR", -N(R")2, -SR" or-SO2R”, wherein each instance of R" independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms.
Alkyl: As used herein, the term "alkyl" means acyclic linear and branched hydrocarbon groups, e.g. "C1-C30 alkyl" refers to alkyl groups having 1-30 carbons. An alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentylhexyl, isohexyl, etc. The term "lower alkyl" means an alkyl group straight chain or branched alkyl having 1 to 6 carbon atoms. Other alkyl groups will be readily apparent to those of skill in the art given the benefit of the present disclosure. An alkyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -CO2H, - CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR" or-SO2R", wherein each instance of R" independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R" independently is unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkyl group is substituted with a-OH group and may also be referred to herein as a "hydroxyalkyl" group, where the prefix denotes the -OH group and "alkyl" is as described herein.
[0144] As used herein, "alkyl" also refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms ("C1-C50 alkyl"). In some embodiments, an alkyl group has 1 to 40 carbon atoms ("C1-C40 alkyl"). In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-C30 alkyl"). In some embodiments, an alkyl group has 1 to 20 carbon atoms ("C1-C20 alkyl"). In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-C10 alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("Ci-Cg alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("Ci-Cg alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("Ci- C5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("Ci alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of Ci-Cg alkyl groups include, without limitation, methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3- pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (Cg). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (Cg) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, the alkyl group is an unsubstituted C1-C50 alkyl. In certain embodiments, the alkyl group is a substituted C1-C50 alkyl.
[0145] Affixing the suffix "-ene" to a group indicates the group is a divalent moiety, e.g., arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.
[0146] Alkylene: The term "alkylene," as used herein, represents a saturated divalent straight or branched chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene and the like. Likewise, the term "alkenylene" as used herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, and the term "alkynylene" herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur in any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide. For example, an alkylene, alkenylene, or alkynylene may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, -CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or - SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is unsubstituted C1-C3 alkyl. In certain embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, an alkylene, alkenylene, or alkynylene does not include any heteroatoms. Alkenyl: As used herein, “alkenyl” means any linear or branched hydrocarbon chains having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, e.g. “C2-C30 alkenyl” refers to an alkenyl group having 2-30 carbons. For example, an alkenyl group includes prop-2-enyl, but-2-enyl, but-3-enyl, 2- methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, the alkenyl comprises 1, 2, or 3 carbon-carbon double bond. In embodiments, the alkenyl comprises a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated. An alkenyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkenyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, - CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or-SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C1- C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkenyl group is substituted with a–OH group and may also be referred to herein as a “hydroxyalkenyl” group, where the prefix denotes the –OH group and “alkenyl” is as described herein. [0147] As used herein, “alkenyl” also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-C50 alkenyl”). In some embodiments, an alkenyl group has 2 to 40 carbon atoms (“C2-C40 alkenyl”). In some embodiments, an alkenyl group has 2 to 30 carbon atoms (“C2-C30 alkenyl”). In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C2-C20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2- C10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-C9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-C7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-C3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include, without limitation, ethenyl (C2), 1- propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C2-C50 alkenyl. In certain embodiments, the alkenyl group is a substituted C2- C50 alkenyl. [0148] Alkynyl: As used herein, “alkynyl” means any hydrocarbon chain of either linear or branched configuration, having one or more carbon-carbon triple bonds occurring in any stable point along the chain, e.g., “C2-C30 alkynyl”, refers to an alkynyl group having 2-30 carbons. Examples of an alkynyl group include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3- methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. In embodiments, an alkynyl comprises one carbon-carbon triple bond. An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkynyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’’, - CO2H, -CO2R’’, -CN, -OH, -OR’’, -OCOR’’, -OCO2R’’, -NH2, -NHR’’, -N(R’’)2, -SR’’ or-SO2R’’, wherein each instance of R’’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is an unsubstituted alkyl (e.g., unsubstituted C1- C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’’ independently is unsubstituted C1-C3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). [0149] As used herein, “alkynyl” also refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-C50 alkynyl”). An alkynyl group that has one or more triple bonds and one or more double bonds is also referred to as an “ene-yne”. In some embodiments, an alkynyl group has 2 to 40 carbon atoms (“C2-C40 alkynyl”). In some embodiments, an alkynyl group has 2 to 30 carbon atoms (“C2-C30 alkynyl”). In some embodiments, an alkynyl group has 2 to 20 carbon atoms (“C2-C20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-C9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-C7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon--carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include, without limitation, ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C2-C50 alkynyl. In certain embodiments, the alkynyl group is a substituted C2-C50 alkynyl. [0150] Aryl: The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein said ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic and wherein each ring in the system contains 4 to 7 ring members. In embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl,” e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl,” e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Exemplary aryls include phenyl, naphthyl, and anthracene. [0151] As used herein, “aryl” also refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C6-C14 aryl. In certain embodiments, the aryl group is a substituted C6-C14 aryl. [0152] Arylene: The term “arylene” as used herein refers to an aryl group that is divalent (that is, having two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene). [0153] Carbocyclyl: As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of a non- aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-C8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C3-C7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-C6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C4-C6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C5-C6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-C10 carbocyclyl”). Exemplary C3-C6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 carbocyclyl groups include, without limitation, the aforementioned C3-C6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-C10 carbocyclyl groups include, without limitation, the aforementioned C3-C8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C3-C10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-C10 carbocyclyl. [0154] In some embodiments, “carbocyclyl” or “carbocyclic” is referred to as a “cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3-C8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C 3-C6, cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C 4-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-C10 cycloalkyl”). Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the aforementioned C5-C6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C3-C10 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C3-C10 cycloalkyl. [0155] Halogen: As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine. [0156] Heteroalkyl: The term “heteroalkyl” is meant a branched or unbranched alkyl, alkenyl, or alkynyl group having from 4 to 50 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. In embodiments, In embodiments, a heteroalkyl group has 4 to 40 carbon atoms (“C4-C40 heteroalkyl”). In some embodiments, a heteroalkyl group has 6 to 30 carbon atoms (“C6-C30 heteroalkyl”). In some embodiments, a heteroalkyl group has 4 to 20 carbon atoms (“C4-C20 heteroalkyl”). Heteroalkyls include tertiary amines, secondary amines, ethers, esters, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl group may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. Examples of heteroalkyls include polyethers, such as methoxymethyl and ethoxyethyl. In embodiments, a heteroalkyl comprises an alkyl group as described herein (e.g., a C4-C50 alkyl or C6-C30 alkyl) which may be unsubstituted or substituted (e.g., comprising a hydroxy group, an oxo group, or an ionizable nitrogen group as described herein), and/or comprises 1 or 2 heteroatoms selected from N and O. In embodiments, a heteroalkyl comprises an oxo substitutent group (e.g., a heteroalkyl comprises an ester group where the oxo is adjacent to an oxygen atom). [0157] Heteroalkylene: The term “heteroalkylene,” as used herein, represents a divalent form of a heteroalkyl group as described herein. [0158] Heteroaryl: The term “heteroaryl,” as used herein, is fully unsaturated heteroatom- containing ring wherein at least one ring atom is a heteroatom such as, but not limited to, nitrogen and oxygen. [0159] As used herein, “heteroaryl” also refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
[0160] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl. [0161] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6- membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7- membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6, 6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
[0162] As used herein, "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14- membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-14 membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclyl") or tricyclic system ("tricyclic heterocyclyl")). and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.
[0163] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.
[0164] Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5- membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione. Exemplary 5- membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5- membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6 -membered heterocyclyl groups containing 2 heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1, 8- naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, lH-benzo[e][l,4]diazepinyl, l,4,5,7-tetrahydropyrano[3,4-b] pyrrolyl, 5,6-dihydro-4H- furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3- dihydro-lH-pyrrolo[2,3-b ]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-lH-pyrrolo- [2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno [3,2- b] pyridinyl, l,2,3,4-tetrahydro-l,6-naphthyridinyl, and the like.
[0165] Heterocycloalkyl: The term "heterocycloalkyl," as used herein, is a non-aromatic ring wherein at least one atom is a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. The heterocycloalkyl group can be substituted or unsubstituted.
[0166] As understood from the above, alkyl, alkenyl, alkynyl, acyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are, in certain embodiments, optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or 'unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group. In general, the term "substituted" means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
[0167] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, - NO2, -N3, -SO2, -SO3H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3+X', -N(ORcc)Rbb, -SeH, -SeRaa, - SH, -SRaa, -SSRCC, -C(=O)Raa, -CO2H, -CHO, -C(ORCC)2, -CO2Raa, -OC(=O)Raa, -OCO2Raa, - C(=O)N(Rbb)2, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, -NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, - C(=NRbb)ORaa, -OC(=NRbb)Raa, - OC(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb)2, - NRbbC(=NRbb)N(Rbb)2, - C(=O)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -SO2ORaa, - OSO2Raa, -S(=O)Raa, -OS(=O)Raa, -Si(Raa)3 -OSi(Raa)3 -C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, - SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRaa, -SC(=O)ORaa, -SC(=O)Raa, -P(=O)2Raa, -OP(=O)2Raa, - P(=O)(Raa)2, -OP(=O)(Raa)2, -OP(=O)(ORCC)2, -P(=O)2N(Rbb)2, -OP(=O)2N(Rbb)2, - P(=O)(NRbb)2, - OP(=O)(NRbb)2, -NRbbP(=O)(ORcc)2, -NRbbP(=O)(NRbb)2, -P(RCC)2, - P(RCC)3, -OP(RCC)2, -OP(RCC)3, - B(Raa)2, -B(ORCC)2, -BRaa(ORcc), C1-C50 alkyl, C2-C5o alkenyl, C2-C5o alkynyl, C3-Ci4 carbocyclyl, 3- 14 membered heterocyclyl, Cg-Ci4 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; or two geminal hydrogens on a carbon atom are replaced with the group =0, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORCC;
[0168] each instance of Raa is, independently, selected from C1-C50 alkyl, C2-C5o alkenyl, C2-C5o alkynyl, C3-Cio carbocyclyl, 3-14 membered heterocyclyl, Cg-Ci4 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
[0169] each instance of Rbb is, independently, selected from hydrogen, -OH, -ORaa, - N(RCC)2, -CN, -C(=O)Raa, -C(=O)N(RCC)2, -CO2Raa, -SO2Raa, -C(=NRcc)ORaa, - C(=NRCC)N(RCC)2, -SO2N(RCC)2, -SO2RCC, - SO2ORCC, -SORaa, -C(=S)N(RCC)2, -C(=O)SRCC, - C(=S)SRCC, -P(=O)2Raa, -P(=O)(Raa)2, -P(=O)2N(RCC)2, - P(=O)(NRCC)2, C1-C50 alkyl, C2-C5o alkenyl, C2-C5o alkynyl, C3-CM carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rbb groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; [0170] each instance of Rcc is, independently, selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; [0171] each instance of Rdd is, independently, selected from halogen, -CN, -NO2, -N3, - SO2H, - SO3H, -OH, -ORee, -ON(Rff)2, -N(Rff)2, -N(Rff)3+X-, -N(ORee)Rff, -SH, -SRee, - SSRee, -C(=O)Ree, -CO2H, - CO2Ree, -OC(=O)Ree, -OCO2Ree, -C(=O)N(Rff)2, - OC(=O)N(Rff)2, -NRffC(=O)Ree, -NRffCO2Ree, - NRffC(=O)N(Rff)2, -C(=NRff)ORee, - OC(=NRff)Ree, -OC(=NRff)ORee, -C(=NRff)N(Rff)2, -OC(=NRff)N(Rff)2, - NRffC(=NRff)N(Rff)2, -NRffSO2Ree, -SO2N(Rff)2, -SO2Ree, -SO2ORee, -OSO2Ree, -S(=O)Ree, - Si(Ree)3, -OSi(Ree)3, -C(=S)N(Rff)2, -C(=O)SRee, -C(=S)SRee, -SC(=S)SRee, -P(=O)2Ree, - P(=O)(Ree)2, - OP(=O)(Ree)2, -OP(=O)(ORee)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form =O or =S; [0172] each instance of Ree is, independently, selected from C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; [0173] each instance of Rff is, independently, selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl and 5-10 membered heteroaryl, or two Rff groups, together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and [0174] each instance of Rgg is, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, - OC1-C50 alkyl, -ON(C1-C50 alkyl)2, -N(C1-C50 alkyl)2, -N(C1-C50 alkyl)3+X-, -NH(C1-C50 alkyl)2+X-, -NH2(C1- C50 alkyl) +X-, -NH3+X-, -N(OC1-C50 alkyl)(C1-C50 alkyl), -N(OH)(C1-C50 alkyl), -NH(OH), -SH, -SC1-C50 alkyl, -SS(C1-C50 alkyl), -C(=O)(C1-C50 alkyl), -CO2H, -CO2(C1-C50 alkyl), -OC(=O)(C1-C50 alkyl), - OCO2(C1-C50 alkyl), -C(=O)NH2, -C(=O)N(C1-C50 alkyl)2, -OC(=O)NH(C1-C50 alkyl), -NHC(=O)(C1-C50 alkyl), -N(C1-C50 alkyl)C(=O)(C1-C50 alkyl), -NHCO2(C1-C50 alkyl), -NHC(=O)N(C1-C50 alkyl)2, - NHC(=O)NH(C1-C50 alkyl), -NHC(=O)NH2, -C(=NH)O(C1-C50 alkyl),-OC(=NH)(C1-C50 alkyl), - OC(=NH)OC1-C50 alkyl, - C(=NH)N(C1-C50 alkyl)2, -C(=NH)NH(C1-C50 alkyl), -C(=NH)NH2, - OC(=NH)N(C1-C50alkyl)2, -OC(NH)NH(C1-C50 alkyl), -OC(NH)NH2, -NHC(NH)N(C1-C50 alkyl)2, - NHC(=NH)NH2, -NHSO2(C1-C50 alkyl), -SO2N(C1-C50 alkyl)2, -SO2NH(C1-C50 alkyl), - SO2NH2,-SO2(C1-C50 alkyl), -SO2O(C1-C50 alkyl), -OSO2(C1-C6 alkyl), -SO(C1-C6 alkyl), -Si(C1-C50 alkyl)3, -OSi(C1-C6 alkyl)3, - C(=S)N(C1-C50 alkyl)2, C(=S)NH(C1-C50 alkyl), C(=S)NH2, -C(=O)S(C1-C6 alkyl), -C(=S)S(C1-C6 alkyl), - SC(=S)S(C1-C6 alkyl), -P(=O)2(C1-C50 alkyl), -P(=O)(C1-C50 alkyl)2, -OP(=O)(C1-C50 alkyl)2, -OP(=O)(OC1- C50 alkyl)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can be joined to form =O or =S; wherein X- is a counterion. [0175] As used herein, the term “halo” or “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I). [0176] As used herein, a “counterion” is a negatively charged group associated with a positively charged quarternary amine in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F-, Cl-, Br-, I-), NO3-, ClO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like). [0177] Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, -OH, -ORaa, -N(Rcc)2, -CN, - C(=O)Raa, - C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)ORaa, - C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, - SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, -C(=S)SRcc, -P(=O)2Raa, -P(=O)(Raa)2, -P(=O)2N(Rcc)2, - P(=O)(NRcc)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, together with the N atom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
[0178] In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
[0179] For example, nitrogen protecting groups such as amide groups (e.g., - C(=O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p- hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.
[0180] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), l-(l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, l,l-dimethyl-2,2-dibromoethyl carbamate (DB-t- BOC), l,l-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), l-methyl-l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t-butylphenyl)-l-methylethyl carbamate (t-Bumeoc), 2-(2'-and 4'- pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),
8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz),
9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2- methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6- chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p- decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1- methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, l-methyl-l(3,5- dimethoxyphenyl)ethyl carbamate, l-methyl-l-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l- phenylethyl carbamate, 1- methyl-l-(4-pyridyl)ethyl carbamate, phenyl carbamate, p- (phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
[0181] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)jRaa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy- 4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchrornan-6-sulfonamide (Pmc), methanesulfonamide (Ms), p-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4- (4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[0182] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(lO)- acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N' -phenylaminothioacyl derivative, N- benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N- phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N- 1, 1,4,4- tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5-triazacyclohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2- (trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9- phenylfluorenylamine (PhF), N-2,7 -dichloro-9-fluorenylmethyleneamine, N- ferrocenylmethylamino (Fem), N-2- picolylamino N'-oxide, N-l,l-dimethylthiomethyleneamine, N- benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2- pyridyl)mesityl]methyleneamine, N-(N' ,N'-dimethylaminomethylene)amine, N,N' - isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivative, N- diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
[0183] In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
[0184] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4- yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro- 7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 1-methyl-l- methoxyethyl, 1-methyl-l-benzyloxyethyl, l-methyl-l-benzyloxy-2-fluoroethyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, l,l-bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9-(9- phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, l,3-benzodisulfuran-2-yl, benzisothiazolyl S,S- dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri -p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4- methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2- trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p- methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-(l,l,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(l,l- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N',N'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
[0185] In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
[0186] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p- methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxido, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4- methoxyphenyl)methyl, 5-dibenzosuberyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4- dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyl oxycarbonylaminomethyl, phenylacetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2- nitro-l-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(Trimethylsilyl)ethyl, 2,2- bis(carboethoxy)ethyl, (l-m-nitrophenyl-2-benzoyl)othyl, 2-phenylsulfonylethyl, 2-(4- methylphenylsulfonyl)-2-methylprop-2-yl, acetyl, benzoyl, trifluoroacetyl, N-[[(p- biphenylyl)isopropoxy]carbonyl]-N-methyl]- y-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, sulfenylthiocarbonate, 3-nitro-2-pyridinesulfenyl sulfide, oxathiolone.
Compounds of the Invention
[0187] Liposomal-based vehicles are considered an attractive carrier for therapeutic agents and remain subject to continued development efforts. While liposomal-based vehicles that comprise certain lipid components have shown promising results with regard to encapsulation, stability and site localization, there remains a great need for improvement of liposomal-based delivery systems. For example, a significant drawback of liposomal delivery systems relates to the construction of liposomes that have sufficient cell culture or in vivo stability to reach desired target cells and/or intracellular compartments, and the ability of such liposomal delivery systems to efficiently release their encapsulated materials to such target cells.
[0188] Efficient delivery of liposome-encapsulated nucleic acids therefore remains an active area of research. Cationic lipid components play an important role in facilitating effective encapsulation of nucleic acids during the loading of liposomes. In addition, cationic lipids may play an important role in the efficient release of the nucleic acid cargo from the liposome into the cytoplasm of a target cell. Various cationic lipids suitable for in vivo use have been discovered. However, there remains a need to identify lipids that can be synthesized efficiently and cheaply without the formation of potentially toxic by-products.
[0189] In particular, there remains a need for improved lipid compounds that demonstrate improved pharmacokinetic properties and which are capable of delivering macromolecules, such as nucleic acids, to a wide variety cell types and tissues with enhanced efficiency. Importantly, there also remains a particular need for novel lipid compounds that are characterized as having reduced toxicity and are capable of efficiently delivering encapsulated nucleic acids and polynucleotides to targeted cells, tissues and organs.
[0190] Described herein a novel class of tricine and citric acid-based cationic lipid compounds for improved in vivo delivery of therapeutic agents, such as nucleic acids. In particular, a tricine and citric acid-based cationic lipid described herein may be used as a cationic lipid, optionally with other lipids, to formulate a lipid-based nanoparticle (e.g., liposome) for encapsulating therapeutic agents, such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic use.
[0191] In embodiments, compounds of the invention as described herein can provide one or more desired characteristics or properties. That is, in certain embodiments, compounds of the invention as described herein can be characterized as having one or more properties that afford such compounds advantages relative to other similarly classified lipids. For example, compounds disclosed herein can allow for the control and tailoring of the properties of liposomal compositions (e.g., lipid nanoparticles) of which they are a component. In particular, compounds disclosed herein can be characterized by enhanced transfection efficiencies and their ability to provoke specific biological outcomes. Such outcomes can include, for example enhanced cellular uptake, endosomal/lysosomal disruption capabilities and/or promoting the release of encapsulated materials (e.g., polynucleotides) intracellularly. Additionally, the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution, and efficiency (e.g., due to the different disassociate rates of the polymer group used). [0192] Exemplary compounds include those described herein. Variables described for a formula may be according to any permitted structure or value as recited for any other formula or embodiment described herein, and any combinations thereof.
Formula (A2)
[0193] In one aspect, the invention features a cationic lipid having a structure according to Formula (A2): or a pharmaceutically acceptable salt thereof, wherein
X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl;
A is a substructure selected from:
B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10;
L1 is a carbonyl, ester, or amide;
L2 is C2-C10 alkylene or a C2-C10 alkenylene;
Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3;
R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-
C30 heteroalkyl comprising a disulfide bond or having a structure that is wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [0194] In embodiments, A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [0195] In embodiments, X is independently O. [0196] In embodiments, X is independently NH. [0197] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkyl. [0198] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkenyl. [0199] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkynyl. [0200] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl). [0201] In embodiments, B is independently an ionizable nitrogen-containing group. [0202] In embodiments, B is independently a permanently charged nitrogen group. [0203] In embodiments, [0204] In embodiments,
[0205] In embodiments,
[0206] In embodiments,
[0207] In embodiments, m is an integer of 2-10.
[0208] In embodiments, n is an integer of 2-10.
[0209] In embodiments, L1 is a carbonyl.
[0210] In embodiments, L1 is an ester. In embodiments, L1 is -OC(O)-. In embodiments, L1 is - (O)CO-.
[0211] In embodiments, L1 is an amide. In embodiments, L1 is -NHC(O)-. In embodiments, L1 is -C(O)NH-
[0212] In embodiments, L2 is C2-C10 alkylene. In embodiments, L2 is unsubstituted C2-C10 alkylene. In embodiments, L2 is substituted C2-C10 alkylene.
[0213] In embodiments, L2 is C2-C10 alkenylene. In embodiments, L2 is unsubstituted C2-C10 alkenylene. In embodiments, L2 is substituted C2-C10 alkenylene.
[0214] In embodiments, Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3.
[0215] In embodiments, R4 is unsubstituted C1-C10 alkylene. Inembodiments, R4 is substituted C1-C10 alkylene.
[0216] In embodiments, X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group. [0217] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4- C30 alkynyl. In embodiments, each of R1, R2, and R3 is unsubstituted. In embodiments, each of R1, R2, and R3 is substituted (e.g., comprising 1-3 substituents as described herein). [0218] In embodiments, each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl. [0219] In embodiments, B is independently , , , , [0220] In embodiments, B is independently , , , , [0221] In embodiments, B is independently , , , ,
[0223] In embodiments, B is independently H
Me .
N-i
[0224] In embodiments, B is independently Me
[0225] In embodiments, B is independently
[0226] In embodiments, B is independently
[0227] In embodiments, B is independently
HN^
[0228] In embodiments, B is independently
[0229] In embodiments, B is independently [0230] In embodiments, B is independently
[0231] In embodiments, B is independently
[0232] In embodiments, B is independently
[0233] In embodiments, B is independently [0234] In embodiments, B is independently
[0235]
[0236]
[0237] In embodiments, B is independently
[0238] In embodiments, B is independently [0239] In embodiments, B is independently [0240] In embodiments, B is independently
HN N—
[0241] In embodiments, B is independently
[0242] In embodiments, B is independently
Formula (Al)
[0243] In one aspect, the invention features a cationic lipid having a structure according to
Formula (Al): or a pharmaceutically acceptable salt thereof, wherein
X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl;
A is a substructure selected from:
B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10;
L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene;
Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3;
R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-
C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
[0244] In embodiments, A is only substructure (a4) when each of R1, R2, and R3 is independently
. M ,,R5 a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is o L , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
[0245] In embodiments, X is independently O.
[0246] In embodiments, X is independently NH.
[0247] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkyl.
[0248] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkenyl.
[0249] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkynyl.
[0250] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., Cg-Cso heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted Cg-Cso heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted Cg-Cso heteroalkyl).
[0251] In embodiments, B is independently an ionizable nitrogen-containing group.
[0252] In embodiments, B is independently a permanently charged nitrogen group. [0253] In embodiments,
[0254] In embodiments,
[0255] In embodiments,
[0256] In embodiments,
[0257] In embodiments, m is an integer of 2-10.
[0258] In embodiments, n is an integer of 2-10.
[0259] In embodiments, L1 is a carbonyl.
[0260] In embodiments, L1 is an ester. In embodiments, L1 is -OC(O)-. In embodiments, L1 is - (O)CO-.
[0261] In embodiments, L1 is an amide. In embodiments, L1 is -NHC(O)-. In embodiments, L1 is -C(O)NH-
[0262] In embodiments, L2 is C2-C10 alkylene. In embodiments, L2 is unsubstituted C2-C10 alkylene. In embodiments, L2 is substituted C2-C10 alkylene.
[0263] In embodiments, L2 is C2-C10 alkenylene. In embodiments, L2 is unsubstituted C2-C10 alkenylene. In embodiments, L2 is substituted C2-C10 alkenylene.
[0264] In embodiments, Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3. [0265] In embodiments, R4 is unsubstituted C1-C10 alkylene. Inembodiments, R4 is substituted
C1-C10 alkylene.
[0266] In embodiments, X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group.
[0267] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4- C30 alkynyl. In embodiments, each of R1, R2, and R3 is unsubstituted. In embodiments, each of R1, R2, and R3 is substituted (e.g., comprising 1-3 substituents as described herein).
[0268] In embodiments, each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
,N4
[0269] In embodiments, B is independently H [0270] In embodiments, B is independently [0271] In embodiments, B is independently , , ,
[0272] In embodiments, B is independently , , ,
[0273] In embodiments, B is independently H
[0274] In embodiments, B is independently
[0275] In embodiments, B is independently
[0276] In embodiments, B is independently
HO
[0277] In embodiments, B is independently [0278] In embodiments, B is independently
[0279] In embodiments, B is independently
[0280] In embodiments, B is independently
[0281] In embodiments, B is independently
[0282] In embodiments, B is independently
[0283] In embodiments, B is independently
[0284] In embodiments, B is independently
[0285] In embodiments, B is independently
[0286] In embodiments, B is independently
[0287] In embodiments, B is independently
[0288] In embodiments, B is independently
[0289] In embodiments, B is independently
[0290] In embodiments, B is independently
[0291] In embodiments, B is independently
[0292] In embodiments, B is independently
Formula (A)
[0293] In one aspect, the invention features a cationic lipid having a structure according to
Formula (A): or a pharmaceutically acceptable salt thereof, wherein
X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl;
A is a substructure selected from: B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10;
L1 is a carbonyl, ester, or amide;
L2 is C2-C10 alkylene or a C2-C10 alkenylene;
Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; and
R4 is C2-C10 alkylene.
[0294] In embodiments, A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or
(O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
[0295] In embodiments, X is independently O.
[0296] In embodiments, X is independently NH.
[0297] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkyl.
[0298] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkenyl.
[0299] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkynyl.
[0300] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., Cg-Cso heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted Cg-Cso heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted Cg-Cso heteroalkyl).
[0301] In embodiments, B is independently an ionizable nitrogen-containing group. [0302] In embodiments, B is independently a permanently charged nitrogen group.
[0303] In embodiments,
[0304] In embodiments,
[0305] In embodiments,
[0306] In embodiments,
[0307] In embodiments, m is an integer of 2-10.
[0308] In embodiments, n is an integer of 2-10.
[0309] In embodiments, L1 is a carbonyl.
[0310] In embodiments, L1 is an ester. In embodiments, L1 is -OC(O)-. In embodiments, L1 is - (O)CO-.
[0311] In embodiments, L1 is an amide. In embodiments, L1 is -NHC(O)-. In embodiments, L1 is -C(O)NH-
[0312] In embodiments, L2 is C2-C10 alkylene. In embodiments, L2 is unsubstituted C2-C10 alkylene. In embodiments, L2 is substituted C2-C10 alkylene.
[0313] In embodiments, L2 is C2-C10 alkenylene. In embodiments, L2 is unsubstituted C2-C10 alkenylene. In embodiments, L2 is substituted C2-C10 alkenylene.
[0314] In embodiments, Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is unsubstituted phenylene. In embodiments, Ar is phenylene comprising 1-4 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-3 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising 1-2 substituents independently selected from halogen, OCH3, and CH3. In embodiments, Ar is phenylene comprising one substitutent selected from halogen, OCH3, and CH3.
[0315] In embodiments, R4 is unsubstituted C2-C10 alkylene. In embodiments, R4 is substituted C2-C10 alkylene.
[0316] In embodiments, X is O and/or B is independently an ionizable nitrogen-containing group. In embodiments, X is O and B is independently an ionizable nitrogen-containing group.
[0317] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4- C30 alkynyl. In embodiments, each of R1, R2, and R3 is unsubstituted. In embodiments, each of R1, R2, and R3 is substituted (e.g., comprising 1-3 substituents as described herein).
[0318] In embodiments, each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising
. M ,.R5 a disulfide bond or having a structure that is ° L , wherein L is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
H Me Et
[0319] In embodiments, B is independently H , , ,
[0320] In embodiments, B is independently , , ,
[0321] In embodiments, B is independently , , ,
[0323] In embodiments, B is independently H
[0324] In embodiments, B is independently
[0325] In embodiments, B is independently
[0326] In embodiments, B is independently
[0327] In embodiments, B is independently [0328] In embodiments, B is independently
[0329] In embodiments, B is independently
[0330] In embodiments, B is independently
[0331] In embodiments, B is independently
[0332] In embodiments, B is independently
[0333] In embodiments, B is independently
[0334] In embodiments, B is independently
[0335] In embodiments, B is independently
[0336] In embodiments, B is independently
[0337] In embodiments, B is independently
[0338] In embodiments, B is independently [0339] In embodiments, B is independently . [0340] In embodiments, B is independently . [0341] In embodiments, B is independently . [0342] In embodiments, B is independently . Formula (I) [0343] In embodiments, a compound has a structure according to Formula (I): or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, B, m, and n is independently according to any combination of embodiments described herein. [0344] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group. [0345] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkyl. [0346] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkenyl. [0347] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkynyl. [0348] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl). [0349] In embodiments, B is independently an ionizable nitrogen containing group. [0350] In embodiments, B is independently a permanently charged nitrogen containing group. [0351] In embodiments, m is 2 and/or n is 2 or 3. [0352] In embodiments, m is 2 and n is 2. [0353] In embodiments, m is 2 and n is 3. [0354] In embodiments, B is independently , , , , [0355] In embodiments, B is independently , , , ,
[0357] In embodiments, B is independently ,
[ ,
Me .
N-i [0359] In embodiments, B is Me
[0360] In embodiments, [0361] In embodiments, a compound is selected from the group consisting of: Compounds (1), (2), (3), (4), (6), (9), (17), (24), (26), (27), (33), and (35). Formula (III) [0362] In embodiments, a compound has a structure according to Formula (III): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, R4, and B is independently according to any combination of embodiments described herein. [0363] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; R4 is C2-C10 alkylene; and B is independently an ionizable nitrogen-containing group. [0364] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkyl. [0365] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkenyl. [0366] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkynyl. [0367] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl). [0368] In embodiments, R4 is unsubstituted C2-C10 alkylene. [0369] In embodiments, R4 is substituted C2-C10 alkylene.
[0370] In embodiments, R4 is -CH2CH2-.
[0371] In embodiments, B is independently > > [0372] In embodiments, B is independently [0373] In embodiments, B is independently , , ,
[0374] In embodiments, B is independently [0375] In embodiments, B is . [0376] In embodiments, a compound is Compound (3). Formula (IV) [0377] In embodiments, a compound has a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, m, n, and B is independently according to any combination of embodiments described herein. [0378] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. [0379] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkyl. [0380] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkenyl. [0381] In embodiments, each of R1, R2, and R3 is independently C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C6-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C6-C30 alkynyl. [0382] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl). [0383] In embodiments, R1, R2, and R3 is C4-C30 heteroalkyl. [0384] In embodiments, m is 2 and/or n is 2 or 3. [0385] In embodiments, m is 2 and n is 2. [0386] In embodiments, m is 2 and n is 3. [0387] In embodiments, B is independently , , , , [0388] In embodiments, B is independently , , , , [0389] In embodiments, B is independently , , , , ,
[0390] In embodiments, B is independently , , ,
Me .
,N4
[0391] In embodiments, B is Me
[0392] In embodiments, a compound is Compound (8) or (39).
Formula (VI) [0393] In embodiments, a compound has a structure according to Formula (VI): or a pharmaceutically acceptable salt thereof, wherein each of R5, L3, n, o, and B is independently according to any combination of embodiments described herein.
[0394] In embodiments, n is an integer of 2, 3, 4, 5, 6, or 7; and
B is independently an ionizable nitrogen-containing group.
[0395] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl.
[0396] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is Ci-Cg alkyl.
H Me Et
[0397] In embodiments, B is independently H , , , ,
[0398] In embodiments, B is independently , , ,
[0399] In embodiments, B is independently , , ,
Me .
N-i [0401] In embodiments, B is Me
[0402] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (112), (113), (114), (115), (116), (117), (126), (127), (128), and (129).
Formula (V) [0403] In embodiments, a compound has a structure according to Formula (V): or a pharmaceutically acceptable salt thereof, wherein each of R5, L3, n, o, and B is independently according to any combination of embodiments described herein.
[0404] In embodiments, n is an integer of 2, 3, or 4; and
B is independently an ionizable nitrogen-containing group. [0405] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl.
[0406] In embodiments, L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
H Me Et
[0407] In embodiments, B is independently H , , , ,
[0408] In embodiments, B is independently
[0410] In embodiments, B is independently [0411] In embodiments, B is . [0412] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (114), and (116). [0413] In embodiments, a compound is selected from the group consisting of: Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), and (40). Formula (VI) [0414] In embodiments, a compound has a structure according to Formula (VI): or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, n, and B is independently according to any combination of embodiments described herein. [0415] In embodiments, each of R1, R2, and R3 is independently C6-C30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. [0416] In embodiments, B is independently , , , [0419] In embodiments, B is independently , , , [0420] In embodiments, B is . [0421] In embodiments, a compound is Compound (21). Formula (VII) [0422] In one aspect, a cationic lipid has a formula according to Formula (VII), , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; each m is an integer of 2-10; each L4 is a carbonyl, ester, or amide; Z is –(CH2)q1–N–(CH2)q2–, wherein q1 and q2 are independently integers of 2-10; or Z is C6H3-Z1, wherein Z1 is a carbonyl, ester, or amide that is covalently attached to the –CH2(CH2)qB moiety; q is an integer of 1-9; and B is independently an ionizable nitrogen-containing group. [0423] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkyl. [0424] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkenyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkenyl. [0425] In embodiments, each of R1, R2, and R3 is independently C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 alkynyl. In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 alkynyl. [0426] In embodiments, each of R1, R2, and R3 is independently C4-C30 heteroalkyl (e.g., C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently unsubstituted C4-C30 heteroalkyl (e.g., unsubstituted C6-C30 heteroalkyl). In embodiments, each of R1, R2, and R3 is independently substituted C4-C30 heteroalkyl (e.g., substituted C6-C30 heteroalkyl). [0427] In embodiments, L4 is a carbonyl. [0428] In embodiments, L4 is an ester. In embodiments, L4 is –OC(O)–. In embodiments, L4 is – (O)CO–. [0429] In embodiments, L4 is an amide. In embodiments, L4 is –NHC(O)–. In embodiments, L4 is -C(O)NH–. [0430] In embodiments, X is O; each L4 is –C(O)O–; m is an integer of 2, 3, or 4; and/or q is 1. [0431] In embodiments, Z is –(CH2)q1–N–(CH2)q2–, and q1 and q2 are each 2. [0432] In embodiments, Z is . . [0433] In embodiments, B is independently , , , [0436] In embodiments, B is independently , , ,
Me . N-i
[0437] In embodiments, B is Me
[0438] In embodiments, a compound is Compound (16) or (31).
[0439] In one aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
[0440] In one aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof.
[0441] In one aspect, the invention features a method of intranasal delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof. [0442] In one aspect, the invention features a method of intranasal delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (A2), Formula (Al) or Formula (A) as described herein, or a pharmaceutically acceptable salt thereof, for example Compound 64, or a compound in Table 6, or a pharmaceutically acceptable salt thereof.
[0443] Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition with the nasal mucosa, nasal turbinates or sinus cavity. The pharmaceutical compositions for administration may be applied in a single administration or in multiple administrations. For example, one dose can be placed in each nostril during administration. For example, bi-dose delivery can be used with the compositions according to the invention. Bi-dose devices contain two sub-doses of a single dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time. Alternatively, a mono-dose device may be used for administering the compositions according to the invention. The composition can be given in one, two, three, four, or more doses, so that the subject is given a first dose (which can be a bi-dose or mono-dose, as described above), and then a second dose is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 days, or 4 ,5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years apart. Exemplary devices for intranasal administration of the compositions according to the invention are spray devices. Suitable commercially available nasal spray devices include Accuspray™ (Becton Dickinson). Nebulizers produce a very fine spray (such as a mist) which can be easily inhaled and are also contemplated herein. Exemplary spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art.
[0444] The invention provides in a further aspect a pharmaceutical kit comprising an intranasal administration device as described herein containing a formulation according to the invention.
[0445] The invention is not necessarily limited to spray delivery of liquid formulations. Compositions according to the invention may be administered in other forms e.g. as a powder. Compounds 41-64 and 90
[0446] In another aspect, a cationic lipid is selected from the group of Compounds 41-64 and
90, or a pharmaceutically acceptable salt thereof:
Compounds 91-111
[0447] In another aspect, a cationic lipid is selected from the group of Compounds 91-111 or a pharmaceutically acceptable salt thereof:
Compounds 112-117
[0448] In another aspect, a cationic lipid is selected from the group of Compounds 112-117 and 126-129, or a pharmaceutically acceptable salt thereof:
Compounds 118-125
[0449] In another aspect, a cationic lipid is selected from the group of Compounds 118-125 or a pharmaceutically acceptable salt thereof:
Formula (B) [0450] In another aspect, the invention features a cationic lipid according to Formula (B). or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. [0451] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B) as described herein, or a pharmaceutically acceptable salt thereof. [0452] In another aspect, the invention features a method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B) as described herein, or a pharmaceutically acceptable salt thereof. [0453] In embodiments, a compound of Formula (B) is selected from the group consisting of Compounds (65)-(89), or a pharmaceutically acceptable salt thereof:
[0454] In embodiments, a compound of Formula (B) is selected from the group consisting of Compounds (65), (66), (67), (70), (71), (74), (75), (77), (81), (84), (87), (88), and (89).
[0455] In embodiments, a compound of Formula (B) is selected from the group consisting of Compounds (68), (69), (72), (73), (76), (78), (79), (80), (82), (83), (85), and (86). [0456] In embodiments, a compound of Formula (B) excludes Compounds (68), (69), (72), (73),
(76), (78), (79), (80), (82), (83), (85), and (86).
[0457] Also provided are compounds of Formula (Bl): wherein Li is as defined for Formula (B); and
R2, R3, and R4 are straight-chain Cg alkylene.
[0458] Also provided are compounds of Formula (Bl):
wherein
Li is straight-chain C5 alkylene; and
R2, R3, and R4 are as defined for Formula (B) [0459] In embodiments, a compound of Formula (B) or (Bl) is selected from the group consisting of Compounds (118)-(125), or a pharmaceutically acceptable salt thereof:
Exemplary Compounds
[0460] Exemplary compounds of the invention include those cationic lipids described in Table 1.
[0461] In embodiments, a cationic lipid is Compound (1) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (2) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (3) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (4) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (6) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (8) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (9) or a pharmaceutically acceptable salt thereof. [0462] In embodiments, a cationic lipid is Compound (11) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (12) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (13) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (14) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (16) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (17) or a pharmaceutically acceptable salt thereof.
[0463] In embodiments, a cationic lipid is Compound (21) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (22) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (24) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (25) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (26) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (27) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (28) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (29) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (30) or a pharmaceutically acceptable salt thereof.
[0464] In embodiments, a cationic lipid is Compound (31) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (32) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (33) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (34) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (35) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (36) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (38) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (39) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (40) or a pharmaceutically acceptable salt thereof.
[0465] Exemplary compounds of the invention include those cationic lipids described in Table
2. Table 2. Exemplary Cationic Lipids
[0466] In embodiments, a cationic lipid is Compound (41) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (42) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (43) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (44) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (45) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (46) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (47) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (48) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (49) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (50) or a pharmaceutically acceptable salt thereof.
[0467] In embodiments, a cationic lipid is Compound (51) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (52) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (53) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (54) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (55) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (56) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (57) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (58) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (59) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (60) or a pharmaceutically acceptable salt thereof.
[0468] In embodiments, a cationic lipid is Compound (61) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (62) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (63) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (64) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (90) or a pharmaceutically acceptable salt thereof.
[0469] Exemplary compounds of the invention include those cationic lipids described in Table
3. Table 3. Exemplary Cationic Lipids
[0470] In embodiments, a cationic lipid is Compound (65) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (66) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (67) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (68) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (69) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (70) or a pharmaceutically acceptable salt thereof.
[0471] In embodiments, a cationic lipid is Compound (71) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (72) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (73) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (74) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (75) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (76) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (77) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (78) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (79) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (80) or a pharmaceutically acceptable salt thereof.
[0472] In embodiments, a cationic lipid is Compound (81) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (82) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (83) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (84) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (85) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (86) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (87) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (88) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (89) or a pharmaceutically acceptable salt thereof.
[0473] Exemplary compounds of the invention include those cationic lipids described in Table 9.
Table 9. Exemplary Cationic Lipids
[0474] In embodiments, a cationic lipid is Compound (91) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (92) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (93) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (94) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (95) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (96) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (97) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (98) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (99) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (100) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (101) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (102) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (103) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (104) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (105) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (106) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (107) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (108) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (109) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (110) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (111) or a pharmaceutically acceptable salt thereof.
[0475] Exemplary compounds of the invention include those cationic lipids described in Table
10.
Table 10. Exemplary Cationic Lipids
[0476] In embodiments, a cationic lipid is Compound (112) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (113) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (114) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (115) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (116) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (117) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (126) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (127) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (128) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (129) or a pharmaceutically acceptable salt thereof.
[0477] Exemplary compounds of the invention include those cationic lipids described in Table
11.
Table 11. Exemplary Cationic Lipids
[0478] In embodiments, a cationic lipid is Compound (118) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (119) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (120) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (121) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (122) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (123) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (124) or a pharmaceutically acceptable salt thereof. In embodiments, a cationic lipid is Compound (125) or a pharmaceutically acceptable salt thereof.
[0479] As further described herein, compositions comprising any compound of the invention include compositions comprising the cationic lipid of any one of the preceding embodiments, one or more non-cationic lipids, one or more cholesterol-based lipids, and/or one or more PEG- modified lipids. In embodiments, this composition is a lipid nanoparticle. In embodiments, the one or more cationic lipid(s) constitute(s) about 30 mol %-60 mol % of the lipid nanoparticle. In embodiments, the one or more non-cationic lipid(s) constitute(s) 10 mol%-50 mol% of the lipid nanoparticle. In embodiments, the one or more PEG-modified lipid(s) constitute(s) 1 mol%-10 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipid constitutes 10 mol%- 50 mol% of the lipid nanoparticle. In embodiments, the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 50%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 55%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 60%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 65%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 70%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 75%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 80%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 85%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 90%. In embodiments, the lipid nanoparticles have an encapsulation percentage for mRNA of at least 95%.
[0480] In embodiments, the composition of any one of the proceeding embodiments is for use in therapy.
[0481] In embodiments, the composition of any one of the proceeding embodiments is for use in a method of treating or preventing a disease amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
[0482] In embodiments, the composition is administered intravenously, intrathecally or intramuscularly, or by pulmonary delivery, optionally through nebulization or use of an inhaler (e.g., a metered dose inhaler, a dry powder inhaler, or a soft mist inhaler).
[0483] Mucosal routes of administration such as oral and intransal administration can elicit immune responses at both the local and distal mucosal sites. Such routes of administration can also give rise to systemic immune responses. As such, there is an interest in utilising routes of administration such as intransal when administering the compositions of the present invention particularly with a view to generating protective immunity. In embodiments, the methods and compositions of the invention may induce mucosal immunity in a subject. In embodiments the methods and compositions of the invention may induce mucosal immunity at distal mucosal sites. In embodiments, the methods and compositions of the invention may generate protective mucosal immunity. By "protective mucosal immunity" is meant immunity or an immune response against an infectious agent which is exhibited by a subject, whereby said immunity or immune response prevents or ameliorates an infection or reduces one or more symptoms thereof. The compositions and methods described herein may elicit an antibody response in a subject. In some embodiments said antibody may be IgA. In some embodiments, said antibody may be IgG.
[0484] During an infection or following a vaccination during which time a subject is exposed to a particular antigen, antigen specific T cells are activated, proliferate and then differentiate into effector cells. These effector cells serve to help clear the infection but most of these cells then go on to die. The remaining memory cells can afford protection and/or an increased response in the event of the subject encountering that same antigen again at a later time. Accordingly, it would be beneficial to identify methods and compositions that are capable of eliciting an antigenspecific T cell response, for example by inducing a CD4 memory T cell population. The compositions and methods described herein may enable a subject to produce an CD4 memory T cell population. In some embodiments, the compositions and methods described herein may induce a CD4 memory T cell population in a subject, optionally wherein said population is induced in the mucosa.
Synthesis of Compounds
[0485] The compounds of the invention as described herein can be prepared according to methods known in the art, including as described herein.
Nucleic Acids
[0486] The compounds of the invention as described herein can be used to prepare compositions useful for the delivery of nucleic acids.
Synthesis of Nucleic Acids
[0487] Nucleic acids according to the present invention may be synthesized according to any known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, mutated T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.
[0488] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7, mutated T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
[0489] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
Modified mRNA
[0490] In some embodiments, mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Modified mRNA includes nucleotide modifications in the RNA. A modified mRNA according to the invention can thus include nucleotide modification that are, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g., 1-methyl-adenine, 2-methyl-adenine, 2- methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio- cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl- guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl- 2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'- methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5- oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g., from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.
Pharmaceutical Formulations of Cationic Lipids and Nucleic Acids
[0491] In certain embodiments, the compounds of the invention as described herein, as well as pharmaceutical and liposomal compositions comprising such lipids, can be used in formulations to facilitate the delivery of encapsulated materials (e.g., one or more polynucleotides such as mRNA) to, and subsequent transfection of one or more target cells. For example, in certain embodiments cationic lipids described herein (and compositions such as liposomal compositions comprising such lipids) are characterized as resulting in one or more of receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and 187ncapsulel87ytosis, fusogenicity, endosomal or lysosomal disruption and/or releasable properties that afford such compounds advantages relative other similarly classified lipids.
[0492] According to the present invention, a nucleic acid, e.g., mRNA encoding a protein (e.g., a full length, fragment or portion of a protein) as described herein may be delivered via a delivery vehicle comprising a compound of the invention as described herein.
[0493] As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle," or grammatical equivalents thereof, are used interchangeably.
[0494] For example, the present invention provides a composition (e.g., a pharmaceutical composition) comprising a compound described herein and one or more polynucleotides. A composition (e.g., a pharmaceutical composition) may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and/or one or more PEG-modified lipids. [0495] In certain embodiments a composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting the one or more target cells with the cationic lipids and/or pharmaceutical compositions disclosed herein (e.g., a liposomal formulation comprising a compound described herein encapsulating one or more polynucleotides) such that the one or more target cells are transfected with the materials encapsulated therein (e.g., one or more polynucleotides). As used herein, the terms "transfect" or "transfection" refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell (e.g., into a target cell). The introduced polynucleotide may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated material (e.g., polynucleotides) taken up by, introduced into, and/or expressed by the target cell which is subject to transfection. In practice, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein demonstrate high transfection efficiencies thereby improving the likelihood that appropriate dosages of the encapsulated materials (e.g., one or more polynucleotides) will be delivered to the site of pathology and subsequently expressed, while at the same time minimizing potential systemic adverse effects or toxicity associated with the compound or their encapsulated contents.
[0496] Following transfection of one or more target cells by, for example, the polynucleotides encapsulated in the one or more lipid nanoparticles comprising the pharmaceutical or liposomal compositions disclosed herein, the production of the product (e.g., a polypeptide or protein) encoded by such polynucleotide may be stimulated and the capability of such target cells to express the polynucleotide and produce, for example, a polypeptide or protein of interest is enhanced. For example, transfection of a target cell by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.
[0497] Further, delivery vehicles described herein (e.g., liposomal delivery vehicles) may be prepared to preferentially distribute to other target tissues, cells or organs, such as the heart, lungs, kidneys, spleen. In embodiments, the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to the target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical and liposomal compositions described herein can be delivered to and/or transfect targeted cells or tissues. In some embodiments, thel89ncapsuleated polynucleotides (e.g., mRNA) are capable of being expressed and functional polypeptide products produced (and in some instances excreted) by the target cell, thereby conferring a beneficial property to, for example the target cells or tissues. Such encapsulated polynucleotides (e.g., mRNA) may encode, for example, a hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.
Liposomal Delivery Vehicles
[0498] In some embodiments, a composition is a suitable delivery vehicle. In embodiments, a composition is a liposomal delivery vehicle, e.g., a lipid nanoparticle.
[0499] The terms "liposomal delivery vehicle" and "liposomal composition" are used interchangeably.
[0500] Enriching liposomal compositions with one or more of the cationic lipids disclosed herein may be used as a means of improving (e.g., reducing) the toxicity or otherwise conferring one or more desired properties to such enriched liposomal composition (e.g., improved delivery of the encapsulated polynucleotides to one or more target cells and/or reduced in vivo toxicity of a liposomal composition). Accordingly, also contemplated are pharmaceutical compositions, and in particular liposomal compositions, that comprise one or more of the cationic lipids disclosed herein.
[0501] Thus, in certain embodiments, the compounds of the invention as described herein may be used as a component of a liposomal composition to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).
[0502] As used herein, liposomal delivery vehicles, e.g., lipid nanoparticles, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue. [0503] In certain embodiments, such compositions (e.g., liposomal compositions) are loaded with or otherwise encapsulate materials, such as for example, one or more biologically-active polynucleotides (e.g., mRNA).
[0504] In embodiments, a composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein, encapsulated within a liposome. In embodiments, a liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol- based lipids and one or more PEG-modified lipids, and wherein at least one cationic lipid is a compound of the invention as described herein. In embodiments, a composition comprises an mRNA encoding for a protein (e.g., any protein described herein). In embodiments, a composition comprises an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein.
[0505] In embodiments, a composition (e.g., a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a compound described herein.
[0506] In embodiments, a nucleic acid is an mRNA encoding a peptide or protein. In embodiments, an mRNA encodes a peptide or protein for use in the delivery to or treatment of the lung of a subject or a lung cell (e.g., an mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein). In embodiments, an mRNA encodes a peptide or protein for use in the delivery to or treatment of the liver of a subject or a liver cell (e.g., an mRNA encodes ornithine transcarbamylase (OTC) protein). Still other exemplary mRNAs are described herein.
[0507] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net positive charge.
[0508] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net negative charge.
[0509] In embodiments, a liposomal delivery vehicle (e.g., a lipid nanoparticle) can have a net neutral charge.
[0510] In embodiments, a lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more compounds of the invention as described herein. [0511] For example, the amount of a compound of the invention as described herein in a composition can be described as a percentage ("wt%") of the combined dry weight of all lipids of a composition (e.g., the combined dry weight of all lipids present in a liposomal composition).
[0512] In embodiments of the pharmaceutical compositions described herein, a compound of the invention as described herein is present in an amount that is about 0.5 wt% to about 30 wt% (e.g., about 0.5 wt% to about 20 wt%) of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition).
[0513] In embodiments, a compound of the invention as described herein is present in an amount that is about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition). In embodiments, a compound of the invention as described herein is present in an amount that is about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in a composition such as a liposomal delivery vehicle.
[0514] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
[0515] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is no more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in a composition (e.g., a liposomal composition).
[0516] In embodiments, a composition (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.1 wt% to about 20 wt% (e.g., about 0.1 wt% to about 15 wt%) of a compound described herein. In embodiments, a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of a compound described herein. In embodiments, a delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of a compound described herein. In embodiments, the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
[0517] The amount of a compound of the invention as described herein in a composition also can be described as a percentage ("mol%") of the combined molar amounts of total lipids of a composition (e.g., the combined molar amounts of all lipids present in a liposomal delivery vehicle).
[0518] In embodiments of pharmaceutical compositions described herein, a compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%) of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle.
[0519] In embodiments, a compound of the invention as described herein is present in an amount that is about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol %, or about 45 mol% to about 60 mol% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle. In embodiments, a compound of the invention as described herein is present in an amount that is about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol% or about 5 mol% to about 25 mol% of the combined molar amounts of all lipids present in a composition such as a liposomal delivery vehicle
[0520] In certain embodiments, a compound of the invention as described herein can comprise from about 0.1 mol% to about 50 mol%, or from 0.5 mol% to about 50 mol%, or from about 1 mol% to about 50 mol%, or from about 5 mol% to about 50 mol%, or from about 10 mol% to about 50 mol%, or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol%, of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).
[0521] In certain embodiments, a compound of the invention as described herein can comprise greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol% of the total amount of lipids in the lipid nanoparticle.
[0522] In certain embodiments, a compound as described can comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol %, less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).
[0523] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined molar amounts of total lipids in a composition (e.g., a liposomal composition).
[0524] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is no more than about 5 mol%, about 10 mol%, about 15 mol%, about
20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about
50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about
80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about
98 mol%, or about 99 mol% of the combined molar amounts of total lipids in a composition (e.g., a liposomal composition).
[0525] In embodiments, the percentage results in an improved beneficial effect (e.g., improved delivery to targeted tissues such as the liver or the lung).
[0526] In a typical embodiment, a composition of the invention (e.g., a liposomal composition) comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol- based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the invention as described herein. For example, a composition suitable for practicing the invention has four lipid components comprising a compound of the invention as described herein as the cationic lipid component, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid. The non-cationic lipid may be DOPE or DEPE. The cholesterol-based lipid may be cholesterol. The PEG-modified lipid may be DMG-PEG2K. [0527] In further embodiments, pharmaceutical (e.g., liposomal) compositions comprise one or more of a PEG-modified lipid, a non-cationic lipid and a cholesterol lipid. In other embodiments, such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids. In yet further embodiments, such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids and one or more cholesterol lipids.
[0528] In embodiments, a composition (e.g., lipid nanoparticle) that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more compounds of the invention as described herein and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.
[0529] In embodiments, a composition (e.g., lipid nanoparticle) that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more compound of the invention as described herein; one or more lipids selected from the group consisting of a cationic lipid, a noncationic lipid, and a PEGylated lipid; and further comprises a cholesterol-based lipid. Typically, such a composition has four lipid components comprising a compound of the invention as described herein as the cationic lipid component, a non-cationic lipid (e.g., DOPE), a cholesterol- based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K).
[0530] In embodiments, a lipid nanoparticle that encapsulates a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more compounds of the invention as described herein, as well as one or more lipids selected from the group consisting of a cationic lipid, a noncationic lipid, a PEGylated lipid, and a cholesterol-based lipid.
[0531] According to various embodiments, the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
Synthesis of Lipid Nanoparticles
[0532] In embodiments, lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include methods described in International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety.
[0533] One exemplary process for lipid nanoparticle formulation is Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 2018/089801). Process A ("A") relates to a conventional method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles. In an exemplary process, an ethanol lipid solution and an aqueous buffered solution of mRNA are prepared separately. A solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) is prepared by dissolving lipids in ethanol. The mRNA solution is prepared by dissolving the mRNA in citrate buffer, resulting in mRNA at a concentration of 0.0833mg/ml in citrate buffer with a pH of 4.5. Then, these two solutions are mixed using a pump system. In some instances, the two solutions are mixed using a gear pump system. In certain embodiments, the two solutions are mixing using a T junction (or "Y" junction). The mixture is then purified by diafiltration with a TFF process. The resultant formulation is concentrated and stored at 2-8 °C until further use.
[0534] A second exemplary process for lipid nanoparticle formulation is Process B of WO 2018/089801 (see, e.g., Example 2 and Figure 2 of WO 2018/089801). Process B ("B") refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA. A range of different conditions, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations, may be employed in Process B. In an exemplary process, lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process. The resultant formulation mixture comprises empty lipid nanoparticles in citrate buffer containing alcohol. The formulation is then subjected to a TFF purification process wherein buffer exchange occurs. The resulting suspension of preformed empty lipid nanoparticles is then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution post-mixing can result in a higher percentage of lipid nanoparticles containing mRNA and a higher total yield of mRNA.
Cationic Lipids
[0535] In addition to any of the compounds of the invention as described herein, a composition may comprise one or more additional cationic lipids. [0536] In some embodiments, liposomes may comprise one or more additional cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.
[0537] Suitable additional cationic lipids for use in the compositions include the cationic lipids as described in the literature.
Helper Lipids
[0538] Compositions (e.g., liposomal compositions) may also comprise one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl -phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof. A noncationic or helper lipid suitable for practicing the invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as a non-cationic or helper lipid.
[0539] In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
[0540] In some embodiments, a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in a liposome is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.
[0541] In some embodiments, a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total noncationic lipids in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non-cationic lipid in a liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.
Cholesterol-based Lipids
[0542] In some embodiments, a composition (e.g., a liposomal composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE), which has the following structure,
[0543] In some embodiments, a cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.
[0544] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.
PEGylated Lipids
[0545] In some embodiments, a composition (e.g., a liposomal composition) comprises one or more further PEGylated lipids. A suitable PEG-modified or PEGylated lipid for practicing the invention is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).
[0546] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1- [succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of compounds of the invention as described herein and, in some embodiments, other lipids together which comprise the liposome. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., CM or Cig).
[0547] Contemplated further PEG-modified lipids (also referred to herein as a PEGylated lipid, which term is interchangeable with PEG-modified lipid) include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of Cg-Czo length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235- 237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
[0548] Further PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, or about 3% to about 5% of the total lipid present in the composition (e.g., a liposomal composition).
Pharmaceutical Formulations and Therapeutic Uses
[0549] Compounds of the invention as described herein may be used in the preparation of compositions (e.g., to construct liposomal compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).
[0550] For example, when a liposomal composition (e.g., a lipid nanoparticle) comprises or is otherwise enriched with one or more of the compounds disclosed herein, the phase transition in the lipid bilayer of the one or more target cells may facilitate the delivery of the encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle) into the one or more target cells.
[0551] Similarly, in certain embodiments compounds of the invention as described herein may be used to prepare liposomal vehicles that are characterized by their reduced toxicity in vivo. In certain embodiments, the reduced toxicity is a function of the high transfection efficiencies associated with the compositions disclosed herein, such that a reduced quantity of such composition may administered to the subject to achieve a desired therapeutic response or outcome.
[0552] Thus, pharmaceutical formulations comprising a compound described and nucleic acids provided by the present invention may be used for various therapeutic purposes. To facilitate delivery of nucleic acids in vivo, a compound described herein and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizing reagents. In some embodiments, a compound described herein can be formulated via pre-mixed lipid solution. In other embodiments, a composition comprising a compound described herein can be formulated using post-insertion techniques into the lipid membrane of the nanoparticles. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.
[0553] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments the administration results in delivery of the nucleic acids to a muscle cell. In some embodiments the administration results in delivery of the nucleic acids to a hepatocyte (/.e., liver cell).
[0554] A common route for administering a liposomal composition of the invention may be intravenous delivery, in particular when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency). Alternatively, depending on the disease or disorder to be treated, the liposomal composition may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, a liposomal composition of the invention is typically administered intramuscularly. Diseases or disorders affecting the eye may be treated by administering a liposomal composition of the invention intravitreally.
[0555] Alternatively or additionally, pharmaceutical formulations of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue (e.g., in a sustained release formulation). Local delivery can be affected in various ways, depending on the tissue to be targeted. Exemplary tissues in which delivered mRNA may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid. In embodiments, the tissue to be targeted in the liver. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
[0556] Compositions described herein can comprise mRNA encoding peptides including those described herein (e.g., a polypeptide such as a protein).
[0557] In embodiments, a mRNA encodes a polypeptide.
[0558] In embodiments, a mRNA encodes a protein.
[0559] Exemplary peptides encoded by mRNA (e.g., exemplary proteins encoded by mRNA) are described herein.
[0560] The present invention provides methods for delivering a composition having full-length mRNA molecules encoding a peptide or protein of interest for use in the treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.
[0561] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or protein for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from an infectious agent, such as a virus.
[0562] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen determined from a subject's own cancer cell, i.e., to provide a personalized cancer vaccine. [0563] In embodiments, a composition comprises an mRNA encoding for cystic fibrosis transmembrane conductance regulator (CFTR) protein.
[0564] In embodiments, a composition comprises an mRNA encoding for ornithine transcarbamylase (OTC) protein.
Delivery Methods
[0565] The route of delivery used in the methods of the invention allows for non-invasive, selfadministration of the compounds of the invention. In some embodiments, the methods involve intratracheal or pulmonary administration by aerosolization, nebulization, or instillation of a compositions comprising mRNA encoding a therapeutic protein in a suitable transfection or lipid carrier vehicles as described above. In some embodiments, the protein is encapsulated with a liposome. In some embodiments, the liposome comprises a lipid, which is a compound of the invention. As used herein below, administration of a compound of the invention includes administration of a composition comprising a compound of the invention.
[0566] Although the local cells and tissues of the lung represent a potential target capable of functioning as a biological depot or reservoir for production and secretion of the protein encoded by the mRNA, applicants have discovered that administration of the compounds of the invention to the lung via aerosolization, nebulization, or instillation results in the distribution of even nonsecreted proteins outside the lung cells. Without wishing to be bound by any particular theory, it is contemplated that nanoparticle compositions of the invention pass, through the lung airwayblood barrier, resulting in translation of the intact nanoparticle to non-lung cells and tissues, such as, e.g., the heart, the liver, the spleen, where it results in the production of the encoded protein in these non-lung tissues. Thus, the utility of the compounds of the invention and methods of the invention extend beyond production of therapeutic protein in lung cells and tissues of the lung and can be used to delivery to non-lung target cells and/or tissues. They are useful in the management and treatment of a large number of diseases, and in particular peripheral diseases which result from both secreted and non-secreted protein and/or enzyme deficiencies (e.g., one or more lysosomal storage disorders). In certain embodiments, the compounds of the invention, used in the methods of the invention result in the distribution of the mRNA encapsulated nanoparticles and production of the encoded protein in the liver, spleen, heart, and/or other non- lung cells. For example, administration of the compounds of the invention, by aerosolization, nebulization, or instillation to the lung will result in the composition itself and its protein product (e.g., functional beta galactosidase protein) will be detectable in both the local cells and tissues of the lung, as well as in peripheral target cells, tissues and organs as a result of translocation of the mRNA and delivery vehicle to non-lung cells.
[0567] In certain embodiments, the compounds of the invention may be employed in the methods of the invention to specifically target peripheral cells or tissues. Following the pulmonary delivery, it is contemplated the compounds of the invention cross the lung airwayblood barrier and distribute into cells other than the local lung cells. Accordingly, the compounds disclosed herein may be administered to a subject by way of the pulmonary route of administration, using a variety of approach known by those skilled in the art (e.g., by inhalation), and distribute to both the local target cells and tissues of the lung, as well as in peripheral nonlung cells and tissues (e.g., cells of the liver, spleen, kidneys, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both the local cells of the lung and the peripheral non-lung cells can serve as biological reservoirs or depots capable of producing and/or secreting a translation product encoded by one or more polynucleotides. Accordingly, the present invention is not limited to the treatment of lung diseases or conditions, but rather can be used as a non-invasive means of facilitating the delivery of polynucleotides, or the production of enzymes and proteins encoded thereby, in peripheral organs, tissues and cells (e.g., hepatocytes) which would otherwise be achieved only by systemic administration. Exemplary peripheral non- lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
[0568] Following administration of the composition to the subject, the protein product encoded by the mRNA (e.g., a functional protein or enzyme) is detectable in the peripheral target tissues for at least about one to seven days or longer following administration of the compound to the subject. The amount of protein product necessary to achieve a therapeutic effect will vary depending on the condition being treated, the protein encoded, and the condition of the patient. For example, the protein product may be detectable in the peripheral target tissues at a concentration (e.g., a therapeutic concentration) of at least 0.025-1.5 pg/ml (e.g., at least 0.050 pg/ml, at least 0.075 pg/ml, at least 0.1 pg/ml, at least 0.2 pg/ml, at least 0.3 pg/ml, at least 0.4 pg/ml, at least 0.5 pg/ml, at least 0.6 pg/ml, at least 0.7 pg/ml, at least 0.8 pg/ml, at least 0.9 pg/ml, at least 1.0 pg/ml, at least 1.1 pg/ml, at least 1.2 pg/ml, at least 1.3 pg/ml, at least 1.4 pg/ml, or at least 1.5 pg/ml), for at least about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer following administration of the compound to the subject.
[0569] It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of an aerosol mist produced by a liquid nebulizer or the use of a dry powder apparatus such as that described in U.S. patent 5,780,014, incorporated herein by reference.
[0570] In certain embodiments, the compounds of the invention may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or upon administration to the subject. Such compounds may be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject. In some embodiments, such devices (e.g., a metered dose inhaler, jet-nebulizer, ultrasonic nebulizer, dry-powder-inhalers, propellant-based inhaler or an insufflator) facilitate the administration of a predetermined mass, volume or dose of the compositions (e.g., about 0.5 mg/kg of mRNA per dose) to the subject. For example, in certain embodiments, the compounds of the invention are administered to a subject using a metered dose inhaler containing a suspension or solution comprising the compound and a suitable propellant. In certain embodiments, the compounds of the invention may be formulated as a particulate powder (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the invention formulated as respirable particles are appropriately sized such that they may be respirable by the subject or delivered using a suitable device (e.g., a mean D50 or D90 particle size less than about 500pm, 400pm, 300pm, 250pm, 200pm, 150pm, 100pm, 75pm, 50pm, 25pm, 20pm, 15pm, 12.5pm, 10pm, 5pm, 2.5pm or smaller). In yet other embodiments, the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, the compounds of the invention are administered to a subject such that a concentration of at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, at least 5.0 mg/kg, at least 6.0 mg/kg, at least 7.0 mg/kg, at least 8.0 mg/kg, at least 9.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, or at least 100 mg/kg body weight is administered in a single dose. In some embodiments, the compounds of the invention are administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg mRNA is administered in one or more doses.
Examples
[0571] Abbreviations
DCM = dichloromethane
DIPEA = N,N-Diisopropylethylamine
DM = dimineralized
DMAP = 4-Dimethylaminopyridine
EDC-HCI = /V-(3-Dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride EDCI = l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ELSD = evaporative light-scattering detection EtOAc = ethyl acetate h = hour r.t. = room temperature SM = starting material TLC = thin layer chromatography
Example 1. Synthesis of JC-TL1-10D-E6-2 (Compound (13))
Synthetic Scheme A
Intermediate [A-31:
[0572] To a stirred solution of hexane-l,6-diol [A-1] (10 g, 0.085 mol) and triethylamine (42.9 g, 0.45 mol) in dichloromethane (150 mL) was cooled to 0 °C, 4-(dimethylamino)pyridin-l-ium (1.03 g, 0.0085 mol) was added followed by propionyl chloride [A-2] (3.93 g, 0.042 mol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by TLC (SM was consumed). Reaction mixture was quenched by cold saturated NaHCOs upto pH 7 , and extracted with DCM (3x100 mL). The resulting organic layer was dried over NajSCU, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiCh: 0-20 % ethyl acetate in hexane), to give the desired 6-hydroxyhexyl propionate [A-3] (4.5 g, 30 %, Yield) as a light yellow oil. [0573] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J= 5.6 Hz, 2H), 3.64 (br, 2H), 2.32 (q, J= 7.6 Hz, 2H), 1.67-1.60 (m, 2H), 1.58-1.54 (m, 2H), 1.44-1.37 (m, 4H), 1.13 (t, J= 7.6 Hz, 3H). Intermediate [A-5]: [0574] To a stirred solution of 6-hydroxyhexyl propionate [A-3] (4.5 g, 0.026 mol) and citric acid [A-4] (1.25 g, 0.0065 mol) in dichloromethane (100 mL) was cooled to 0 oC, EDC.HCl (4.95 g, 0.026 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.78 g, 0.0065 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (50 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product tris(6-(propionyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [A-5] (0.57 g, 13 %, Yield) as a pale yellow liquid. [0575] 1H NMR (400 MHz, CDCl3): δ 4.21 (t, J= 6.8 Hz, 2H), 4.12-4.04 (m, 10H), 2.88 (d, J= 15.6 Hz, 2H), 2.79 (d, J= 15.6 Hz, 2H), 2.32 (q, J= 7.6 Hz, 6H), 1.71-1.67 (m, 2H), 1.64-1.62 (m, 10H), 1.38-1.36 (m, 12H), 1.13 (t, J= 7.8Hz, 9H). [0576] ELSD analysis: Purity 98.66 %, Calculated C33H56O13 = 660.37, Observed = 661.10 (m/z, M+H+).
JC-TL1-10D-E6-2 (Compound (13)) Compound (13) [0577] To a stirred solution of tris(6-(propionyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [A-5] (0.57 g, 0.00086 mol) and 3-(dimethylamino)propanoic acid [6] (0.404 g, 0.00345 mol) in dichloromethane (15 mL) was cooled to 0 oC, EDC.HCl (0.662 g, 0.00345 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.105 g, 0.00086 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product tris(6-(propionyloxy)hexyl) 2-((3-(dimethylamino)propanoyl)oxy)propane- 1,2,3-tricarboxylate [JC-TL1-10D-E6-2] (Compound (13)) (0.115 g, 17.4 %, Yield) as a yellow liquid. [0578] 1H NMR (400 MHz, DMSO-d6): δ 4.05-3.97 (m, 12H), 3.16 (d, J= 15.2Hz, 2H), 3.08 (d, J= 15.2 Hz, 2H), 2.46-2.37 (m, 4H), 2.28 (q, J= 7.6 Hz, 6H), 2.10 (s, 6H), 1.56-1.53 (m, 12H), 1.31-1.30 (m, 12H), 1.01 (t, J= 6.8Hz, 9H). [0579] ELSD analysis: Purity 99.66 %, Calculated C38H65NO14 = 759.44, Observed = 760.50 (m/z, M+H+).
Example 2. Synthetic Protocol for M1266-J03983-009 (JC-TL1-12D-E4-6) (Compound (12))
Synthetic Scheme B
B-3
[0580] To a stirred solution of butane-l,4-diol [B-1] (10 g, 0.111 mol) and heptanoic acid [B-2] (14.4 g, 0.111 mol) in dichloromethane (200 mL) was cooled to 0 °C, EDC.HCI (25.5 g, 0.133 mol), was added followed by 4-(dimethylamino)pyridin-l-ium (1.35 g, 0.011 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (200 mL) was added to the reaction mixture and extracted with DCM (3x300 mL). The resulting organic layer was dried over NajSCU, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-20 % ethyl acetate in n-hexane) to obtain pure product 4-hydroxybutyl heptanoate [B-3] (6.2 g, 27 % Yield) as a yellow liquid. Results: [0581] 1H NMR (400 MHz, CDCl3): δ 4.09 (t, J= 6.0 Hz, 2H), 3.69-3.65 (m, 2H), 2.28 (t, J= 7.6 Hz, 2H), 1.73-1.59 (m, 6H), 1.28 (br, 6H), 0.87-0.85 (m, 3H). Intermediate [B-5]: [0582] To a stirred solution of 4-hydroxybutyl heptanoate [B-3] (6.18 g, 0.0306 mol) and citric acid [B-4] (1.47 g, 0.00765 mol) in dichloromethane (150 mL) was cooled to 0 oC, EDC.HCl (5.84 g, 0.0306 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.9 g, 0.00765 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane) to obtain pure product tris(4-(heptanoyloxy)butyl) 2-hydroxypropane-1,2,3- tricarboxylate [B-5] (1.53 g, 27 % Yield) as a colourless liquid. [0583] 1H NMR (400 MHz, CDCl3): δ 4.25 (t, J= 6.0 Hz, 2H), 4.14-4.07 (m, 10H), 2.88 (d, J= 15.6 Hz, 2H), 2.80 (d, J= 15.6 Hz, 2H), 2.29 (t, J= 7.6 Hz, 6H), 1.85-1.68 (m, 12H), 1.64-1.56 (m, 6H), 1.34- 1.22 (m, 18H), 0.88 (t, J= 7.8Hz, 9H). [0584] ELSD analysis: Purity 98.99 %, Calculated C39H68O13 = 744.46, Observed = 745.25 (m/z, M+H+).
JC-TL1-12D-E4-6 (Compound (12)) (Compound (12)) [0585] To a stirred solution of tris(4-(heptanoyloxy)butyl) 2-hydroxypropane-1,2,3- tricarboxylate [5] (1.53 g, 0.00205 mol) and 3-(dimethylamino)propanoic acid [B-6] (0.962 g, 0.00822 mol) in dichloromethane (30 mL) was cooled to 0 oC, EDC.HCl (1.56 g, 0.00822 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.251 g, 0.00205 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane) to obtain pure product tris(4-(heptanoyloxy)butyl) 2-((3-(dimethylamino)propanoyl)oxy)propane- 1,2,3-tricarboxylate [JC-TL1-12D-E4-6] (Compound (12)) (0.3 g, 18 %, Yield) as a brown liquid. [0586] 1H NMR (400 MHz, CDCl3): δ 4.18 (t, J= 6.0Hz, 2H), 4.10-4.08 (m, 10H), 3.30 (d, J= 16.0Hz, 2H), 3.23 (d, J= 15.6Hz, 2H), 2.57 (t, J= 7.2Hz, 2H), 2.48 (t, J= 7.2Hz, 2H), 2.29 (t, J= 7.2Hz, 6H), 2.21 (s, 6H), 1.72-1.67 (m, 12H), 1.64-1.59 (m, 7H), 1.34-1.25 (m, 17H), 0.88 (t, J= 6.4Hz, 9H). [0587] ELSD analysis: Purity 99.83 %, Calculated C44H77NO14 = 843.53, Observed = 844.65 (m/z, M+H+).
Example 3. Synthetic Protocol for M1266-J03983-027 (JC-TL1-12D-E6-4) (Compound (40)) Synthetic Scheme C Intermediate [C-3]: [0588] To a stirred solution of hexane-1,6-diol [C-1] (9.8 g, 0.08 mol) and triethylamine (41.8 g, 0.4 mol) in dichloromethane (150 mL) was cooled to 0 oC, 4-(dimethylamino)pyridin-1-ium (1.0 g, 0.008 mol) was added followed by pentanoyl chloride [C-2] (5.0 g, 0.04 mol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by TLC (SM was consumed). Reaction mixture was quenched by cold saturated NaHCO3 upto pH 7, and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-20 % ethyl acetate in hexane), to give the desired 6-hydroxyhexyl pentanoate [C-3] (6.2 g, 36 %, Yield) as a light yellow oil. [0589] 1H NMR (400 MHz, CDCl3): δ 4.06 (t, J= 6.8 Hz, 2H), 3.64 (t, J= 6.8 Hz, 2H), 2.29 (t, J= 7.2 Hz, 2H), 1.66-1.54 (m, 6H), 1.42-1.31 (m, 6H), 0.92-0.82 (m, 3H). Intermediate [C-5]:
[0590] To a stirred solution of 6-hydroxyhexyl pentanoate [C-3] (5.3 g, 0.026 mol) and Citric acid [C-4] (1.26 g, 0.0065 mol) in dichloromethane (100 mL) was cooled to 0 °C, EDC.HCI (5.02 g, 0.026 mol), was added followed by 4-(dimethylamino)pyridin-l-ium (0.8 g, 0.0065 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x200 mL). The resulting organic layer was dried over NajSCU, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product tris(6-(pentanoyloxy)hexyl) 2-hydroxypropane-l,2,3- tricarboxylate [C-5] (1.61 g, 33 %, Yield) as a pale yellow liquid.
[0591] ELSD analysis: Purity 99.62 %, Calculated CagHggOia = 744.46, Observed = 767.20 (m/z,
M+Na+).
JC-TL1-12D-E6-4 (Compound (40)) Compound (40)
[0592] To a stirred solution of tris(6-(pentanoyloxy)hexyl) 2-hydroxypropane-l,2,3- tricarboxylate [C-5] (1.61 g, 0.0022 mol) and 3-(dimethylamino)propanoic acid [C-6] (1.013 g, 0.0086 mol) in dichloromethane (30 mL) was cooled to 0 °C, EDC.HCI (1.69 g, 0.0086 mol), was added followed by 4-(dimethylamino)pyridin-l-ium (0.26 g, 0.0022 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product JC-TL1-12D-E6-4 (Compound (40) (0.225 g, 12 %, Yield) as a pale yellow liquid. [0593] 1H NMR (400 MHz, CDCl3): δ 4.14 (t, J= 6.8 Hz, 2H), 4.08-4.03 (m, 10H), 3.30 (d, J= 15.6 Hz, 2H), 3.21 (d, J= 15.6 Hz, 2H), 2.60-2.44 (m, 4H), 2.29 (t, J= 7.6 Hz, 6H), 2.21 (s,6H), 1.63-1.56 (m, 18H), 1.37-1.29 (m, 18H), 0.91 (t, J= 7.2 Hz, 9H). [0594] ELSD analysis: Purity 98.58 %, Calculated C44H77NO14 = 843.53, Observed = 844.25 (m/z, M+Na+). Example 4. Synthetic Protocol for M1266-J03983-025 (JC-TL1-16D-E5-9) (Compound (29)) Synthetic Scheme D Intermediate [D-3]: [0595] To a stirred solution of pentane-1,5-diol [D-1] (10 g, 0.0961 mol) and decanoic acid [D- 2] (16.5 g, 0.0961 mol) in dichloromethane (200 mL) was cooled to 0 oC, EDC.HCl (22.0 g, 0.115 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (1.17 g, 0.009 mol) and stirred for 16 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-20 % ethyl acetate in n- hexane), to obtain pure product 5-hydroxypentyl decanoate [D-3] (5.4 g, 22 % Yield) as a pale yellow liquid. [0596] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J= 6.4 Hz, 2H), 3.68-3.63 (m, 2H), 2.28 (t, J= 7.6 Hz, 2H), 1.68-1.58 (m, 6H), 1.47-1.42 (m, 2H), 1.29-1.25 (m, 12H), 0.89-0.87 (m, 3H). Intermediate [5]: [0597] To a stirred solution of 5-hydroxypentyl decanoate [D-3] (5.37 g, 0.0208 mol) and Citric acid [D-4] (1.0 g, 0.0052 mol) in dichloromethane (100 mL) was cooled to 0 oC, EDC.HCl (4.0 g, 0.0208 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.635 g, 0.0052 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product tris(5-(decanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [D-5] (0.7 g, 14 % Yield) as a pale yellow liquid. [0598] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J= 6.8 Hz, 2H), 4.11-4.04 (m, 10H), 2.88 (d, J= 15.6 Hz, 2H), 2.79 (d, J= 15.6 Hz, 2H), 2.28 (t, J= 7.6 Hz, 6H), 1.74-1.59 (m, 16H), 1.46-1.36 (m, 6H), 1.29- 1.26 (m, 38H), 0.89-0.86 (m, 9H). JC-TL1-16D-E5-9 (Compound (29)) tricarboxylate [D-5] (0.7 g, 0.00078 mol) and 3-(dimethylamino)propanoic acid [D-6] (0.36 g, 0.00312 mol) in dichloromethane (20 mL) was cooled to 0 oC, EDC.HCl (0.59 g, 0.00312 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.09 g, 0.00078 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (10 mL) was added to the reaction mixture and extracted with DCM (3x25 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product JC-TL1-16D-E5-9 (Compound (29) (0.11 g, 14 %, Yield) as a colourless liquid. [0600] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J= 6.8Hz, 2H), 4.09-4.04 (m, 10H), 3.30 (d, J= 15.6Hz, 2H), 3.22 (d, J= 15.6Hz, 2H), 2.60-2.56 (m, 2H), 2.50-2.46 (m, 2H), 2.28 (t, J= 7.6Hz, 6H), 2.22 (s, 6H), 1.69-1.59 (m, 18H), 1.44-1.37 (m, 6H), 1.29-1.26 (m, 36H), 0.87 (t, J= 6.4Hz, 9H). [0601] ELSD analysis: Purity 98.73 %, Calculated C56H101NO14 = 1011.72, Observed = 1012.40 (m/z, M+H+).
Example 5. Synthetic Protocol for M1266-J03984-031 (TL1-10D-E5-3) (Compound (38)) Synthetic Scheme E Intermediate [E-3]: [0602] To a stirred solution of pentane-1,5-diol [E-1] (10 g, 0.096 mol) and triethylamine (48.5 g, 0.48 mol) in dichloromethane (100 mL) was cooled to 0 oC, 4-(dimethylamino)pyridin-1-ium (1.17 g, 0.0096 mol) was added followed by butyryl chloride [E-2] (5.09 g, 0.048 mol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by TLC (SM was consumed). Reaction mixture was quenched by cold saturated NaHCO3 upto pH 7, and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-20 % ethyl acetate in hexane), to give the desired 5-hydroxypentyl butyrate [E-3] (4.2 g, 25 %, Yield) as a colourless oil. [0603] 1H NMR (400 MHz, CDCl3): δ 4.08 (t, J= 6.8 Hz, 2H), 3.68-3.63 (m, 2H), 2.28 (t, J= 7.2 Hz, 2H), 1.70-1.58 (m, 5H), 1.47-1.40 (m, 2H), 1.25 (t, J=5.2Hz, 1H), 0.94 (t, J= 7.6 Hz, 3H). Intermediate [E-5]: [0604] To a stirred solution of 5-hydroxypentyl butyrate [E-3] (4.16 g, 0.0239 mol) and Citric acid [4] (1.15 g, 0.0059 mol) in dichloromethane (50 mL) was cooled to 0 oC, EDC.HCl (4.60 g, 0.0239 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.73 g, 0.0059 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product tris(5-(butyryloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [E-5] (1.05 g, 27 %, Yield) as a pale yellow liquid. [0605] ELSD analysis: Purity 97.05 %, Calculated C33H56O13 = 660.37, Observed = 661.10 (m/z, M+H+). JC-TL1-10D-E5-3 (Compound (38)) (Compound (38)) [0606] To a stirred solution of tris(5-(butyryloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [E-5] (1.0 g, 0.0015 mol) and 3-(dimethylamino)propanoic acid [E-6] (0.929 g, 0.006 mol) in dichloromethane (20 mL) was cooled to 0 oC, EDC.HCl (1.16 g, 0.006 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.184 g, 0.0015 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product JC-TL1-10D-E5-3 (Compound (38)) (0.410 g, 35 %, Yield) as a pale yellow liquid. [0607] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J= 6.8Hz, 2H), 4.12-4.03 (m, 10H), 3.29 (d, J= 15.6 Hz, 2H), 3.21 (d, J= 15.6 Hz, 2H), 2.59-2.56 (m, 2H), 2.49-2.44 (m, 2H), 2.27 (t, J= 7.6Hz, 6H), 2.21 (s, 6H), 1.71-1.60 (m, 18H), 1.44-1.37 (m, 6H), 0.94 (t, J= 7.6 Hz, 9H). [0608] ELSD analysis: Purity 99.79 %, Calculated C38H65NO14 = 759.44, Observed = 760.15 (m/z, M+H+). Example 6. Synthetic Protocol for M1266-J03996-029 (TL1-14D-E8-4) (Compound (28)) Intermediate-F-3 [0609] To a stirred solution of 1, 8-octanediol (10 g, 0.084 mol) and triethylamine (45ml, 0.34 mol) in dichloromethane (200 ml) was cooled to 0°C, then added pentanoyl chloride (4.09 g, 0.034 mol) drop wise. Reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL) and dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [F-3] (5.1 g, 32.23%) as yellow liquid. [0610] 1H NMR (400 MHz, CDCl3): δ 4.04 (t, J = 6.4 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.61 (m, 5H), 1.36 (m, 12H), 0.90 (t, J = 6.4 Hz, 3H). Intermediate-F-5 [0611] To a stirred solution of 8-hydroxyoctyl pentanoate [Int-3] (4.5 g, 0.195 mol) and citric acid (0.937 g, 0.00488 mol) in dichloromethane (20 ml) was added EDCI (3.75 g, 0.0195 mol) followed by addition of DMAP (0.60 g, 0.00488 mol). Reaction mass was stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was diluted with DCM (25.0 mL), washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 20 % ethyl acetate in n-heptane to get desired product [F-5] (0.8 g, 19.80%) as yellow colour liquid. [0612] LCMS analysis: Purity 99 %, Calculated C45H80O13 = 828.56, Observed = 829.25 (m/z, M+H+). TL1-14D-E8-4 (Compound (28)) Compound (28) [0613] To a stirred solution tris(8-(pentanoyloxy)octyl) 2-hydroxypropane-1,2,3-tricarboxylate [F-5] (0.8 g, 0.00964 mol) and N,Ndimethylpropionic acid (0.451 g, 0.00385 mol) in dichloromethane (10 ml ) was added EDCI (0.740 g, 0.00385 mol,) followed by addition of DMAP (0.117g, 0.000964 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was diluted with DCM (25.0 mL), washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain desired product TL1- 14D-E8-4 (0.30 g, 25%) as yellow colour liquid. [0614] 1H NMR (400 MHz, DMSO-d6): δ 4.13 (t, J = 6.4 Hz, 2H), 4.06 (m, 10H), 3.31-3.19 (m, 4H), 2.58 (t, J = 7.2 Hz, 2H) 2.48 (t, J = 7.2 Hz, 2H) 2.29 (t, J = 7.6 Hz, 6H), 2.21 (s, 6H), 1.64-1.56 (m, 17H), 1.38-1.31 (m, 31H), 0.91 (t, J = 7.2, 9H). [0615] LCMS analysis: Purity 99.34 %, Calculated C50H89NO14 = 927.63, Observed = 928.25 (m/z, M+H+).
Example 7. Synthetic Protocol for M1266-J03996-019 (TL1-16D-E6-8) (Compound (57)) Synthetic Scheme G [0616] A stirred solution of 1,6-hexanediol (10 g, 0.084 mol) and trimethylamine (55.92 ml, 0.42 mol) in dichloromethane (200 ml) was cooled to 0°C. Added nonanoyl chloride (7.42 g, 0.042 mol) drop wise. Reaction mass was allowed to stirred at RT for 16 h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100.0 mL), dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [G-3] (5.0 g, 22.90%) as yellow liquid. [0617] 1H NMR (400 MHz, CDCl3): δ 4.06 (t, J = 6.4 Hz, 2H), 3.64 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.66-1.59 (m, 6H), 1.43-1.20 (m, 14H), 0.89 (t, J = 6.4 Hz, 3H). Intermediate G-5
[0618] To a stirred solution of 6-hydroxyhexyl nonanoate [G-3] (4.5 g, 0.0174 mol) and citric acid (1.06 g, 0.0055 mol) in dichloromethane (100 ml) was added EDCI (4.25 g, 0.022 mol) followed by DMAP (0.076 g, 0.0055 mol). Reaction mass was stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 ml), dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain desired product [G-5] 0.7 g, (13.39%) as yellow colour liquid mass.
[0619] LCMS analysis: Purity 90.81%, Calculated C5iH920i3 = 912.65, Observed = 913.35 (m/z,
M+H+).
TL1-16D-E6-8 (Compound (57)) (Compound (57))
[0620] To a stirred solution of tris(6-(nonanoyloxy)hexyl) 2-hydroxypropane-l,2,3- tricarboxylate [G-5] (0.7 g, 0.000766 mol) and N,N-dimethylpropionic acid [6] (0.358 g, 0.00306 mol) in dichloromethane (25 ml) was added EDCI (0.590 g, 0.003061 mol,) followed by addition of DMAP (0.11g, 0.00090 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM consumed). Reaction mass was quenched with DM water (100 ml), extracted with DCM (2x 50.0 mL), dried over sodium sulphate and concentrated. Crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [TL1-16D-E6-8] 0.250 g, (32.25%) as yellow colour liquid mass. [0621] 1H NMR (400 MHz, DMSO-d6): δ 4.21 -4.15 (t, J = 6.4 Hz, 2H), 4.08 (m, 10H), 3.31-3.19 (m, 4H), 2.58 (t, J = 7.2 Hz, 2H) 2.48 (m, 2H) 2.29 (t, J = 7.6 Hz, 6H), 2.218 (s, 6H),1.61 (br, 18H), 1.37 (bs, 12H), 1.28-1.26 (m, 30H) 0.88 (t, J = 7.2, 9H). [0622] LCMS analysis: Purity 96.13 %, Calculated C56H101NO14 = 1011.72, Observed = 1012.35 (m/z, M+H+). Example 8. Synthetic Protocol for M1266-J03996-031(TL1-16D-E8-6) (Compound (54)) Synthetic Scheme H [0623] To a stirred solution of 1,8, octanediol (10 g, 0.084 mol) and triethylamine (45ml, 0.34 mol) in dichloromethane (200 ml) was cooled to 0°C. Added heptenoyl chloride (5.05 g, 0.034 mol) as drop wise. Reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water 01 L, dichloromethane layer was separated and dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain [H-3] 5.2 g, (29.42%) as yellow colour liquid mass. [0624] 1H NMR (400 MHz, CDCl3): δ 4.04 (t, J = 6.4 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.62 (m, 6H), 1.38 (m, 15H), 0.87 (t, J = 6.4 Hz, 3H). Intermediate H-5 [0625] To a stirred solution of 8-hydroxyoctyl heptanoate [H-3] (5.0 g, 0.143 mol) and citric acid (0.928 g, 0.00483 mol) in dichloromethane (100 ml) was added EDCI (3.71 g, 0.0193 mol) followed by addition of DMAP (0.58 g, 0.0193 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to obtain desired product [Int-5] (0.820 g, 18.63%) as yellow colour liquid mass. [0626] LCMS analysis: Purity 98.36 %, Calculated C51H92O13 = 912.65, Observed = 913.35 (m/z, M+H+). TL1-16D-E8-6 (Compound (54)) [0627] To a stirred solution tris(8-(heptanoyloxy)octyl) 2-hydroxypropane-1,2,3-tricarboxylate [H-5] (0.820 g, 0.00897 mol) and N,Ndimethylpropionic acid [Int-6] (0.420 g, 0.00359 mol) in dichloromethane (50 ml ), was added EDCI (0.690 g, 0.00359 mol,) followed by addition of DMAP (0.109g, 0.000897 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 20 % ethyl acetate in n-heptane to obtain desired product [TL1-16D-E8-6] 0.250 g, (27.77%) as yellow colour liquid mass. [0628] 1H NMR (400 MHz, DMSO-d6): δ 4.127(t, J = 6.4 Hz, 2H), 4.061(m, 10H), 3.310-3.190(m, 4H), 2.583(t, J = 7.2 Hz, 2H) 2.478 (t, J = 7.2 Hz, 2H) 2.84(t, J = 7.6 Hz, 6H), 2.215 (s, 6H), 1.699 (s, 1H), 1.607 (m, 17H), 1.311(m, 44H), 0.877(t, J = 7.2, 9H). [0629] LCMS analysis: Purity 98.95 %, Calculated C56H101NO14 = 1011.72, Observed = 1012.30 (m/z, M+H+). Example 9. Synthetic Protocol for M1266-J03996-021 (TL1-18D-E6-10) (Compound (56)) Synthetic Scheme I [0630] A stirred solution of 1,6-hexanediol (10 g, 0.084 mol) and triethylamine (55.92 ml, 0.42 mol) in dichloromethane (200 ml), was cooled to 0°C and added undecanoyl chloride (8.59 g, 0.042 mol) drop wise. Reaction mass was allowed to stirred at RT for 16 h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate. Distilled out solvent under reduced pressure and resulting crude was purified over silica using 10% ethyl acetate in n-heptane. Obtained pure wt 7.3 g of I-3, (30.12%) as yellow colour liquid mass. [0631] 1H NMR (400 MHz, CDCl3): δ 4.06 (t, J=6.4 Hz, 2H), 3.64 (t, J = 6.4 Hz, 2H), 2.28 (t, J= 7.6 Hz, 2H), 1.63 (m, 5H), 1.43 (m, 4H), 1.25 (m, 15H), 0.87 (t, J = 6.4 Hz, 3H). Intermediate I-5 [0632] To a stirred solution of 6-hydroxyhexyl undecanoate [I-3] (7.0 g, 0.02443 mol) and citric acid [4] (1.48 g, 0.0077 mol) in dichloromethane (100 ml) was added EDCI (5.95 g, 0.0310 mol) followed by addition of DMAP (0.946 g, 0.0077 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water (500 ml), dried over sodium sulphate and concentrated. Crude was purified over silica using 10-15 % ethyl acetate in n-heptane to get desired product [I-5] 0.550 g, (7.25%) as yellow liquid mass. [0633] LCMS analysis: Purity 99.54 %, Calculated C57H104O13 = 996.75, Observed = 1019.40 (m/z, M+H++23). TL1-18D-E6-10 (Compound (56)) (Compound (56)) [0634] To a stirred solution of tris(6-(undecanoyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [I-5] (0.550 g, 0.00055 mol) and N,N-dimethylpropionic acid [I-6] (0.258 g, 0.002207 mol) in dichloromethane (25 ml ) was added EDCI (0.423 g, 0.002207 mol,) followed by addition of DMAP (0.07g, 0.00055 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (25 ml) and dried over sodium sulphate and concentrated. Crude was purified over silica using 10% ethyl acetate in n-heptane. Obtained pure product, [TL1-18D-E6-10] 0.150 g, (24.83%) as yellow liquid. [0635] 1H NMR (400 MHz, CDCl3): δ 4.14 (t, J = 6.4 Hz, 2H), 4.08 (m, 10H), 3.31-3.19 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H) 2.48 (t, J = 7.2 Hz, 2H) 2.28 (t, J = 7.6 Hz, 6H), 2.22 (s, 6H), 1.61 (m, 18H), 1.37 (br, 12H), 1.28 (m, 42H) 0.87 (t, J = 7.2, 9H). [0636] LCMS analysis: Purity 99.87 %, Calculated C62H113NO14 = 1095.82, Observed = 1096.40 (m/z, M+H+). Example 10. Synthetic Protocol for TL1-18D-E8-8 (Compound (53)) Synthetic Scheme J [0637] A stirred solution of octane diol (10 g, 0.068 mol) in dichloromethane (200 ml) was cooled to 0°C and then added triethylamine (45.26 ml, 0.34 mol). Reaction mass was stirred for 05 minute then added DMAP (4.17 g, 0.034 mol) followed by nonanoyl chloride (6.0 g, 0.034 mol) drop wise. Resulting reaction mass was allowed to stirred at RT for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get [J-3] (5.2 g, 26.55%) as yellow colour liquid mass. [0638] 1H NMR (400 MHz, CDCl3): δ 4.04 (t, J = 6.4 Hz, 2H), 3.65 (m, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.57 (m, 4H), 1.280 (m, 20H), 0.888 (t, J = 6.4 Hz, 3H). Intermediate J-5 [0639] To a stirred solution of 8-hydroxyoctyl nonanoate [J-3] (5.0 g, 0.0174 mol) and citric acid (1.06 g, 0.0055 mol) in dichloromethane (100 ml) was added EDCI (4.25 g, 0.022 mol) followed by addition of DMAP (0.076 g, 0.0055 mol). Reaction mass was stirred for 16 h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was diluted with DCM (100.0 mL) and washed with DM water (100.0 mL), dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get [I-5] (0.9 g, 15.7%) as yellow colour liquid mass. [0640] 1H NMR (400 MHz, DMSO-d6): δ 6.92 (s, 1H), 4.20-4.15 (m, 4H), 4.11 (m, 8H), 4.01 (s, 2H), 3.95 (s, 2H), 2.28 (t, J = 7.6 Hz, 6H), 1.63 (m, 18H), 1.33 (m, 52H) 0.83 (m, 9H). TL1-18D-E8-8 (Compound (53)) (Compound (53)) [0641] To a stirred solution of tris(8-(nonanoyloxy)octyl) 2-hydroxypropane-1,2,3-tricarboxylate [I-5] (0.9 g, 0.00090 mol) and N,N-dimethylpropionic acid (0.422 g, 0.0036 mol) in § dichloromethane (10 ml) was added EDCI (0.7 g, 0.00361 mol,) followed by addition of DMAP (0.11g, 0.00090 mol). Reaction mass was stirred for 16 h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was diluted with (50.0 mL) and washed with DM water (100 ml) dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get final [TL1-18D-E8-8] (0.288 g, 29.12%) as yellow colour liquid mass. [0642] 1H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.8 Hz, 2H), 4.01 (m, 10H), 3.31-3.27 (d, J = 15.6 Hz, 2H), 3.23-3.19 (d, J = 15.6 Hz, 2H), 2.60-2.56 (m, 2H), 2.49-2.46 (m, 2H), 2.28 (t, J = 7.6 Hz.6H), 2.21 (s, 6H), 1.66-1.57 (m, 18H), 1.31-1.26 (m, 54H), 1.24 (brs, 28H), 0.87 (t, J = 6.8 Hz, 9H). [0643] LCMS analysis: Purity 99.37 %, Calculated C62H113NO14 = 1095.82, Observed = 1096.40 (m/z, M+H+).
Example 11. Synthetic Protocol for M1266-J03983-035 (JC-TL1-18D-E4-12) (Compound (22)) Synthetic Scheme K [0644] To a stirred solution of butane-1,4-diol [K-1] (10 g, 0.111 mol) and tridecanoic acid [K- 2] (23.7 g, 0.111 mol) in dichloromethane (300 mL) was cooled to 0 oC, EDC.HCl (25.5 g, 0.133 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (6.7 g, 0.055 mol) and stirred for 16 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-20 % ethyl acetate in n- hexane), to obtain pure product 4-hydroxybutyl tridecanoate [K-3] (11.8 g, 35 % Yield) as a pale yellow liquid. [0645] 1H NMR (400 MHz, CDCl3): δ 4.10 (t, J= 6.4 Hz, 2H), 3.71-3.66 (m, 2H), 2.28 (t, J= 7.6 Hz, 2H), 1.76-1.57 (m, 6H), 1.27-1.11 (m, 18H), 0.89-0.85 (m, 3H). Intermediate [K-5]: [0646] To a stirred solution of 4-hydroxybutyl tridecanoate [K-3] (11.8 g, 0.04123 mol) and Citric acid [K-4] (2.0 g, 0.0103 mol) in dichloromethane (150 mL) was cooled to 0 oC, EDC.HCl (7.90 g, 0.0412 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (1.25 g, 0.0103 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product tris(4-(tridecanoyloxy)butyl) 2-hydroxypropane- 1,2,3-tricarboxylate [K-5] (1.1 g, 10 % Yield) as a pale yellow liquid. [0647] ELSD analysis: Purity 99.74 %, Calculated C57H104O13 = 996.75, Observed = 1019.30 (m/z, M+Na+).
JC-TL1-18D-E4-12 (Compound (22)) (Compound (22)) [0648] To a stirred solution of tris(4-(tridecanoyloxy)butyl) 2-hydroxypropane-1,2,3- tricarboxylate [K-5] (1.1 g, 0.0011 mol) and 3-(dimethylamino)propanoic acid [K-6] (0.51 g, 0.0044 mol) in dichloromethane (20 mL) was cooled to 0 oC, EDC.HCl (0.84 g, 0.0044 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.134 g, 0.0011 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (10 mL) was added to the reaction mixture and extracted with DCM (3x25 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product tris(4-(tridecanoyloxy)butyl) 2-((3- (dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate [JC-TL1-18D-E4-12] (Compound (22)) (0.2 g, 16 %, Yield) as a colourless liquid. [0649] 4.16 (m, 2H), 4.10-4.08 (m, 10H), 3.30 (d, J= 15.6Hz, 2H), 3.23 (d, J= 15.6Hz, 2H), 2.59-2.56 (m, 2H), 2.49-2.46 (m, 2H), 2.28 (t, J= 7.6Hz, 6H), 2.21 (s, 6H), 1.70-1.69 (m, 12H), 1.62-1.59 (m, 7H), 1.31-1.28 (m, 53H), 0.87 (t, J= 6.8Hz, 9H). [0650] ELSD analysis: Purity 99.91 %, Calculated C62H113NO14 = 1095.82, Observed = 1096.45 (m/z, M+H+).
Example 12. Synthetic Protocol for M1266-J03996-027 (TL1-12D-E8-2) (Compound (11)) Synthetic Scheme L [0651] To a stirred solution of 1, 8-octanediol (10 g, 0.084 mol) and triethylamine (45ml, 0.34 mol) in dichloromethane (200 ml) was cooled to 0°C, then added propionoyl chloride (3.14 g, 0.034 mol) drop wise. Reaction mass was allowed to stirred at RT for 16 h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water (100.0 mL), dried over sodium sulphate and concentrated under reduced pressure. Crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [L-3] (7.3 g; 30.12%) as yellow liquid. [0652] 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.4 Hz, 2H), 3.62 (q, J = 6.0 Hz, 2H), 2.30 (q, J = 7.6 Hz, 2H), 1.61 (m, 3H), 1.35 (s, 9H), 1.11 (t, J = 6.4 Hz, 3H). Intermediate L-5 [0653] To a stirred solution of 8-hydroxyoctyl propionate [L-3] (5.3 g, 0.062 mol) and citric acid (1.25 g, 0.00655 mol) in dichloromethane (20 ml) was added EDCI (5.03 g, 0.0262 mol) followed by addition of DMAP (0.80 g, 0.00635 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (Sm was consumed). Reaction mass was washed with DM water (50 ml), dried over sodium sulphate and concentrated. Crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [Int-5] (1.1 g, 22.68%) as yellow liquid. [0654] 1H NMR (400 MHz, CDCl3): δ 4.059(t, J = 6.8 Hz, 6H), 3.643(d, J = 3.2 Hz, 6H), 2.282(t, J = 7.6 Hz, 6H), 1.629(m, 18H), 1.331 (m, 22H) 0.829(m, 9H). [0655] LCMS analysis: Purity 98.35 %, Calculated C39H68O13 = 744.47, Observed = 745.20 (m/z, M+H+). TL1-12D-E8-2 (Compound (11)) (Compound (11)) [0656] To a stirred solution of tris(8-(propionyloxy)octyl) 2-hydroxypropane-1,2,3- tricarboxylate [L-5] (1.1 g, 0.00147 mol) and N,Ndimethylpropionic acid (0.691 g, 0.00590 mol) in dichloromethane (10 ml ) was added EDCI (1.13 g, 0.005906 mol,) followed by addition of DMAP (0.180g, 0.00147 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water 20 ml, dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [TL1-12D-E8-2] (Compound ( (0.300 g, 25%) as yellow colour liquid. [0657] 1H NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.4 Hz, 2H), 4.08 (t, J = 6.8 Hz, 10H), 3.31-3.18 (m, 4H), 2.61 (t, J = 7.2 Hz, 2H) 2.50 (t, J = 7.2 Hz, 2H) 2.34-2.28 (q, J = 7.6 Hz, 6H), 2.24 (s, 6H), 1.61 (m, 12H), 1.31 (brs, 24H), 1.13 (t, J = 7.2, 9H). [0658] LCMS analysis: Purity 99.77 %, Calculated C44H77NO14 = 843.53, Observed = 844.15 (m/z, M+H+). Example 13. Synthetic Protocol for M1266-J03996-055 (TL1-14D-E5-7-2i) (Compound (48)) Synthetic Scheme M [0659] To a stirred solution of 1,5, pentanediol (20 g, 0.190 mol) and triethylamine (90ml, 0.68 mol) in dichloromethane (200 ml) was cooled to 0°C. Added 3-pentyloctanoyl chloride (22.13 g, 0.095 mol) drop wise. Reaction mass was allowed to RT and stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (100 mL), dried over sodium sulphate and concentrated under reduced pressure. Resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [M-3] (17.0 g, 60.71%) as yellow colour liquid. [0660] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.4 Hz, 2H), 3.66 (t, J = 6.4 Hz, 2H), 2.23 (d, J = 6.8 Hz, 2H), 1.83 (s,1H), 1.69-1.58 (m, 4H), 1.47-1.41 (m, 2H), 1.25 (s, 17H), 0.88 (t, J = 6.4 Hz, 6H). [0661] LCMS analysis: Purity 99.96 %, Calculated C18H36O3 = 300.27, Observed = 301.30 (m/z, M+H+). Intermediate M-5 [0662] To a stirred solution of 5-hydroxypentyl 3-pentyloctanoate [M-3] (17 g, 0.056 mol) and citric acid (2.72 g, 0.0141 mol) in dichloromethane (60 ml) was added EDCI (10.82 g, 0.0.056 mol) followed by DMAP (1.72 g, 0.0141 mol). Reaction mass was stirred for 16h. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mass was washed with DM water (50 ml), dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 10% ethyl acetate in n-heptane to get desired product [Int-5] (12 g, 85%) as yellow colour liquid mass. [0663] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.8 Hz, 2H), 4.05 (m, 10H), 2.89-2.77 (m, 4H), 2.23-2.21 (d, J = 7.2 Hz, 6H), 1.83 (m, 3H), 1.72-1.61 (m, 12H) 1.56 (m, 6H), 1.39 (m, 8H), 1.32-1.26 (m, 40H), 0.85 (m, 18H). TL1-14D-E5-7-2i (Compound (48)) (Compound (48)) [0664] To a stirred solution of tris(5-((3-pentyloctanoyl) oxy) pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [Int-5] (2.5 g, 0.00240 mol) and N,N-dimethyl propionic acid [Int-6] (1.12 g, 0.00962 mol) in dichloromethane (100 ml ) was added EDCI (2.0 g, 0.00962 mol,) followed by addition of DMAP (0.30 g, 0.0024 mol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM was consumed). Reaction mixture was washed with DM water (50 ml) and dried over sodium sulphate. Distilled out solvent and resulting crude was purified over silica using 30% ethyl acetate in n-heptane to get desired product [TL1-14D-E5-7-2i] 1.2 g, (44.44%) as yellow colour liquid mass. [0665] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.4 Hz, 2H), 4.08 (m, 10H), 3.31-3.19 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 2.47 (t, J = 7.2 Hz, 2H), 2.22-2.21 (m, 12H), 1.83 (m, 3H), 1.69-1.60 (m, 12H), 1.42 (m, 6H), 1.30-1.22 (m, 48H), 0.87 (t, J = 7.2, 18H). [0666] LCMS analysis: Purity 99.83 %, Calculated C65H119NO14 = 1137.86, Observed = 1138.45 (m/z, M+H+).
Example 14. Synthetic Protocol for M1266-450487 ( JC-ATL-011 ) (Compound (6)) Synthetic Scheme N [0667] To a solution of citric acid (25.0 g, 0.130 mol) and octan-1-ol (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol,) was added, and the resulting mixture was stirred at 100 oC for 24h. The reaction progress was monitored by TLC (5% EtOAc/hexanes). The reaction mixture was cooled to r.t, diluted with water (500 mL) and extracted with ethyl acetate (500 mL x 3). The organic layer was dried over anhydrous Na2SO4 and solvent was evaporated. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give compound [N-5] (40.0 g, 58.22 %) as a colourless liquid [0668] 1H NMR (400 MHz, DMSD-d6): δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). [0669] LCMS analysis: Purity 97.38%, Calculated C30H56O7 = 528.40, Observed = 529.2 (m/z, M+H+). JC-ATL-011 (Compound (6)) (Compound (6)) [0670] To a stirred solution of 1,2,3-trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [N-5] (0.5 g, 946 µmol) in dichloromethane (30 mL, 469 mmol), was added triethylamine (287 mg, 3 eq., 2.84 mmol) and 4-(dimethylamino)pyridin-1-ium (116 mg, 946 µmol) at 0 °C. Reaction mass was stirred for 10 minute at same temperature, followed by addition of ditrichloromethyl carbonate (281 mg, 946 µmol) and 2-(dimethylamino)ethan-1-ol (84.3 mg, 946 µmol), the resulting reaction mixture was stirred at room temperature for 16 hrs. The progress of reaction was monitor by TLC. The resulting reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (3x30 mL). Whole organic was combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography by using 0-5% MeOH in DCM as eluent. The pure fraction was combined and concentrated under reduced pressure to obtain [JC-ATL-011] (Compound (6)) (410 mg, yield- 67.37%) as a yellow liquid. [0671] δ 4.20 (t, J = 6.0 Hz, 2H), 4.16-4.14 (d, J = 6.8 Hz, 2H), 3.35- 3.31 (d, J = 15.6 Hz, 2H), 3.24-3.20 (d, J = 15.6 Hz, 2H), 2.58 (d, J = 6 Hz, 2H), 2.27 (s, 6H), 1.67-1.56 (m, 6H), 1.27 (brs, 30H), 0.87 (t, J = 6.4 Hz, 9H). [0672] LCMS analysis: Purity 99.87 %, Calculated C35H65NO9 = 644.47, Observed = 644.75 (m/z, M+H+).
Example 15. Synthetic Protocol for M1266-449313 ( JC-ATL-012 ) (Compound (25)) Synthetic Scheme O [0673] To a stirred solution of pentane-1,5-diol (6.5 g, 0.9 eq., 62.4 mmol) in dichloromethane (50 mL, 781 mmol) was added octatonic acid (10 g, 69.3 mmol). Reaction mass was cooled to 0°C. Added ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (16.7 g, 1.3 eq., 87.4 mmol), and 4-(dimethylamino)pyridin-1-ium (1.71 g, 0.2 eq., 13.9 mmol). The reaction mixture was stirred at r.t. for 48h. Reaction progress was monitored by TLC / ELSD, after consumption of starting material, the reaction mixture was washed with saturated NaHCO3 and brine solution. After evaporation of organic layer, resulting crude was purified by flash column chromatography to get compound O-3 (6.0 g; yield: 37.56 %) as colourless liquid. [0674] δ 4.08 (t, J = 6.4 Hz, 2H), 3.67 (s, 2H), 2.30 (t, J = 7.2 Hz, 2H), 1.71-1.58 (m, 6H), 1.44 (m, 2H), 1.29 (s, 10H), 0.89 (t, J = 6.8 Hz, 3H). Intermediate O-5 [0675] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [compound O-3] (250 mg, 1.3 mmol) in dichloromethane (5 mL, 78.1 mmol), was added N,N-dimethylpyridin-4-amine (159 mg, 1.3 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (998 mg, 4 eq., 5.2 mmol). Reaction mass stirred 10 min at 25°C, then added 5- hydroxypentyl octanoate (1.35 g; 5.86 mmol). Reaction mixture was stirred for 16 hr at r.t. under nitrogen atmosphere. Reaction progress monitored by TLC / ELSD, after consumption of starting material reaction mass diluted with water and dichloromethane. Organic layer was separated, dried over sodium sulphate. Solvent was distilled out under reduced pressure. Crude was purified with column chromatography using 15-20% ethyl acetate, to get compound O-5 (0.9 g; yield: 83.42 %) as a light yellow colour less liquid. [0676] 1H NMR (400 MHz, CDCl3): δ 4.21 (m, 2H), 4.08 (m, 10H), 2.90-2.77 (dd, J = 15.6 Hz.4H), 2.29 (t, J = 7.6 Hz, 6H), 1.61(m, 12H), 1.41 (m, 8H), 1.28(brs, 28H), 0.85 (t, J = 6.4 Hz, 9H). JC-ATL-012 (Compound (25)) (Compound (25)) [0677] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [compound O-5] (0.5 g, 603 µmol) in dichloromethane (6 mL, 93.7 mmol), triethylamine (183 mg, 3 eq., 1.81 mmol) and 4-(dimethylamino)pyridin-1-ium (74.3 mg, 603 µmol) was successively added at 0 °C and stirred for 10 minute at same temperature, followed by addition of ditrichloromethyl carbonate (179 mg, 603 µmol) and 2-(dimethylamino)ethan-1-ol (53.8 mg, 603 µmol) was added. The resulting reaction mixture was stirred at room temperature for 16 hrs and progress of reaction was monitored by TLC / ELSD. The resulting reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (3x30 mL). Combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography by using 0-5% MeOH in DCM as eluent. The pure fraction was combined and concentrated under reduced pressure to obtain [JC-ATL-012] (Compound (25)) (225 mg. Yield 39.51%) as a yellow liquid. [0678] 1H NMR (400 MHz, CDCl3): δ 4.195 (m, 4H), 4.08 (m, 10H), 3.34-3.30 (d, J = 15.6 Hz, 2H), 3.24-3.20 (d, J = 15.6 Hz, 2H), 2.57 (t, J = 5.6 Hz, 2H), 2.28 (t, J = 8.8 Hz, 12H), 1.72-1.57(m, 19H), 1.44 (m, 6H), 1.28(brs, 24H), 0.87 (t, J = 6.4 Hz, 9H). LCMS analysis: Purity 99.74 %, Calculated C50H89NO15 = 943.62, Observed = 944.60 (m/z, M+H+). Example 16. Synthetic Protocol for M1266-674154 (TL1-14D-E5-7-6i) (Compound (36)) Synthetic Scheme P Intermediate P-3 [0679] To the stirred solution of 7-methyloctanoic acid (12 g, 75.8 mmol) in dichloromethane (0.2 L), N,N-dimethylpyridin-4-amine (9.26 g, 75.8 mmol) and ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (29.1 g, 2 eq., 152 mmol) were added slowly at 0 oC, after 10 min pentane-1,5-diol (15.8 g, 2 eq., 152 mmol) was added. Reaction mixture was allowed to stirred at RT for 18 h. Progress of reaction was monitored by ELSD / TLC. Reaction mixture was diluted with cold water (500.0 mL) and extracted with dichloromethane (3x 100 ml). Combined organic layer was washed with fresh water (2x 100 mL) and brine solution (2x 100.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude was purified with column chromatography using 10% Ethyl acetate in Hexanes, to give 5-hydroxypentyl 7-methyloctanoate [Int-3] (12 g, 49.1 mmol) as colourless clear liquid. [0680] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.68-3.63 (dd, J = 5.2 Hz, 2H), 2.29 (t, J = 7.6 Hz, 2H), 1.64 (m, 5H), 1.58- 1.41(m, 3H), 1.29 (m, 5H), 1.16(m, 2H), 0.851 (d, J = 6.8 Hz, 6H). Intermediate P-5 [0681] The solution of 2-hydroxypropane-1,2,3-tricarboxylic acid (2.44 g, 12.7 mmol) in dichloromethane (0.1 L) was cooled to 0°C, then added N,N-dimethylpyridin-4-amine (1.55 g, 12.7 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.73 g, 4 eq., 50.7 mmol) slowly at room temperature, after 10 min, 5-hydroxypentyl 7-methyloctanoate [P-3] (12.4 g, 4 eq., 50.7 mmol) was added drop wise. Reaction mixture was allowed to stirred at 25°C for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with cold water (100.0 mL) and extracted with dichloromethane (3x 100 mL). Combined organic layer was washed with fresh water (2x 50 mL) and brine solution (2x 50.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude was purified with column chromatography using 10% Ethyl acetate in Hexanes, to give 1,2,3-tris({5-[(7- methyloctanoyl)oxy]pentyl}) 2-hydroxypropane-1,2,3-tricarboxylate [P-5] (4.5 g, 5.17 mmol) as colourless clear liquid. [0682] δ 4.22 (t, J = 5.6 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.29 (t, J = 7.6 Hz, 6H), 1.64 (m, 16H), 1.50(m, 4H), 1.49 (m, 6H), 1.28(m, 13H), 1.15 (m, 6H), 0.85 (d, J = 6.8 Hz, 18H). TL1-14D-E5-7-6i (Compound (36)) (Compound (36)) [0683] A stirred solution of 1,2,3-tris({5-[(7-methyloctanoyl)oxy]pentyl}) 2-hydroxypropane- 1,2,3-tricarboxylate [P-5] (5.2 g, 5.97 mmol) in dichloromethane (61.2 mL, 956 mmol) was cooled to 0°C.3-(dimethylamino)propanoic acid (2.8 g, 4 eq., 23.9 mmol) was added and stirred for 10 minute.{3-[cyano(ethyl)amino]propyl}dimethylazanium chloride (4.58 g, 4 eq., 23.9 mmol) was added at same temperature, followed by N,N-dimethylpyridin-4-amine (729 mg, 5.97 mmol). Reaction mass allowed to stirred at room temperature for 16 h. Progress of reaction was monitored by ELSD / TLC. Reaction mixture was diluted with cold water (500.0 mL) and extracted with dichloromethane (2x 150 ml). Combined organic layer was washed with fresh water (2x 100 mL) and brine solution (2x 100.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude was purified by column chromatography using 50% Ethyl acetate in Hexanes, to give [TL1-14D-E5-7-6i] (Compound (36)) (1.85 g, 1.91 mmol) as brown colour liquid mass. [0684] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.8 Hz, 2H), 4.08 (m, 10H), 3.31-3.27 (d, J = 15.6 Hz, 2H), 3.23-3.19 (d, J = 15.6 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.6 Hz, 6H), 2.22 (s, 6H), 1.69-1.5(m, 18H), 1.51 (m, 3H), 1.42 (m, 6H), 1.29 (m, 12H), 1.14 (m, 6H), 0.87 (d, J = 6.8 Hz, 18H). [0685] LCMS analysis: Purity 98.45 %, Calculated C53H95NO14 = 969.68, Observed = 970.68 (m/z, M+H+). Example 17. Synthetic Protocol for M1266-480517 (TL1-12D-001) (Compound (33)) Synthetic Scheme Q [0686] To a stirred solution of 4-(tert-butoxy)-4-oxobutanoic acid [Q-1] (10 g, 0.0574 mol) and 2-(dimethylamino)ethan-1-ol [Q-2] (6.14 g, 0.0689 mol) in dichloromethane (100 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (13.9 g, 0.0723 mol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.41 g, 0.0115 mol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired tert-butyl (2-(dimethylamino)ethyl) succinate [Q-3] (9.0 g, 63.91% yield) as yellow liquid. [0687] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.0 Hz, 2H), 2.59-2.41 (m, 6H), 2.24 (s, 6H), 1.41 (s, 9H) Intermediate [Q-4]: [0688] To a stirred solution of tert-butyl (2-(dimethylamino)ethyl) succinate [Q-3] (9 g, 0.036 mol) in DCM (100 mL), Trifluoroacetic acid (10 mL, 0.132 mol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with triethylamine upto pH 7. The resulting organic layer concentrated under reduced pressure and the crude directly used in next step. [0689] ELSD analysis: Purity 99.15 %, Calculated C8H15NO4 = 189.10, Observed = 189.80 (m/z, M+H+). Intermediate [Q-7]: [0690] To a stirred solution of octanoic acid [Q-6] (10 g, 69.3 mmol) and pentane-1,5-diol [Q-5] (6.5 g, 62.4 mmol) in dichloromethane (200 mL, 781 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (16.7 g, 87.4 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.71 g, 13.9 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture, and extracted with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [Q-7] (6.0 g, 26.0 mmol, 37.56% yield) as yellow liquid. [0691] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 4H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [Q-9]: [0692] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [Q-8] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [Q-7] (6 g, 26 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired tris(5- (octanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [Q-9] (2.1 g, 2.53 mmol, 38.9% yield) as yellow liquid. [0693] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). [0694] ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). TL1-12D-001 (Compound (33)) (Compound (33)) [0695] To a stirred solution of 4-[2-(dimethylamino)ethoxy]-4-oxobutanoic acid [Q-4] (1.92 g, 10.1 mmol) and 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3-tricarboxylate [Q-9] (2.1 g, 2.53 mmol) in dichloromethane (15 mL, 234 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (1.94 g, 10.1 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (312 mg, 2.53 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexane), to give the desired product [TL1-12D-001] (Compound (33)) (115 mg, 4.54 % yield) as a transparent liquid. [0696] 1H-NMR (400 MHz, DMSO-d6)- δ 4.08-3.97 (m, 14H), 3.17-3.13 (d, J = 15.6 Hz, 2H), 3.10- 3.06 (d, J = 15.2 Hz, 2H), 2.46-2.43 (m, 6H), 2.27-2.24 (m, 6H), 2.14 (s, 6H), 1.58-1.48 (m, 18H), 1.35-1.23 (m, 30H), 0.86-0.83 (m, 9H). [0697] ELSD analysis: Purity 99.90 %, Calculated C53H93NO16 = 999.65, Observed = 1000.50 (m/z, M+H+).
Example 18. Synthetic Protocol for M1266-497687 (TL1-10D-006) (Compound (27)) Synthetic Scheme R [0698] To a stirred solution of 4-(tert-butoxy)-4-oxobutanoic acid [R-1] (1.5 g, 0.0086 mol) and 2-(dimethylamino)ethan-1-ol [R-2] (0.92 g, 0.0103 mol) in dichloromethane (20 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (3.30 g, 0.0172 mol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.0 g, 0.0086 mol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (20 mL) was added to the reaction mixture, and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired tert-butyl (2-(dimethylamino)ethyl) succinate [R-3] (1.5 g, 71% yield) as pale yellow liquid. [0699] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.0 Hz, 2H), 2.59-2.48 (m, 6H), 2.24 (s, 6H), 1.41 (s, 9H) Intermediate [R-4]: [0700] To a stirred solution of tert-butyl (2-(dimethylamino)ethyl) succinate [R-3] (1.5 g, 0.006 mol) in DCM (15 mL), Trifluoroacetic acid (1.4 g, 0.012 mol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with triethylamine upto pH 7. The resulting organic layer concentrated under reduced pressure and the crude directly used in next step. [0701] ELSD analysis: Purity 99.15 %, Calculated C8H15NO4 = 189.10, Observed = 189.80 (m/z, M+H+). Intermediate [R-7]: [0702] To a stirred solution of 5-methylhexanoic acid [R-6] (10 g, 76.8 mmol) and pentane-1,5- diol [R-5] (8 g, 76.8 mmol) in dichloromethane (150 mL) was cooled to 0 oC, EDC.HCl (18.6 g, 96.8 mmol), was added followed by 4-(dimethylamino)pyridin-1-ium (1.89 g, 15.4 mmol) and stirred for 16 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography using 0-20 % ethyl acetate in n-hexane to obtain pure product 5-hydroxypentyl 5-methylhexanoate [R-7] (6.1 g, 37%) as a pale yellow liquid. [0703] 1H-NMR (400 MHz, CDCl3)- δ 4.07 (t, J = 6.4 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.69-1.53 (m, 8H), 1.51-1.39 (m, 2H), 1.24-1.16 (m, 2H), 0.87 (d, J = 6.8 Hz, 6H). Intermediate [R-9]: [0704] To a stirred solution of 5-hydroxypentyl 5-methylhexanoate [R-7] (6 g, 27.7 mmol), and 2-hydroxypropane-1,2,3-tricarboxylic acid [R-8] (1.33 g, 6.93 mmol) in dichloromethane (150 mL) was cooled to 0 oC, EDC.HCl (5.32 g, 27.7 mmol), was added followed by 4- (dimethylamino)pyridin-1-ium (854 mg, 6.93 mmol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane), to obtain pure product 1,2,3- tris({5-[(5-methylhexanoyl)oxy]pentyl}) 2-hydroxypropane-1,2,3-tricarboxylate [R-9] (1.9 g, 35%) as a pale yellow liquid. [0705] 1H-NMR (400 MHz, CDCl3)- δ 4.21 (t, J = 6.4 Hz, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.27 (t, J = 7.6 Hz, 6H), 1.68-1.51 (m, 22H), 1.41-1.38 (m, 6H), 1.21-1.16 (m, 6H), 0.88 (d, J = 6.4 Hz, 18H). (Compound (27)) [0706] To a stirred solution of 1,2,3-tris({5-[(5-methylhexanoyl)oxy]pentyl}) 2-hydroxypropane- 1,2,3-tricarboxylate [R-9] (1.9 g, 2.41 mmol) and 4-[2-(dimethylamino)ethoxy]-4-oxobutanoic acid [R-4] (1.83 g, 9.66 mmol) in dichloromethane (20 mL) was cooled to 0 oC, EDC.HCl (1.85 g, 9.66 mmol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.247 g, 2.41 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture and extracted with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n- hexane), to obtain pure product [JC- TL1-10D-006] (Compound (27)) (0.5 g, 22%) as a brown liquid. [0707] 1H-NMR (400 MHz, CDCl3)- δ 4.19 (t, J = 4.8 Hz, 2H), 4.14 (t, J = 6.8 Hz, 2H), 4.09-4.04 (m, 10H), 3.30-3.19 (dd, J = 15.6, 12.8 Hz, 4H), 2.63-2.62 (m, 4H), 2.60-2.59 (br, 2H), 2.30-2.29 (m, 4H), 2.27-2.25 (m, 7H), 1.71-1.51 (m, 21H), 1.44-1.36 (m, 6H), 1.21-1.12 (m, 6H), 0.88 (d, J = 6.4 Hz, 18H). ELSD analysis: Purity 99.89 %, Calculated C50H87NO16 = 957.60, Observed = 958.45 (m/z, M+H+).
Example 19. Synthetic Protocol for M1266-499030 (TL1-14D-003) (Compound (24)) Synthetic Scheme S [0708] To a stirred solution of 4-(tert-butoxy)-4-oxobutanoic acid [S-1] (10 g, 0.0574 mol) and 2-(dimethylamino)ethan-1-ol [S-2] (6.14 g, 0.0689 mmol) in dichloromethane (100 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (13.9 g, 0.0723 mol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.41 g, 0.0115 mol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired tert-butyl (2-(dimethylamino)ethyl) succinate [S-3] (9.0 g, 63.91% yield) as pale yellow liquid. Results: [0709] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.0 Hz, 2H), 2.59-2.41 (m, 6H), 2.24 (s, 6H), 1.41 (s, 9H) Intermediate [S-4]: [0710] To a stirred solution of tert-butyl (2-(dimethylamino)ethyl) succinate [S-3] (9 g, 0.036 mol) in DCM (100 mL), Trifluoroacetic acid (10 mL, 0.132 mol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with triethylamine upto pH 7. The resulting organic layer concentrated under reduced pressure and the crude directly used in next step. Results: ELSD analysis: Purity 99.15 %, Calculated C8H15NO4 = 189.10, Observed = 189.80 (m/z, M+H+). Intermediate [S-7]: [0711] To a stirred solution of decanoic acid [S-6] (10 g, 58 mmol) and pentane-1,5-diol [5] (5.44 g, 52.2 mmol) in dichloromethane (200 mL) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (14 g, 73.1 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.43 g, 11.6 mmol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture, and extracted with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl decanoate [S-7] (3.6 g, 24% yield) as yellow liquid. Results: 1H-NMR (400 MHz, CDCl3)- δ 4.09 (t, J = 6.4 Hz, 2H), 3.67 (q, J = 6.0 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.70-1.56 (m, 6H), 1.49-1.43 (m, 2H), 1.31-1.23 (m, 12H), 0.91 (m, 3H). ELSD analysis: Purity 99.43 %, Calculated C15H30O3 = 258.22, Observed = 259.15 (m/z, M+H+). Intermediate [S-9]: [0712] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [S-8] (651 mg, 3.39 mmol) and 5-hydroxypentyl decanoate [S-7] (3.5 g, 13.5 mmol) in dichloromethane (50 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (2.6 g, 13.5 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (417 mg, 3.39 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by TLC (SM was consumed). Water (20 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 1,2,3-tris[5-(decanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [S-9] (1.97 g, 2.16 mmol) as pale yellow liquid. [0713] 1H-NMR (400 MHz, CDCl3)- δ 4.22 (t, J = 6.4 Hz, 2H), 4.11-4.04 (m, 10H), 2.88 (dd, J = 15.6, 19.2 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.73-1.59 (m, 18H), 1.43-1.36 (m, 6H), 1.29-1.26 (m, 36H), 0.88 (d, J = 6.8 Hz, 9H). [0714] ELSD analysis: Purity 98.31%, Calculated C51H92O13 = 912.65, Observed = 935.90 (m/z, M+Na+). JC-TL1-14D-003 (Compound (24)) (Compound (24)) [0715] To a stirred solution of 4-[2-(dimethylamino)ethoxy]-4-oxobutanoic acid [S-4] (1.51 g, 8.32 mmol) and 1,2,3-tris[5-(decanoyloxy)pentyl] 2-hydroxypropane-1,2,3-tricarboxylate [S-9] (1.9 g, 2.41 mmol), in dichloromethane (50 mL) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (1.6 g, 8.32 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (254 mg, 2.08 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexane), to give the desired product [JC-TL1-14D-003] (Compound (24)) (0.6 g, 26%) as a yellow oil [0716] 1H-NMR (400 MHz, CDCl3)- δ 4.20 (t, J = 5.6 Hz, 2H), 4.14 (t, J = 6.8 Hz, 2H), 4.09-4.03 (m, 10H), 3.28 (d, J = 15.6 Hz, 2H), 3.21 (d, J = 15.6 Hz, 2H), 2.65-2.60 (m, 6H), 2.30-2.26 (m, 12H), 1.71-1.56 (m, 18H), 1.44-1.36 (m, 6H), 1.29-1.25 (m, 36H), 0.87 (d, J = 6.8 Hz, 9H). [0717] ELSD analysis: Purity 99.72 %, Calculated C59H105NO16 = 1083.74, Observed = 1084.50 (m/z, M+H+).
Example 20. Synthetic Protocol for M1266-507720 (TL1-12D-002) (Compound (35)) Synthetic Scheme T [0718] To a stirred solution of 4-(tert-butoxy)-4-oxobutanoic acid [T-1] (10 g, 0.0574 mol) and 2-(dimethylamino)ethan-1-ol [T-2] (6.14 g, 0.0689 mmol) in dichloromethane (100 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (13.9 g, 0.0723 mol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.41 g, 0.0115 mol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired tert-butyl (2-(dimethylamino)ethyl) succinate [T-3] (9.0 g, 63.91% yield) as yellow liquid. [0719] -NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.0 Hz, 2H), 2.59-2.41 (m, 6H), 2.24 (s, 6H), 1.41 (s, 9H) Intermediate [T-4]: [0720] To a stirred solution of tert-butyl (2-(dimethylamino)ethyl) succinate [T-3] (9 g, 0.036 mol) in DCM (100 mL), Trifluoroacetic acid (10 mL, 0.132 mol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with triethylamine upto pH 7. The resulting organic layer concentrated under reduced pressure and the crude directly used in next step. [0721] ELSD analysis: Purity 99.15 %, Calculated C8H15NO4 = 189.10, Observed = 189.80 (m/z, M+H+). Intermediate [T-7]: [0722] To a stirred solution of 7-methyloctanoic acid [T-6] (5 g, 31.6 mmol) and pentane-1,5- diol [T-5] (3.29 g, 31.6 mmol) in dichloromethane (100 mL, 781 mmol) was cooled to 0 oC, N-(3- Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (7.63 g, 39.8 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (778 mg, 6.32 mmol)and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-20% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl 7-methyloctanoate [T-7] (4.4 g, 57% yield) as yellow liquid. [0723] 1H-NMR (400 MHz, CDCl3)- δ 4.06 (t, J = 6.4 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.67-1.57 (m, 6H), 1.52-1.40 (m, 4H), 1.30-1.26 (m, 4H), 1.15-1.14 (m, 2H), 0.85 (d, J = 6.8, 6H). Intermediate [9]: [0724] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [T-8] (0.6 g, 3.12 mmol) and 5-hydroxypentyl 7-methyloctanoate [T-7] (3.05 g, 12.5 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, EDC hydrochloride (2.39 g, 12.5 mmol), was added followed by 4-(dimethylamino)pyridin-1-ium (385 mg, 3.12 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired 1,2,3-tris({5-[(7-methyloctanoyl)oxy]pentyl}) 2-hydroxypropane-1,2,3-tricarboxylate [T-9] (1 g, 36.75% yield) as pale yellow liquid. [0725] ELSD analysis: Purity 99.50 %, Calculated C48H86O13 = 870.61, Observed = 893.35 (m/z, M+Na+). TL1-12D-002 (Compound (35)) Compound (35) [0726] To a stirred solution of 4-[2-(dimethylamino)ethoxy]-4-oxobutanoic acid [T-4] (869 mg, 4.59 mmol) and 1,2,3-tris({5-[(7-methyloctanoyl)oxy]pentyl}) 2-hydroxypropane-1,2,3- tricarboxylate [T-9] (1 g, 1.15 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, EDC hydrochloride (880 mg, 4.59 mmol), was added followed by 4-(dimethylamino)pyridin-1-ium (141 mg, 1.15 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired product [TL1-12D- 002] (Compound (35))(140 mg, 11.7% yield) as pale yellow liquid. [0727] 1H-NMR (400 MHz, CDCl3)- δ 4.20 (t, J = 5.6 Hz, 2H), 4.15-4.12 (m, 2H), 4.09-4.04 (m, 10H), 3.28 (d, J = 15.6 Hz, 2H), 3.21 (d, J = 15.6 Hz, 2H), 2.65-2.62 (m, 6H), 2.31-2.26 (m, 10H), 1.71-1.56 (m, 18H), 1.52-1.46 (m, 3H), 1.44-1.36 (m, 6H), 1.32-1.25 (m, 14H), 1.18-1.10 (m, 6H), 0.85 (d, J = 6.4, 18H). [0728] ELSD analysis: Purity 99.89 %, Calculated C56H99NO16 = 1041.70, Observed = 1042.50 (m/z, M+H+).
Example 21. Synthetic Protocol for M1266-534954 (TL1-12D-011) (Compound 55) Synthetic Scheme U Intermediate [U-2]: [0729] To a stirred solution of 2-iodoethan-1-ol [U-1] (5 g, 0.0290 mol) in dichloromethane (100 mL), 1H-imidazole (1.97 g, 0.0290 mol) was added at room temperature. The resulting reaction mixture was cooled to 0 °C and tert-butyl(chloro)dimethylsilane (8.76 g, 0.0580 mol) was added portion wise at same temperature, then allowed to stirred for 16 h at room temperature. Progress of reaction was monitored by ELSD/TLC. Water (50 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-10 % ethyl acetate in n-hexane), to obtain pure product [2] (5.0 g, 60.1 %, Yield) as a colourless liquid. [0730] 1H-NMR (400 MHz, CDCl3): δ 3.83 (t, J = 5.6 Hz, 2H), 3.19 (t, J = 6.8 Hz, 2H), 0.90 (s, 9H), 0.10 (s, 6H). Intermediate [3]: [0731] To a stirred solution of 3-(methylamino)propanoic acid [U-3] (5 g, 48.5 mmol), and potassium hydroxide (5.44 g, 97 mmol) in 1,4-dioxane (25 mL, 293 mmol) and water (25 mL, 1.39 mol) added di-tert-butyl dicarbonate (10.6 g, 48.5 mmol) at room temperature for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, mixture was concentrated under reduced pressure. The crude material was acidified by 10 % NaHSO4 upto pH 2-3, and extracted with ethyl acetate (3x200 mL). The resulting organic layer was dried over anhydrous sodium sulphate concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain pure product 3-{[(tert-butoxy)carbonyl](methyl)amino}propanoic acid [U-4] (9 g, 91.33 %, Yield) as a colourless liquid. [0732] 1H-NMR (400 MHz, CDCl3): δ 3.50 (t, J = 6.8 Hz, 2H), 2.87 (s, 3H), 2.59 (bs, 2H), 1.48 (s, 9H). Intermediate [U-7]: [0733] To a stirred solution of octanoic acid [U-6] (10 g, 69.3 mmol) and pentane-1,5-diol [U-5] (6.5 g, 62.4 mmol) in dichloromethane (200 mL, 781 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (16.7 g, 87.4 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.71 g, 13.9 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (100 mL) was added to the reaction mixture, and extracted with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [U-7] (6.0 g, 26.0 mmol, 37.56% yield) as pale yellow liquid. [0734] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 4H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [U-9]: [0735] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [U-8] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [U-7] (6 g, 26 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired tris(5- (octanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [U-9] (2.1 g, 38.9% yield) as pale yellow liquid. [0736] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.89-0.85 (m, 9H). [0737] ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [U-10]: [0738] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [9] (2.96 g, 3.57 mmol) and 3-{[(tert-butoxy)carbonyl](methyl)amino}propanoic acid [U-4] (2.9 g, 14.3 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (2.74 g, 14.3 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (439 mg, 3.57 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert- butoxy)carbonyl](methyl)amino}propanoyl)oxy]propane-1,2,3-tricarboxylate [U-10] (3 g, 82.5 % yield) as a light yellow liquid. [0739] 1H-NMR (400 MHz, CDCl3)- δ 4.21-4.04 (m, 12H), 3.47 (t, J = 7.2Hz, 2H), 3.28 (d, J = 15.6Hz, 2H), 3.20 (d, J = 15.6Hz, 2H), 2.83 (bs, 3H), 2.56 (t, J = 7.2Hz, 2H), 2.28 (t, J = 7.2Hz, 6H), 1.71-1.59 (m, 18H), 1.47-1.39 (m,15H), 1.36-1.27 (m, 24H), 0.89-0.85 (m, 9H). Intermediate [U-11]: [0740] Trifluoroacetic acid (4.98 mL, 65.1 mmol) was added into a solution of 1,2,3-tris[5- (octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl](methyl)amino}propanoyl)oxy]propane-1,2,3- tricarboxylate [U-10] (3 g, 2.96 mmol) in dichloromethane (40 mL, 625 mmol) at 0 oC, allow to stirred for until reaction completion. The progress of reaction was monitored by TLC. After completion the reaction, reaction mixture was concentrated under reduced pressure to remove excess trifluoroacetic acid. The reaction mass was quenched with saturated NaHCO3 solution up to pH 8, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired 1,2,3-tris[5- (octanoyloxy)pentyl] 2-{[3-(methylamino)propanoyl]oxy}propane-1,2,3-tricarboxylate [U-11] (2.85 g, 93 % yield) as pale yellow liquid. [0741] 1H-NMR (400 MHz, CDCl3)- δ 4.21-4.16 (m, 2H), 4.10-4.04 (m, 10H), 3.25-3.16 (m, 6H), 2.85-2.81 (m, 3H), 2.72 (s, 3H), 2.28 (t, J = 7.6 Hz, 6H), 1.70-1.59 (m, 18H), 1.43 (m, 6H), 1.29-1.27 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). Intermediate [U-12]: [0742] To stirred a solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3- (methylamino)propanoyl]oxy}propane-1,2,3-tricarboxylate [U-11] (1 g, 1.09 mmol) in acetonitrile (15 mL, 287 mmol), dipotassium carbonate (756 mg, 5.47 mmol), and tert-butyl(2- iodoethoxy)dimethylsilane [U-2] (470 mg, 1.64 mmol) was added at room temperature, allow to stir at 60 oC for 16 h. The progress of reaction was monitored by TLC and ELSD data. After completion the reaction, reaction mass was cooled, filter by sintered glass funnel. The filtrate was concentrated under reduced pressure and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired compound [U- 12] (0.4 g, 34% yield) as pale yellow liquid. [0743] ELSD analysis: Purity 99.58 %, Calculated C57H105NO15Si = 1071.73, Observed = 1072.90 (m/z, M+H+). TL1-12D-011 [0744] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-({2-[(tert- butyldimethylsilyl)oxy]ethyl}(methyl)amino)propanoyl]oxy}propane-1,2,3-tricarboxylate [U-12] (0.4 g, 373 µmol) in tetrahydrofuran (10 mL, 123 mmol), hydrogen pyridine fluoride (202 µL, 2.24 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with cold aqueous sodium bicarbonate solution upto pH 8, and extract with ethyl acetate (3x50 mL). The resulting organic layer was dried over Na2SO4, concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10 % methanol in dichloromethane), to obtain the desired produced TL1-12D-011 (0.2 g, 56% yield) as a light yellow colour liquid. Results: [0745] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.4 Hz, 2H), 4.09-4.04 (m, 10H), 3.60 (t, J = 5.2Hz, 2H), 3.29 (d, J = 15.6 Hz, 2H), 3.20 (d, J = 15.6Hz, 2H), 2.75 (t, J = 6.8Hz, 2H), 2.57-2.50 (m, 4H), 2.30-2.26 (m, 8H), 1.72-1.59 (m, 20H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.87 (t, J = 6.8Hz, 9H). [0746] ELSD analysis: Purity 99.70 %, Calculated C51H91NO15 = 957.64, Observed = 958.25 (m/z, M+H+). Example 22. Synthetic Protocol for M1266-539235 (TL1-12D-027) (Compound 75) Synthetic Scheme V [0747] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N, N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [V-3] (16 g, 69.5 mmol) as light yellow colour liquid. [0748] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H ), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H).
[0749] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [V-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [V-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0750] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). TL1-12D-027 (Compound (75)) Compound (75) [0751] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate (1 g, 1.21 mmol) and 4-(dimethylamino)butanoic acid (633 mg, 4 eq., 4.82 mmol) in dichloromethane (25.0 mL) was added EDCI (925 mg, 4 eq., 4.82 mmol) and N,N-dimethylpyridin- 4-amine (147 mg, 1.21 mmol) at 0°C. Reaction mass allowed to RT and stirred for 16 hr. Progress of reaction was monitored by ELSD/TLC. To the resulting reaction mixture water (100 ml) was added and extracted with DCM (2x50.0 mL), dried over anhy sodium sulphate and concentrated. Crude was purified over silica using 3% MeOH in DCM to obtain 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[4-(dimethylamino)butanoyl]oxy}propane-1,2,3-tricarboxylate [TL1-12D-027] (Compound (75)) (68 µg, 7.22e-8 mol) as colour less liquid mass. [0752] 4.15 (t, J = 6.4 Hz, 2H), 4.07 (m, 10H), 3.29-3.25 (d, J = 15.6 Hz, 2H), 3.21-3.17 (d, J = 15.6 Hz, 2H), 2.50 (m, 16H), 1.83 (bs, 2H), 1.61 (m, 14H), 1.40 (m, 6H), 1.27 (brs, 28H), 0.87 (t, J = 10.8 Hz, 9H). [0753] LCMS analysis: Purity 99.35 %, Calculated C51H91NO14 = 941.64, Observed = 942.35 (m/z, M+H+). Example 23. Synthetic Protocol for M1266-536156 (TL1-12D-028) (Compound (50)) Synthetic Scheme W Compound W-3 [0754] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N,N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [W-3] (16 g, 69.5 mmol) as light yellow colour liquid. [0755] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 6H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Compound W-5 [0756] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [W-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [W-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0757] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). TL1-12D-028 (Compound (50)) Compound (50) [0758] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [Int-5] (0.2 g, 241 µmol) in dichloromethane (10 mL) was added 5- (dimethylamino)pentanoic acid (35 mg, 241 µmol), EDCI (185 mg, 4 eq., 965 µmol) and N,N- dimethylpyridin-4-amine (29.5 mg, 241 µmol) at RT. Reaction mass was stirred for 16 h at RT. Progress of reaction mass was monitored by ELSD and TLC. Rreaction mass was diluted with 25.0 mL of DCM and washed with water (25.0 mL). Organic layer was dried over anhy sodium sulphate and concentrated. Crude was purified over silica using 3% MeOH in DCM to obtain pure product [TL1-12D-028] (Compound (50) (120 mg, 125 µmol) as brown colour mass. [0759] NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.8 Hz, 2H), 4.074 (m, 10H), 3.29-3.25 (d, J = 15.6 Hz, 2H), 3.21-3.17 (d, J = 15.6 Hz, 2H), 2.44 (bs, 8H), 2.30 (t, J = 7.2 Hz.6H), 1.65 (m, 18H), 1.40 (m, 6H), 1.27 (brs, 28H), 0.87 (t, J = 2.8 Hz, 9H). [0760] LCMS analysis: Purity 99.83 %, Calculated C52H93NO14 = 955.66, Observed = 956.35 (m/z, M+H+). Example 24. Synthetic Protocol for M1266-548305 (TL1-12D-011-A) (Compound (62)) Synthetic Scheme X TBDMS-CI Imidazole Br^^^^OTBDMS
X-10
Intermediate [X-2]: [0761] To a stirred solution of 3-bromopropan-1-ol [X-1] (5 g, 36 mmol) in dichloromethane (100 mL), 1H-imidazole (4.9 g, 71.9 mmol) was added at room temperature. The resulting reaction mixture was cooled to 0 °C and tert-butyl(chloro)dimethylsilane (10.8 g, 71.9 mmol) was added portion wise at same temperature, then allowed to stirred for 16 h at room temperature. Progress of reaction was monitored by ELSD/TLC. Water (50 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0-10 % ethyl acetate in n-hexane), to obtain pure product [X-2] (4.1 g, 45 %, Yield) as a colourless liquid. [0762] 1H-NMR (400 MHz, CDCl3): δ 3.73 (t, J = 6.0 Hz, 2H), 3.51 (t, J = 6.4 Hz, 2H), 2.06-2.00 (m, 2H), 0.89 (s, 9H), 0.06 (s, 6H). Intermediate [X-4]: [0763] To stirred a solution of 3-(methylamino)propanoic acid [X-3] (5 g, 48.5 mmol), and potassium hydroxide (5.44 g, 97 mmol) in 1,4-dioxane (25 mL, 293 mmol) and water (25 mL, 1.39 mol) added di-tert-butyl dicarbonate (10.6 g, 48.5 mmol) at room temperature for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, mixture was concentrated under reduced pressure. The crude material was acidified by 10 % NaHSO4 upto pH 2-3. Water (20 mL) was added to the reaction mixture and extracted with ethyl acetate (3x100 mL). The resulting organic layer was dried over anhydrous sodium sulphate concentrated under reduced pressure to obtain crude product which was purified by flash column chromatography (0- 100 % ethyl acetate in n-hexane), to obtain pure product 3-{[(tert- butoxy)carbonyl](methyl)amino}propanoic acid [X-4] (9 g, 91.33 %, Yield) as a colourless liquid. [0764] 1H-NMR (400 MHz, CDCl3): δ 3.50 (t, J = 6.8 Hz, 2H), 2.87 (s, 3H), 2.59 (bs, 2H), 1.48 (s, 9H). Intermediate [X-7]: [0765] To a stirred solution of octanoic acid [X-6] (10 g, 69.3 mmol) and pentane-1,5-diol [X-5] (6.5 g, 62.4 mmol) in dichloromethane (200 mL, 781 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (16.7 g, 87.4 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.71 g, 13.9 mmol) and the reaction mixture stirred at r.t. for 16 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (2x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [X-7] (6.0 g, 26.0 mmol, 37.56% yield) as pale yellow liquid. [0766] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 4H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [X-9]: [0767] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [X-8] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [X-7] (6 g, 26 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired tris(5- (octanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [X-9] (2.1 g, 38.9% yield) as pale yellow liquid. [0768] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). [0769] ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [X-10]: [0770] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [X-9] (2.96 g, 3.57 mmol) and 3-{[(tert-butoxy)carbonyl](methyl)amino}propanoic acid [X-4] (2.9 g, 14.3 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC, ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (2.74 g, 14.3 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (439 mg, 3.57 mmol) and the reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert- butoxy)carbonyl](methyl)amino}propanoyl)oxy]propane-1,2,3-tricarboxylate [X-10] (3 g, 82.5 % yield) as a light yellow liquid. [0771] 1H-NMR (400 MHz, CDCl3)- δ 4.21-4.04 (m, 12H), 3.47 (t, J = 7.2Hz, 2H), 3.28 (d, J = 15.6Hz, 2H), 3.20 (d, J = 15.6Hz, 2H), 2.83 (bs, 3H), 2.56 (t, J = 7.2Hz, 2H), 2.30-2.26 (m, 6H), 1.71- 1.59 (m, 18H), 1.47-1.39 (m,15H), 1.36-1.27 (m, 24H), 0.89-0.85 (m, 9H). [0772] Trifluoroacetic acid (4.98 mL, 65.1 mmol) was added into a solution of 1,2,3-tris[5- (octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl](methyl)amino}propanoyl)oxy]propane-1,2,3- tricarboxylate [X-10] (3 g, 2.96 mmol) in dichloromethane (40 mL, 625 mmol) at 0 oC, allow to stirred for until reaction completion. The progress of reaction was monitored by TLC. After completion the reaction, reaction mixture was concentrated under reduced pressure to remove excess trifluoroacetic acid. The reaction mass was quenched with saturated NaHCO3 solution up to pH 8, and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired 1,2,3-tris[5- (octanoyloxy)pentyl] 2-{[3-(methylamino)propanoyl]oxy}propane-1,2,3-tricarboxylate [X-11] (2.85 g, 93 % yield) as pale yellow liquid. [0773] 1H-NMR (400 MHz, CDCl3)- δ 4.21-4.16 (m, 2H), 4.10-4.04 (m, 10H), 3.25-3.16 (m, 6H), 2.85-2.81 (m, 3H), 2.72 (s, 3H), 2.28 (t, J = 7.6 Hz, 6H), 1.70-1.59 (m, 18H), 1.43 (m, 6H), 1.29-1.27 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). Intermediate [X-12]: [0774] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-(methylamino) propanoyl]oxy}propane-1,2,3-tricarboxylate [X-11] (1.5 g, 1.64 mmol) in acetonitrile (20 mL, 383 mmol), dipotassium carbonate (680 mg, 4.92 mmol), and 2-[(3-bromopropoxy)dimethylsilyl]-2- methylpropan-1-ylium [X-2] (623 mg, 2.46 mmol) was added at room temperature, allow to stir at 60 oC for 16 h. The progress of reaction was monitored by TLC and ELSD data. After completion the reaction, reaction mass was cooled, filter by sintered glass funnel. The filtrate was concentrated under reduced pressure and the crude was purified by flash column chromatography (SiO2: 0-5% Methanol in Dichloromethane), to give the desired 1,2,3-tris[5- (octanoyloxy)pentyl] 2-{[3-({3-[(tert- butyldimethylsilyl)oxy]propyl}(methyl)amino)propanoyl]oxy}propane-1,2,3-tricarboxylate [X-12] (1.0 g, 56 % yield) as pale yellow liquid. [0775] ELSD analysis: Purity 97.32 %, Calculated C58H107NO15Si = 1085.74, Observed = 1086.90 (m/z, M+H+). TL1-12D-011-A (Compound (62)) Compound (62) [0776] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-({3-[(tert- butyldimethylsilyl)oxy]propyl}(methyl)amino)propanoyl]oxy}propane-1,2,3-tricarboxylate (0.9 g, 828 µmol) in tetrahydrofuran (20 mL, 246 mmol), pyridine hydrofluoride (498 µL, 5.52 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with cold aqueous sodium bicarbonate solution upto pH 8, and extracted with ethyl acetate (3x50 mL). The resulting organic layer was dried over Na2SO4, concentrated under reduced pressure, and the crude was purified by flash column chromatography (SiO2: 0-10 % methanol in dichloromethane), to obtain the desired produced [TL1-12D-011-A] (Compound (62) (0.108 g, 46.56% yield) as a light yellow colour liquid. [0777] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.8 Hz, 2H), 4.10-4.04 (m, 10H), 3.78 (bs, 1H), 3.29-3.17 (m, 4H), 2.70 (br, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.70-1.59 (m, 18H), 1.55 (bs, 6H), 1.44-1.37 (m, 6H), 1.30-1.25 (m, 26H), 0.87 (t, J = 6.4Hz, 9H). [0778] ELSD analysis: Purity 99.62 %, Calculated C52H93NO15 = 971.65, Observed = 972.50 (m/z, M+H+).
Example 25. Synthetic Protocol for M1266-527861 (TL1-12D-019) (Compound (17)) Synthetic Scheme Y [0779] To a stirred solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N,N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cooled to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with ethyl acetate (200 ml). Organic layer was washed with brine solution (500.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure, get crude. Crude was purified with column chromatography using 5-10% Ethyl acetate in Hexanes, to give compound Y-3 (16 g, 69.5 mmol) as light yellow colour liquid. [0780] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 6H), 1.44 (m, 2H), 1.29 (s, 8H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate Y-5 [0781] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under inert atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (100.0 mL) and dichloromethane (2x 50.0 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure. Crude was purified with flash chromatography using 10-15% ethyl acetate in hexane to give compound Y-5 (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0782] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 5H), 1.29 (m, 25H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate Y-7 [0783] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [5] (0.1 g, 121 µmol) in dichloromethane (2.5 mL, 39 mmol) added N,N- dimethylpyridin-4-amine (14.7 mg, 121 µmol) and EDC.HCl (92.5 mg, 4 eq., 482 µmol) were added at room temperature under nitrogen atmosphere and stirred 30 min at RT, then 4-{[(tert- butoxy)carbonyl]amino}butanoic acid (98 mg, 4 eq., 482 µmol) was added in to reaction mass. Reaction mass was stirred over night at RT. Reaction progress was monitored by ELSD. Reaction mixture was diluted with water (50.0 mL) and extracted with DCM (50 ml). Organic layer was washed with brine solution (500.0 mL). Organic layer was dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure to get compound Y-7 (0.2 g, 50.5 µmol) as off white semi solid crude. Which was proceeded to next step without further purification. Intermediate Y-8 [0784] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(4-{[(tert-butoxy) carbonyl]amino}butanoyl)oxy] propane-1,2,3-tricarboxylate [7] (0.2 g, 1.0eq) in dichloromethane (10 ml ) was cooled to 0°C. Added trifloroacetic acid (0.2 ml , 1v/v) and reaction mass was stirred for 2h at RT. Progress of reaction was monitored by TLC. Once the starting was consumed, solvent was removed under reduced pressure and resulting crude (0.2 g) proceeded for next step without further purification. TL1-12D-019 (Compound (17)) Compound (17) [0785] To a stirred solution of 3-(dimethylamino)propanoic acid (92.4 mg, 4 eq., 789 µmol) in dichloromethane (4 mL, 62.5 mmol) was added EDC.HCl (151 mg, 4 eq., 789 µmol) and N,N- dimethylpyridin-4-amine (24.1 mg, 197 µmol), reaction mass was stirred 30 min then added 1,2,3- tris[5-(octanoyloxy)pentyl] 2-[(3-aminopropanoyl)oxy]propane-1,2,3-tricarboxylate; trifluoroacetic acid [8] (0.2 g, 197 µmol). Reaction mass was stirred for 16 h, reaction progress was monitored by ELSD. Starting material was consumed, distil out solvent under reduced pressure to get crude. The resulting crude was purified with Prep ELSD purification and get compound [TL1- 12D-019] (Compound (17)) (0.1 g, 0.1 mmol) as a white solid. [0786] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.8 Hz, 2H), 4.074 (m, 10H), 3.29-3.25 (d, J = 15.6 Hz, 2H), 3.21-3.17 (d, J = 15.6 Hz, 2H), 2.44 (bs, 8H), 2.30 (t, J = 7.2 Hz.6H), 1.65 (m, 22H), 1.40 (m, 6H), 1.27 (brs, 28H), 0.87 (t, J = 2.8 Hz, 9H). [0787] LCMS analysis: Purity 99.66 %, Calculated C53H94N2O15 = 998.67, Observed = 999.50 (m/z, M+H+).
Example 26. Synthetic Protocol for M1266-560527 (TL1-12-008) (Compound (89)) Synthetic Scheme Z [0788] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N, N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [Z-3] (16 g, 69.5 mmol) as light yellow colour liquid. [0789] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate Z-5 [0790] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [Z-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [Z-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0791] δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate Z-7 [0792] To a stirred solution of 3-{[(tert-butoxy)carbonyl]amino}propanoic acid [Z-6] (2.28 g, 4 eq., 12.1 mmol) in dichloromethane (50 mL, 781 mmol) was added N,N-dimethylpyridin-4-amine (368 mg, 3.02 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (2.6 g, 4.5 eq., 13.6 mmol), then reaction mass was stirred and added 1,2,3-tris[5- (octanoyloxy)pentyl] 2-hydroxypropane-1,2,3-tricarboxylate [Z-5] (2.5 g, 3.02 mmol) at RT. Reaction mass was stirred for 36 h at RT. Reaction progress was monitored by TLC and ELSD. Reaction mass was diluted with DCM (100.0 mL) and washed with water (2x 50.0 mL). Organic layer was separated and dried with sodium sulphate and distil out under reduced pressure. [0793] The resulting crude was purified with flash chromatography using 15% ethyl acetate in heptane, to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert- butoxy)carbonyl]amino}propanoyl)oxy]propane-1,2,3-tricarboxylate [Int-7] (1.85 g, 1.85 mmol) as a clear liquid. [0794] LCMS analysis: Purity 70%, Calculated C53H93NO16 = 999.65, Observed = 1000.30 (m/z, M+H+). Intermediate Z-8 [0795] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl] amino}propanoyl) oxy]propane-1,2,3-tricarboxylate (1.8 g, 1.8 mmol) in DCM, added trifluoroacetic acid (5 mL) at 0°C. Reaction mass was allowed to RT and stirred at RT for 4 h. Reaction progress was monitored by TLC. Reaction mass was distil out under reduced pressure , to give1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-aminopropanoyl)oxy]propane-1,2,3-tricarboxylate trifluoroacetic acid (1.83 g, 1.8 mmol) as a colour less semi solid. This compound was used in next step without further purification. [0796] LCMS analysis: Purity 64.94 %, Calculated C48H85NO14 = 899.60, Observed = 900.85 (m/z, M+H+). TL1-12D-008 (Compound (89)) Compound (89) [0797] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3- aminopropanoyl)oxy]propane-1,2,3-tricarboxylate hydrochloride [Z-8] (0.5 g, 534 µmol) in acetonitrile (8 mL, 153 mmol), were added iodoethane (833 mg, 10 eq., 5.34 mmol) and dipotassium carbonate (221 mg, 3 eq., 1.6 mmol). Reaction mass stirred for 48 h at 55°C. Progress of reaction was monitored by ELSD, after consumption of starting material, reaction mass allowed to come at RT and filter through sintered funnel, solid was wash with Ethyl acetate. Filtrate was distilled out under reduced pressure. Crude was purified with prep HPLC, to give [TL1-12D-008] (Compound (89)) (0.1 g, 93.4 µmol) as a yellow colour liquid. [0798] 1H NMR (400 MHz, DMSO-d6): δ 4.07 (t, J = 6.4 Hz, 2H), 4.01 (m, 10H), 3.28 (m, 2H), 3.10 (m, 8H), 2.74-2.70 (m, 2H) 2.26 (t, J = 7.2, 6H), 1.61-1.57 (m, 12H), 1.53 (t, J = 6.8 Hz, 6H), 1.35 (m, 6H), 1.24 (brs, 24H), 1.17 (t, J = 7.2 Hz, 6H), 0.85 (t, J = 6.8 Hz, 9H). [0799] LCMS analysis: Purity 99.70 %, Calculated C52H93NO14 = 955.66, Observed = 956.45 (m/z, M+H+). Example 27. Synthetic Protocol for M1266-560531 (TL1-12D-009) (Compound (61)) Synthetic Scheme AA [0800] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N, N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [AA-3] (16 g, 69.5 mmol) as light yellow colour liquid. [0801] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate AA-5 [0802] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [AA-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [AA-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0803] δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate AA-7 [0804] To a stirred solution of 3-{[(tert-butoxy)carbonyl]amino}propanoic acid [AA-6] (2.28 g, 4 eq., 12.1 mmol) in dichloromethane (50 mL, 781 mmol) was added N,N-dimethylpyridin-4-amine (368 mg, 3.02 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (2.6 g, 4.5 eq., 13.6 mmol), then reaction mass was stirred and added 1,2,3-tris[5- (octanoyloxy)pentyl] 2-hydroxypropane-1,2,3-tricarboxylate [Int-5] (2.5 g, 3.02 mmol) at RT. Reaction mass was stirred for 36 h at RT. Reaction progress was monitored by TLC and ELSD. Reaction mass was diluted with DCM (100.0 mL) and washed with water (2x 50.0 mL). Organic layer was separated and dried with sodium sulphate and distil out under reduced pressure. The resulting crude was purified with flash chromatography using 15% ethyl acetate in heptane, to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl]amino}propanoyl)oxy]propane- 1,2,3-tricarboxylate [AA-7] (1.85 g, 1.85 mmol) as a clear liquid. [0805] LCMS analysis: Purity 70%, Calculated C53H93NO16 = 999.65, Observed = 1000.30 (m/z, M+H+). Intermediate AA-8 [0806] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl] amino}propanoyl) oxy]propane-1,2,3-tricarboxylate [AA-7] (1.8 g, 1.8 mmol) in D7M, added trifluoroacetic acid (5 mL) at 0°C. Reaction mass was allowed to RT and stirred at RT for 4 h. Reaction progress was monitored by TLC. Reaction mass was distil out under reduced pressure to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-aminopropanoyl)oxy]propane-1,2,3-tricarboxylate trifluoroacetic acid (1.83 g, 1.8 mmol) as a colour less semi solid. This compound was used in next step without further purification. [0807] LCMS analysis: Purity 64.94 %, Calculated C48H85NO14 = 899.60, Observed = 900.85 (m/z, M+H+). TL1-12D-009 (Compound (61)) Compound (61) [0808] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3- aminopropanoyl)oxy]propane-1,2,3-tricarboxylate hydrochloride [AA-8] (0.5 g, 534 µmol) in acetonitrile (8 mL, 153 mmol), were added 1-iodopropane (907 mg, 10 eq., 5.34 mmol) and dipotassium carbonate (221 mg, 3 eq., 1.6 mmol). Reaction mass stirred for 48 h at 55°C. Progress of reaction was monitored by ELSD, after consumption of starting material, reaction mass allowed to come at RT and filter through sintered funnel, solid was wash with Ethyl acetate. Filtrate was distilled out under reduced pressure. Crude was purified with prep HPLC, to give [TL1-12D-009] (Compound (61)), TFA salt (62 mg, 56.4 µmol) as a yellow colour liquid. [0809] 1H NMR (400 MHz, DMSO-d6): δ 4.07 (t, J = 6.0 Hz, 2H), 4.01 (m, 10H), 3.28 (m, 2H), 3.20-3.17 (d, J = 15.2 Hz, 2H), 3.144-3.10 (d, J = 15.6 Hz, 2H), 3.04 (m, 4H), 2.85 (t, J = 7.2 Hz, 2H), 2.26 (t, J = 7.6, 6H), 1.59-1.49 (m, 22H), 1.35 (m, 6H), 1.24 (brs, 24H), 0.85 (t, J = 6.8 Hz, 15H). [0810] LCMS analysis: Purity 99.13 %, Calculated C54H97NO14 = 983.69, Observed = 984.65 (m/z, M+H+).
Example 28. Synthetic Protocol for M1266-577919 (TL1-12D-030) (Compound (64)) Synthetic Scheme BB [0811] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N, N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [BB-3] (16 g, 69.5 mmol) as light-yellow colour liquid. [0812] δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate BB-5 [0813] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [BB-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [BB-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0814] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate BB-7 [0815] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate (1.0 g, 1.21 mmol) [BB-5] in dichloromethane (12.5 mL, 195 mmol) was added N,N- dimethylpyridin-4-amine (147 mg, 1.21 mmol) and 6-bromohexanoic acid [BB-6] (941 mg, 4 eq., 4.82 mmol) successively at room temperature and stirred for 10 minute at same temperature, followed by addition of ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (925 mg, 4 eq., 4.82 mmol) .The resulting reaction mixture was stirred at room temperature for 16 h. Progress of reaction was monitored by TLC / ELSD. The resulting reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2x30 mL). Combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by flash chromatography on silica gel using 10- 30 % gradient of Ethyl acetate in heptane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(6- bromohexanoyl)oxy]propane-1,2,3-tricarboxylate (160 mg, 159 µmol) as a colourless liquid. [0816] LCMS analysis: Purity 78%, Calculated C51H89BrO14 = 1004.54, Observed = 1005.30 (m/z, M+H+). TL1-12D-030 (Compound (64)) Compound (64) [0817] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(6- bromohexanoyl)oxy]propane-1,2,3-tricarboxylate (1.1 g, 0.57 eq., 1.09 mmol) in tetrahydrofuran (22 mL, 270 mmol) , dimethylamine (1.74 g, 20 eq., 38.6 mmol) was successively added at room temperature and stirred for 10 minute at same temperature. The resulting reaction mixture was stirred at 55°C temperature for 16 hrs monitor by TLC. The resulting reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2x30 mL). The resulting whole organic were combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by Prep HPLC. The pure fraction were combined and concentrated under reduced pressure to obtain as [TL1-12D-030] (Compound (64) (260 mg, 268 µmol) a colourless liquid. [0818] 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.0 Hz, 2H), 4.01 (m, 10H), 3.28-3.26 (d, J = 15.6 Hz, 2H), 3.18-3.14 (d, J = 15.6 Hz, 2H), 2.76-2.75(d, J = 4.8Hz, 6H) 2.33 (m, 2H), 2.28 (t, J = 7.2 Hz. 6H), 1.65 (m, 22H), 1.35 (m, 6H), 1.24 (brs, 28H), 0.87 (t, J = 6.8 Hz, 9H). [0819] LCMS analysis: Purity 97.59 %, Calculated C53H95NO14 = 969.68, Observed = 970.65 (m/z, M+H+). Example 29. Synthetic Protocol for M1266-596266 (TL1-12D-031) (Compound (63)) Synthetic Scheme CC [0820] To a stirrer solution of pentane-1,5-diol (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N, N-dimethylpyridin- 4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cool to 0-5°C, then added octanoyl chloride (16 g, 98.4 mmol) dropwise. Reaction mixture was allowed to r.t. and stirred for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with DCM (2x 500.0 mL). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude purified with column chromatography using 5-10% ethyl acetate in hexane, to give 5-hydroxypentyl octanoate [CC-3] (16 g, 69.5 mmol) as light-yellow colour liquid. [0821] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate CC-5 [0822] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [CC-3] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol). Reaction mass stirred 10 min at RT, then added 5-hydroxypentyl octanoate (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 hr at RT under nitrogen atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (200.0 mL) and dichloromethane (2x 100 mL). Organic layer separated, dried over sodium sulphate, distil out under reduced pressure, get crude. Crude was purified by flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- hydroxypropane-1,2,3-tricarboxylate [CC-5] (3.5 g, 4.22 mmol) as a light yellow colour liquid. [0823] δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate CC-7 [0824] To a stirred solution 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [CC-5] (1 g, 1.21 mmol) and 7-bromoheptanoic acid (1.01 g, 4 eq., 4.82 mmol) in dichloromethane (20 mL) was added EDC.HCL (925 mg, 4 eq., 4.82 mmol) followed by 4- (dimethylamino)pyridin-1-ium (149 mg, 1.21 mmol). Reaction mass was stirred for 16h at RT. Progress of reaction was monitored by ELSD/TLC (SM consumed). Reaction mass was diluted with DCM (25.0 mL) and washed with DM water (50 ml) and dried over sodium sulphate and concentrated. Resulting crude was purified over silica using 10 % ethyl acetate in n-heptane to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(7-bromoheptanoyl)oxy]propane-1,2,3-tricarboxylate [Int-7] (0.4 g, 392 µmol) as yellow gummy mass. [0825] LCMS analysis: Purity 92.40 %, Calculated C52H91BrO14 = 1018.56, Observed = 1020.00 (m/z, M+H+). TL1-12D-031 (Compound (63)) Compound (63) [0826] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(7- bromoheptanoyl)oxy]propane-1,2,3-tricarboxylate (0.8 g, 0.23 eq., 784 µmol) in tetrahydrofuran (17.8 mL, 218 mmol) and dimethylamine (1.55 g, 10 eq., 34.3 mmol) was successively added at room temperature and stirred for 10 minute at same temperature. The resulting reaction mixture was stirred at 55°C temperature for 16 hrs monitor by TLC. The resulting reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2x30 mL). The resulting whole organic were combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by Prep HPLC. The pure fraction was combined and concentrated under reduced pressure to obtain the desired product [TL1-12D-031] (Compound (63)) as a colourless liquid. [0827] 4.05 (t, J = 6.0 Hz, 2H), 4.01 (m, 10H), 3.18-3.14 (d, J = 15.6 Hz, 2H), 3.08-3.04 (d, J = 15.6 Hz, 2H), 3.02-2.97 (m, 2H), 2.76-2.75(d, J = 5.2 Hz, 6H) 2.28 (t, J = 7.6 Hz.8H), 1.55 (m, 22H), 1.35 (m, 6H), 1.24 (brs, 28H), 0.87 (t, J = 6.8 Hz, 9H). [0828] LCMS analysis: Purity 99.65 %, Calculated C54H97NO14 = 983.69, Observed = 984.90 (m/z, M+H+). Example 30. Synthesis of Compound (26) Synthetic Scheme DD [0829] Part 1 – To a 100 mL round bottom flask was added tertiary alcohol (DD-1) (2.00 g, 3.78 mmol), mono-tert-butyl succinate (1.98 g, 3.0 equiv), DMAP (462 mg, 1.0 equiv), DIPEA (5.92 mL, 9.0 equiv), and anhydrous CH2Cl2 (15 mL). To this stirring mixture at room temperature was added EDC (2.18 g, 3.0 equiv) in one batch. The reaction was stirred at room temperature for 16 h and was monitored by TLC using 20% EtOAc in hexanes. After significant consumption of (DD-1) as determined by TLC, the reaction mixture was diluted with CH2Cl2 and washed with sat. NaHCO3 solution, brine, and dried with sodium sulfate. Crude material is then purified using 0-20% EtOAc in hexanes where the desired compound (DD-2) eluded out at 100% hexanes and 10% EtOAc in hexanes. All the fractions containing (DD-2) were combined despite some containing (DD-1) as an impurity. The combined material was concentrated to dryness to provide intermediate (DD-2) as viscous oil. [0830] Part 2 – To a 50 mL round bottom flask containing the solution of intermediate (DD-2) in anhydrous CH2Cl2 (3 mL) was added TFA (3 mL) drop wise at room temperature. The reaction was stirred at room temperature until complete consumption of (DD-2) as determined by TLC analysis. After 3 h, the reaction was completed, and some toluene was added to the reaction mixture before it was concentrated down to dryness under reduced pressure. The leftover residue was purified using 0-30% EtOAc in hexanes to deliver the desired intermediate (DD-3) as viscous colorless oil (2.20 g, 98% over two steps). NMR (400 MHz, CDCl3) δ 4.16 – 4.04 (m, 6H), 3.24 (q, J = 15.5 Hz, 4H), 2.66 (s, 4H), 1.67 – 1.56 (m, 6H), 1.33 – 1.23 (m, 30H), 0.90 – 0.85 (m, 9H). [0831] To the scintillation vial, the acid [DD-3] (200 mg, 0.318 mmol), dimethyl aminoethanol [DD-4a] (85 mg, 0.954 mmol), DMAP (39.0 mg, 0.318 mmol), CH2Cl2 (4 mL), and lastly EDC (91.5 mg, 0.477 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of [DD-3]. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. The crude material was purified using 0-10% MeOH in DCM to afford the product [DD-5b] (Compound (26)) as colorless viscous oil (290 mg, 85% yield). [0832] Analysis: Mass Expected = 700.5 Mass Observed = 700.5
Example 31. Synthesis of Compound (41): Synthetic Scheme EE-1 Procedure [0833] To a 100 mL RBF containing the aliphatic alcohol (2.24 g, 14.2 mmol) was added citric acid (816 mg, 4.25 mmol) and TsOH (269 mg, 1.42 mmol). The solids were then washed down with toluene (10 mL) and heated at 110oC overnight equipped with Dean-Stark apparatus. The remaining residue was slightly diluted with hexanes and loaded onto a large silica gel column. Purification using 0% to 20% EtOAc in hexanes afforded the product EE-3A as clear light-yellow oil (2.20 g, 85% yield). Analysis [0834] [M+H]+ Expected = 613.5 Observed 613.5 Synthetic Scheme EE-2 Procedure [0835] To the scintillation vial, the alcohol [EE-3A] (1.0 g, 1.63 mmol), acid [EE-4] (752 mg, 4.89 mmol), DMAP (199.0 mg, 1.63 mmol), CH2Cl2 (8 mL), DIPEA (2.56 mL, 14.7 mmol), and lastly EDC (938 mg, 4.89 mmol) were added in one batch. The reaction was stirred at room temperature for four days until consumption of EE-3A. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. Material was purified using 0%, 20%, and followed by 40% EtOAc in hexanes to elude out product (on TLC plate it takes 80% EtOAc in hexanes to elude). Fractions containing pure product as determined by MS were combined and concentrated. The final product EE-5 (Compound (41), 420 mg (36%) as a pale yellow and viscous oil. Analysis [0836] [M+H]+ Expected = 712.6 Observed 712.6 Example 32. Synthesis of Compound (47) Synthetic Scheme FF Procedure [0837] To the scintillation vial, the alcohol [GG-7b] (724 mg, 1.39 mmol), acid [GG-10] (638 mg, 4.16 mmol), DMAP (169.0 mg, 1.39 mmol), DIPEA (2.17 mL, 12.5 mmol), CH2Cl2 (11 mL), and lastly EDC (797 mg, 4.16 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of GG-7b. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. Material was purified using 0%, 20%, and followed by 40% EtOAc in hexanes to elude out product (on TLC plate it takes 80% EtOAc in hexanes to elude). Fractions containing pure product as determined by MS were combined and concentrated to dryness to give viscous yellow oil as product GG-11, (Compound (47)), (411 mg, 48% yield). Analysis [0838] [M+H]+ Expected = 622.4 Observed 622.5 Example 33. Synthesis of Compound (52) Synthetic Scheme GG Procedure [0839] To the scintillation vial, the alcohol [HH-7c] (829 mg, 1.47 mmol), acid [HH-8] (676 mg, 4.40 mmol), DMAP (179.0 mg, 1.47 mmol), CH2Cl2 (12 mL), and lastly EDC (844 mg, 4.40 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of 7c. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. Material was purified using 0%, 20%, and followed by 40% EtOAc in hexanes to elude out product (on TLC plate it takes 80% EtOAc in hexanes to elude). Fractions containing pure product as determined by MS were combined and concentrated. The final product HH-9 [TL-005] (Compound (52)), 499 mg (51%) as a pale yellow and viscous oil. [0840] [M+H]+ Expected = 664.5 Observed 664.5 Example 34. Synthesis of Compound (2) Synthetic Scheme HH Procedure [0841] To the scintillation vial, the acid [II-9b] (250 mg, 0.401 mmol), dimethyl aminopropanol [II-10] (82.8 mg, 0.803 mmol), DMAP (49.0 mg, 0.401 mmol), CH2Cl2 (4 mL), and lastly EDC (154 mg, 0.803 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of 9b. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. The crude material was purified using 0-30% MeOH in DCM to afford the product II-11 (Compound (2)), 146 mg (51%) as a colorless viscous oil. [0842] Analysis: Mass expected 708.5 Observed 708.9 Example 35. Synthesis of Compound (4) Synthetic Scheme II Procedure [0843] To the scintillation vial, the acid [3] (300 mg, 0.477 mmol), ethanolamine [JJ-4] (284.24 mg, 2.38 mmol), DMAP (58.24 mg, 0.477 mmol), CH2Cl2 (6 mL), DIPEA (0.498 mL, 2.86 mmol), and lastly EDC (137.18 mg, 0.715 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of 3. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. The crude material was purified using 5% MeOH in DCM first (10% MeOH/DCM had too high of an RF) and then purified using 5% MeOH in EtOAc to afford the product JJ-5 (Compound (4)), 187 mg (54%) as a colorless viscous oil. [0844] Analysis: Mass expected: 729.50 | Observed: 729.50 Example 36. Synthesis of Compound (31) Synthetic Scheme JJ Procedure [0845] To the scintillation vial, the acid [3] (660 mg, 1.05 mmol), diethanolamine [KK-4] (90.5, 0.475 mmol), DMAP (128.22 mg, 1.05 mmol), CH2Cl2 (6 mL), DIPEA (1.09 mL, 6.3 mmol), and lastly EDC (301.8 mg, 1.57 mmol) were added in one batch. The reaction was stirred at room temperature until consumption of SM. Reaction was diluted with CH2Cl2 and washed with NaHCO3 solution, brine, and dried with sodium sulfate. The crude material was purified using 7-10% MeOH in DCM and then re-purified using 0-10% MeOH/EtOAc to result in the product KK-5 [ETH-013] (Compound (31)), 671 mg (45%) as light-yellow viscous oil. [0846] Analysis: Mass expected: 1410.98| Observed: 1410.92 Example 37. Synthesis of Compound (21): Synthetic Scheme KK 2-(hexyldisulfaneyl)ethan-1-ol (LL-3) [0847] To a solution of LL-1 (1.0 g, 5.34 mmol) and LL-2 (1.26 g, 10.68 mmol) in chloroform was added TEA (2.98 mL, 21.36 mmol). The resulting mixture was stirred at room temperature for 3 h. TLC analysis indicated completion of the reaction (MS: no ionization). The reaction mixture was concentrated, and the crude residue was purified to get the pure product 3 (409 mg, 40%). [0848] ESI-MS analysis: Calculated C8H19OS2, [M + H]+ = 195.09, Observed = No ionization, reaction was monitored by TLC Tris(2-(hexyldisulfaneyl)ethyl) 2-hydroxypropane-1,2,3-tricarboxylate (LL-5) [0849] To a solution of citric acid (LL-4) (60 mg, 0.31 mmol) and 2-(hexyldisulfaneyl)ethan-1-ol (LL-3) (200 mg, 1.03 mmol) in 2.5 mL of dichloromethane was added DMAP (38 mg, 0.31 mmol) and EDC (239 mg, 1.25 mmol), and the resulting mixture was stirred at room temperature for overnight. MS analysis indicated completion of the reaction. Then reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get tri-ester (LL-5) (140 mg, 62%). [0850] ESI-MS analysis: Calculated C30H60NO7S6, [M + NH4]+ = 738.27, Observed = 738.2 Tris(2-(hexyldisulfaneyl)ethyl) 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3- tricarboxylate (Compound (21)) Compound (21)) [0851] To a solution of tri-ester (LL-5) (140 mg, 0.19 mmol) and 3-(dimethylamino)propanoic acid (LL-6) (89 mg, 0.58 mmol) in 3.0 mL of dichloromethane was added DMAP (24 mg, 0.19 mmol) and EDC (112 mg, 0.58 mmol), and the resulting mixture was stirred at room temperature for overnight. MS analysis indicated completion of the reaction. Then reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get Compound (21) (46 mg, 29%). Results: ESI-MS analysis: Calculated C35H66NO8S6, [M + H]+ = 820.31, Observed = 820.2
Example 38. Synthesis of Compound (34): Synthetic Scheme LL 5-hydroxypentyl decanoate (MM-3) [0852] To a solution of pentane-1,5-diol (MM-1) (2.5 mL, 24.00 mmol) in tetrahydrofuran (40 mL) was added decanoyl chloride (2) (2.5 mL, 12.00 mmol) at 0 oC. To that was slowly added triethylamine (1.67 mL, 12.00 mmol) over 10 minutes, then reaction mixture was warmed to room temperature and stirred for 16 h. MS analysis indicated completion of the reaction. The reaction mixture suspension was diluted with dichloromethane washed with sat. NaHCO3 solution, water and brine. The organic layer was dried over Na2SO4 and concentrated. The crude residue was purified to get the desired mono-product (2.0 g, 64%). [0853] ESI-MS analysis: Calculated C15H31O3, [M + H]+ = 259.23, Observed = 259.2 Tris(5-(decanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate (MM-5) [0854] To a solution of citric acid (MM-4) (0.5 g, 2.60 mmol) and 5-hydroxypentyl decanoate (MM-3) (2.2 g, 8.58 mmol) in 12 mL of dichloromethane was added DMAP (0.32 g, 2.60 mmol) and EDC (1.99 g, 10.41 mmol), and the resulting mixture was stirred at room temperature for overnight. MS analysis indicated completion of the reaction. Then reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get the intermediate (MM-5) (0.66 g, 28%). [0855] ESI-MS analysis: Calculated C51H96NO13, [M + NH4]+ = 930.69, Observed = 930.6 Tris(5-(decanoyloxy)pentyl) 2-((4-(dimethylamino)butanoyl)oxy)propane-1,2,3- tricarboxylate Compound (34) Compound (34) [0856] To a solution of intermediate (MM-5) (360 mg, 0.39 mmol) and 4- (dimethylamino)butanoic acid (MM-6) (198 mg, 1.18 mmol) in 7.0 mL of dichloromethane were added DMAP (48 mg, 0.39 mmol), DIPEA (0.55 mL, 3.15 mmol), and EDC (112 mg, 0.58 mmol), and the resulting mixture was stirred at room temperature for overnight. After 16 h, MS analysis of the reaction mixture indicated ~1:1 ratio of the desired product and starting material 5. The reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get the desired product Compound (34) (65 mg, 45%) and recovered 236 mg of intermediate 5. [0857] ESI-MS analysis: Calculated C57H104NO14, [M + H]+ = 1026.75, Observed = 1026.5 Example 39. Synthesis (Compound (30)): Synthetic Scheme MM 4-hydroxybutyl nonanoate (NN-3) [0858] To a solution of butane-1,4-diol (NN-1) (5.0 mL, 56.59 mmol) in tetrahydrofuran (150 mL) was added nonanoyl chloride (NN-2) (5.1 mL, 28.29 mmol) in tetrahydrofuran (50 mL) at 0 oC. To that was slowly added triethylamine (3.9 mL, 28.29 mmol) and stirred at 0 oC for 2 h. TLC and MS analysis indicated completion of the reaction. The reaction mixture was diluted with dichloromethane washed with sat. NaHCO3 solution, water and brine. The organic layer was dried over Na2SO4 and concentrated. The crude residue was purified to get the desired mono-product (3.83 g, 58%). [0859] ESI-MS analysis: Calculated C13H27O3, [M + H]+ = 231.20, Observed = No ionization, reaction was monitored by TLC Tris(4-(nonanoyloxy)butyl) 2-hydroxypropane-1,2,3-tricarboxylate (NN-5) [0860] To a solution of citric acid (NN-4) (0.5 g, 2.60 mmol) and 4-hydroxybutyl nonanoate (NN- 3) (1.98 g, 8.58 mmol) in 12 mL of dichloromethane was added DMAP (0.32 g, 2.60 mmol) and EDC (1.99 g, 10.41 mmol), and the resulting mixture was stirred at room temperature for overnight. MS analysis indicated completion of the reaction. Then reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get the intermediate (NN-5) (0.76 g, 35%). [0861] ESI-MS analysis: Calculated C45H84NO13, [M + NH4]+ = 846.59, Observed = 846.5 Tris(4-(nonanoyloxy)butyl) 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3- tricarboxylate (Compound (30)) [0862] To a solution of intermediate (NN-5) (760 mg, 0.92 mmol) and 3- (dimethylamino)propanoic acid (6) (422 mg, 2.75 mmol) in 15 mL of dichloromethane were added DMAP (112 mg, 0.92 mmol), DIPEA (1.27 mL, 7.33 mmol), and EDC (527 mg, 2.75 mmol), and the resulting mixture was stirred at room temperature for overnight. After 16 h, MS analysis of the reaction mixture indicated ~1:1 ratio of the desired product and starting material 5. The reaction mixture was diluted with DCM and washed with sat. NaHCO3 solution, water and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified to get the desired product (Compound (30)) (46 mg, 13%) and recovered 450 mg of intermediate 5. [0863] ESI-MS analysis: Calculated C50H90NO14, [M + H]+ = 928.64, Observed = 928.5 Example 40. Synthesis of TL1-12D-037 (Compound (90)): Synthetic Scheme NN [0864] To a stirred solution of 7-hydroxyheptanoic acid [OO-1] (4.5 g, 30.8 mmol) and hexan-1-ol [OO-2] (15.7 g, 5 eq., 0.154 mol) in dichloromethane (90 ml) was added {3- [cyano(ethyl)amino] propyl}dimethylazanium chloride (8.85 g, 1.5 eq., 46.2 mmol) followed by addition of N,N-dimethylpyridin-4-amine (3.76 g, 1.0 eq., 30.8 mmol) under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16h. The reaction was diluted with water (100 mL), its organic layer was separated, and aqueous layer was extracted with dichloromethane 2×100 mL. Combined organic layer was washed with brine solution (100 mL), dried over sodium sulphate, and concentrated under reduced pressure. The resulting crude was purified over silica using 5-10% ethyl acetate in n-heptane as eluent system to give product hexyl 7-hydroxyheptanoate [OO-3] (2.1 g, Yield – 29.62%) as a colourless liquid. [0865] 1H-NMR (400 MHz, CDCl3)- 4.05 (t, J = 6.4 Hz, 2H), 3.66-3.61 (dd, J = 6.4 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.67-1.54 (m, 6H), 1.37-1.20 (m, 10H), 0.89 (t, J= 7.2 Hz, 3H). Intermediate [OO-5]: [0866] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [OO-4] (4 g, 20.8 mmol) in dichloromethane (300 mL) was added {3-[cyano(ethyl)amino]propyl}dimethylazanium chloride (16 g, 4 eq., 83.3 mmol) followed by addition of N,N-dimethylpyridin-4-amine (5.09 g, 2 eq., 41.6 mmol) under nitrogen atmosphere. The reaction mass was stirred for 15 min at room temperature and Hexyl 7-hydroxyheptanoate [OO-3] (16.8 g, 3.5 eq., 72.9 mmol) was added in to reaction mixture. Reaction mixture was stirred for 48 h at room temperature. The resulting reaction mixture was quenched with water (500 mL), organic layer was separated, and aqueous layer was extracted with dichloromethane 2×100 mL. The resulting organic layer was combined, dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by silica using 10-20% ethyl acetate in n-heptane as eluent system. The pure fractions were combined and concentrated under reduced pressure to obtain as tri[6- (hexyloxycarbonyl)hexyl] 2-hydroxy-1,2,3-propanetricarboxylate [OO-5] (3.5 g, Yield – 20.34%). [0867] 1H-NMR (400 MHz, CDCl3)- 4.19 (t, J = 6.8 Hz, 2H), 4.08-4.03 (m, 10H), 2.89-2.76 (dd, J = 15.6 Hz.4H), 2.28 (t, J = 7.2 Hz, 6H), 1.69-1.57 (m, 18H), 1.36-1.27 (m, 30H), 0.88 (t, J = 7.2 Hz, 9H). ELSD analysis: Purity 99.63%, Calculated: C45H80O13= 828.55, Observed = 829.50 (m/z, M+H+). Tl1-12D-037 (Compound (90)): [0868] To a stirred solution of 3-(dimethylamino) propanoic acid [OO-6] (1.58 g, 4 eq., 13.5 mmol), in dichloromethane (30 mL, 469 mmol) was successively added {3-[cyano(ethyl)amino] propyl}dimethylazanium chloride (2.59 g, 4 eq., 13.5 mmol) followed by addition of 4- (dimethylamino)pyridin-1-ium (832 mg, 2 eq., 6.75 mmol) at room temperature. Reaction mixture was stirred for 15 minute at room temperature and 1,2,3-tris[7-(hexyloxy)-7-oxoheptyl] 2- hydroxypropane-1,2,3-tricarboxylate [5] (2.8 g, 3.38 mmol) was added in to reaction mixture. The resulting reaction mixture was stirred at room temperature for 16 h. The resulting reaction mixture was quenched with water 100 mL and extracted with dichloromethane 2 × 30 mL. The resulting organic layer was combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by silica using 40-50% ethyl acetate in n- heptane as eluent system. The pure fractions were combined and concentrated under reduced pressure to obtain as 1,2,3-tris[7-(hexyloxy)-7-oxoheptyl] 2-{[3-(dimethylamino)propanoyl] oxy}propane-1,2,3-tricarboxylate [TL1-12D-037] (2.2 g, 2.37 mmol, Yield – 70.18 %) a light brown liquid. [0869] 1H-NMR (400 MHz, DMSO-d6): 4.04-3.97 (m, 12H), 3.17-3.02 (dd, J = 15.2 Hz.4H), 2.45- 2.39 (dd, J = 6.0 Hz.4H), 2.26 (t, J = 7.6 Hz, 6H), 2.10 (s, 6H), 1.55-1.47 (m, 18H), 1.269-1.261 (br d, J = 3.2 Hz, 30H), 0.85 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.47 %, Calculated C50H89NO14 = 927.62, Observed = 928.50 (m/z, M+H+). Example 40A. Synthesis of TL1-12D-024 (Compound 100): Synthetic Scheme OO Intermediate [PP-3]: [0870] To a stirred solution of octanoic acid [PP-2] (100.0 g, 693 mmol) and pentane-1,5-diol [PP-1] (65.0 g, 624 mmol) in dichloromethane (2000 mL) was cooled to 0 oC and ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (167.0 g, 874 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (17.1 g, 139 mmol). The reaction mixture was stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture and extracted with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [PP-3] (60.0 g, 37.56% Yield) as a yellow liquid. 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71-1.58 (m, 5H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [PP-5]: [0871] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [PP-4] (12.5 g, 65.1 mmol) and 5-hydroxypentyl octanoate [PP-3] (60.0 g, 260 mmol) in dichloromethane (500 mL) was cooled to 0 oC, ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (49.9 g, 260 mmol) was added followed by 4-(dimethylamino)313yridine-1-ium (8.02 g, 65.1 mmol). The reaction mixture was stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes) to give the desired tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [PP-5] (21.0 g, yield 38.9%) as a yellow liquid. [0872] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [PP-7]: [0873] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [PP-5] (10 g, 12.1 mmol) in triethylamine (6.10 g, 60.3 mmol) added 3- bromopropanoyl chloride (1.21 mL, 12.1 mmol) was successively at 0 °C and stirred for 16 h, at same 25 oC temperature. The progress of reaction was monitored by ELSD/TLC (SM was consumed). The reaction mass was diluted with ethyl acetate, filtered through celite and washed with ethyl acetate to get crude product. The crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired [PP-7] (2.0 g, 18.7% yield) as yellow liquid. [0874] NMR (400 MHz, CDCl3): δ 6.42 (d, J = 17.2 Hz, 1H), 6.12-6.05 (m, 1H), 5.89 (d, J = 10.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2 H), 4.09-4.03 (m, 10H), 3.34 (d, J = 15.2 Hz, 2H), 3.25 (d, J = 15.6Hz, 2H), 2.28 (t, J = 7.2 Hz, 6 H), 1.70-1.59 (m, 18H), 1.43-1.35 (m, 6H), 1.28 (s, 24H), 0.87 (t, J = 6.8 Hz, 9 H). ELSD analysis: Purity 99.84%, Calculated C48H82O14 = 882.57, Observed = 883.40 (m/z, M+H+). TL1-12D-024 (Compound 100): [0875] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [PP-7] (140 mg, 159 µmol) in tetrahydrofuran (5 mL, 61.4 mmol) was added 1- methylpiperazine [PP-8] (15.9 mg, 159 µmol). Reaction mass was stirred at RT for 48 h. Progress of the reaction was monitored by TLC. Reaction mass was distilled out under reduced pressure and resulting crude material was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexanes), to give the desired 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-(4-methylpiperazin- 1-yl)propanoyl]oxy}propane-1,2,3-tricarboxylate [TL1-12D-024] (0.1 g, yield 64.15%) as yellow liquid. [0876] 1H-NMR (400 MHz, CDCl3)- δ 4.15 (t, J = 6.4 Hz, 2H), 4.09-4.04 (m, 10H), 3.27 (d, J = 16.0 Hz, 2H), 3.22 (d, J = 15.6 Hz, 2H), 2.70-2.66 (m, 2H), 2.55-2.48 (m, 6H), 2.35 (s, 2H), 2.28 (t, J = 7.6 Hz, 6H), 2.04 (bs, 5H), 1.69-1.57 (m, 18H), 1.44-1.37 (m, 6H), 1.32-1.27 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.32 %, Calculated C53H94N2O14 = 982.67, Observed = 983.65 (m/z, M+H+). Example 40B: Synthesis of of (TL1-12D-029) (Compound 101): Synthetic Scheme PP [0877] To a stirred solution of octanoic acid [QQ-2] (10 g, 69.3 mmol) and pentane-1,5-diol [QQ-1] (6.5 g, 62.4 mmol) in dichloromethane (200 mL) was cooled to 0 oC and ({[3- (dimethylamino) propyl]imino}methylidene)(ethyl)amine hydrochloride (16.7 g, 87.4 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.71 g, 13.9 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture, and extract with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [QQ-3] (6.0 g, 26.0 mmol, yield 37.56%) as pale-yellow liquid. [0878] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 4H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [QQ-5]: [0879] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [QQ-4] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [QQ-3] (6 g, 26 mmol) in dichloromethane (50 mL) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired compound tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [QQ-5] (2.1 g, yield 38.9%) as a yellow liquid. [0880] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [QQ-7]: [0881] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [QQ-5] (1.19 g, 1.44 mmol) and 4-(tert-butoxy)-4-oxobutanoic acid [QQ-6] (1 g, 5.74 mmol) in dichloromethane (50 mL) was cooled to 0 oC and EDC. HCl (1.1 g, 5.74 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (177 mg, 1.44 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x50 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 1,2,3- tris[5-(octanoyloxy)pentyl] 2-{[4-(tert-butoxy)-4-oxobutanoyl]oxy}propane-1,2,3-tricarboxylate [QQ-7] (1.0 g, 1.01 mmol, yield 70.72 %) as a pale yellow liquid. [0882] ELSD analysis: Purity 99.59 %, Calculated C53H92O16 = 984.64, Observed = 1002.65 (m/z, M+NH4+). Intermediate [QQ-8]: [0883] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[4-(tert-butoxy)-4- oxobutanoyl]oxy} propane-1,2,3-tricarboxylate [QQ-7] (0.8 g, 812 µmol) in dichloromethane (25 mL, 390 mmol) was added trifluoroacetic acid (741 mg, 6.5 mmol) at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with triethylamine up to pH 7. The resulting organic layer concentrated under reduce pressure to give 4-((18-(((5-(octanoyloxy)pentyl)oxy)carbonyl)-8,16,20,28-tetraoxo-9,15,21,27- tetraoxapentatriacontan-18-yl)oxy)-4-oxobutanoic acid 2,2,2-trifluoroacetic acid [QQ-8] (700 mg, crude), which was used in next step as such. [0884] ELSD analysis: Purity 94.19 %, Calculated C49H84O16 = 928.58, Observed = 951.55 (m/z, M+Na+). TL1-12D-029 (Compound 101): [0885] To a stirred solution of 4-{[1,5-bis({[5-(octanoyloxy)pentyl]oxy})-3-({[5- (octanoyloxy)pentyl] oxy}carbonyl)-1,5-dioxopentan-3-yl]oxy}-4-oxobutanoic acid [QQ-8] (651 mg, 0.7 mmol) and (2-aminoethyl)dimethylamine (80.3 mg, 911 µmol) [QQ-9] in dichloromethane (15 mL, 234 mmol) was cooled to 0 oC and [bis(dimethylamino)methylidene]({3H-[1,2,3]triazolo[4,5- b]pyridin-3-yl})oxidanium (247 mg, 1.05 mmol) was added followed by ethylbis(propan-2-yl)amine (272 mg, 2.1 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). The reaction mixture was concentrated and resulting crude material was purified by Prep HPLC. The pure fraction were combined and concentrated under reduced pressure to obtained 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[2- (dimethylamino)ethyl]carbamoyl} propanoyl)oxy]propane-1,2,3-tricarboxylate 2,2,2- trifluoroacetic acid [TL1-12D-029] (0.3 g, yield 42.86 %) as a Light yellow liquid. [0886] 1H-NMR (400 MHz, CDCl3)- δ 4.12 (t, J = 6.4 Hz, 2H), 4.09-4.03 (m, 10H), 3.64 (bs, 2H), 3.27-3.17 (m, 6H), 2.86 (s, 6H), 2.65 (t, J = 6.4 Hz, 2H), 2.45 (t, J = 6.4 Hz, 2H), 2.28 (t, J = 7.6 Hz, 6H), 1.70-1.58 (m, 18H), 1.44 (m, 6H), 1.31-1.28 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.39 %, Calculated C53H94N2O15 = 998.67, Observed = 999.65 (m/z, M+H+). Example 40C. Synthesis of TL1-12D-016 (Compound 105): Synthetic Scheme QQ
[0887] To a stirred solution of octanoic acid [RR-2] (100.0 g, 693 mmol) and pentane-1,5-diol [RR-1] (65.0 g, 624 mmol) in dichloromethane (2000 mL) was cooled to 0 oC and ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (167.0 g, 874 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (17.1 g, 139 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture, and extract with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give 5-hydroxypentyl octanoate [RR-3] (60.0 g, yield 37.56% Yield) as yellow liquid. [0888] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 5H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [RR-5]: [0889] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [RR-4] (12.5 g, 65.1 mmol) and 5-hydroxypentyl octanoate [RR-3] (60.0 g, 260 mmol) in dichloromethane (500 mL) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (49.9 g, 260 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (8.02 g, 65.1 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [RR-5] (21.0 g, 38.9% yield) as a yellow liquid. [0890] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [RR-7]: [0891] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [RR-5] (10 g, 12.1 mmol) in triethylamine (6.10 g, 60.3 mmol) added 3- bromopropanoyl chloride (1.21 mL, 12.1 mmol) was successively at 0 °C and stirred for 16 h, at same 25 oC temperature. TLC shows SM consumed and formed new spot. The reaction mass was diluted with ethyl acetate, filtered through celite and washed with ethyl acetate to get crude product. The crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired [RR-7] (2.0 g, yield 18.7%) as a yellow liquid. [0892] NMR (400 MHz, CDCl3): δ 6.42 (d, J = 17.2 Hz, 1H), 6.12-6.05 (m, 1H), 5.89 (d, J = 10.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2 H), 4.09-4.03 (m, 10H), 3.34 (d, J = 15.2 Hz, 2H), 3.25 (d, J = 15.6Hz, 2H), 2.28 (t, J = 7.2 Hz, 6 H), 1.70-1.59 (m, 18H), 1.43-1.35 (m, 6H), 1.28 (s, 24H), 0.87 (t, J = 6.8 Hz, 9 H). ELSD analysis: Purity 99.84%, Calculated C48H82O14 = 882.57, Observed = 883.40 (m/z, M+H+). TL1-12D-016 (Compound 105):
[0893] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [RR-7] (350 mg, 396 µmol) in tetrahydrofuran (5 mL, 61.4 mmol) was added 2- [(2-hydroxyethyl)amino]ethan-1-ol [RR-8] (38 µL, 396 µmol). Reaction mass was stirred at RT for 48 h. Progress of the reaction was monitored by TLC. Reaction mass was distilled out under reduced pressure and resulting crude material was purified by Prep HPLC. The pure fraction were combined and concentrated under reduced pressure to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2- ({3-[bis(2-hydroxyethyl)amino]propanoyl}oxy)propane-1,2,3-tricarboxylate [TL1-12D-016] (350 mg, 89.36% Yield ) as a yellow color liquid. [0894] 1H-NMR (400 MHz, CDCl3)- δ 4.17 (t, J = 6.8 Hz, 2H), 4.10-4.04 (m, 10H), 4.02-4.00 (m, 4H), 3.60 (t, J = 7.2 Hz, 2H), 3.36-3.34 (m, 4H), 3.24 (d, J = 15.6 Hz, 2H), 3.15 (d, J = 15.6 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 6.4 Hz, 6H), 1.70-1.58 (m, 18H), 1.44-1.36 (m, 6H), 1.31-1.24 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.73 %, Calculated C52H93NO16 = 987.65, Observed = 988.65 (m/z, M+H+). Example 40D. Synthesis of TL1-12D-015 (Compound 103): Synthetic Scheme RR Intermediate [SS-3]: [0895] To a stirred solution of octanoic acid [SS-2] (100.0 g, 693 mmol) and pentane-1,5-diol [SS-1] (65.0 g, 624 mmol) in dichloromethane (2000 mL) was cooled to 0 oC and ({[3- (dimethylamino)propyl] imino}methylidene)(ethyl)amine hydrochloride (167.0 g, 874 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (17.1 g, 139 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture, and extract with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give 5-hydroxypentyl octanoate [SS-3] (60.0 g, yield 37.56%) as a yellow liquid. [0896] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 5H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [SS-5]: [0897] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [SS-4] (12.5 g, 65.1 mmol) and 5-hydroxypentyl octanoate [SS-3] (60.0 g, 260 mmol) in dichloromethane (500 mL) was cooled to 0 oC, and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (49.9 g, 260 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (8.02 g, 65.1 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3-tricarboxylate [SS-5] (21.0 g, yield 38.9%) as a yellow liquid. [0898] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [SS-7]: [0899] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [SS-5] (10 g, 12.1 mmol) in triethylamine (6.10 g, 60.3 mmol) added 3- bromopropanoyl chloride (1.21 mL, 12.1 mmol) was successively at 0 °C and stirred for 16 h, at 25 oC temperature. TLC showed SM consumed and formation of new spot. The reaction mass was filtered through celite and washed with ethyl acetate to get crude product. The crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired compound [SS-7] (2.0 g, yield 18.7%) as a yellow liquid. [0900] 1H NMR (400 MHz, CDCl3): δ 6.42 (d, J = 17.2 Hz, 1H), 6.12-6.05 (m, 1H), 5.89 (d, J = 10.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2 H), 4.09-4.03 (m, 10H), 3.34 (d, J = 15.2 Hz, 2H), 3.25 (d, J = 15.6Hz, 2H), 2.28 (t, J = 7.2 Hz, 6 H), 1.70-1.59 (m, 18H), 1.43-1.35 (m, 6H), 1.28 (s, 24H), 0.87 (t, J = 6.8 Hz, 9 H). ELSD analysis: Purity 99.84%, Calculated C48H82O14 = 882.57, Observed = 883.40 (m/z, M+H+). TL1-12D-015 (Compound 103): [0901] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [SS-7] (712 mg, 806 µmol) in tetrahydrofuran (20 mL, 246 mmol) was added 1-(methylamino)propan-2-ol [SS-8] (71.9 mg, 806 µmol). Reaction mass was stirred at RT for 48 h. Progress of the reaction was monitored by TLC. Reaction mass was distilled out under reduced pressure and resulting crude material was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexanes), to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-({3-[(2- hydroxypropyl)(methyl)amino] propanoyl}oxy)propane-1,2,3-tricarboxylate [TL1-12D-015] (0.3 g, Yield 38.27%) as a yellow liquid. [0902] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.4 Hz, 2H), 4.09-4.04 (m, 10H), 3.79 (bs, 1H), 3.31-3.19 (m, 4H), 2.89-2.79 (m, 1H), 2.63-2.58 (m, 1H), 2.50-2.44 (m, 2H), 2.30-2.26 (m, 10H), 1.72-1.57 (m, 20H), 1.44-1.38 (m, 6H), 1.29-1.27 (m, 24H), 1.10 (d, J = 6.0 Hz, 3H), 0.87 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.75 %, Calculated C52H93NO15 = 971.65, Observed = 972.60 (m/z, M+H+). Example 40E. Synthesis of TL1-12D-014 (Compound 104): Synthetic Scheme SS Intermediate [TT-3]: [0903] To a stirred solution of pentane-1,5-diol (64 g, 615 mmol) in dichloromethane (0.5 L, 7.81 mol) was added octanoyl chloride (50 g, 307 mmol) and triethylamine (156 g, 1.54 mol). The reaction mixture stirred at r.t. for 24 h. The progress of reaction was monitored by TLC (SM was consumed). Reaction mixture was quenched by cold saturated NaHCO3 upto pH 7 and extracted with DCM (2x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-20 % ethyl acetate in hexane), to afford 5-hydroxypentyl octanoate (45 g, yield 63.5 %) as a pale-yellow liquid. [0904] 1H-NMR (400 MHz, CDCl3)- δ 4.07 (t, J = 6.4 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 2.28 (m, J = 7.6 Hz, 2H), 1.69-1.56 (m, 4H), 1.47-1.41 (m, 2H), 1.39-1.2 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate [TT-5]: [0905] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [TT-4] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [TT-3] (6 g, 26 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give tris(5-(octanoyloxy)pentyl) 2-hydroxypropane- 1,2,3-tricarboxylate [TT-5] (2.1 g, yield 38.9%) as a pale yellow liquid. [0906] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.89-0.85 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [TT-7]: [0907] To a stirred solution of 2-iodoethan-1-ol [TT-6] (5 g, 0.0290 mol) in dichloromethane (100 mL), 1H-imidazole (1.97 g, 0.0290 mol) was added at room temperature. The resulting reaction mixture was cooled to 0 °C and tert-butyl(chloro)dimethylsilane (8.76 g, 0.0580 mol) was added portion wise at same temperature. The reaction mixture was stirred for 16 h at room temperature. Progress of reaction was monitored by ELSD/TLC. Water (50 mL) was added to the reaction mixture and extract with DCM (3x100 mL). The resulting organic layer was dried over anhydrous sodium sulphate and concentrated under reduce pressure to obtain crude product which was purified by flash column chromatography (0-10 % ethyl acetate in n-hexane), to give pure product [TT-7] (5.0 g, yield 60.1 %) as a colorless liquid. [0908] 1H-NMR (400 MHz, CDCl3): δ 3.83 (t, J = 5.6 Hz, 2H), 3.19 (t, J = 6.8 Hz, 2H), 0.90 (s, 9H), 0.10 (s, 6H). Intermediate [TT-10]: [0909] To a stirred solution of cyclobutanamine [TT-8] (5 g, 70.3 mmol) in acetonitrile (250 mL, 4.79 mol), dipotassium carbonate (29.1 g, 211 mmol), and ethyl 3-bromopropanoate [TT-9] (12.7 g, 70.3 mmol) was added at room temperature, and reaction was allow to stir at 60 oC for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, reaction mass was cooled, filter by sintered glass funnel. The filtrate was concentrated under reduced pressure and the crude was purified by flash column chromatography (SiO2: 0-10% Methanol in Dichloromethane), to give ethyl 3-(cyclobutylamino)propanoate [TT-10] (4.5 g, 37.38% Yield) as pale yellow liquid. [0910] 1H-NMR (400 MHz, CDCl3): δ 4.10 (q, J = 7.2 Hz, 2H), 3.26-3.19 (m, 1H), 2.77(t, J = 6.8 Hz, 2H), 2.46 (t, J = 6.8 Hz, 2H), 2.21-2.14 (m, 3H), 1.75-1.63 (m, 3H), 1.24-1.20 (m, 3H). Intermediate [TT-11]: [0911] To a stirred suspension of ethyl 3-(cyclobutylamino)propanoate [TT-10] (4 g, 23.4 mmol) in ethanol (40 mL, 685 mmol), lithium(1+) hydrate hydroxide (1.08 g, 25.7 mmol) was added at room temperature, The reaction mixture was stirred reaction mixture for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, reaction mixture was concentrated under reduced pressure. The crude material was acidified by 1 N HCl up to pH 5 and extracted with ethyl acetate. The resulting organic layer was dried over anhydrous sodium sulphate and concentrated under reduce pressure to obtain 3-(cyclobutylamino)propanoic acid [TT-11] (3.34 g, yield 99.86%), compound was used for next step. [0912] ELSD analysis: Purity 77 %, Calculated C7H13NO2 = 143.09, Observed = 144.25 (m/z, M+H+). Intermediate [TT-12]: [0913] To a stirred solution of 3-(cyclobutylamino)propanoic acid [TT-11] (3.2 g, 22.32 mmol), and potassium hydroxide (2.5 g, 44.72 mmol) in methanol (20 mL, 494 mmol) and water (20 mL, 1110 mmol) was added di-tert-butyl dicarbonate (7.32 g, 33.52 mmol) at room temperature and stirred for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, mixture was concentrated under reduced pressure. The crude material was acidified by 10 % NaHSO4 up to pH 2-3 and extracted with ethyl acetate (3x200 mL). The resulting organic layer was dried over anhydrous sodium sulphate concentrated under reduced pressure to obtained crude product which was purified by flash column chromatography (0-100 % ethyl acetate in n-hexane), to obtain 3-{[(tert-butoxy)carbonyl](cyclobutyl)amino}propanoic acid [TT-12] (4.0 g, yield 73.56 %) as a colorless liquid. [0914] 1H-NMR (400 MHz, CDCl3): δ 4.29 (bs, 1H), 3.51-3.48 (m, 2H), 2.57 (t, J = 7.2 Hz, 2H), 2.17-2.10 (m, 2H), 2.09-1.99 (m, 2H), 1.66-1.54 (m, 2H), 1.45 (s, 9H) ppm. Intermediate [TT-13]: [0915] To a stirred solution of 3-{[(tert-butoxy)carbonyl](cyclobutyl)amino}propanoic acid [TT- 12] (2.11 g, 8.68 mmol) and 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [TT-5] (1.8 g, 2.17 mmol) in dichloromethane (45 mL, 703 mmol) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (1.66 g, 8.68 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (1.07 g, 8.68 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture and extracted with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes) to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert- butoxy)carbonyl](cyclobutyl)amino}propanoyl)oxy]propane-1,2,3-tricarboxylate [TT-14] (1.9 g, yield 83%) as pale yellow liquid. [0916] ELSD analysis: Purity 97.56 %, Calculated C57H99NO16 = 1053.70, Observed = 1076.65 (m/z, M+Na+). Intermediate [TT-14]: [0917] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{[(tert-butoxy)carbonyl] (cyclobutyl)amino}propanoyl)oxy]propane-1,2,3-tricarboxylate [TT-13] (1.9 g, 1.8 mmol) in dichloromethane (20 mL, 312 mmol), trifluoroacetic acid (1.38 mL, 10 eq., 18 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 6 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). After completion the reaction, mixture was concentrated under reduced pressure. The crude material was quenched by NaHCO3 upto pH 8 and extracted with ethyl acetate (3x50 mL). The resulting organic layer was dried over anhydrous sodium sulphate, concentrated under reduced pressure to obtain 1,2,3-tris[5- (octanoyloxy)pentyl] 2-{[3-(cyclobutylamino)propanoyl]oxy}propane-1,2,3-tricarboxylate [TT-14] (850 mg, yield 49.43 %) as a yellow liquid, which was used in next step as such. [0918] ELSD analysis: Purity 99.45 %, Calculated C52H91NO14 = 953.64, Observed = 954.60 (m/z, M+H+). Intermediate [TT-15]: [0919] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3- (cyclobutylamino)propanoyl] oxy}propane-1,2,3-tricarboxylate [TT-14] (750 mg, 786 µmol) in acetonitrile (15 mL), dipotassium carbonate (543 mg, 3.93 mmol), and tert-butyl(2- iodoethoxy)dimethylsilane [TT-7] (270 mg, 943 µmol) was added at room temperature. The reaction mixture was stirred at 60 oC for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, reaction mass was cooled, filter by sintered glass funnel. The filtrate was concentrated under reduced pressure and the crude was purified by flash column chromatography (SiO2: 0-10% Methanol in Dichloromethane), to give 1,2,3-tris[5- (octanoyloxy)pentyl] 2-{[3-({2-[(tert-butyldimethylsilyl)oxy]ethyl}(cyclobutyl)amino)propanoyl] oxy}propane-1,2,3-tricarboxylate [TT-15] ( 290 g, 33.16% Yield) as colorless liquid. [0920] ELSD analysis: Purity 76.81 %, Calculated C60H109NO15Si = 1111.76, Observed = 1112.75 (m/z, M+H+). [TL1-12D-014] (Compound 104):
[0921] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-({2-[(tert- butyldimethylsilyl)oxy]ethyl} (cyclobutyl)amino)propanoyl]oxy}propane-1,2,3-tricarboxylate [TT- 15] (330 mg, 297 µmol) in tetrahydrofuran (4 mL, 49.1 mmol) was added pyridine hydrofluoride (0.2 mL, 1.48 mmol) at 0°C. The resulting reaction mixture was stirred for 2 h at room temperature. Progress of reaction mass was monitored by ELSD/TLC (SM was consumed). The resulting reaction mixture was quenched with cold aqueous sodium bicarbonate solution (20 mL) and extract with ethyl acetate (3x25 mL). The resulting organic layer was dried over Na2SO4 and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10 % methanol in dichloromethane), to obtain desired produced [TL1-12D-014] (86 mg, yield 29.04 %) as a yellow color liquid. [0922] 1H-NMR (400 MHz, CDCl3)- δ 4.16 (t, J = 6.8 Hz, 2H), 4.09-4.04 (m, 10H), 3.54-3.52 (m, 2H), 3.30 (d, J = 15.6, 2H), 3.22 (d, J = 15.6, 2H), 3.14-3.06 (m, 1H), 2.77-2.74 (m, 2H), 2.52-2.49 (m, 2H), 2.45-2.42 (m, 2H), 2.28 (t, J = 7.6 Hz, 6H), 2.05-1.98 (m, 2H), 1.91-1.82 (m, 2H), 1.71 (m, 20H), 1.44-1.37 (m, 6H), 1.32-1.24 (m, 24H), 0.87 (t, J = 6.4 Hz, 9H) ppm. ELSD analysis: Purity 98.79 %, Calculated C54H95NO15 = 997.67, Observed = 998.55 (m/z, M+H+). Example 40F. Synthesis of TL1-12D-013 (Compound 108): Synthetic Protocol TT
[0923] To a stirred solution of cyclopropanamine [UU-1] (1 g, 17.5 mmol), and 2-iodoethan-1-ol [UU-2] (3.01 g, 17.5 mmol) in acetonitrile (20 mL, 383 mmol) at 60 oC for 16 h. The progress of reaction was monitored by TLC. After completion the reaction, reaction mass was filtered by sintered funnel. The filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography (0-20 % MeOH/Dichloromethane), to afford 2- (cyclopropylamino)ethan-1-ol [UU-3] (1.6 g, yield 90 %) as a pale yellow liquid. [0924] 1H-NMR (400 MHz, CDCl3): δ 4.08 (t, J = 5.2 Hz, 2H), 3.35 (br, 2H), 2.75 (br, 1H), 0.99-0.95 (m, 4H). Intermediate [UU-6]: [0925] To a stirred solution of pentane-1,5-diol [UU-4] (64 g, 615 mmol) in dichloromethane (0.5 L, 7.81 mol), was added octanoyl chloride [UU-5] (50 g, 307 mmol) and triethylamine (156 g, 1.54 mol). The reaction mixture stirred at r.t. for 24 h. The progress of reaction was monitored by TLC (SM was consumed). Reaction mixture was quenched by cold saturated NaHCO3 up to pH7 and extracted with DCM (2x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-20 % ethyl acetate in hexane), to afford 5-hydroxypentyl octanoate [UU-6] (45 g, 63.5 % Yield) as a pale-yellow liquid. [0926] 1H-NMR (400 MHz, CDCl3)- δ 4.07 (t, J = 6.4 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 2.28 (m, J = 7.6 Hz, 2H), 1.69-1.56 (m, 4H), 1.47-1.41 (m, 2H), 1.39-1.2 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate [UU-8]: [0927] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [UU-7] (1.25 g, 6.51 mmol) and 5-hydroxypentyl octanoate [UU-6] (6 g, 26 mmol) in dichloromethane (50 mL, 781 mmol) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (4.99 g, 26 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (802 mg, 6.51 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired tris(5-(octanoyloxy)pentyl) 2- hydroxypropane-1,2,3-tricarboxylate [UU-8] (2.1 g, 38.9% yield) as pale yellow liquid. [0928] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.89-0.85 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [UU-10]: [0929] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [UU-8] (5 g, 6.05 mmol) in triethylamine (3.05 g, 18.15 mmol) was added 3- bromopropanoyl chloride [UU-9] (605 µL, 6.05 mmol) was successively at 0 °C and stirred for 16h at same 25 oC temperature, TLC shows SM consumed and formed new spot. The reaction mass was diluted, filtered through celite and washed with ethyl acetate to get crude product. The crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give tris(5-(octanoyloxy)pentyl) 2-(acryloyloxy)propane-1,2,3-tricarboxylate [UU-10] (1.0 g, yield 18.7%) as yellow liquid. [0930] 1H NMR (400 MHz, CDCl3): δ 6.44-6.40 (d, J = 17.2 Hz, 1H), 6.12-6.05 (dd, J = 10.4 Hz, J = 6.8 Hz, 1H), 5.98-5.88 (d, J = 10.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2 H), 4.09-4.03 (m, 10 H), 3.36-3.23 (dd, J = 15.2 Hz, J = 15.6Hz, 4H), 2.30-2.26 (t, J = 7.2 Hz, 6 H), 1.70-1.55 (m, 16H), 1.43-1.35 (m, 6H), 1.28 (s, 26H), 0.87 (t, J = 6.8 Hz, 9 H). TL1-12D-013 (Compound 108): [0931] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [UU-10] (350 mg, 396 µmol) in tetrahydrofuran (2 mL, 24.6 mmol), was added 2-(cyclopropylamino)ethan-1-ol [UU-3] (60.1 mg, 594 µmol). Reaction mass was stirred at RT for 48 h. Progress of the reaction was monitored by TLC. After completion of reaction, concentrated the reaction mixture under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexanes), to give 1,2,3-tris[5- (octanoyloxy)pentyl] 2-({3-[cyclopropyl(2-hydroxyethyl)amino]propanoyl}oxy)propane-1,2,3- tricarboxylate [TL1-12D-013] (0.11g, 28.4 % yield) as a colorless liquid. [0932] 1H-NMR (400 MHz, CDCl3)- δ 4.18 (t, J = 6.4 Hz, 2H), 4.09-4.04 (m, 10H), 3.58 (s, 2H), 3.30 (d, J = 15.6 Hz, 2H), 3.20 (d, J = 15.6 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.73 (t, J = 5.2 Hz, 2H), 2.56 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 7.6 Hz, 6H), 1.72-1.59 (m, 20H), 1.45-1.36 (m, 6H), 1.29-1.27 (m, 23H), 0.87 (t, J = 6.8 Hz, 9H), 0.51-0.42 (m, 4H). ELSD analysis: Purity 99.71 %, Calculated C53H93NO15 = 983.65, Observed = 984.50 (m/z, M+H+). Example 40G. Synthesis of TL1-12D-018 (Compound 109): Synthetic Protocol UU [0933] To a stirrer solution of tert-butyl N-(2-aminoethyl)carbamate [VV-1] (2 g, 12.5 mmol) in tetrahydrofuran (0.4 L, 4.91 mol) added triethylamine (1.75 mL, 12.5 mmol) and iodomethane (1.77 g, 12.5 mmol) dropwise. reaction mass was stirrer for 48 h at RT. Reaction progress was monitored by TLC. As starting material consumed, reaction mass was distilling out under reduced pressure. The resulting crude was purified with column chromatography using 0-5% methanol in DCM, to give tert-butyl N-[2-(methylamino)ethyl]carbamate [VV-2] (0.8 g, 30 % Yield) as a brown color semi solid. [0934] 1H NMR (400 MHz, CDCl3): δ 3.70-3.56 (m,2H), 2.76 (s, 3H), 1.45 (s, 9H), 1.28-1.23 (m, 2H). ELSD Purity - 81.71% Intermediate [VV-5]: [0935] To a stirred solution of pentane-1,5-diol [VV-3] (50 g, 4.9 eq., 480 mmol) in dichloromethane (205 mL, 3.2 mol), was added triethylamine (69.1 mL, 5 eq., 492 mmol) and N,N- dimethylpyridin-4-amine (6.01 g, 0.5 eq., 49.2 mmol) at RT. Reaction mass cooled to 0-5°C, then dropwise added octanoyl chloride [VV-4] (16 g, 98.4 mmol). Reaction mixture allowed to stir at RT for 16 h. Progress of reaction was monitored by TLC. Reaction mixture was diluted with water (2.0 Lit) and extracted with ethyl acetate (200 ml). Organic layer was washed with brine solution (500.0 mL), dried over anhydrous sodium sulphate, filtered and concentrate under reduced pressure. Crude was purified over silica gel with column chromatography using 5-10% Ethyl acetate in hexanes to give 5-hydroxypentyl octanoate [VV-5] (16 g, 70.6 % yield) as light yellow colour liquid. [0936] 1H NMR (400 MHz, CDCl3): δ 4.07 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.69-1.58 (m, 8H), 1.44 (m, 2H), 1.29 (s, 10H), 0.87 (t, J = 7.2 Hz, 3H). Intermediate [VV-7]: [0937] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [VV-6] (2.5 g, 13 mmol) in dichloromethane (50 mL, 781 mmol), was added N,N-dimethylpyridin-4-amine (1.59 g, 13 mmol) and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (9.98 g, 4 eq., 52 mmol) and stirred for 10 min at RT, then added 5-hydroxypentyl octanoate [VV-5] (10.5 g, 3.5 eq., 45.5 mmol). Reaction mixture was stirred for 16 h at RT under inert atmosphere. Reaction progress was monitored by TLC, after consumption of starting material reaction mass diluted with water (100.0 mL) and extracted with dichloromethane (2x 100.0 mL). Organic layer was separated, dried over sodium sulphate, distil out under reduced pressure. Crude purified with flash chromatography using 10-15% ethyl acetate in hexane to give 1,2,3-tris[5- (octanoyloxy)pentyl] 2-hydroxypropane-1,2,3-tricarboxylate [VV-7] (3.5 g, 4.22 mmol) as a light yellow color liquid. [0938] 1H NMR (400 MHz, CDCl3): δ 4.22 (t, J = 6.4 Hz, 2H), 4.09 (m, 10H), 2.89-2.77 (dd, J = 15.6 Hz, 4H), 2.28 (t, J = 7.6 Hz, 6H), 1.64 (m, 18H), 1.43 (m, 6H), 1.29 (m, 24H), 0.87 (7, J = 7.2 Hz, 9H). Intermediate [VV-9]: [0939] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [VV-7] (1 g, 1.21 mmol) in triethylamine (610 mg, 5 eq., 6.03 mmol), was added 3- bromopropanoyl chloride [VV-8] (121 µL, 1.21 mmol) 0 °C and stirred for 16 h at 25 oC temperature. Progress of TLC shows SM consumed and formed new spot. The reaction mass was filtered through celite and washed with ethyl acetate to get crude product. crude was purified by using flash column under 15-25% ethyl acetate in hexane to give 1,2,3-tris[5-(octanoyloxy) pentyl] 2-(prop-2-enoyloxy)propane-1,2,3-tricarboxylate [VV-9] (0.20 g, 18.78 % yield). [0940] ELSD: Purity 99.84 %, Calculated C48H82O14 = 882.57, Observed = 883.40 (m/z, M+H+). Intermediate [VV-10]: [0941] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate (850 mg, 962 µmol) in tetrahydrofuran (20.6 mL, 254 mmol). Added tert-butyl N-[2-(methylamino) ethyl] carbamate (335 mg, 2 eq., 1.92 mmol). Reaction mass was stirred at RT. Progress of the reaction was monitored by TLC. Reaction mass was distilling out under reduced pressure, get crude. The resulting crude was purified with column chromatography using 5% methanol in Dichloromethane, to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-({3-[(2-{[(tert- butoxy)carbonyl]amino}ethyl)amino]propanoyl}oxy)propane-1,2,3-tricarboxylate (200 mg, 189 µmol) as a yellow color liquid. [0942] ELSD: Purity 99.15 %, Calculated C56H100N2O16 = 1056.71, Observed = 1057.71 (m/z, M+H+). TL1-12D-018 (Compound 109): [0943] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-({3-[(2-{[(tert- butoxy)carbonyl]amino}ethyl)(methyl)amino]propanoyl}oxy)propane-1,2,3-tricarboxylate [VV-10] (450 mg, 426 µmol) in dichloromethane (10 mL, 156 mmol), was added triflororaceticacid (97 mg, 2 eq., 851 µmol) at 0 °C. Reaction mass allowed to stir at RT for 2 h. Progress of the reaction was monitored by TLC and ELSD. SM was consumed. Reaction mass was lyophilized to obtain 1,2,3- tris[5-(octanoyloxy)pentyl] 2-({3-[(2-aminoethyl)(methyl)amino]propanoyl}oxy)propane-1,2,3- tricarboxylate [TL1-12D-018] (250 mg, 261 µmol) as brown liquid. [0944] 4.18 (t, J = 6.4 Hz, 2H), 4.09-4.04 (q, J = 9.2 Hz, 10H), 3.62- 3.50 (d, J = 8.0 Hz, 4H), 3.45 (t, J = 6.4 Hz, 2H), 3.25-3.14 (dd, J = 15.6 Hz, J = 15.6 Hz, 4H), 2.88 (m, 4H), 2.30 (t, J = 7.6 Hz.6H), 1.73-1.55 (m, 18 H), 1.45-1.36 (m, 6H), 1.32-1.25 (brs, 24H), 0.87 (t, J = 6.4 Hz, 9H). ELSD Purity 97.62 %, Calculated C51H92N2O14.C2HF3O2 = 957.65, Observed = 957.50 (m/z, M+H+). Example 40H. Synthesis of JC-TL1-16D-E4-10 (Compound 129) Synthetic Protocol VV Intermediate [WW-3]: [0945] To a stirred solution of butane-1,4-diol [WW-1] (10 g, 0.111 mol) and undecanoic acid [WW-2] (20.6 g, 0.111 mol) in dichloromethane (200 mL) was cooled to 0 oC, EDC.HCl (25.5 g, 0.133 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (1.35 g, 0.011 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (200 mL) was added to the reaction mixture and extract with DCM (3x300 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure to obtained crude product which was purified by flash column chromatography (0-20 % ethyl acetate in n-hexane) to obtained pure product 4-hydroxybutyl undecanoate [WW-3] (6.0 g, 21 % Yield) as a pale-yellow liquid. [0946] 1H NMR (400 MHz, CDCl3): δ 4.10 (t, J= 6.4 Hz, 2H), 3.67 (br, 2H), 2.30-2.26 (m, 2H), 1.75- 1.68 (m, 2H), 1.66-1.57 (m, 4H), 1.44-1.25 (m, 14H), 0.88 (t, J= 7.2 Hz, 3H). Intermediate [WW-5]:
[0947] To a stirred solution of 4-hydroxybutyl undecanoate [WW-3] (5.37 g, 0.0208 mol) and Citric acid [WW-4] (1.0 g, 0.0052 mol) in dichloromethane (150 mL) was cooled to 0 oC, EDC.HCl (4.0 g, 0.0208 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.635 g, 0.0052 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (100 mL) was added to the reaction mixture and extract with DCM (3x200 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure to obtained crude product which was purified by flash column chromatography (0-30 % ethyl acetate in n-hexane) to obtained pure product tris(4-(undecanoyloxy)butyl) 2-hydroxypropane- 1,2,3-tricarboxylate [WW-5] (1.77 g, 37 % Yield) as a yellow liquid. [0948] ELSD analysis: Purity 96.78 %, Calculated C51H92O13 = 912.65, Observed = 935.35 (m/z, M+Na+). JC-TL1-16D-E4-10 (Compound 129): [0949] To a stirred solution of tris(4-(undecanoyloxy)butyl) 2-hydroxypropane-1,2,3- tricarboxylate [WW-5] (0.91 g, 0.0010 mol) and 3-(dimethylamino)propanoic acid [WW-6] (0.466 g, 0.004 mol) in dichloromethane (15 mL) was cooled to 0 oC, EDC.HCl (0.765 g, 0.004 mol), was added followed by 4-(dimethylamino)pyridin-1-ium (0.122 g, 0.0010 mol) and stirred for 48 h at room temperature. The progress of reaction was monitored by TLC (SM was consumed). Water (50 mL) was added to the reaction mixture and extract with DCM (3x25 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure to obtained crude product which was purified by flash column chromatography using (0-100 % ethyl acetate in n-hexane) to obtained pure product tris(4-(undecanoyloxy)butyl) 2-((3- (dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate [JC-TL1-12D-E4-10] (0.385 g, 38.5 %, Yield) as a brown liquid. [0950] Results: 1H NMR (400 MHz, CDCl3): δ 4.18 (t, J= 6.0 Hz, 2H), 4.10-4.08 (m, 10H), 3.30 (d, J= 15.6 Hz, 2H), 3.23 (d, J= 15.6 Hz, 2H), 2.59-2.54 (m, 2H), 2.51-2.49 (m, 2H), 2.28 (t, J= 7.6 Hz, 6H), 2.21 (s, 6H), 1.73-1.67 (m, 12H), 1.62-1.57 (m, 8H), 1.32-1.25 (m, 40H), 0.89-0.85 (m, 9H). ELSD analysis: Purity 99.23 %, Calculated C56H101NO14 = 1011.72, Observed = 1012.35 (m/z, M+H+). Example 40I. Synthesis of JC-24-TL-001 (Compound 118) Synthetic Protocol WW [0951] To a solution of citric acid [XX-2] (25.0 g, 0.130 mol) and octan-1-ol [XX-1] (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol,) was added, and the resulting mixture was stirred at 100 oC for 24 h. The reaction progress was monitored by TLC (5% Ethyl acetate/hexanes). The reaction mixture was cooled to RT, diluted with water (500 mL) and extracted with ethyl acetate (500 mL x 3). The organic layer dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [XX-3] (40.0 g, Yield- 58.18 %) as a colourless liquid. [0952] 1H NMR (400 MHz, CDCl3): δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 97.38 %, Calculated C30H56O7 = 528.77, Observed = 529.2 (m/z, M+H+). Intermediate [XX-5]: [0953] To a stirred solution of 6-bromohexanoic acid [XX-4] (4.0 g, 4 eq., 20.5 mmol) in dichloromethane (50 mL), N,N-dimethyl-4-pyridylamine (2.5 g, 20.5 mmol, 4 eq.,) and EDC.HCl (3.93 g, 20.5 mmol, 4 eq.,) were successively added at room temperature and allowed to stir. After 10 minutes trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [XX-3] (2.64 g, 5.13 mmol) was added at room temperature and allowed to stir for 48h. Reaction progress was monitored by TLC/ELSD. SM was consumed. The resulting reaction mixture was quenched with water (100 mL) and extracted with dichloromethane (2x30 mL). All organic were combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography using 10-20% ethyl acetate in n-heptane. The pure fraction were combined and concentrated under reduced pressure to obtain trioctyl 2-((6-bromohexanoyl)oxy)propane-1,2,3- tricarboxylate [XX-5] (3.1 g, 4.39 mmol, yield 85.67%) as a colourless liquid. [0954] ELSD: Purity 90.57 %, Calculated Mass for C36H65BrO8 = 704.38, Observed = 705.30 (m/z, M+H+). JC-24-TL-001 (Compound 118): [0955] To a stirred solution of trioctyl 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [XX-5] (1.0 g, 1.42 mole, 1.0 eq.) in tetrahydrofuran (20 mL, 246 mmol), N,N-dimethylamine 2M solution [XX-6] (1.77 ml, 3.54 mole, 2.5 eq.) was successively added at room temperature. The resulting reaction mixture was stirred at 55°C temperature for 8 hrs. Progress of reaction was monitored by ELSD. ELSD data shows formation of desired compound. Reaction mixture was quenched over water- 100 ml. Stirred and separated the bottom organic layer. Removed the solvent under reduced pressure and resulting crude was purified over silica using 5-10%MeOH in DCM to get desired product as the HBR salt. The compound was dissolved in 30ml dry dichloromethane, followed by addition of Potassium carbonate (2.0 eq) to solution. The mixture was stirred for 90 minutes, then Filtered through 0.2 mm membrane filter. Recovered solvent under reduced pressure to get desired trioctyl 2-((6-(dimethylamino)hexanoyl)oxy)propane-1,2,3- tricarboxylate [JC-24-TL-001] (0.330 g, Yield- 35.10%) as a yellow liquid. [0956] 1H NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.8 Hz, 2H), 4.07- 4.04 (m, 4H), 3.29 - 3.25 (d, J = 15.4 Hz, 2H), 3.20 – 3.16 (d, J = 15.4 Hz, 2H), 2.57 (m, 2H), 2.47 (s, 6H), 2.32 (t, J = 7.32 Hz, 2H), 1.70-1.54 (m, 10H), 1.38 - 1.26 (m, 32H), 0.87 (t, J = 6.52 Hz, 9H). ELSD: Purity 99.70 %, Calculated Mass for C38H71NO8 = 669.52, Observed = 670.80 (m/z, M+H+). CAD Purity: 96.50 %. Example 40J. Synthesis of JC-24-TL-002 (Compound 119) Synthetic Protocol XX Intermediate [ZZ-3]: [0957] To a solution of citric acid [ZZ-2] (25.0 g, 0.130 mol) and octan-1-ol [ZZ-1] (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol) was added, and the resulting mixture was stirred at 100 oC for 24 h. TLC (5% Ethyl acetate/hexanes) monitored the reaction progress. The reaction mixture was cooled to RT, diluted with water (500 mL), and extracted with ethyl acetate (500 mL x 3). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [ZZ-3] (40.0 g, Yield- 58.18 %) as a colourless liquid. [0958] 1H NMR (400 MHz, CDCl3): δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 97.38 %, Calculated C30H56O7 = 528.77, Observed = 529.2 (m/z, M+H+). Intermediate [ZZ-5]: [0959] To a stirred solution of trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [ZZ-3] (1g, 104 µmol) in dichloromethane (40 mL, 625 mmol) and Triethylamine (3.16 µL, 3 eq., 22.7 mmol) was added 4-bromobutyryl chloride [ZZ-4] (2.81 g, 2 eq., 15.1 mmol) at 0 °C under nitrogen atmosphere. The resulting reaction mixture was allowed to stir at room temperature for 16 h. The progress of reaction was monitored by TLC & ELSD (SM was consumed). After completion, reaction mass was extracted with DCM (2x 200ml). The combined organic layer were dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude. The crude was then purified over silica (0-20% EtOAC/Hexane) to get the desired product trioctyl 2-((4- bromobutanoyl)oxy)propane-1,2,3-tricarboxylate [ZZ-5] (1.0 g, Yield-78%) as colourless liquid. [0960] 1H NMR (400 MHz, CDCl3): δ 4.20 – 4.13(m, 2H), 4.11 – 4.09 (m, 6H), 3.36 – 3.33 (d, J = 15.20 Hz, 2H), 3.27 – 3.23 (d, J = 15.20 Hz, 2H), 2.54 (t, J = 7.2 Hz, 2H), 1.67- 1.61 (m, 6H), 1.29 (m, 32H), 0.90 (t, J = 6.8 Hz, 9H). JC-24- TL-002 (Compound 119): [0961] To a stirred solution of trioctyl 2-((4-bromobutanoyl)oxy)propane-1,2,3-tricarboxylate [ZZ-5] (1 g, 1.48 mmol) in tetrahydrofuran (20.4 mL, 251 mmol), pyrrolidine (262 mg, 2.5 eq., 3.69 mmol) was added at room temperature. After 10 min, the resulting reaction mixture allowed to stir at 55°C temperature for 16 h. The progress of reaction was monitor by ELSD. Reaction mixture was concentrated at high vacuum and purified by column chromatography by using (5-10%MeOH in DCM) to get trioctyl 2-((4-(pyrrolidin-1-yl)butanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL- 002] (150 mg, yield- 15.22%) as a yellow liquid. [0962] 1H NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.76 Hz, 2H), 4.10 - 4.01 (m, 4H), 3.27 – 3.25 (d, J = 15.36 Hz, 2H), 3.18 – 3.14 (d, J = 15.36 Hz, 2H), 2.94 (brs, 4H), 2.42 (t, J = 6.56 Hz, 2H), 2.10-2.02 (m, 6H), 1.65-1.58 (m, 6H), 1.35-1.2 (m, 32H), 0.87 (t, J = 6.56 Hz, 9H). ELSD analysis: Purity 99.66 %, Calculated C38H69NO8= 667.50, Observed = 668.50 (m/z, M+H+). CAD purity: 96.14 %. Example 40K. Synthesis of JC-24-TL-003 (Compound 120) Synthetic Protocol ZZ
Intermediate [AB-3]: [0963] To a solution of citric acid [AB-2] (25.0 g, 0.130 mol) and octan-1-ol [AB-1] (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol) was added, and the resulting mixture was stirred at 100 oC for 24 h. TLC (5% Ethyl acetate/hexanes) monitored the reaction progress. The reaction mixture was cool to RT, diluted with water (500 mL) and extracted with ethyl acetate (500 mL x 3). The organic layer dried over anhydrous sodium sulphate and evaporated under reduced pressure. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [AB-3] (40.0 g, Yield- 58.18 %) as a colourless liquid. [0964] δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 97.38 %, Calculated C30H56O7 = 528.77, Observed = 529.2 (m/z, M+H+). Intermediate [AB-5]: [0965] To the stirred solution of 5-bromovaleric acid (4 g, 4 eq., 22.1 mmol) in dichloromethane (60 mL, 937 mmol), were added N,N-dimethyl-4-pyridylamine (1.35 g, 2 eq., 11 mmol) and EDC.HCl (4.24 g, 4 eq., 22.1 mmol) at room temperature under nitrogen atmosphere. After 10 minutes, trioctyl 2-hydroxy-1,2,3-propanetricarboxylate (2.92 g, 5.52 mmol) was added to the resulting reaction mass and allowed to stir at room temperature for 48 h. The progress of reaction was monitor by TLC. The reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2x 30 mL). The combined organic layer was and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by silica gel flash column chromatography using 10-20% ethyl acetate in n-heptane. The pure fraction were combined and concentrated under reduced pressure to obtain trioctyl 2-((5- bromopentanoyl)oxy)propane-1,2,3-tricarboxylate [AB-5] (2.2 g, Yield 57.57% ) as a colourless liquid. [0966] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.8 Hz, 2H), 4.09- 4.06 (m, 4H), 3.32 - 3.28 (d, J = 15.2 Hz, 2H), 3.23 – 3.19 (d, J = 15.6 Hz, 2H), 2.37 (t, J = 7.6 Hz, 2H), 1.93- 1.89 (m, 2H), 1.88-1.75 (m, 2H), 1.67-1.57 (m, 8H), 1.33 - 1.28 (m, 30H), 0.87 (t, J = 7.2 Hz, 9H). ELSD analysis: Purity 91.45 %, Calculated C35H63BrO8= 690.37, Observed = 691.25 (m/z, M+H+). JC-24- TL-003 (Compound 120): [0967] To a stirred solution of trioctyl 2-((5-bromopentanoyl)oxy)propane-1,2,3-tricarboxylate [AB-5] (1 g, 1.45 mmol) in tetrahydrofuran (20 mL, 246 mmol), pyrrolidine [AB-6] (257 mg, 2.5 eq., 3.61 mmol) was added at room temperature and stirred for 10 minute. The resulting reaction mixture was allowed to stir at 55 °C temperature for 8 h. Reaction progress was monitored by ELSD. Reaction mixture was concentrated under reduced pressure and purified by silica gel flash column chromatography using 5-10% MeOH in DCM to afford HBR salt of desired product. Obtained product salt taken in dry dichloromethane (3 ml), and added Potassium carbonate (2.0 eq) to the resulting solution and stirred for 90 minute. The resulting solution filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to get trioctyl 2-((5- (pyrrolidin-1-yl)pentanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL-003] (0.250 g, yield- 25.35%) as a yellow liquid. [0968] 1H NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.8 Hz, 2H), 4.07- 4.04 (m, 4H), 3.29 - 3.25 (d, J = 15.4 Hz, 2H), 3.20 – 3.16 (d, J = 15.4 Hz, 2H), 2.73 (brs, 4H), 2.35 (t, J = 7.12 Hz, 2H), 1.94 (brs, 4H), 1.79-1.57 (m, 10H), 1.40 - 1.16 (m, 32H), 0.87 (t, J = 7.04 Hz, 9H). ELSD analysis: Purity 99.62 %, Calculated C39H71NO8= 681.52, Observed = 682.45 (m/z, M+H+). CAD Purity: 97.94 %. Example 40L. Synthesis of JC-24-TL-004 (Compound 121) Synthetic Protocol AB [0969] To a solution of citric acid [AC-2] (25.0 g, 0.130 mol) and octan-1-ol [AC-1] (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol,) was added, and the resulting mixture was stirred at 100 oC for 24 h. The reaction progress was monitored by TLC (5% Ethyl acetate/hexanes). The reaction mixture was cooled to RT, diluted with water (500 mL) and extracted with ethyl acetate (500 mL x 3). The organic layer dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [AC-3] (40.0 g, Yield- 58.18 %) as a colourless liquid. [0970] 1H NMR (400 MHz, CDCl3): δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 97.38 %, Calculated C30H56O7 = 528.77, Observed = 529.2 (m/z, M+H+). Intermediate [AC-5]: [0971] To a stirred solution of 6-bromohexanoic acid [AC-4] (4.0 g, 4 eq., 20.5 mmol) in dichloromethane (50 mL), N,N-dimethyl-4-pyridylamine (2.5 g, 20.5 mmol, 4 eq.,) and EDC.HCl (3.93 g, 20.5 mmol, 4 eq.,) were added successively at room temperature. After 10 minutes, trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [AC-3] (2.64 g, 5.13 mmol) was added at room temperature and the resulting reaction mixture was stirred at room temperature for 48 h. Reaction progress was monitor by TLC/ELSD until consumption of the starting material. The resulting reaction mixture was quenched with water (100 mL) and extracted with dichloromethane (2x30 mL). All organic were combined and dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by column chromatography using 10-20% ethyl acetate in n-heptane. The pure fraction were combined and concentrated under reduced pressure to obtain trioctyl 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [AC-5] (3.1 g, 4.39 mmol, yield 85.67%) as a colourless liquid. [0972] ELSD: Purity 90.57 %, Calculated Mass for C36H65BrO8 = 704.38, Observed = 705.30 (m/z, M+H+). JC-24- TL-004 (Compound 121): [0973] To a stir solution of trioctyl 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [AC- 5] (1 g, 1.45 mmol) in tetrahydrofuran (20 mL, 246 mmol), pyrrolidine [AC-6] (257 mg, 2.5 eq., 3.61 mmol) was added at room temperature. After 10 min the resulting reaction mixture was allowed to stir at 55°C temperature for 16 h. The progress of reaction monitored by ELSD. Reaction mixture was concentrated under reduced pressure and purified by silica gel flash column chromatography (5-10%MeOH in DCM) to afford HBR salt of desired product. The product salt was taken up in dry dichloromethane (5 ml), and added Potassium carbonate (2.0 eq) to the resulting solution and stirred for 90 minute. The resulting solution filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to get trioctyl 2-((6-(pyrrolidin- 1-yl)hexanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24- TL-004] (0.110 g, Yield 11.13%) as a yellow liquid. [0974] NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.8 Hz, 2H), 4.08 - 4.03 (m, 4H), 3.29 - 3.25 (d, J = 15.4 Hz, 2H), 3.20 – 3.16 (d, J = 15.4 Hz, 2H), 2.75 (brs, 4H), 2.32 (t, J = 7.28 Hz, 2H), 1.98 (brs, 4H), 1.74 (brs, 2H), 1.66-1.57 (m, 8H), 1.37-1.26 (m, 34H), 0.87 (t, J = 7.0 Hz, 9H). ELSD: Purity 99.95 %, Calculated Mass for C40H73NO8= 695.53, Observed = 696.35 (m/z, M+H+). Cad Purity: 95.21 %. Example 40M. Synthesis of JC-24-TL-005 (Compound 122) Synthetic Protocol AC Intermediate [AD-3]: [0975] To a solution of citric acid [AD-2] (25.0 g, 0.130 mol) and octan-1-ol [AD-1] (67.7 g, 0.52 mol) in toluene (500 mL), PTSA (13.0 g, 0.065 mol,) was added, and the resulting mixture was stirred at 100 oC for 24 h. The reaction progress was monitored by TLC (5% Ethyl acetate/hexanes). The reaction mixture was cooled to RT, diluted with water (500 mL) and extracted with ethyl acetate (500 mL x 3). The organic layer dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude was purified by column chromatography using 60-120 mesh silica and eluted with 5% ethyl acetate in hexane to give trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [AD-3] (40.0 g, Yield- 58.18 %) as a colourless liquid. [0976] 1H NMR (400 MHz, CDCl3): δ 5.61 (s, 1H), 4.01 (t, J = 6.8 Hz, 2H), 3.96 (t, J = 6.4 Hz, 4H), 2.87 (d, J = 15.2 Hz, 2H), 2.72 (d, J = 15.2 Hz, 2H), 1.57-1.49 (m, 6H), 1.24 (brs, 30H), 0.85 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 97.38 %, Calculated C30H56O7 = 528.77, Observed = 529.2 (m/z, M+H+). Intermediate [AD-5]: [0977] To the stirred solution of 7-bromoheptanoic acid [AD-4] (4 g, 4 eq., 19.1 mmol) in dichloromethane (49.6 mL), were added N,N-dimethyl-4-pyridylamine (1.17 g, 2 eq., 9.57 mmol) and EDC.HCl (4.58 g, 5 eq., 23.9 mmol) successively at room temperature. After 10 minute, trioctyl 2-hydroxypropane-1,2,3-tricarboxylate [AD-3] (2.53 g, 4.78 mmol) was then added, and the resulting reaction mixture was stirred at room temperature for 48 h. The progress of reaction was monitor by TLC. The resulting reaction mixture quenched with water (100 mL) and extracted with dichloromethane (2x100 mL). The resulting organic layer dried over Na2SO4 and concentrated under reduced pressure. The resulting crude material was purified by silica gel flash column chromatography using 10-20% ethyl acetate in n-heptane. The pure fraction were combined and concentrated under reduced pressure to obtain trioctyl 2-((7-bromoheptanoyl)oxy)propane- 1,2,3-tricarboxylate [AD-5] (2.3 g, yield 66.81%) as a colourless liquid. [0978] ELSD: Purity 95.50 %, Calculated Mass for C37H67BrO8 = 718.40, Observed = 719.35 (m/z, M+H+). JC-24- TL-005 (Compound 122): [0979] To a stirred solution of trioctyl 2-((7-bromoheptanoyl)oxy)propane-1,2,3-tricarboxylate [AD-5] (1 g, 1.39 mmol) in tetrahydrofuran (2.8 mL, 34.3 mmol), pyrrolidine [AD-6] (247 mg, 2.5 eq., 3.47 mmol) was added at room temperature and stirred for 10 minutes. The resulting reaction mixture allowed to stir at 55 °C temperature for 8 h. Reaction progress was monitor by ELSD. Reaction mixture was concentrated under reduced pressure and purified by silica gel flash column chromatography using 5-10% MeOH in DCM to afford HBR salt of desired product. The product salt was taken up in dry dichloromethane (3 ml), added Potassium carbonate (2.0 eq) to the resulting solution, and stirred for 90 minute. The resulting solution was then filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to get trioctyl 2-((7- (pyrrolidin-1-yl)heptanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL-005] (0.175 g, yield- 17.73%) as a yellow liquid. [0980] 1H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.8 Hz, 2H), 4.07- 4.01 (m, 4H), 3.30 - 3.26 (d, J = 15.4 Hz, 2H), 3.21 – 3.17 (d, J = 15.4 Hz, 2H), 2.92-2.66 (m, 6H), 2.30 (t, J = 7.48 Hz, 2H), 1.93 (brs, 4H), 1.74-1.53 (m, 10H), 1.36-1.20 (m, 34H), 0.87 (t, J = 7.0 Hz, 9H). ELSD: Purity 99.55 %, Calculated Mass for C41H75NO8= 709.55, Observed = 710.40 (m/z, M+H+). CAD purity: 97.96 %. Example 40N. Synthesis of JC-24-TL-006 (Compound 112) Synthetic Protocol AD
[0981] To a stirred solution of 6-bromo-1-hexanol [AE-2] (25 g, 138 mmol) in dichloromethane (237 mL, 3.71 mol) was added triethylamine (48.1 mL, 2.5 eq., 345 mmol). Reaction mass was cooled to 0 °C, and heptanoyl chloride [AE-1] (20.5 g, 138 mmol) was added as drop wise. Reaction mass allowed to stir at RT for 16 h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction mass was diluted with water (500 ml) and was extracted with dichloromethane (100 ml). The combined organic layer washed with aq. sodium bicarbonate solution (1000 ml), dried over sodium sulphate, and concentrated under reduced pressure. The crude was purified over silica using 5-10% ethyl acetate to give product 6-bromohexyl heptanoate [AE-3] (30 g, Yield- 74 %) as colourless liquid. [0982] NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 2.7 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.90-1.82 (m, 2H), 1.67-1.57 (m, 4H), 1.50-1.35 (m, 4H), 1.35-1.32 (m, 6H), 0.87 (t, J = 6.4 Hz, 3H). Intermediate [AE-5]: [0983] To a stirred solution of 6-bromohexyl heptanoate [AE-3] (50.4 g, 3.3 eq., 172 mmol) and 2-hydroxy-1,2,3-propanetricarboxylic acid [AE-4] (10 g, 52 mmol) in dimethylformamide (0.5 L, 6.46 mol) was added TEA (43.9 mL, 6 eq., 312 mmol). The reaction mixture was heated to 50 °C for 16h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the SM, the reaction mixture was diluted with water (500 ml) and extracted with diethyl ether (3x 100 ml). The separated organic layer was dried over Na2SO4 and concentrated under reduce pressure to obtained crude product. Resulting crude was purified over silica using 20% ethyl acetate in n- hexane to obtained tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3-tricarboxylate [AE-5] (28.0 g, Yield- 65 %) as colour less liquid. ELSD analysis: Purity 99.88 %, Calculated C45H80O13 = 828.56, Observed = 829.60 (m/z, M+H+). Intermediate [AE-7]: [0984] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [AE-5] (4 g, 4.82 mmol) and 6-bromohexanoic acid [AE-6] (3.76 g, 4 eq., 19.3 mmol) in dichloromethane (50 mL, 781 mmol) was added EDC.HCl (3.7 g, 4 eq., 19.3 mmol) followed by addition of N,N-dimethyl-4-pyridylamine (1.18 g, 2 eq., 9.65 mmol). Reaction mixture was stirred for 16 h at room temperature, wherein ELSD/TLC confirmed consumption of the SM. Water (500ml) added to the reaction mixture and extracted with DCM (3x100 ml). The separated organic layer was dried over Na2SO4, filtered, and concentrated under reduce pressure. Resulting crude was purified over silica using 5% ethyl acetate in n-hexane to obtained pure product tris(6- (heptanoyloxy)hexyl) 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [AE-7] (2.9 g, yield 57.94%) as colour less liquid. [0985] ELSD analysis: Purity 99.40 %, Calculated C51H89BrO14 = 1004.54, Observed = 1005.50 (m/z, M+H+). JC-24-TL-006 (Compound 112): [0986] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-((6-bromohexanoyl)oxy)propane- 1,2,3-tricarboxylate [AE-7] (1 g, 994 µmol) in tetrahydrofuran (10 mL, 123 mmol) was added dimethylamine (1.24 mL, 2M solution in THF) [AE-8] (112 mg, 2.5 eq., 2.48 mmol). Reaction mass was then heated to 55 °C for 16h, with progress of the reaction being monitored by ELSD/TLC. Reaction mass cooled to room temperature and the concentrated under reduce pressure to obtained crude product. The resulting crude material was purified over silica using flash chromatography and 5% MeOH in DCM to obtain product as HBR salt. The salt was dissolved in dry dichloromethane (3 ml), potassium carbonate (2.0 eq) added to the solution, and allow to stir for 90 minutes. The solution was filtered through 0.2 mm membrane filter. The solvent was removed under reduced pressure to obtain tris(6-(heptanoyloxy)hexyl) 2-((6- (dimethylamino)hexanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL-006] (450 mg, Yield- 46.66%) as light yellow liquid. [0987] NMR (400 MHz, CDCl3): δ 4.14 (t, J = 6.72 Hz, 2H), 4.08 - 4.03 (m, 10H), 3.29 - 3.25 (d, J = 15.48 Hz, 2H), 3.20-3.16 (d, J = 15.48 Hz, 2H), 2.63 (brs, 2H), 2.51 (brs, 4H), 2.34-2.26 (m, 8H), 1.72 – 1.57 (m, 22H), 1.42-1.34 (m, 15 H), 1.33-1.24 (m, 19H), 0.89 (t, J = 7.0 Hz, 9H). ELSD analysis: Purity 99.88 %, Calculated C53H95NO14 = 969.68, Observed = 970.50 (m/z, M+H+). CAD purity = 95.65 %. Example 40O. Synthesis of JC-24-TL-007 (Compound 113) Synthetic Protocol AE
[0988] To a stirred solution of 6-bromo-1-hexanol [AF-2] (25 g, 138 mmol) in dichloromethane (237 mL, 3.71 mol) was added triethylamine (48.1 mL, 2.5 eq., 345 mmol). Reaction mass was cooled to 0 °C, and heptanoyl chloride [AF-1] (20.5 g, 138 mmol) was added as drop wise. Reaction mass allowed to stir at RT for 16 h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction mass was diluted with water (500 ml) and was extracted with dichloromethane (100 ml). The combined organic layer washed with aq. sodium bicarbonate solution (1000 ml), dried over sodium sulphate, and concentrated under reduced pressure. The crude was purified over silica using 5-10% ethyl acetate to give product 6-bromohexyl heptanoate [AF-3] (30 g, Yield- 74 %) as colourless liquid. [0989] NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 2.7 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.90-1.82 (m, 2H), 1.67-1.57 (m, 4H), 1.50-1.35 (m, 4H), 1.35-1.32 (m, 6H), 0.87 (t, J = 6.4 Hz, 3H). Intermediate [AF-5]: [0990] To a stirred solution of 6-bromohexyl heptanoate [AF-3] (50.4 g, 3.3 eq., 172 mmol) and 2-hydroxy-1,2,3-propanetricarboxylic acid [AF-4] (10 g, 52 mmol) in dimethylformamide (0.5 L, 6.46 mol) was added TEA (43.9 mL, 6 eq., 312 mmol). Reaction mas was heated to 50 °C for 16h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the SM, the reaction mixture was diluted with water (500 ml) and extracted with diethyl ether (3x 100 ml). The separated organic layer was dried over Na2SO4 and concentrated under reduce pressure to obtained crude product. Resulting crude was purified over silica using 20% ethyl acetate in n- hexane to obtained tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3-tricarboxylate [AF-5] (28.0 g, Yield- 65 %) as colour less liquid. ELSD analysis: Purity 99.88 %, Calculated C45H80O13 = 828.56, Observed = 829.60 (m/z, M+H+). Intermediate [AF-7]: [0991] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [AF-5] (4 g, 4.82 mmol) and 6-bromohexanoic acid [AF-6] (3.76 g, 4 eq., 19.3 mmol) in dichloromethane (50 mL, 781 mmol) was added EDC.HCl (3.7 g, 4 eq., 19.3 mmol) followed by addition of N,N-dimethyl-4-pyridylamine (1.18 g, 2 eq., 9.65 mmol). Reaction mixture was stirred for 16 h at room temperature, wherein ELSD/TLC confirmed consumption of the SM. Water (500ml) added to the reaction mixture and extracted with DCM (3x100 ml). The separated organic layer was dried over Na2SO4, filtered, and concentrated under reduce pressure. Resulting crude was purified over silica using 5% ethyl acetate in n-hexane to obtained pure product tris(6- (heptanoyloxy)hexyl) 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [AF-7] (2.9 g, yield 57.94%) as colour less liquid. ELSD analysis: Purity 99.40 %, Calculated C51H89BrO14 = 1004.54, Observed = 1005.50 (m/z, M+H+). JC-24-TL-007 (Compound 113): [0992] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-((6-bromohexanoyl)oxy)propane- 1,2,3-tricarboxylate [AF-7] (1 g, 994 µmol) in tetrahydrofuran (10 mL, 123 mmol) was added pyrrolidine [AF-8] (177 mg, 2.5 eq., 2.48 mmol). Reaction mass was then heated to 55 °C for 16h, with progress of the reaction being monitored by ELSD/TLC. Reaction mass was cooled to room temperature and concentrated under reduce pressure to obtained crude product. The resulting crude product was purified over silica using flash chromatography and 5% MeOH in DCM to obtain product as HBR salt. The salt was dissolved in dry dichloromethane (3 ml), potassium carbonate (2.0 eq) added to the solution, and allow to stir for 90 minutes. The solution was filtered through 0.2 mm membrane filter. The solvent was removed under reduced pressure to obtain tris(6-(heptanoyloxy)hexyl) 2-((6-(pyrrolidin-1-yl)hexanoyl)oxy)propane-1,2,3- tricarboxylate [JC-24-TL-007] (330 mg, yield- 33.32%) as light yellow liquid. [0993] NMR (400 MHz, CDCl3): δ 4.13 (t, J = 6.72 Hz, 2H), 4.09 - 4.02 (m, 10H), 3.29 - 3.25 (d, J = 15.48 Hz, 2H), 3.20 – 3.16 (d, J = 15.48 Hz, 2H), 2.71 (brs, 4H), 2.33-2.26 (m, 8H), 1.95 (s, 4H), 1.78-1.56 (m, 24H), 1.43-1.36 (m, 14 H), 1.32-1.23 (m, 18H), 0.89 (t, J = 7.0 Hz, 9H). ELSD analysis: Purity 99.85 %, Calculated C55H97NO14= 995.69, Observed = 996.85 (m/z, M+H+). Cad purity = 93.84 %. Example 40P. Synthesis of JC-24-TL-008 (Compound 114) Synthetic Protocol AF
[0994] To a stirred solution of 6-bromo-1-hexanol [AG-2] (25 g, 138 mmol) in dichloromethane (237 mL, 3.71 mol) was added triethylamine (48.1 mL, 2.5 eq., 345 mmol). Reaction mass was cooled to 0 °C, and heptanoyl chloride [AG-1] (20.5 g, 138 mmol) was added as drop wise. Reaction mass allowed to stir at RT for 16 h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction mass was diluted with water (500 ml) and was extracted with dichloromethane (100 ml). The combined organic layer washed with aq. sodium bicarbonate solution (1000 ml), dried over sodium sulphate, and concentrated under reduced pressure. The crude was purified over silica using 5-10% ethyl acetate to give product 6-bromohexyl heptanoate [AG-3] (30 g, Yield- 74 %) as colourless liquid. [0995] 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 2.7 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.90-1.82 (m, 2H), 1.67-1.57 (m, 4H), 1.50-1.35 (m, 4H), 1.35-1.32 (m, 6H), 0.87 (t, J = 6.4 Hz, 3H). Intermediate [AG-5]: [0996] To a stirred solution of 6-bromohexyl heptanoate [AG-3] (50.4 g, 3.3 eq., 172 mmol) and 2-hydroxy-1,2,3-propanetricarboxylic acid [AG-4] (10 g, 52 mmol) in dimethylformamide (0.5 L, 6.46 mol) was added TEA (43.9 mL, 6 eq., 312 mmol). Reaction mas was heated to 50 °C for 16h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the SM, the reaction mixture was diluted with water (500 ml) and extracted with diethyl ether (3x 100 ml). The separated organic layer was dried over Na2SO4 and concentrated under reduce pressure to obtained crude product. Resulting crude was purified over silica using 20% ethyl acetate in n- hexane to obtained tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3-tricarboxylate [AG-5] (28.0 g, Yield- 65 %) as colour less liquid. ELSD analysis: Purity 99.88 %, Calculated C45H80O13 = 828.56, Observed = 829.60 (m/z, M+H+). Intermediate [AG-7]: [0997] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-hydroxypropane-1,2,3- tricarboxylate [AG-5] (2 g, 2.41 mmol) and 5-bromovaleric acid [AG-6] (1.75 g, 4 eq., 9.65 mmol) in dichloromethane (25 mL, 390 mmol), were added EDC.HCl (1.85 g, 4 eq., 9.65 mmol) followed by N,N-dimethyl-4-pyridylamine (589 mg, 2 eq., 4.82 mmol). Reaction mixture was stirred for 16h at room temperature, wherein ELSD/TLC confirmed consumption of the SM. Reaction mixture diluted with water (500 ml) and extracted with DCM (3x100 ml). The separated organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. Resulting crude product was purified over silica using 5% ethyl acetate in n-hexane to obtained tris(6- (heptanoyloxy)hexyl) 2-((5-bromopentanoyl)oxy)propane-1,2,3-tricarboxylate [AG-7] (720 mg, yield- 30 %) as colourless liquid. ELSD analysis: Purity 99.20 %, Calculated C50H87BrO14= 990.53, Observed = 991.40 (m/z, M+H+). JC-24-TL-008 (Compound 114): [0998] To a stirred solution of tris(6-(heptanoyloxy)hexyl) 2-((5-bromopentanoyl)oxy)propane- 1,2,3-tricarboxylate [AG-7] (720 mg, 726 µmol) in tetrahydrofuran (10 mL, 123 mmol) was added pyrrolidine (103 mg, 2 eq., 1.45 mmol). Reaction mass was then heated to 55 °C for 16h, with progress of the reaction being monitored by ELSD/TLC. Reaction mass was cooled to room temperature and distilled out solvent to get crude product. The resulting crude material was purified over silica using flash chromatography (5% MeOH in DCM) to obtain product as HBR salt. The salt was dissolved in dry dichloromethane (3 ml), potassium carbonate (2.0 eq) added to the solution, and allow to stir for 90 minutes. After this time, the solution was filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to obtain tris(6- (heptanoyloxy)hexyl) 2-((5-(pyrrolidin-1-yl)pentanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL- 008] (220 mg, yield- 30.86 %) as colourless liquid. [0999] 1H-NMR (400 MHz, CDCl3): δ 4.14 (t, J = 6.68 Hz, 2H), 4.09-4.03 (m, 10H), 3.28-3.24 (d, J = 15.44 Hz, 2H), 3.19-3.15 (d, J = 15.44 Hz, 2H), 2.93 (brs, 4H), 2.36 (t, J = 7.0 Hz, 2H), 2.28 (t, J = 7.52 Hz, 6H), 2.05 (s, 4H), 1.69-1.57 (m, 24H), 1.42-1.36 (m, 12 H), 1.29-1.25 (m, 18H), 0.87 (t, J = 7.0 Hz, 9H). ELSD analysis: Purity 99.85 %, Calculated C54H95NO14 = 981.68, Observed = 982.85 (m/z, M+H+). CAD Purity = 93.90 %. Example 40Q. Synthesis of JC-24-TL-009 (Compound 115) Synthetic Protocol AG
[01000] To a stirred solution of 5-bromo-1-pentanol [AH--2] (25 g, 150 mmol) in dichloromethane (257 mL, 4.02 mol) was added triethylamine (52.2 mL, 2.5 eq., 374 mmol). Reaction mass was cooled to 0 °C and octanoyl chloride [AH-1] (25.5 mL, 150 mmol) was added drop wise. Reaction mass allowed to stir at RT for 16 h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction mass was diluted with water (500 ml) and extracted with dichloromethane (2x100 ml). Combined organic layer was washed with sodium bicarbonate solution (1000 ml). Organic layer was dried over sodium sulphate, filtered, and concentrated under reduced pressure. The crude was purified over silica get by using flash chromatography and 5-10% ethyl acetate to give 5-bromopentyl octanoate [AH- 3] (31 g, yield 71.26 %) as colourless liquid. [01001] Note: Two batches of 25.0 g (total 50 g) were performed yielding total 62 g of desired product. 4.07 (t, J = 6.52 Hz, 2H), 3.40 (t, J = 6.72Hz, 2H), 2.31-2.27 (t, J = 2.68 Hz, 2H), 1.92 - 1.85 (m, 2H), 1.69 – 1.59 (m, 4H), 1.54 – 1.43 (m, 2H), 1.34-1.22 (m, 8H), 0.87 (t, J = 6.76 Hz, 3H). Intermediate [AH-5]:
[01003] To a stirred solution of 5-bromopentyl octanoate [AH-3] (61.1 g, 3.8 eq., 187 mmol) and 2-hydroxy-1,2,3-propanetricarboxylic acid [AH-4] (9.6 g, 50 mmol) in dimethylformamide (480 mL, 6.2 mol) was added TEA (69.6 mL, 10 eq., 0.5 mol). The reaction mixture was stirred for 16 h at 60 °C, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction was cooled to RT and then quenched with water (2000 mL) and extracted with diethyl ether (2x 500 mL). The organic layer dried over sodium sulphate, filtered, and concentrated under reduced pressure. Resulting crude was purified over silica using 20% ethyl acetate in n-hexane to obtained tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [AH-5] (21 g, yield-50.69%) as light yellow colour liquid. 4.21 (t, J = 6.68 Hz, 2H), 4.12-4.03 (m, 10H), 2.89- 2.85 (d, J = 15.50 Hz, 2H), 2.80-2.76 (d, J = 15.6 Hz, 2H), 2.30 (t, J = 6.4 Hz, 6H), 1.75 -1.56 (m, 18H), 1.45 -1.37 (m, 6H), 1.31-1.26 (m, 22H), 0.87 (t, J = 9.0 Hz, 9H). Intermediate [AH-7]: [01005] To a stirred solution of 6-bromohexanoic acid [AH-6] (1.32 g, 4 eq., 6.75 mmol) in dichloromethane (17.5 mL, 273 mmol), were added N,N-dimethyl-4-pyridylamine (413 mg, 2 eq., 3.38 mmol) and EDC.HCl (1.62 g, 5 eq., 8.44 mmol) at room temperature and stirred for 10 minute at same temperature. After this time, tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [AH-5] (1.4 g, 1.69 mmol) was added, and the resulting reaction mixture allowed to stir at room temperature for 36h, with progress of reaction being monitor by ELSD. The reaction mixture was quench with water (50 mL) and extracted with dichloromethane (2x 30 mL). The combined organic layer dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude material purified by using silica gel flash column chromatography (5-10% ethyl acetate in heptane). The pure fraction were combined and concentrated under reduced pressure to obtained tris(5-(octanoyloxy)pentyl) 2-((6-bromohexanoyl)oxy)propane-1,2,3-tricarboxylate [AH-7] (0.8 g, yield 47.09%) as a colourless liquid. [01006] ELSD analysis: Purity 99.87 %, Calculated C51H89BrO14= 1006.16, Observed = 1007.50 (m/z, M+H+). JC-24- TL-009 (Compound 115): [01007] To a stirred solution of tris(5-(octanoyloxy)pentyl) 2-((6-bromohexanoyl)oxy)propane- 1,2,3-tricarboxylate [AH-7] (0.8 g, 795 µmol) in tetrahydrofuran (10 mL, 123 mmol) was added pyrrolidine [AH-8] (113 mg, 2 eq., 1.59 mmol) at room temperature. The reaction mixture was heated up to 55°C for 16h, with progress of the reaction being monitored by ELSD/TLC. The reaction mixture was concentrated at 45°C under reduced pressure, and purified by silica gel column chromatography using 2-5% MeOH in DCM to get product as HBr salt. The salt was dissolved in dry dichloromethane (3 ml), potassium carbonate (2.0 eq) added to the solution, and allowed to stir for 90 minutes. The resulting solution was filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to afforded tris(5-(octanoyloxy)pentyl) 2- ((6-(pyrrolidin-1-yl)hexanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL-009] (174 mg, yield 21.96% ) as yellow liquid. [01008] 1H NMR (400 MHz, CDCl3): δ 4.14 (t, J = 6.64 Hz, 2H), 4.08- 4.03 (m, 9H), 3.29 - 3.25 (d, J = 15.52 Hz, 2H), 3.20-3.16 (d, J = 15.52 Hz, 2H), 2.66 (brs, 5H), 2.33-2.26 (m, 8H), 2.92 (brs, 4H), 1.71-1.58 (m, 22H), 1.44 -1.34 (m, 10H), 1.29-1.28 (brs, 24H), 0.87 (t, J = 6.4 Hz, 9H). ELSD analysis: Purity 99.74 %, Calculated C55H97NO14 = 995.69, Observed = 996.45 (m/z, M+H+). CAD Purity = 93.40 %. Example 40R. Synthesis of JC-24-TL-010 (Compound 116) Synthetic Protocol AH [01009] To a stirred solution of 5-bromo-1-pentanol [AI-2] (25 g, 150 mmol) in dichloromethane (257 mL, 4.02 mol) was added triethylamine (52.2 mL, 2.5 eq., 374 mmol). Reaction mass was cooled to 0 °C and octanoyl chloride [AI-1] (25.5 mL, 150 mmol) was added drop wise. Reaction mass allowed to stir at RT for 16 h, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction mass was diluted with water (500 ml) and extracted with dichloromethane (2x100 ml). Combined organic layer was washed with sodium bicarbonate solution (1000 ml). Organic layer was dried over sodium sulphate, filtered, and concentrated under reduced pressure. The crude was purified over silica get by using flash chromatography and 5-10% ethyl acetate to give 5-bromopentyl octanoate [AI-3] (31 g, yield 71.26 %) as colourless liquid. [01010] Note: Two batches of 25.0 g (total 50 g) were performed yielding total 62 g of desired product. [01011] 1H NMR (400 MHz, CDCl3): 4.07 (t, J = 6.52 Hz, 2H), 3.40 (t, J = 6.72Hz, 2H), 2.31-2.27 (t, J = 2.68 Hz, 2H), 1.92 - 1.85 (m, 2H), 1.69 – 1.59 (m, 4H), 1.54 – 1.43 (m, 2H), 1.34-1.22 (m, 8H), 0.87 (t, J = 6.76 Hz, 3H). Intermediate [AI-5]: [01012] To a stirred solution of 5-bromopentyl octanoate [AI-3] (61.1 g, 3.8 eq., 187 mmol) and 2-hydroxy-1,2,3-propanetricarboxylic acid [AI-4] (9.6 g, 50 mmol) in dimethylformamide (480 mL, 6.2 mol) was added TEA (69.6 mL, 10 eq., 0.5 mol). The reaction mixture was stirred for 16 h at 60 °C, with the progress of reaction being monitored by ELSD/TLC. After consumption of the starting material, the reaction was cooled to RT and then quenched with water (2000 mL) and extracted with diethyl ether (2x 500 mL). The organic layer dried over sodium sulphate, filtered, and concentrated under reduced pressure. Resulting crude was purified over silica using 20% ethyl acetate in n-hexane to obtained tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [AI-5] (21 g, yield-50.69%) as light yellow colour liquid. 4.21 (t, J = 6.68 Hz, 2H), 4.12-4.03 (m, 10H), 2.89- 2.85 (d, J = 15.50 Hz, 2H), 2.80-2.76 (d, J = 15.6 Hz, 2H), 2.30 (t, J = 6.4 Hz, 6H), 1.75 -1.56 (m, 18H), 1.45 -1.37 (m, 6H), 1.31-1.26 (m, 22H), 0.87 (t, J = 9.0 Hz, 9H). Intermediate [AI-7]: [01014] To a stirred solution of 5-bromovaleric acid [AI-6] (1.75 g, 4 eq., 9.65 mmol) in dichloromethane (25 mL, 390 mmol), were added N,N-dimethyl-4-pyridylamine (589 mg, 2 eq., 4.82 mmol), and EDC.HCl (2.31 g, 5 eq., 12.1 mmol) at room temperature and stirred for 10 minute at same temperature. After this time, tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [AI-5] (2 g, 2.41 mmol) was added to the resulting reaction mixture and allowed to stir at room temperature for 48h, with monitoring by TLC/ELSD. After consumption of the starting material, the reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2x 30 mL). The combined organic layer dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude material was purified by using silica gel flash column chromatography and 5-10% ethyl acetate in heptane. The pure fraction were combined and concentrated under reduced pressure to obtained tris(5-(octanoyloxy)pentyl) 2- ((5-bromopentanoyl)oxy)propane-1,2,3-tricarboxylate [AI-7] (1 g, yield 41.78%) as a colourless liquid. ELSD analysis: Purity 98.70 %, Calculated C50H87BrO14 = 990.53, Observed = 993.35 (m/z, M+H+). JC-24-TL-010 (Compound 116): [01015] To a stirred solution of tris(5-(octanoyloxy)pentyl) 2-((5-bromopentanoyl)oxy)propane- 1,2,3-tricarboxylate [AI-7] (1 g, 1.01 mmol) in tetrahydrofuran (8.33 mL, 102 mmol) was added pyrrolidine [AI-8] (179 mg, 2.5 eq., 2.52 mmol) at room temperature. The reaction mixture was heated up to 55°C for 16h, with progress of the reaction being monitored by ELSD/TLC. Reaction mixture was concentrated at 45°C under reduced pressure and purified by silica gel column chromatography (2-5% MeOH in DCM) to get product as HBr salt. The salt was dissolved in dry dichloromethane (3 ml), potassium carbonate (2.0 eq) added to the solution, and allowed to stir for 90 minutes. The resulting solution filtered through 0.2 mm membrane filter. Filtrate was evaporated under reduced pressure to get tris(5-(octanoyloxy)pentyl) 2-((5-(pyrrolidin-1- yl)pentanoyl)oxy)propane-1,2,3-tricarboxylate [JC-24-TL-010] (114 mg, yield -11.51%) as a yellow liquid. [01016] 1H NMR (400 MHz, CDCl3): δ 4.15 (t, J = 6.6 Hz, 2H), 4.12-4.03 (m, 10H), 3.28-3.24 (d, J = 15.48 Hz, 2H), 3.19-3.11 (d, J = 15.48 Hz, 2H), 2.84 (brs, 4H), 2.36 (t, J = 7.16 Hz, 2H), 2.28 (t, J = 7.56 Hz, 6H), 2.01 (brs, 4H), 1.85-1.83 (m, 2H), 1.71-1.58 (m, 22H), 1.44-1.36 (m, 6H), 1.29-1.27 ( m, 24H), 0.87 (t, J = 7.0 Hz, 9H). ELSD analysis: Purity 99.77 %, Calculated C54H95NO14= 981.68, Observed = 982.45 (m/z, M+H+).CAD Purity = 92.00 %. Example 40S. Synthesis of TL1-12D-025 (Compound 126) Synthetic Protocol AI [01017] To a stirred solution of octanoic acid [AJ-2] (100.0 g, 693 mmol) and pentane-1,5-diol [AJ-1] (65.0 g, 624 mmol) in dichloromethane (2000 mL) was cooled to 0 oC and ({[3- (dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (167.0 g, 874 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (17.1 g, 139 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (200 mL) was added to the reaction mixture, and extract with DCM (3x500 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-10% Ethyl acetate in Hexane), to give the desired 5-hydroxypentyl octanoate [AJ-3] (60.0 g, yield 37.56%) as a yellow liquid. [01018] 1H-NMR (400 MHz, CDCl3)- δ 4.10-4.07 (m, 2H), 3.67 (bs, 2H), 2.32-2.28 (m, 2H), 1.71- 1.58 (m, 5H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 9H), 0.90-0.87 (m, 3H). Intermediate [AJ-5]: [01019] To a stirred solution of 2-hydroxypropane-1,2,3-tricarboxylic acid [AJ-4] (12.5 g, 65.1 mmol) and 5-hydroxypentyl octanoate [AJ-3] (60.0 g, 260 mmol) in dichloromethane (500 mL) was cooled to 0 oC and ({[3-(dimethylamino)propyl]imino}methylidene)(ethyl)amine hydrochloride (49.9 g, 260 mmol) was added followed by 4-(dimethylamino)pyridin-1-ium (8.02 g, 65.1 mmol). The reaction mixture stirred at r.t. for 48 h. The progress of reaction was monitored by ELSD/TLC (SM was consumed). Water (50 mL) was added to the reaction mixture, and extract with DCM (3x100 mL). The resulting organic layer was dried over Na2SO4, and concentrated under reduce pressure, and the crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired tris(5-(octanoyloxy)pentyl) 2-hydroxypropane-1,2,3- tricarboxylate [AJ-5] (21.0 g, yield 38.9%) as yellow liquid. [01020] 1H-NMR (400 MHz, CDCl3)- δ 4.23-4.20 (m, 2H), 4.10-4.04 (m, 10H), 2.89-2.77 (dd, J = 15.6, 19.2 Hz, 4H), 2.30-2.26 (m, 6H), 1.75-1.56 (m, 18H), 1.45-1.36 (m, 6H), 1.29-1.28 (m, 24H), 0.88 (m, 9H). ELSD analysis: Purity 97.90 %, Calculated C45H80O13 = 828.56, Observed = 829.35 (m/z, M+H+). Intermediate [AJ-7]: [01021] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-hydroxypropane-1,2,3- tricarboxylate [AJ-5] (10 g, 12.1 mmol) in triethylamine (6.10 g, 60.3 mmol) was added 3- bromopropanoyl chloride (1.21 mL, 12.1 mmol) was successively at 0 °C and stirred for 16h at same 25 oC temperature. TLC shows SM consumed and formed new spot. The reaction mass was filtered through celite and washed with ethyl acetate to get crude product. The crude was purified by flash column chromatography (SiO2: 0-30% Ethyl acetate in Hexanes), to give the desired [AJ-7] (2.0 g, yield 18.7%) as yellow liquid. [01022] 1H NMR (400 MHz, CDCl3): δ 6.42 (d, J = 17.2 Hz, 1H), 6.12-6.05 (m, 1H), 5.89 (d, J = 10.4 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2 H), 4.09-4.03 (m, 10H), 3.34 (d, J = 15.2 Hz, 2H), 3.25 (d, J = 15.6Hz, 2H), 2.28 (t, J = 7.2 Hz, 6 H), 1.70-1.59 (m, 18H), 1.43-1.35 (m, 6H), 1.28 (s, 24H), 0.87 (t, J = 6.8 Hz, 9 H). ELSD analysis: Purity 99.84%, Calculated C48H82O14 = 882.57, Observed = 883.40 (m/z, M+H+). Intermediate [AJ-8]: [01023] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-(prop-2-enoyloxy)propane- 1,2,3-tricarboxylate [AJ-7] (0.5 g, 566 µmol) in tetrahydrofuran (5 mL, 61.4 mmol), added 4-[(tert- butoxy)carbonyl]piperazin-1-ium [AJ-8] (117 mg, 623 µmol). Reaction mass was stirred at RT for 48 h. Progress of the reaction was monitored by TLC. Reaction mass was distilled out under reduced pressure and resulting crude material was purified by flash column chromatography (SiO2: 0-100% Ethyl acetate in Hexanes), to give 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{4-[(tert- butoxy)carbonyl]piperazin-1-yl}propanoyl)oxy]propane-1,2,3-tricarboxylate [AJ-9] (0.5 g, yield 88%) as a yellow liquid. [01024] ELSD analysis: Purity 95.97 %, Calculated C57H100N2O16 = 1068.71, Observed = 1069.75 (m/z, M+H+). TL1-12D-025 (Compound 126):
[01025] To a stirred solution of 1,2,3-tris[5-(octanoyloxy)pentyl] 2-[(3-{4-[(tert- butoxy)carbonyl]piperazin-1-yl}propanoyl)oxy]propane-1,2,3-tricarboxylate [AJ-9] (0.5 g, 468 µmol) in dichloromethane (5 mL, 78.1 mmol), trifluoroacetic acid (179 µL, 2.34 mmol) was added at 0 °C. The resulting reaction mixture was stirred for 6 h at room temperature. The progress of reaction mass was monitored by ELSD/TLC (SM was consumed). Reaction mass was lyophilized and obtained 1,2,3-tris[5-(octanoyloxy)pentyl] 2-{[3-(piperazin-1-yl)propanoyl]oxy}propane-1,2,3- tricarboxylate [TL1-12D-025] (325 mg, yield 71.71%) as a Yellow liquid. [01026] NMR (400 MHz, CDCl3): δ 4.20 (t, J = 6.8 Hz, 2H), 4.09 (m, 10H), 3.69 (s, 8H), 3.48-3.47 (m, 2H), 3.22 (s, 4H), 2.91-2.88 (m, 2H), 2.28 (t, J = 7.6 Hz, 6 H), 1.71-1.56 (m, 18H), 1.43-1.37 (m, 6 H), 1.31-1.28 (m, 24H), 0.87 (t, J = 6.8 Hz, 9H). ELSD analysis: Purity 99.64%, Calculated C52H92N2O14 = 968.65, Observed = 969.65 (m/z, M+H+). Example 41. Lipid nanoparticle formulation [01027] Ionizable lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include methods described in International Publication No. WO 2018/089801, which is hereby incorporated by reference in its entirety. [01028] The lipid nanoparticles in the examples of the present invention were formulated using Process A of WO 2018/089801 (see, e.g., Example 1 and Figure 1 of WO 2018/089801). Process A (“A”) relates to a method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles. In an exemplary process, an ethanolic solution of a mixture of lipids (cationic lipid, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipid) at a fixed lipid to mRNA ratio were combined with an aqueous buffered solution of target mRNA at an acidic pH under controlled conditions to yield a suspension of uniform LNPs. After ultrafiltration and diafiltration into a suitable diluent system, the resulting nanoparticle suspensions were diluted to final concentration, filtered, and stored frozen at −80°C until use. [01029] Lipid nanoparticle formulations of Table 4 using certain cationic lipids as described herein were prepared by Process A. All of the lipid nanoparticle formulations comprised hEPO mRNA and the different lipids (Cationic Lipid: DMG-PEG2000: Cholesterol: DOPE/DSPC) in the mol % ratios specified in Table 4.
Table 4. Exemplary Lipid Nanoparticle Formulations
* The N/P ratio is defined as the ratio of the number of nitrogen in cationic lipid to the number of phosphate in nucleic acid.
Example 42. Delivery of human EPO mRNA by intramuscular administration [01030] Lipid screening studies were conducted with female BALB/cJ mice 6-8 weeks of age and formulations 1 or 2 (MC3 only) above. Mice were dosed with 0.1 pg in 30 pL of LNPs by a single intramuscular (IM) injection into the gastrocnemius leg muscle. Blood samples were taken 6 and 24 hours post injection and hEPO levels were measured in the blood serum of the mice using an ELISA assay according to the manufacture's protocol. W02022/099003 Al also describes an in vivo assay for intramuscular administration (e.g. on page 46, paragraph [00206]). Exemplary data are provided in Table 5.
Table s. Exemplary in vivo protein expression (ng/mL) following intramuscular injection
Example 43. Delivery of Firefly Luciferase (FFL) mRNA by intranasal administration
[01031] Lipid nanoparticle formulation 4 listed in Table 4 comprising FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol and DOPE was administered in mice via pipetting the formulations at lOpg/Animal and 15pl per nostril. On Day 2, 24 hours post dose (±5%), all animals underwent a luminescent imaging session using I VIS with separate ROIs on the nose and lungs. Whole body imaging was performed 10-15 minutes following D-Luciferin administration. All animals were dosed with 0.2 mL of 15 mg/mL D-luciferin solution via intraperitoneal (IP) injection. Anesthesia was performed by isoflurane during the procedure and animals were placed sternal recumbency (face-down). The intranasal vaccine drug product may be administered via nasal spray. Exemplary data are provided in Table 6, which describes the average radiance in p/s/cm2/sr (the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (sr))
Table 6. Exemplary in vivo protein expression following intranasal administration
Example 44. Delivery of Firefly Luciferase (FFL) mRNA by pulmonary administration
[01032] Lipid nanoparticle formulation 4 listed in Table 4 comprising FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol and DOPE was administered to male CD1 mice (6-8 weeks old) by a single intratracheal administration via Catheter (50pl/animal) while under anesthesia. At approximately 24 hours post-dose, the animals were dosed with luciferin at 150 mg/kg (60 mg/ml) by intraperitoneal injection at 2.5ml/kg. After 5-15 minutes, all animals were imaged using an I VIS imaging system to measure luciferase production in the lung. Table 7 provides the the average radiance in p/s/cm2/sr and shows that lipid nanoparticles comprising the cationic lipids described herein are effective in delivering FFL mRNA in vivo based on positive luciferase activity. Table 7. Exemplary in vivo protein expression following pulmonary administration
Example 45. Intranasal dosing in a mouse influenza virus challenge model
[01033] To test the efficacy of intranasal closing in a mouse influenza virus challenge model, BALB/c mice were inoculated intranasally with 10 ug of Lipid Nanoparticles (LNPs) encapsulating CA09 HA mRNA (these LNPs comprised either Compound 64 or the lipid OF-02 [OF-02 is published in Fenton et al., 2016, Adv. Mater., 28:2939-43]) or intramuscularly with 1 ug of LNPs encapsulating CA09 HA mRNA (these LNPs comprised OF-02 only) at days 0 and 21, along with a negative control group with two doses of phosphate-buffered saline (PBS) buffer administered intranasally. At day 35, all mice were challenged intranasally with A/California/07/09 (H1N1), at 2.5 E+5 TCID50. All mice administered LNPs comprising lipid Compound 64 intranasally or LNPs comprising OF-02 intramuscularly had no mortality 14 days post-challenge (see Figures 1 and 2). However, those in the PBS control group suffered significant and rapid weight loss which led to 100% mortality by day 6 (see Figures 1 and 2). Mice administered LNPs comprising OF-02 intranasally had some protection against mortality, with 43% surviving at day 14 post-challenge. These results demonstrated high efficacy of LNPs comprising Compound 64 administered intranasally in a lethal mouse influenza challenge model.
Table 8: Lipid Nanoparticle (LNP) formulation details for example 45
Example 46. Delivery of Firefly Luciferase (FFL) mRNA by intranasal administration
Lipid nanoparticle formulation 3 listed in Table 4 comprising FFL mRNA, cationic lipid, DMG- PEG2000, cholesterol and DOPE was administered in mice via pipetting the formulations at lOpg/Animal and 15pl per nostril. On Day 2, 24 hours post dose (±5%), all animals underwent a luminescent imaging session using I VIS. Whole body imaging was performed 10-15 minutes following D-Luciferin administration. All animals were dosed with 0.2 mL of 15 mg/mL D-luciferin solution via intraperitoneal (IP) injection. Anesthesia was performed by isoflurane during the procedure and animals were placed 10 sternal recumbency (face-down). Exemplary data are provided in Table 9, which describes the average radiance in p/s/cm2/sr (the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (srj).
Table 9. Exemplary in vivo protein expression following intranasal administration
Example 47. Comparison of intranasal administration of Compound 64 with intramuscular administration of OF-02
To test the immune responses of mice to intranasal dosing, BALB/c mice were inoculated intranasally with 10 ug of Compound 64 LNPs encapsulating CA09 HA mRNA or intramuscularly with 1 ug of OF-02 LNPs encapsulating CA09 HA mRNA at days 0 and 21, along with a negative control group with 10% trehalose buffer administered intranasally.
Distant mucosal activation (IgG/lgA ELISA in vaginal washes)
ELISA Assays
Immunoglobulin A (IgA) ELISA
For the IgA ELISA, Influenza Hl A/Cal/09 protein was captured on 96 well polystyrene plates (Nunc MaxiSorp 439454) at a concentration of 1 pg/mL in carbonate-bicarbonate buffer. The plates were coated with 50 pL/well of antigen (1 μg/mL) and incubated overnight (16 ± 4 hours) at 2-8°C. After overnight incubation, the antigen coated plates were washed 5 times with washing buffer (PBS, 0.05% Tween20), and blocked with blocking solution (PBS, 10% BSA). The plates were incubated for 30 minutes at room temperature. Test samples, naive control, and reference samples (if available) were diluted (serum - 1:10 or 1:20; BAL, nasal wash, vaginal wash - 1:5) followed by a 4- fold serial dilution in sample diluent (PBS, 1% BSA, 0.05% Tween 20) and added to wells in duplicates, followed by incubation at room temperature for 60 minutes. Plates were washed 5 times with washing buffer, and goat anti-mouse HRP IgA (Bethyl A90-103P) was added at a dilution of 1:10,000. Plates were incubated 30 minutes at room temperature, then washed 5 times with washing buffer. TMB substrate (Sera Care 5120-0075) was added to each plate, and the reaction was stopped after 15 minutes of incubation at room temperature away from light, with TMB stop solution (Sera Care 5120-0020). Plates were read at 450 nm with a Thermo Labsystems Multiskan spectrophotometer. The anti-HA specific antibody titers are expressed as a reciprocal of the highest serum dilution with an absorbance value >0.3. Immunoglobulin G (IgG) ELISA For the IgG ELISA, Influenza H1 A/Cal/09 protein (Provided by Sanofi) was captured on 96 well polystyrene plates (Nunc MaxiSorp 439454) at a concentration of 1 µg/mL in carbonate- bicarbonate buffer. The plates were coated with 50 µL/well of antigen and incubated overnight (16 ± 4 hours) at 2-8°C. After overnight incubation, the antigen coated plates were washed 3 times with washing buffer (PBS 0.05% Tween20) and blocked with blocking solution (1.0% BSA in PBS). Test samples, naïve control and reference sample were diluted (serum - 1:10 or 1:20; BAL, nasal wash, vaginal wash - 1:5) followed by a 1:5 dilution in sample diluent (PBS 1% BSA 0.05% Tween 20) and added to wells in duplicates, followed by incubation at room temperature for 120 minutes. Anti- mouse HRP IgG (Invitrogen 314300) was added at a dilution of 1:10,000, followed by incubation at room temperature for 60 minutes. Plates were washed 5 times with washing buffer. Excess HRP IgG was washed with PBS. TrueBlue substrate (SERA CARE 10344659) was added to each plate and the reaction was stopped after 10 minutes of incubation away from light, with HCL. Plates were read at 450 nm with a Thermo Labsystems Multiskan spectrophotometer. The anti-HA specific antibody titers are expressed as a reciprocal of the highest serum dilution with an absorbance value >0.3. Lung CD4 memory T cells Lung Tissue Processing for Flow Cytometry Analysis: Lung tissue was transferred to a 6-well dish and mince using a sterile scalpel. The minced lung tissue was transferred to a labelled 50 mL conical tube. The 6-well dish was washed with 5 mL of Digestion Buffer (5 mg/mL Collagenase, Type 1 + 0.2 mg/mL DNase I in BSA Buffer (PBS + 0.5% BSA)) and added to 50 mL tube containing tissue sample. The tissue sample was incubated at 37°C for 30 minutes in an orbital shaker at 150 rpm. The reaction was stopped with 10 mL of cold BSA Buffer and the sample filtered into a new 50 mL conical tuber using a 70 µm strainer. The tissue was pressed through a strainer using the plunger end of a syringe. The strainer was washed with 5 mL of cold BSA Buffer and the cells centrifuged at 400 x g for 10 minutes at 4oC; the supernatant was discarded. The red blood cells were lysed with ACK Lysis Buffer for 1.5 minutes on ice. The cells were washed with cold BSA Buffer and centrifuged at 400 x g for 10 minutes at 4°C; the supernatant was discarded. The cell pellet was resuspended in 1 mL of FBS Buffer (PBS + 5% FBS). The cell count was enumerated with AOPI staining solution using a Nexcelom Cellometer. The cell count for each sample was recorded. Samples were stored in a 37°C incubator until ready for flow cytometry staining. Splenic T cell activation Spleen Processing for Flow Cytometry Analysis: Spleen was transferred to a 6-well dish and injected with 1 mL of Digestion Buffer (0.1% (w/v) Collagenase, Type IV + 20 μg/mL DNase I in HBSS containing 1% FBS) using a 23-gauge needle. The tissue was transferred to a labelled 15 mL conical tube containing 4 mL of Digestion Buffer. The tissue was digested for 25 minutes at 37°C. The tissue was minced with scissors and incubated for 10 minutes at 37°C. After incubation, 1 mM/mL of EDTA was added to each tube to stop the enzymatic reaction. The tissue plus solution was transferred to a 100 μm strainer affixed to a labelled 50 mL conical tube. With the plunger end of a syringe, the tissue was pressed through the strainer. The strainer was washed with 10 mL of 1x PBS. The cells were centrifuged at 1200 rpm for 10 minutes at 4°C; the supernatant was discarded. The red blood cells were lysed with ACK Lysis Buffer for 5 minutes on ice. The cell suspension was washed with with cold 1x PBS and transferred to a 100 µm strainer affixed to a new, labelled 15 mL conical tube. The cells were centrifuged at 1200 rpm for 10 minutes at 4°C; the supernatant was discarded. The cell pellet was resuspended in 3 mL of FBS Buffer. The cell count was enumerated with AOPI staining solution using a Nexcelom Cellometer. The cell count for each sample was recorded. Samples were stored in a 37°C incubator until ready for flow cytometry staining. Flow Cytometry Staining Samples were stained with LIVE/DEAD Near IR in PBS, followed by Fc-receptor blockade with anti- CD16/CD32 antibody in FBS Stain Buffer for 10 minutes at 4°C, and stained for 30 minutes on ice with antibody cocktail in Brilliant Stain Buffer (BD Biosciences). Excess antibodies were washed away with FBS Stain Buffer and cells fixed with 2-4% paraformaldehyde. Following fixation, cells are resuspended in 200 µL of FBS Stain Buffer and stored at 4°C. Conclusions As demonstrated in Fig 3, in these studies it was discovered that intranasal administration of Compound 64 induced lower serum IgG and serum influenza hemagglutination (HAI) titer values than intramuscular administration of OF-02. However, intranasal administration of Compound 64 was found to induce higher IgA in the lung (Fig.3) and also in a distant mucosal site (vagina) as well as a higher IgG at the distant mucosal site (see Fig.3). Furthermore, intranasal administration of Compound 64 induced greater splenic T cell activation and production of CD4 memory T cells in the lung (Fig.4). [01034] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. [01035] All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.
NUMBERED EMBODIMENTS 1. A compound having a structure according to (a) Formula (A2): or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; A is a substructure selected from: , , B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl; (b) Formula (A1): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl; or (c) Formula (A): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C2-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. 2. The compound of numbered embodiment 1, wherein X is O and/or B is independently an ionizable nitrogen-containing group. 3. The compound of numbered embodiment 1 or 2, wherein each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, or C4-C30 alkynyl. 4. The compound of numbered embodiment 1 or 2, wherein each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. 5. The compound of any one of numbered embodiments 1-4, wherein B is independently
, . 6. The compound of numbered embodiment 1, having a structure according to Formula (I): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group. 7. The compound of numbered embodiment 6, wherein B is independently an ionizable nitrogen containing group. 8. The compound of numbered embodiment 6 or 7, wherein m is 2 and/or n is 2 or 3. 9. The compound of any one of numbered embodiments 6-8, wherein B is independently , . 10. The compound of numbered embodiment 9, wherein B is . 11. The compound of numbered embodiment 6, wherein said compound is selected from the group consisting of: Compounds (1), (2), (3), (4), (6), (9), (17), (24), (26), (27), (33), and (35). 12. The compound of numbered embodiment 1, having a structure according to Formula (III): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; R4 is C2-C10 alkylene; and B is independently an ionizable nitrogen-containing group. 13. The compound of numbered embodiment 12, wherein R4 is –CH2CH2–. 14. The compound of numbered embodiment 12 or 13, wherein B is independently
or . 15. The compound of numbered embodiment 14, wherein B is . 16. The compound of numbered embodiment 12, wherein said compound is Compound (3). 17. The compound of numbered embodiment 1, having a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. 18. The compound of numbered embodiment 17, wherein each of R1, R2, and R3 is C4-C30 heteroalkyl. 19. The compound of numbered embodiment 17 or 18, wherein m is 2 and/or n is 2 or 3. 20. The compound of any one of numbered embodiments 17-19, wherein B is independently
. 21. The compound of numbered embodiment 20, wherein B is . 22. The compound of numbered embodiment 17, wherein said compound is Compound (8) or (39). 23. The compound of numbered embodiment 1, having a structure according to (a) Formula (V1) or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, 4, 5, 6, or 7; and B is independently an ionizable nitrogen-containing group; or (b) Formula (V): , or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. 24. The compound of numbered embodiment 23, wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl. 25. The compound of numbered embodiment 23, wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl; or wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl. 26. The compound of any one of numbered embodiments 23-25, wherein B is independently , . 27. The compound of numbered embodiment 26, wherein B is . 28. The compound of numbered embodiment 23, wherein said compound is selected from the group consisting of: (a) Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), and (40); or (b) Compounds (11), (12), (13), (14), (22), (25), (28), (29), (30), (32), (34), (36), (38), (40), (112), (113), (114), (115), (116), and (117). 29. The compound of numbered embodiment 1, having a structure according to Formula (VI): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. 30. The compound of numbered embodiment 29, wherein B is independently
. 31. The compound of numbered embodiment 30, wherein B is . 32. The compound of numbered embodiment 29, wherein said compound is Compound (21). 33. A compound having a formula according to Formula (VII), or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; each m is an integer of 2-10; each L4 is a carbonyl, ester, or amide; Z is –(CH2)q1–N–(CH2)q2–, wherein q1 and q2 are independently integers of 2-10; or Z is C6H3-Z1, wherein Z1 is a carbonyl, ester, or amide that is covalently attached to the –CH2(CH2)qB moiety; q is an integer of 1-9; and B is independently an ionizable nitrogen-containing group. 34. The compound of numbered embodiment 33, wherein X is O; each L4 is –C(O)O–; m is an integer of 2, 3, or 4; and/or q is 1. 35. The compound of numbered embodiment 33 or 34, wherein Z is –(CH2)q1–N–(CH2)q2–, and q1 and q2 are each 2. 36. The compound of numbered embodiment 33 or 34, wherein Z is . . 37. The compound of any one of numbered embodiments 33-36, wherein B is independently or
, . 38. The compound of numbered embodiment 37, wherein B is . 39. The compound of numbered embodiment 33, wherein said compound is Compound (16) or (31). 40. A compound selected from the group of (a) Compounds 41-64 and 90, or a pharmaceutically acceptable salt thereof: (b) Compounds 91-111, or a pharmaceutically acceptable salt thereof:
(c) Compounds 112-117 or 126-129, or a pharmaceutically acceptable salt thereof:
(d) Compounds 118-125 or a pharmaceutically acceptable salt thereof:
41. A composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is the compound of any one of numbered embodiments 1-40.
42. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound of any one of numbered embodiments 1-
40. 43. The composition of numbered embodiment 42, further comprising one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.
44. The composition of numbered embodiment 42 or 43, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide. 45. The composition of any one of numbered embodiments 41-44, comprising an mRNA that encodes a peptide or polypeptide for use in vaccine.
46. The composition of numbered embodiment 45, wherein the mRNA encodes an antigen. 47. The composition of numbered embodiment 46, wherein the antigen is from an infectious agent.
48. The composition of any one of numbered embodiments 41-47, formulated for a route of administration that is oral, rectal, vaginal, transmucosal, pulmonary, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal.
49. The composition of numbered embodiment 48, wherein said pulmonary delivery is intratracheal or inhaled. 50. The composition of numbered embodiment 48, wherein said route of administration is intranasal, optionally wherein the liposome comprises the cationic lipid compound 64, or a cationic lipid in Table 6, or a pharmaceutically acceptable salt thereof.
51. The composition of numbered embodiment 48, wherein said route of administration is intramuscular. 52. The composition of numbered embodiment 48, wherein said route of administration is pulmonary.
53. A method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids,
optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): , or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. 54. A method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B): , or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. 55. The method of numbered embodiment 54, wherein the composition further comprises one more lipids selected from the group consisting of one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. 56. The method of numbered embodiment 54 or 55, wherein the nucleic acid is an mRNA encoding a peptide or polypeptide. 57. The method of any one of numbered embodiments 53–56, wherein the composition comprises an mRNA that encodes a peptide or polypeptide for use in vaccine. 58. The method of numbered embodiment 57, wherein the mRNA encodes an antigen. 59. The method of numbered embodiment 58, wherein the antigen is from an infectious agent. 60. The method of any one of numbered embodiments 53-59, wherein the compound is selected from the group consisting of (a) Compounds (65)-(89), or a pharmaceutically acceptable salt thereof:
61. The method of any one of numbered embodiments 53-60, wherein said delivery is intranasal.
62. The method of any one of numbered embodiments 53-60, wherein said delivery is intramuscular.
63. The method of any one of numbered embodiments 53-60, wherein said delivery is pulmonary.
64. A composition comprising the cationic lipid of any one of numbered embodiments 1-40, and further comprising:
(i) one or more non-cationic lipids,
(ii) one or more cholesterol-based lipids, and
(iii) one or more PEG-modified lipids.
65. The composition of numbered embodiment 64, wherein the composition is a lipid nanoparticle, optionally a liposome.
66. The composition of numbered embodiment 65, wherein the one or more cationic lipid(s) constitute(s) about 30 mol %-60 mol % of the lipid nanoparticle.
67. The composition of numbered embodiment 65 or 66, wherein the one or more noncationic lipid(s) constitute(s) about 10 mol %-50 mol % of the lipid nanoparticle.
68. The composition of any one of numbered embodiments 65-67, wherein the one or more
PEG-modified lipid(s) constitute(s) about 1 mol %-10 mol % of the lipid nanoparticle. 69. The composition of any one of numbered embodiments 65-68, wherein the cholesterol- based lipid constitutes about 10 mol %-50 mol% of the lipid nanoparticle.
70. The composition of any one of numbered embodiments 65-69, wherein the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.
71. The composition of any one of numbered embodiments 65-70, wherein the lipid nanoparticle encapsulates an mRNA encoding a peptide or protein, optionally for use in a vaccine.
72. The composition of numbered embodiment 71, wherein the lipid nanoparticles have an encapsulation percentage for mRNA of
(i) at least 50%;
(ii) at least 55%;
(iii) at least 60%;
(iv) at least 65%;
(v) at least 70%;
(vi) at least 75%;
(vii) at least 80%;
(viii) at least 85%;
(ix) at least 90%; or
(x) at least 95%.
73. The composition of numbered embodiment 71 or 72 for use in therapy.
74. The composition of numbered embodiment 71 or 72 for use in a method of treating or preventing a disease amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen and/or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
75. The composition for use according to numbered embodiment 73 or 74, wherein the composition is administered intravenously, intrathecally, intramuscularly, intranasally, sublingually, or by pulmonary delivery, optionally through nebulization. 76. The composition for use according to any one of numbered embodiments 73-75, wherein the composition is administered intranasally.
77. The composition of any one of numbered embodiments 45, 46, 47, 71, or 72, wherein the composition is for use in a method of inducing mucosal immunity, preferably wherein said mucosal immunity is induced at one or more distal mucosal sites.
78. The composition of any one of numbered embodiments 45, 46, 47, 71, or 72, wherein the composition is for use in a method of inducing a CD4 memory T cell population, optionally wherein said population is induced in the mucosa.
79. The composition for use according to numbered embodiment 77 or 78, wherein the composition is administered intravenously, intrathecally, intramuscularly, intranasally, sublingually, or by pulmonary delivery, optionally through nebulization.
80. The composition for use according to numbered embodiment 79, wherein the composition is administered intranasally.
81. A method for treating or preventing a disease wherein said method comprises administering to a subject in need thereof the composition of numbered embodiment 71 or 72 and wherein the disease is amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen and/or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.
82. A method of inducing muscosal immunity, preferably wherein said mucosal immunity is induced at one or more distal mucosal sites in a subject, wherein said method comprises administering a composition of any one of numbered embodiments 45, 46, 47, 71, or 72 to the subject.
83. A method of inducing a CD4 memory T cell population in a subject, optionally wherein said population is induced in the mucosa, wherein said method comprises administering the composition of any one of numbered embodiments 45, 46, 47, 71, or 72 to the subject.
84. The method of numbered embodiment 81, 82 or 83, wherein the composition is administered intravenously, intrathecally, intramuscularly, intranasally, sublingually, or by pulmonary delivery, optionally through nebulization.
85. The method of numbered embodiment 81 or 84, wherein the composition is administered intranasally. 430 86. A composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): , or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group, wherein the composition is for use in (a) a method of inducing muscosal immunity, preferably wherein said mucosal immunity is induced at one or more distal mucosal sites; or 431 (b) a method of inducing a CD4 memory T cell population, optionally wherein said population is induced in the mucosa. 87. A method of (a) of inducing muscosal immunity, preferably wherein said mucosal immunity is induced at one or more distal mucosal sites in a subject; or (b) of inducing a CD4 memory T cell population in a subject, optionally wherein said population is induced in the mucosa, wherein said method comprises administering to the subject a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG-modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, 432 and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. 88. The composition for use according to numbered embodiment 86 or the method of numbered embodiment 87 wherein the composition is administered intravenously, intrathecally, intramuscularly, intranasally, sublingually, or by pulmonary delivery, optionally through nebulization, preferably wherein the composition is administered intranasally.

Claims

433 CLAIMS WHAT IS CLAIMED IS: 1. A compound having a structure according to Formula (A2): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C6 alkyl; or 434 Formula (A1): , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C1-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4-C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl; or Formula (A): 435 , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group; m is an integer of 2-10; n is an integer of 2-10; L1 is a carbonyl, ester, or amide; L2 is C2-C10 alkylene or a C2-C10 alkenylene; Ar is phenylene optionally comprising 1-4 substituents independently selected from halogen, OCH3, and CH3; R4 is C2-C10 alkylene; and wherein A is only substructure (a4) when each of R1, R2, and R3 is independently a C4- C30 heteroalkyl comprising a disulfide bond or having a structure that is , wherein L3 is OC(O), CO2, or (O)CO; o is an integer of 2-5; and R5 is C4-C24 alkyl; or L3 is OC(O), CO2, or (O)CO; o is an integer of 6-12; and R5 is C1-C5 alkyl.
436 2. The compound of claim 1, having a structure according to Formula (I): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group or a permanently charged nitrogen group. 3. The compound of claim 1, having a structure according to Formula (III): , or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; R4 is C2-C10 alkylene; and B is independently an ionizable nitrogen-containing group. 4. The compound of claim 1, having a structure according to Formula (IV): , or a pharmaceutically acceptable salt thereof, wherein 437 each of R1, R2, and R3 is independently C6-C30 alkyl, C6-C30 alkenyl, C6-C30 alkynyl, or C4-C30 heteroalkyl; m is an integer of 2-10; n is an integer of 2-10; and B is independently an ionizable nitrogen-containing group. 5. The compound of claim 1, having a structure according to (a) Formula (V1): , or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, 4, 5, 6, or 7; and B is independently an ionizable nitrogen-containing group; or (b) Formula (V): or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. 6. The compound of claim 1, having a structure according to Formula (VI): , 438 or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, and R3 is independently C6-C30 heteroalkyl comprising a disulfide group; n is an integer of 2, 3, or 4; and B is independently an ionizable nitrogen-containing group. 7. A compound having a formula according to Formula (VII), , or a pharmaceutically acceptable salt thereof, wherein X is independently O or NH; each of R1, R2, and R3 is independently C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, or C4-C30 heteroalkyl; each m is an integer of 2-10; each L4 is a carbonyl, ester, or amide; Z is –(CH2)q1–N–(CH2)q2–, wherein q1 and q2 are independently integers of 2-10; or Z is C6H3-Z1, wherein Z1 is a carbonyl, ester, or amide that is covalently attached to the –CH2(CH2)qB moiety; q is an integer of 1-9; and B is independently an ionizable nitrogen-containing group.
439 8. A compound selected from the group of (a) Compounds 41-64 and 90, or a pharmaceutically acceptable salt thereof:
(b) Compounds 91-111, or a pharmaceutically acceptable salt thereof:
(c) Compounds 112-117 or 126-129, or a pharmaceutically acceptable salt thereof:
(d) Compounds 118-125, or a pharmaceutically acceptable salt thereof:
9. A composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound of any one of claims 1-8.
10. A method of pulmonary, intranasal, or intramuscular delivery of a composition comprising an mRNA encoding a protein or polypeptide, encapsulated within a liposome, wherein the liposome comprises one or more cationic lipids, optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and optionally one or more PEG- modified lipids, wherein at least one cationic lipid is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group.
11. A method of pulmonary, intranasal, or intramuscular delivery of a composition comprising a nucleic acid encapsulated within a liposome, wherein the liposome comprises a cationic lipid that is a compound having a structure according to Formula (B): or a pharmaceutically acceptable salt thereof, wherein each n is independently 0 or 1; X1A is independently O or NR1A; R1A is H or C1-C6 alkyl; X1B is a covalent bond, C(O), CH2CO2, or CH2C(O); one of X2A and X2B is O and the other is a covalent bond; one of X3A and X3B is O and the other is a covalent bond; one of X4A and X4B is O and the other is a covalent bond; R1 is independently L1-B1, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R2 is independently L2-B2, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R3 is independently L3-B3, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; R4 is independently L4-B4, C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L1, L2, L3, and L4 are each independently C1–C30 alkylene; C2–C30 alkenylene; or C2–C30 alkynylene; each of B1, B2, B3, and B4 is independently an ionizable nitrogen-containing group, and wherein the cationic lipid comprises at least one ionizable nitrogen-containing group. 12. The method of claim 10 or 11, wherein the compound is selected from the group consisting of (a) Compounds (65)-(89), or a pharmaceutically acceptable salt thereof:
(b) Compounds 118-125, or a pharmaceutically acceptable salt thereof:
13. The method of any one of claims 10-12, wherein said delivery is intranasal.
14. The method of any one of claims 10-12, wherein said delivery is intramuscular.
15. The method of any one of claims 10-12, wherein said delivery is pulmonary.
EP24731386.9A 2023-06-12 2024-06-12 Tricine and citric acid-based cationic lipids Pending EP4724418A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP23305930 2023-06-12
EP23306902 2023-11-02
EP23306900 2023-11-02
PCT/EP2024/066219 WO2024256458A1 (en) 2023-06-12 2024-06-12 Tricine and citric acid-based cationic lipids

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Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4401796A (en) 1981-04-30 1983-08-30 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5780014A (en) 1995-04-14 1998-07-14 Inhale Therapeutic Systems Method and apparatus for pulmonary administration of dry powder alpha 1-antitrypsin
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US5744335A (en) 1995-09-19 1998-04-28 Mirus Corporation Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein
AU2017357758B2 (en) 2016-11-10 2023-11-16 Translate Bio, Inc. Improved process of preparing mRNA-loaded lipid nanoparticles
CA3144457A1 (en) * 2019-06-21 2020-12-24 Translate Bio, Inc. Tricine and citric acid lipids
JP2024515668A (en) * 2021-04-19 2024-04-10 トランスレイト バイオ， インコーポレイテッド Improved compositions for delivery of mRNA

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