Ionizable lipids comprising cyclic moieties for the delivery of nucleic acids and other therapeutic agents

Abstract

Provided herein are novel sulfur-containing lipids having a structure of Formula (A) or a salt thereof. The compounds may be formulated in a lipid nanoparticle for use in the delivery of charged cargo such as nucleic acid.

Description

IONIZABLE LIPIDS COMPRISING CYCLIC MOIETIES FOR THE DELIVERY OF NUCLEIC ACIDS AND OTHER THERAPEUTIC AGENTSTECHNICAL FIELD

[0001] Provided herein are ionizable lipids comprising cyclic moieties that may be formulated in a delivery vehicle so as to facilitate the encapsulation of a wide range of therapeutic agents or prodrugs therein, such as, without limitation, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceutical drugs and salts thereof.BACKGROUND

[0002] Nucleic acid-based therapeutics have enormous potential in medicine. To realize this potential, however, the nucleic acid must be delivered to a target site in a patient. This presents challenges since nucleic acid is rapidly degraded by enzymes in the plasma upon administration. Even if the nucleic acid is delivered to a disease site, there still remains the challenge of intracellular delivery. To address these problems, lipid nanoparticles have been developed that protect nucleic acid from such degradation and facilitate delivery across cellular membranes to gain access to the intracellular compartment, where the relevant translation machinery resides.

[0003] A key component of a lipid nanoparticle (LNP) is an ionizable lipid. The ionizable lipid is typically positively charged at low pH, which facilitates association with the negatively charged nucleic acid. However, the ionizable lipid is neutral at physiological pH, making it more biocompatible in biological systems. Further, it has been suggested that after the LNPs are taken up by a cell by endocytosis, the ionizability of these lipids at low pH enables endosomal escape. This in turn enables the nucleic acid to be released into the intracellular compartment.

[0004] An earlier example of an LNP product approved for clinical use and reliant on ionizable lipid is Onpattro®. Onpattro® is a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. Onpattro® is reliant on an ionizable lipid referred to as “DLin-MC3-DMA” or more commonly “MC3”, 1 (Figure 1), by investigators. Furthermore, MC3 represents an evolution of a structurally related ionizable lipid, referred to by investigators as “KC2”, 2 (Figure 1). MC3 is considered a state-of-the art ionizable lipid for the delivery of siRNA, requiring about 3 times less siRNA than KC2. As a consequence, MC3 is currently regarded as the benchmark against which the potency of new lipids is evaluated. Still, KC2 remains a valuable research tool.

[0005] Ionizable lipids are also crucial components of certain COVID-19 vaccines. To illustrate, the Pfizer / BioNTech and Modema vaccines rely on LNPs to deliver mRNA to the cytoplasm of liver cells. This mRNA encodes for the highly immunogenic Sars-Cov-2 spike protein. Once inside the host cell, the mRNA is transcribed to produce antigenic proteins. The Pfizer / BioNTech vaccine comprises an ionizable lipid referred to as “ALC-0315”, 3 (Scheme 1), while the Modema vaccine comprises an ionizable lipid referred to as “SM- 102”, 4.Scheme 1

[0006] All of the above lipids were optimized for delivery of therapeutic nucleic acids to the liver. However, there remains a need to develop new lipids for the delivery of charged cargo, such as nucleic acids, to other organs, such as the spleen, lungs, bone marrow, skin, etc. The delivery of therapeutics beyond the liver would expand the clinical utility of LNPs to target disease conditions that affect tissues and organs beyond the liver. There is also an ongoing need to develop LNPs with improved delivery of nucleic acid or other charged cargo to the liver.

[0007] The present disclosure seeks to address one or more of the above identified problems and / or provides useful alternatives to known products and / or compositions for the delivery of nucleic acid or other charged cargo.DEFINITIONS

[0008] As used herein, the term "ionizable lipid" refers to a lipid that, at a given pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., a CLogP) that is greater than 8, 9, 10 or 11.

[0009] As used herein, the term "ionizable, cationic amino lipid" refers to a lipid that, at physiological pH, is in an electrostatically neutral form and comprises a nitrogen atom in its head group that accepts a proton, thereby becoming electrostatically positively charged at a pH below its pKa.

[0010] As used herein, “type 1 ionizable head” or “MC-type ionizable head” refers to a moiety that has ahead group of the lipid of Formula I below, or equivalents thereof, with n ranging from 1 to 5:Formula I

[0011] As used herein, “type 2 ionizable head” refers to a moiety that has a head group of the lipid of Formula II below, or equivalents thereof, with n ranging from 1 to 5:Formula II

[0012] As used herein, “type 3 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula III below, or equivalents thereof, with m and n independently ranging from 1 to 5:Formula III

[0013] As used herein, “type 4 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula IV below, or equivalents thereof, with R = Ci-Ce alkyl or cycloalkyl, and with m and n independently ranging from 2 to 5:R— (CH2)m-N-(CH2)n— OHFormula IV

[0014] As used herein, “type 5 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula V below, or equivalents thereof, with m and n independently ranging from 1 to 5:O O— cH2) o^(CH2,"-fFormula V

[0015] As used herein, “type 6 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula VI below, or equivalents thereof and with m ranging from 1 to 5, and n, independently, ranging from 2 to 5, wherein R is Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl:Formula VI

[0016] As used herein, “type 7 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula VII below, or equivalents thereof, with R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, and with n ranging from 1 to 5 wherein a methylene of (CH2)nis optionally substituted with a sulfur or oxygen atom:■AA- (CH2)n-OHFormula VII

[0017] As used herein, “type 8 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula VIII below, or equivalents thereof, with R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, and with n ranging from 1 to 5:Formula VIII

[0018] As used herein, “type 9 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula IX below, or equivalents thereof, with m and n independently ranging from 1 to 5:Formula IX

[0019] As used herein, “type 10 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula X below, or equivalents thereof, wherein the curved lines represent atoms of a ring structure comprising the N atom, wherein the ring structure has from 2 to 8 C atoms, and wherein j ranges from 0 to 5:Formula X

[0020] As used herein, “type 11 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula XI below, or equivalents thereof, with R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, wherein the circle represents a homocyclic or heterocyclic ring comprising from 3 to 8 atoms, and wherein j ranges from 0 to 5:Formula XI

[0021] As used herein, “type 12 ionizable head” refers to a moiety that is the head group of the structure as defined by Formula XII below, or equivalents thereof, wherein R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl and wherein j ranges from 1 to 5:Formula XII

[0022] The terms, “protonatable amino head group”, “ionizable head group” or “head group” are used interchangeably herein and refer to a moiety of the ionizable, cationic amino lipid that comprises the nitrogen atom in its head group that accepts a proton, thereby becoming electrostatically positively charged at a pH below its pKa. The protonatable amino head group has a central carbon or nitrogen atom to which each of the two lipophilic chains are directly bonded.

[0023] An example of the type 2 ionizable head group is provided below and has a central carbon atom to which each lipophilic chain is directly bonded as indicated by the * symbol:[lipophilic chain][lipophilic chain]

[0024] In addition, an example of the type 12 ionizable head group is provided below in which a central carbon atom is indicated by the * symbol in the structure below:[lipophilic chain][lipophilic chain]

[0025] As used herein, the term “lipophilic chain” refers to an optionally substituted alkyl group bonded to the central carbon atom of the head group of the lipid, the alkyl group comprising at least 6 carbon atoms and optionally comprising C=C double bonds, wherein the parent compound of the alkyl group has a CLogP of at least 6.

[0026] For example, the known lipids MC3, 1, and KC2, 2, have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene, which has a CLogP of 9.25:lipiphilic chain(s):(6Z,9Z)-octadeca-6,9-diene: CLogP: 9.25

[0027] Lipid ALC-0315, 3, has a pair of lipophilic chains derived from hexyl 2- hexyldecanoate, which has a CLogP of 10.01 :lipiphilic chain(s)

[0028] Lipid SM-102, 4, has one lipophilic chain derived from undecyl hexanoate, which has a CLogP of 7.59, and one lipophilic chain derived from heptadecane-9-yl octanoate, which has a CLogP of 11.6: l hexanoategP: 7.588 lipiphilic chain(s) heptadecan-9-yl octanoate CLogP: 11.6

[0029] As used herein, “imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein” with reference to an ionizable lipid, refers to the apparent pKa of the lipid nanoparticle when the ionizable lipid is formulated therein. The apparent pKa is measured in a lipid nanoparticle having a composition of ionizable lipid / DSPC / cholesterol / PEG2ooo-DMG (50:10:38.5:1.5 mol:mol) and prepared as described in WO 2023 / 184038, which is incorporated herein by reference.

[0030] The apparent pKais measured using a 6-( / ?-Toluidino)-2 -naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; Jayaraman et al., 2012, Angew. Chem Int. Ed., 51:8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). In the adapted method, a series of buffers are prepared spanning a pH range of 2.5-10.9 in varying pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.12 mM TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polystyrene 96-well plate, to yield a final concentration of 6.25 and 12 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using a SpectraMax M5™ microplate reader at Zc =321 nm, Zcm=445 nm. The fluorescence is subsequently plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0031] As used herein, the term “alkyl” or “alkyl group” is a Ci to C40 carbon-containing chain that is linear, cyclic (monocyclic or polycyclic) and / or branched and that optionally comprises C=C double bonds (e.g., 0-2 double bonds) and / or is or comprises one or more substituent ring structures, and that is optionally substituted.

[0032] As used herein, the term “Cm to Cnalkyl” or “Cm to Cnalkyl group” refers to a linear, cyclic and / or branched carbon chain having a total minimum of m carbon atoms and up to n carbon atoms, and that is optionally unsaturated and optionally substituted. For example, a “Ci to C3 alkyl” or “Ci to C3 alkyl group” is an alkyl having between 1 and 3 carbon atoms.

[0033] The term “ring structure” is a 3- to 22 -membered monocyclic or polycyclic alkyl ring that is optionally substituted and optionally unsaturated. In some non-limiting examples, the ring structure is a 3- to 16-membered monocyclic or polycyclic alkyl ring that is optionally substituted. In further examples, the ring structure is a 3- to 8-membered monocyclic or polycyclic alkyl ring that is optionally substituted.

[0034] The term “monocyclic” is an optionally substituted alkyl group that is a single ring or that comprises a single ring substituent.

[0035] The term “polycyclic” is optionally substituted alkyl group that is, or comprises as a substituent(s), two or more ring structures that are chemically bonded to each other or two or more discrete ring structures.

[0036] The term “optionally substituted” with reference to an alkyl or alkyl group means that at least one hydrogen atom of the alkyl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl is interrupted (i.e., a -(CH)2- group replaced) by a non-carbon atom or one or more substituents, including but not limited to those comprising heteroatoms selected from O, S and NR', wherein R' is as defined below. Non-limiting examples of atoms or substituents that may replace a hydrogen atom include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; — OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) include cycloalkyl groups (mono or polycyclic); — O — ; — (C=O)O — ; — O(C=O)-; —C(=0); — 0(C=0)0-; — S(0)x-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR'— , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0037] As used herein, the term “helper lipid” means a compound selected from: a sterol such as cholesterol or a derivative thereof; a diacylglycerol or a derivative thereof, such as a glycerophospholipid, including phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and the like; and a sphingolipid, such as a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or reduced analogues thereof, that lack a double bond in the sphingosine unit. An example of a diacylglycerol derivative is a glycerophospholipid-cholesterol conjugate in which one of the acyl chains is substituted with a moiety comprising cholesterol. The term encompasses lipids that are either naturally-occurring or synthetic.

[0038] As used herein, the term “delivery vehicle” includes any preparation in which the lipid described herein is capable of being formulated and includes but is not limited to delivery vehicles comprising one or a combination of the foregoing helper lipids.

[0039] As used herein, the term “nanoparticle” is any suitable particle in which the ionizable lipid can be formulated and that may comprise one or more of the helper lipids. The ionizable lipid is co-formulated with additional lipid components, such as the one or more helper lipids. The term includes, but is not limited to, particles with one or more bilayers, monolayers that are continuous or discontinuous, including multilamellar vesicles, unilamellar vesicles and particles with a core having an electron-dense region. The term also includes polymer-lipid hybrids, including particles in which the lipid is attached to a polymer.

[0040] As used herein, the term “encapsulated,” with reference to incorporating a cargo molecule (e.g., nucleic acid such as mRNA) within a delivery vehicle refers to any association of the cargo with any component or compartment of the delivery vehicle such as a nanoparticle.

[0041] The term “pharmaceutically acceptable salt” with reference to a form of the lipid of the disclosure in a protonated form (i.e., charged) and / or as part of a pharmaceutical formulation in which an LNP is formulated refers to a salt prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids.

[0042] As used herein, the term “Cmto Cnalkyl” or “Cmto Cnalkyl group” refers to a linear or branched carbon chain having a total minimum of m carbon atoms and up to n carbon atoms, and that is optionally unsaturated and optionally substituted. For example, a “Ci to C3 alkyl” or “Ci to C3 alkyl group” is an alkyl having between 1 and 3 carbon atoms.

[0043] The term “ring structure”, “ring” or “cyclic alkyl group” is a 3- to 22 -membered monocyclic or polycyclic alkyl ring that is optionally substituted and optionally unsaturated. In some non-limiting examples, the ring structure, ring or cyclic alkyl group is a 3- to 16- membered monocyclic or polycyclic alkyl ring that is optionally substituted. In further examples, the ring structure is a 3- to 8-membered monocyclic or polycyclic alkyl ring that is optionally substituted.

[0044] The term “monocyclic” is an optionally substituted alkyl group that is a single ring or that comprises a single ring substituent.

[0045] The term “polycyclic” is optionally substituted alkyl group that is, or comprises as a substituent(s), two or more ring structures that are chemically bonded to each other or two or more discrete ring structures.

[0046] The term “optionally substituted” with reference to an alkyl or alkyl group means that at least one hydrogen atom of the alkyl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl group is interrupted (i.e., a -(CH)2- group replaced) by a non-carbon atom or one or more substituents, including but not limited to those comprising heteroatoms selected from O, S and / or NR', wherein R' is as defined below. Non-limiting examples of atoms or substituents that may replace a hydrogen atom include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R';O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) include cycloalkyl groups (e.g., mono or polycyclic); — O — ; — (C=O)O — ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; — NR'S(O)XR’— ; and — S(O)XNR'— , wherein R' ateach occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0047] As used herein, the term “helper lipid” means a compound selected from: a sterol such as cholesterol or a derivative thereof; a diacylglycerol or a derivative thereof, such as a glycerophospholipid, including phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and the like; and a sphingolipid, such as a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or reduced analogues thereof, that lack a double bond in the sphingosine unit. An example of a diacylglycerol derivative is a glycerophospholipid-cholesterol conjugate in which one of the acyl chains is substituted with a moiety comprising cholesterol. The term encompasses lipids that are either naturally occurring or synthetic.

[0048] As used herein, the term “delivery vehicle” includes any preparation in which the lipid described herein is capable of being formulated and includes but is not limited to delivery vehicles comprising helper lipids.

[0049] As used herein, the term “nanoparticle” is any suitable particle in which the lipid can be formulated and that may comprise one or more helper lipid components. The one or more lipid components may include an ionizable lipid prepared by the method described herein and / or may include additional lipid components, such as the one or more helper lipid components. The term includes, but is not limited to, vesicles with one or more bilayers, including multilamellar vesicles, unilamellar vesicles and vesicles with an electron-dense core. The term also includes polymer-lipid hybrids, including particles in which the lipid is attached to a polymer.

[0050] As used herein, the term “encapsulated,” with reference to incorporating a cargo molecule (e.g., mRNA) within a delivery vehicle refers to any association of the cargo with any component or compartment of the delivery vehicle such as a nanoparticle.

[0051] The term “sterol” refers to a naturally-occurring or synthetic compound having a gonane skeleton and that has a hydroxyl moiety attached to one of its rings, typically the A- ring.

[0052] The term “pharmaceutically acceptable salt” with reference to a form of the lipid of the disclosure in a protonated form (i.e., charged) and / or as part of a pharmaceutical formulation in which an LNP is formulated refers to a salt prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids.SUMMARY

[0053] The present disclosure describes ionizable, cationic amino lipids that include at least one sulfur atom and incorporate a ring structure in at least one lipophilic chain.

[0054] According to one aspect of the disclosure, there is provided an ionizable, cationic amino lipid or a pharmaceutically acceptable salt thereof having a structure of FORMULA A:FORMULA A wherein R1is H or a substituent selected from,C1-C10, alkyl,Cs-Cs cycloalkyl,C1-C10 alkoxy,C1-C10 alkylthio, or an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C10 alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl, and the dash represents a bond to a ring atom of the ring structure having the n at its center, the ring structure having the n at its center comprising a total of n atoms, wherein n is 3 to 20, 5 to 8, or 4 to 10, the ring structure being carbocyclic or heterocyclic, wherein m is 0 to 6,G is an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C14 alkyl, a C3-C16 cycloalkyl, or a C3-C16 alkylcycloalkyl, and the dash represents a bond to the CH group, wherein n is 1 to 4,A1is absent, a heteroatom that is optionally O or S, or an ester moiety, -C(O)-O-, wherein the C of the ester moiety is bonded to the (CH2)nand the O is bonded to the (CH2)P, or the C of the ester moiety is bonded to the (CH2)Pand the O is bonded to the (CH2)n, p is 2 to 10, and q is 2 to 10,A2is absent, a heteroatom optionally selected from O or S, or an ester moiety, C(O)-O, wherein the C of the ester moiety is bonded to the (CH2)qand O is bonded to an atom of the R2, or the C is bonded to (CH2)qand O is bonded to an atom of the R2,R2is a linear, branched or cyclic, optionally substituted, Ci to Cio alkyl, optionally wherein 1 to 3 carbon atoms form a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom; or wherein R2is a moiety of FORMULA A.l:FORMULA A.1 wherein t is 0 to 6, and u is 1 to 4, wherein 1-3 carbon atoms of an alkyl of at least one R1, R2or R3optionally shares a double bond of E or Z configuration with an adjacent atom, or shares a triple bond with an adjacent atom,G is the ester -COO-R’ or R’-COO- as defined above, the circle of Formula A.l with the n in its center represents the ring structure as defined above for Formula A comprising a total of n atoms, with n ranging from 3 to 20, or 4 to 10, R3is H or a substituent independently selected from a structure defined in R1above, A3is either C or N, and if A3is C, thenW1and Y are either bonded to each other or not bonded to each other as indicated by the dashed bond; and if W1and Y are bonded to each other thenW1is O or S;W2is O or S;X is CH;Y is (CH2)m, wherein m is 1 or 2; andZ is a group selected from one of structures a-c below, wherein the wavy line represents the bond to X,a. •~ '(CH2)n-N / / tyPe2 ionizable head, wherein n of the type 2 ionizable head is 1 to 5; b. type 3 ionizable head, wherein m and n ofthe type 3 ionizable head are independently 1 to 5; andR c. •~v'(CH2)m— N— (CH2)— OH type 4 ionizable head, wherein m and n of the type 4 ionizable head are independently 2 to 5 and wherein the R of the type 4 ionizable head is Ci-Ce alkyl or cycloalkyl, if W1and Y are not bonded to each other, then:W1is H; andW2is O, S, NH or NR3, wherein R3is a Ci to C4 alkyl optionally substituted with an OH group; and groupW^'Z, is selected from one of structures d-1 below, whereinY the wavy line represents the bond to W2: d. ifW2is O, type 1 ionizable head, wherein n of the type1 ionizable head is 1 to 5; O, type 5 ionizable head, whereinm and n of the type 5 ionizable head are independently 1 to 5;(CH2)n-OH if W2is O, type 6 ionizable head, wherein m of the type 6 ionizable head is 1 to 5, and independently n of the type 6 ionizable head is 2 to 5, wherein R of the type 6 ionizable head is Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl; g. (CH2)n-OH if W2is NH or NR3, type 7 ionizable head, wherein n of the type 7 ionizable head is 1 to 5; h. (CH2)n_N\ if W2is NH or NR3, type 8 ionizable head, wherein n of the type 8 ionizable head is 1 to 5; ando is O, type 9 ionizable head, whereinthe ring structure of the type 10 ionizable head has 2 to 8 C atoms, and wherein j of the type 10 ionizable head is 0 to 5; k if W2is NH or NR2, type 11 ionizable head,wherein the circle of the type 11 ionizable head represents a homocyclic or heterocyclic ring comprising from 3 to 8 atoms, and wherein j of the type 11 ionizable head is 0 to 5; and o'R1 if W2is O, type 12 ionizable head, and wherein j of thetype 12 ionizable head is 1 to 5 and wherein R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, if A3is N, thenW1and Y are absent;W2and X together form a group of structure (CRaRb)p, wherein Raand Rbare independently selected from H, C1-C5 alkyl, cycloalkyl, and wherein p is 2 to 6, 3 to 6 or 4 to 6; andZ is OH, SO2NH2 or NR’R”, wherein R’ and R” are independently C1-C5 alkyl, or cycloalkyl, or wherein R’ and R” form a heterocyclic group that incorporates the N atom to which R’ and R” are bound.

[0055] According to an embodiment of the foregoing aspect, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof as described above, wherein the ring structure with the n of Formula A or Formula A.1 at its center is the heterocyclic and wherein the heterocyclic comprises carbon atoms and at least one nitrogen, oxygen, and sulfur atom.

[0056] According to an embodiment of the foregoing aspect, there is provided a cationic amino lipid or the pharmaceutically acceptable salt thereof, wherein the ring structure with the n of Formula A or Formula A.1 at its center is the carbocyclic and wherein the carbocyclic is a 4 to 7 membered ring or a 5 to 6 membered ring. In some embodiments, the carbocyclic ring structure of at least Formula A is a 4 to 7-membered alkyl ring. In some embodiments, the carbocylic ring of at least Formula A is a substituted or unsubstituted cyclohexyl moiety. In some embodiments, the carbocylic ring of at least Formula A is an unsubstituted cyclohexyl moiety.

[0057] According to an embodiment of the foregoing aspect, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof, wherein the ring structure of Formula A or Formula A.1 having the n in its center comprises at least one substituent bonded to a ring atom thereof, the substituent selected from Ci to Cio alkyl, aryl, alkoxy, alkylthio, or an ester having a structure as defined by -COO-R’ or R’-COO-or an amide having a structure as defined by -CONR’R” or R’-CON(R”)-, wherein R’ and R” are, independently, optionally substituted Ci to Ci6 alkyl or cycloalkyl and the dash represents the bond to an atom of the ring structure and optionally R’ and R” share a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom. According to a further embodiment of the foregoing aspect or any embodiment thereof, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof, wherein the A3is N, and Z is NR’N”, wherein R’ and R” form the heterocyclic group that incorporates the N atom to which R’ and R” are bound and wherein the heterocyclic group is pyrrolidine, piperidine or morpholine.

[0058] According to a further aspect, there is provided an ionizable, cationic amino lipid or a pharmaceutically acceptable salt thereof comprising: a protonatable amino head group; two lipophilic chains, wherein the protonatable amino head group has a central carbon or nitrogen atom to which each of the two lipophilic chains are directly bonded; at least one of the two lipophilic chains has a structure of FORMULA A.2:FORMULA A.2 wherein R1is H or a substituent selected from,Ci-Cio, alkyl,Cs-Cs cycloalkyl,C1-C10 alkoxy,C1-C10 alkylthio, or an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C10 alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl, and the dash represents a bond to a ring atom of the ring structure having the n at its center, the ring structure having the n at its center comprising a total of n atoms, wherein n is 3 to 20, or 4 to 10, the ring structure being carbocyclic or heterocyclic, wherein m is 0 to 6,G is an ester that is -COO-R’ or R’-COO-, wherein R’ is an optionally substituted alkyl selected from a linear or branched C1-C14 alkyl, C3-C16 or C4-C10 cycloalkyl, or C3-C16 alkylcycloalkyl, and the dash represents a bond to the CH group, wherein n of (CH)2 is 1 to 4,A1is absent, a heteroatom that is optionally O or S, or an ester moiety, -C(O)-O-, wherein the C of the ester moiety is bonded to the (CH2)nand the O is bonded to the (CH2)P, or the C of the ester moiety is bonded to the (CH2)Pand the O is bonded to the (CH2)n, p is 2 to 10, each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the ionizable, cationic amino lipid (i) imparts an apparent pKaof between 6 and 7.5 to a lipid nanoparticle when formulated therein; and has (ii) a ClogP of at least 11.

[0059] According to another embodiment, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof as described in the foregoing aspect, wherein a second one of the two lipophilic chains bonded to the central carbon or nitrogen atom of the head group has a structure as defined by FORMULA A.3:R2— A2-(CH2)q~vvFORMULA A.3 q is 2 to 10; wherein A2is absent, a heteroatom that is optionally O or S, or an ester moiety, C(O)-O, wherein the C of the ester moiety is bonded to the (CH2)qand the O of the ester moiety is bonded to an atom of the R2, or the C is bonded to (CH2)qand the O of the ester moiety is bonded to an atom of the R2,wherein R2is a linear, branched or cyclic, optionally substituted, Ci to Cio alkyl, optionally wherein 1 to 3 carbon atoms form a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom; or wherein R2is a moiety of FORMULA A.1:FORMULA A.1 wherein the wavy line represents a bond to A2, t is 0 to 6, u is 1 to 4, wherein the alkyl of at least one R1, R2or R3optionally shares a double bond of E or Z configuration with an adjacent atom, or shares a triple bond with an adjacent atom, G is the ester -COO-R’ or R’-COO- as defined above, the circle of Formula A.1 with the n in its center represents the ring structure as defined above for Formula A comprising a total of n atoms, with n ranging from 3 to 20 or 4 to 10.

[0060] According to another embodiment, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof as described in any one the foregoing aspects or embodiments thereof, wherein the R’ of the G that is an ester (-COO-R’ or R’- COO-) is a C2-C14 alkyl or cycloalkyl, C3-C14 alkyl or cycloalkyl, C3-C10 alkyl or cycloalkyl or C4-C10 alkyl or cycloalkyl.

[0061] According to another embodiment, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof, having a structure selected from any one of compounds T-01 to T-12 as defined hereinafter or a pharmaceutically acceptable salt thereof. According to another embodiment, there is provided an ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof, having a structure selected from any one of compounds T-05, T-07, T-08 or T-09 as defined hereinafter or a pharmaceutically acceptable salt thereof.

[0062] According to another aspect, there is provided a lipid nanoparticle comprising the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof as described in any one the foregoing aspects or embodiments thereof, a nucleic acid and a helper lipid.

[0063] In one embodiment, the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

[0064] According to another aspect, there is provided a method for administering a nucleic acid to a subject in need thereof, the method comprising administering the lipid nanoparticle of the foregoing aspect or embodiment thereof comprising the nucleic acid to the subject.

[0065] According to another aspect, there is provided a method for delivering a nucleic acid molecule to a cell, the method comprising contacting the lipid nanoparticle described in the foregoing aspect or embodiment with the cell in vivo or in vitro.

[0066] According to another aspect, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof described in any of the foregoing aspects or embodiments thereof in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

[0067] According to another aspect, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof described in any of the foregoing aspects or embodiments thereof to deliver a nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

[0068] In some embodiments, the nucleic acid is an mRNA, DNA vector, siRNA or antisense oligonucleotide.

[0069] In some embodiments, a lipid nanoparticle comprising the ionizable, cationic amino lipid described herein is for delivery of a cargo to the liver and / or spleen. In some embodiments, such LNP has improved delivery relative to the same LNP formulated with a nor-MC3 control as described in Example 2 hereinafter.

[0070] Other objects, features, and advantages of the present disclosure will be apparent to those of skill in the art from the following detailed description and figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIGURE 1 is a bar graph showing entrapment (%), particle size and poly dispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising the ionizable lipids nor-MC3, T-05, T-07, T-08, and T-09. The LNPs are composed of 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG and the amine-to-phosphate ratio (N / P) was 6.

[0072] FIGURE 2A shows luminescence intensity / mg in the liver for the mRNA- containing LNPs comprising the ionizable lipid nor-MC3, T-05, T-07, T-08, and T-09 after 4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0073] FIGURE 2B shows luminescence intensity / mg in the spleen for the mRNA- containing LNPs comprising the ionizable lipid nor-MC3, T-05, T-07, T-08, and T-09 after4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).DETAILED DESCRIPTION

[0074] Various aspects and embodiments of the disclosure are directed to ionizable lipids having structures of FORMULA A or pharmaceutically acceptable salts thereof.

[0075] Formulations comprising such lipids find use in the delivery of nucleic acid to any target site of interest. In some embodiments, such lipids have been found to be particularly efficacious for the delivery of mRNA when formulated in a suitable delivery vehicle. In further embodiments, such lipids can be easily synthesized and prepared by processes having improved economics relative to known methods for making ionizable lipids.

[0076] Embodiments disclosed herein relate an ionizable, cationic amino lipid or a pharmaceutically acceptable salt thereof having a structure as defined below:wherein R1is H or optionally a substituent selected from,Ci-Cio, alkyl,Cs-Cs cycloalkyl,C1-C10 alkoxy,C1-C10 alkylthio, or an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C10 alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl, and the dash represents a bond to a ring atom of the ring structure having the n at its center, the ring structure having the n at its center comprising a total of n atoms, wherein n is 3 to 20, or 4 to 10, the ring structure being carbocyclic or heterocyclic, wherein m is 0 to 6,G is an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C14 alkyl, a C3-C16 cycloalkyl, or a C3-C16 alkylcycloalkyl, and the dash represents a bond to the CH group, wherein n is 1 to 4,A1is absent, a heteroatom that is optionally O or S, or an ester moiety, -C(O)-O-, wherein the C of the ester moiety is bonded to the (CH2)nand the O is bonded to the (CH2)P, or the C of the ester moiety is bonded to the (CH2)Pand the O is bonded to the (CH2)n, p is 2 to 10, q is 2 to 10,A2is absent, a heteroatom optionally selected from O or S, or an ester moiety, C(O)-O, wherein the C of the ester moiety is bonded to the (CH2)qand O is bonded to an atom of the R2, or the C is bonded to (CH2)qand O is bonded to an atom of the R2,R2is a linear, branched or cyclic, optionally substituted, Ci to Cio alkyl, optionally wherein 1 to 3 carbon atoms form a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom; or wherein R2is a moiety of FORMULA A.l:FORMULA A.1 wherein t is 0 to 6, u is 1 to 4, wherein the alkyl of at least one R1, R2or R3optionally shares a double bond of E or Z configuration with an adjacent atom, or shares a triple bond with an adjacent atom, G is the ester -COO-R’ or R’-COO- as defined above, the circle of Formula A.l with the n in its center represents the ring structure as defined above for Formula A comprising a total of n atoms, with n ranging from 3 to 20, or 4 to 10, R3is H or a substituent independently selected from a structure defined in R1above, A3is either C or N, and if A3is C, thenW1and Y are either bonded to each other or not bonded to each other as indicated by the dashed bond; and if W1and Y are bonded to each other thenW1is O or S;W2is O or S;X is CH;Y is (CH2)m, wherein m is 1 or 2; andZ is a group selected from one of structures a-c below, wherein the wavy line represents the bond to X, a. (CH2)n_N tyPe2 ionizable head; type 3 ionizable head; andtype 4 ionizable head, if W1and Y are not bonded to each other, then:W1is H;W2is O, S, NH or NR3, wherein R3is a Ci to C4 alkyl optionally substituted with an OH group; andGroup, wherein the wavy line represents the bond to W2, isY selected from one of structures d-1 below, wherein the wavy line represents the bond to W2:g. (CH2)n-OH if W2is NH or NR2, type 7 ionizable head;k. if W2is NH or NR2, type 11 ionizable head; and l. type 12 ionizable head;if A3is N, thenW1and Y are absent;W2and X together form a group of structure (CRaRb)p, wherein Raand Rbare independently selected from H, C1-C5 alkyl, cycloalkyl, and wherein p is 2 to 6, 3 to 6 or 4 to 6; andZ is OH, SO2NH2 or NR’R”, wherein R’ and R” are independently C1-C5 alkyl, or cycloalkyl, or wherein R’ and R” form a heterocyclic group that incorporates the N atom to which R’ and R” are bound.Methods to produce lipids of Formula A

[0077] Exemplary, but by no means limiting, lipids of Formula A are compounds T-l-T-14 below. Lipids of Formula A or pharmaceutically acceptable salts thereof can be prepared using any suitable method known to those of skill in the art. Especially advantageous methods for the synthesis of the lipids are set forth in Schemes 3-14 below. However, the synthetic routes outlined in Scheme 3-14 must in no way be construed as limiting. Indeed, those skilled in the art will appreciate that alternative starting materials can be employed in the same sequences, leading to analogues of compound T-l-T-14 as defined by Formula A.

[0078] Therefore, synthetic schemes 3-14 set forth below are merely illustrative of select embodiments.

[0079] Compounds T-01-T-10 above are lipids of Formula A wherein A3is a carbon atom. As described in co-owned and co-pending applications WO 2022 / 246555, WO 2023 / 147657, WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 130421, U.S. provisional application No. 63 / 664,796 filed on June 27, 2024, U.S. provisional application No. 63 / 664,792 filed on June 27, 2024, each of which is incorporated herein by reference, a lipid of Formula A wherein A3is a carbon atom can be prepared from ketone 2.1 (Scheme 2).

[0080] The above applications provide several methods to prepare ketones 2.1 comprising structurally diverse lipophilic chains. The ketone carbonyl group in 2.1 can subsequently be transformed into an ionizable head group of type 1-12 through appropriate synthesis steps. The synthetic steps that are necessary to install a head group of type 1-12 on ketone 2.1 are described in detail in co-owned and co-pending PCT applications WO 2024 / 065043, WO 2023 / 215989, WO 2024 / 065041, WO 2024 / 065042, and PCT / CA2023 / 051727, incorporated herein by reference.Scheme 2

[0081] Ketones of general structure 2.1 that are required to produce representative, but by no means limiting, lipids T-01-T-10 are 3.1-3.7 (Scheme 3).precursor of T-09precursor of T-10Scheme 3

[0082] Ketones 3.1, 3.2, 3.3, 3.5, 3.6 and 3.7 are esters of dihydroxyketones 4.1-4.4, while3.4 can be obtained from 4.5. Accordingly, the synthesis of 3.1-3.7 starts with the preparation of dihydroxyketones 4.1-4.5,Scheme 4

[0083] As described in detail in co-owned and co-pending PCT applications WO2022 / 246555, WO 2023 / 147657, WO 2024 / 130421, and U.S. provisional application No.63 / 776,506 filed on March 24, 2025, 4.1-4.4 can be prepared through a Mukaiyama-Claisen self-condensation of appropriate esters, as set forth in Scheme 5.Scheme 5

[0084] As described in detail in the above-cited co-owned and co-pending WO2023 / 147657 and in Nabi, A., et al., J. Org. Chem. 2024, 89, 12775, incorporated herein byreference, dihydroxyketone 4.5 can be made by Mukaiyama-Claisen self-condensation of caprolactone (Scheme 6).Scheme 6

[0085] The above-cited co-owned and co-pending applications teach that dihydroxyketones 4.1-4.5 can be transformed into diester or monoester derivatives. Thus, treatment with at least two molar equivalents of a carboxylic acid in the presence of a condensing agent, for example a carbodiimide such as EDCI, and optionally a catalyst such as 4- (dimethylamino)pyridine (“DMAP”), produces diester derivatives. The preparation of 3.1- 3.3 and 3.5-3.7 by this method is shown in Scheme 7.Scheme 7

[0086] On the other hand, treatment with about one molar equivalent of a carboxylic acid in the presence of a condensing agent, for example a carbodiimide such as EDCI, and optionally a catalyst such as DMAP, produces a monoester derivative. For example, dihydroxyketone 4.5 can thus be transformed into 8.1 (Scheme 8). The free OH group in themonoester product can be further esterified with a different carboxylic acid under analogous conditions, leading to the formation of a diester comprising two different acyl groups.

[0087] Alternatively, the OH can be activated for displacement, for example, by conversion into a halide or a sulfonate ester such as mesylate, tosylate, and the like, and then substituted with an appropriate nucleophile. This is exemplified in Scheme 8 with the preparation of 3.4 from 8.1. The latter is converted into mesylate 8.2, which is then transformed into thioacetate 8.3.Scheme 8

[0088] The reaction of the latter with an epoxide, for example, 2-(2-cyclohexylethyl)- oxirane, in the presence of an agent that selectively releases the acetyl group from 8.3, for example, NaOMe, leads to the formation of 8.4. Esterification of the OH group with 3- cyclohexylpropanoic acid in the presence of a condensing agent, for example a carbodiimide such as EDCI and optionally in the presence of DMAP, produces 3.4.

[0089] The synthesis of lipid T-01 from 3.1 (Scheme 9) illustrates a method for the introduction of a type 1 ionizable head group. Thus, 3.1 is transformed into alcohol 9.1 by treatment with a reagent that selectively reduces ketone carbonyls faster than ester carbonyls. For example, 9.1 can be prepared by reaction of 3.1 with a boron hydride in an appropriate solvent, for instance, sodium borohydride in ethanol. Esterification of 9.1 with 4-(dimethylamino)butanoic acid or its hydrochloride in the presence of a condensing agent, for example a carbodiimide such as EDCI, and optionally in the presence of a catalysts such as DMAP, produces lipid T-01.Scheme 9

[0090] By the same method, ketone 3.2 can be converted into lipid T-02. The skilled artisan will further recognize that related type 1 head groups can be installed as described in coowned and co-pending U.S. provisional application No. 63 / 664,792 filed on June 27, 2024.

[0091] The synthesis of lipid T-03 illustrates a method for the introduction of a type 2 ionizable head group. As described in co-owned and co-pending U.S. provisional application No. 63 / 776,506 filed on March 24, 2025, this can be achieved starting with the ketalization of ketone 3.1 with, for example, 4-chloro-l,2-butanediol in the presence of an acid catalyst, for example, pyridinium para-toluenesulfonate (PPTS), in an appropriate solvent, for example, toluene, at a suitable temperature, for example, at reflux, and preferably with continuous removal of water, for example, with a Dean-Stark trap (Scheme 10). Reaction of the resulting 10.1 with dimethylamine in an appropriate solvent, such asTHF, at a temperature between ambient and 120 °C, optionally under conditions of micro wave irradiation, produces T-03.Scheme 10

[0092] By the same method, ketone 3.2 can be converted into lipid T-04. The skilled artisan will further recognize that related type 2 head groups can be installed as described in coowned and co-pending U.S. provisional application No. 63 / 664,792 fded on June 27, 2024.

[0093] The synthesis of lipid T-05 illustrates a method for the introduction of a type 7 ionizable head group. This can be achieved, for example, starting with the reductive amination of 3.3 with 4-((tert-butyldimethylsilyl)oxy)butan-l -amine in a solvent such as CH2CI2 or 1,2-di chloroethane, in the presence of a reducing agent such as, for example, NaBH(OAc)3, and optionally in the presence of an acid catalyst such as acetic acid (Scheme 11). Subsequent reductive methylation amine 11.1 thus obtained, for example, with aqueous formaldehyde solution in a solvent such as, for example, THF, in the presence of a reducing agent such as, for example, NaBH(OAc)3, and optionally in the presence of an acid catalyst such as acetic acid, produces 11.2. Release of the silyl protecting group from 11.2 by treatment with a source of fluoride ion, for example, HF-pyridine complex, gives T-05.Scheme 11

[0094] By the same method, ketone 3.4 can be converted into T-06, 3.2 into T-07, 3.5 into T-08, 3.6 into T-09, and 3.7 into T-lo. The skilled artisan will further recognize that related type 7 ionizable head groups can be installed as described in co-owned and co-pending U.S. provisional application No. 63 / 664,792 fded on June 27, 2024. Such application also provides alternative methods to introduce other ionizable head groups as defined earlier, yet not present in the exemplary, but non-limiting, embodiments of this disclosure.

[0095] Compounds T-ll-T-12 above are lipids of Formula A wherein A3is a nitrogen atom. A lipid of Formula A wherein A3is a nitrogen atom, such as representative, but nonlimiting, compounds T-ll-T-12, can be prepared by methods that are described in detail in co-owned and co-pending PCT applications WO 2024 / 065043, PCT / CA2023 / 050287 and U.S. provisional application No. 63 / 664,788 filed on June 27, 2024, which are incorporated herein by reference. The above applications provide several methods to prepare lipids of the type T-ll-T-12 comprising structurally diverse lipophilic chains. U.S. provisional application No. 63 / 664,788 provides an especially advantageous method for the synthesis of T-ll-T-12 starting with compound 12.1 (Scheme 12). Accordingly, conversion of 12.1 into tosylate 12.2 and subsequent reaction of the latter with thioacetate 12.3 in the presence ofNaOMe produces 12.4. Treatment of 12.4 with a source of fluoride ion, for example, pyridine-HF complex, causes release of the silyl group and formation of alcohol 12.5, which can be transformed into tosylate 12.6. Reaction of the latter with thioacetate 12.7 in the presence of NaOMe gives 12.8. Compounds such as 12.3 and 12.7 can be prepared as described in detail in the above-mentioned co-owned and co-pending application U.S. provisional application No. 63 / 664,788 filed on June 27, 2024. The Boc group can be released from 12.8 by treatment with acid, for example, trifluoroacetic acid (TFA), to produce amine 12.9, or its TFA salt, which can be transformed into T-ll or T-12.BocoScheme 12

[0096] As shown in Scheme 13, reductive amination of 12.9 with aldehyde 13.1 in the presence of a reducing agent, for example, a boron hydride such as NaBH(OAc)s, andoptionally in the presence of an acid, for example, acetic acid, results in formation of 13.2, which can be converted into T-ll by treatment with a source of fluoride ion, for example, pyridine-HF complex. Furthermore, 12.9 can be transformed into T-12 by ^-alkylation with alkyl bromide 13.3 in an appropriate polar solvent, for example, acetonitrile, in the presence of a base, for example, K2CO3, and at a suitable temperature between 40 and 120 °C.Scheme 13

[0097] The person skilled in the art will appreciate that modifications of the synthetic sequences outlined in the above-mentioned applications, for example, the use alternative building blocks in such synthetic sequences, can produce diverse lipids of Formula A wherein A3is a nitrogen atom.Formulation of the above lipids in a delivery vehicle

[0098] The lipids of the disclosure may be formulated in a variety of drug delivery vehicles (also referred to herein as a “delivery vehicle”) known to those of ordinary skill in the art. An example of a delivery vehicle is a lipid nanoparticle, which includes liposomes, lipoplexes, polymer nanoparticles comprising lipids, polymer-based nanoparticles, emulsions, and micelles.

[0099] In one embodiment, a lipid having the structure of Formula A of the disclosure is formulated in a delivery vehicle by mixing them with additional lipids, including helperlipids, such as vesicle forming lipids and optionally an aggregation inhibiting lipid, such as a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid).

[0100] As set forth previously, a helper lipid includes a sterol, a diacylglycerol, a ceramide or derivatives thereof.

[0101] Examples of sterols include cholesterol, or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, and the like.

[0102] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, a DSPC- cholesterol conjugate or mixtures thereof. These lipids may be synthesized or obtained from natural sources, such as from egg. The DSPC-cholesterol conjugate is a lipid in which one of the acyl chains is substituted with a cholesterol moiety link to the head group by a succinate linker.

[0103] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[0104] Delivery vehicles incorporating the lipids of the disclosure can be prepared using a wide variety of well described formulation methodologies known to those of skill in the art, including but not limited to extrusion, ethanol injection and in-line mixing. In one embodiment, the preparation method is an in-line mixing technique in which aqueous and organic solutions are mixed using a rapid-mixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36:133347, each of which is incorporated herein by reference in its entirety.

[0105] The delivery vehicle can also be a nanoparticle that is a lipoplex that comprises a lipid core stabilized by a surfactant. Vesicle-forming lipids may be utilized as stabilizers. The lipid nanoparticle in another embodiment is a polymer-lipid hybrid system that comprises a polymer nanoparticle core surrounded by stabilizing lipid. Nanoparticles comprising lipids of the disclosure may alternatively be prepared from polymers withoutlipids. Such nanoparticles may comprise a concentrated core of a therapeutic agent that is surrounded by a polymeric shell or may have a solid or a liquid dispersed throughout a polymer matrix.

[0106] Lipids described herein can also be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or an oil core. An emulsion can be lipid- stabilized. For example, an emulsion may comprise an oil fdled core stabilized by an emulsifying component such as a monolayer or bilayer of lipids.

[0107] Lipids described herein may be incorporated into a micelle. Micelles are selfassembling particles composed of amphipathic lipids or polymeric components that are utilized for the delivery of agents present in the hydrophobic core.Delivery of nucleic acid, genetic material, proteins, peptides or other charged agents

[0108] Lipids disclosed herein may facilitate the incorporation of a compound or molecule (referred to herein also as “cargo” or “cargo molecule”) bearing a net negative or positive charge into the delivery vehicle and subsequent delivery to a target cell in vitro or in vivo.

[0109] In one embodiment, the cargo molecule is genetic material, such as a nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), micro RNA (miRNA), messenger RNA (mRNA) or DNA such as vector DNA or linear DNA. The nucleic acid length can vary and can include nucleic acid of 5-50,000 nucleotides in length. The nucleic acid can be in any form, including single stranded DNA or RNA, double stranded DNA or RNA, or hybrids thereof. Single stranded nucleic acid includes antisense oligonucleotides.

[0110] In one embodiment, the cargo is an mRNA, which includes a polynucleotide that encodes at least one peptide, polypeptide or protein. The mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in copending U.S. provisional Application No. 63 / 195,269, fded April 1, 2022, which is incorporated herein by reference.

[0111] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.

[0112] In those embodiments in which an mRNA is a chemically synthesized molecule, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is orcomprises 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, C 5 -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, 2-thiocytidine, pseudouridine, and 5 -methylcytidine); 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'-N-phosphoramidite linkages).

[0113] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments 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 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0114] In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0115] The present disclosure may be used to encapsulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length.

[0116] Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0117] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0118] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of locationin a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0119] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0120] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA for in vitro studies or for in vivo studies in animal models to assess biodistribution.

[0121] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[0122] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. A wide variety of different siRNA molecules may be used to target a specific gene or transcript. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-stranded and has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[0123] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing.The snRNA molecules may have therapeutic importance in diseases that are an outcome of splicing defects.

[0124] In another embodiment, the cargo is a DNA vector as described in co-owned and co-pending WO 2022 / 251959, which is incorporated herein by reference. The DNA vectors may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide. Accordingly, the nucleotide polymers can be nucleotide sequences including genomic DNA, cDNA, or RNA.

[0125] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain doublestranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replicating systems such as vector DNA.

[0126] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodi ester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0127] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moieties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronically similar structures, including aza-sugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[0128] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A non-limiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11): 1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically- regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[0129] The nucleic acids used in the present method can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[0130] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs.

[0131] In another embodiment, the DNA vector is a nanoplasmid or a mini circle.

[0132] Gene editing systems can also be incorporated into delivery vehicles comprising the charged lipid. This includes a Cas9-CRISPR, TALEN and zinc finger nuclease gene editing system. In the case of Cas9-CRISPR, a guide RNA (gRNA), together with a plasmid or mRNA encoding the Cas9 protein may be incorporated into a delivery vehicle comprising the lipids described herein. Optionally, a ribonucleoprotein complex may be incorporated into a delivery vehicle comprising the lipid described herein. Likewise, the disclosure includes embodiments in which genetic material encoding DNA binding and cleavage domains of a zinc finger nuclease or TALEN system are incorporated into a delivery vehicle together with the lipids of the disclosure.

[0133] While a variety of nucleic acid cargo molecules are described above, it will be understood that the above examples are non-limiting and the disclosure is not to be considered limiting with respect to the particular cargo molecule encapsulated in the delivery vehicle.

[0134] For example, the lipids described herein may also facilitate the incorporation of proteins and peptides into a delivery vehicle, which includes ribonucleoproteins. This includes both linear and non-linear peptides, proteins or ribonucleoproteins.

[0135] While pharmaceutical compositions are described above, the lipids described herein can be a component of any nutritional, cosmetic, cleaning or foodstuff product. Pharmaceutical formulations

[0136] The ionizable lipids of the disclosure may be present in a salt form. The salt is typically a pharmaceutically acceptable salt. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, and zinc. In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and the like.

[0137] In some embodiments, the delivery vehicle comprising the cargo molecule is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0138] In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral or in-utero administration. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0139] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0140] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

[0141] The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.

[0142] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated.EXAMPLESMaterials

[0143] The lipid l,2-distearoyl-5«-glycero-3-phosphorylcholine (DSPC) was purchased from Lipoid Inc. (Newark, NJ). l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and lOx Phosphate Buffered Saline (pH 7.4) were purchased from Sigma Aldrich (St Louis, MO). The ionizable amino-lipid was synthesized as previously described in WO 2022 / 246555, which is incorporated herein by reference.

[0144] An mRNA encoding firefly luciferase synthesized by NanoVation Therapeutics (Vancouver, BC) was used to analyse luciferase activity.MethodsPreparation of lipid nanoparticles (LNP) containing mRNA

[0145] Lipids T-05, T-07, T-08, T-09 and T-10 described herein, DSPC, cholesterol, and PEG2000-DMG, were dissolved in ethanol at the appropriate ratios to a final concentration of 10 or 20 mM total lipid. Nucleic acid (mRNA) was dissolved in an appropriate buffer such as 25 mM sodium acetate pH 4 or sodium citrate pH 4 to a concentration necessary to achieve the appropriate amine-to-phosphate ratios. The aqueous and organic solutions were mixed using a rapid-mixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36:133347 (each incorporated herein by reference) at a flow rate ratio of 3: 1 (v / v; respectively) and a total flow rate of 20 mL / min. The resultant mixture was dialyzed directly against 1000-fold volume of PBS pH 7.4. All formulations were concentrated using an Amicon™ centrifugal filter unit and analysed using the methods described below.Analysis of LNP

[0146] Particle size analysis of LNPs in PBS was carried out using backscatter measurements of dynamic light scattering with a Malvern Zetasizer™ (Worcestershire, UK). The reported particle sizes correspond to the number-weighted average diameters (nm). Total lipid concentrations were determined by extrapolation from the cholesterol content, which was measured using the Cholesterol E-Total Cholesterol Assay (Wako Diagnostics, Richmond, VA) as per the manufacturer’s recommendations. Encapsulation efficiency of the formulations was determined using the Quant-iT RiboGreen™ Assay kit (Invitrogen, Waltham, MA). Briefly, the total mRNA content in solution was measured by lysing lipid nanoparticles in a solution of TE containing 2% Triton Tx-100, and free DNA vector in solution (external to LNP) was measured based on the RiboGreen™ fluorescence in a TE solution without Triton. Total mRNA content in the formulation was determined using a modified Bligh-Dyer extraction procedure. Briefly, LNP formulations containingmRNA were dissolved in a mixture of chloroform, methanol, and PBS that results in a single phase and the absorbance at 260 nm measured using a spectrophotometer.In vivo analysis in CD-I mice

[0147] LNP-mRNA encoding firefly luciferase were injected intravenously (tail-vein) into 6-8 week old CD-I mice. Four hours following injection, the animals were euthanized and the liver and spleen and isolated. Tissue was homogenized in Gio Lysis™ buffer and a luciferase assay performed using the Steady Gio Luciferase™ assay kit (as per manufacturers recommendations).Organic synthesis of lipids T-01-T-12.

[0148] Unless otherwise specified, all reagents and solvents were commercial products and were used without further purification. All reactions were performed under a nitrogen atmosphere. Reaction mixture from aqueous workups were dried by passing over a plug of anhydrous Na2SO4 held in a filter tube and concentrated under reduced pressure on a rotary evaporator. Thin-layer chromatography was performed on silica gel plates coated with silica gel (Merck 60 F254 plates) and column chromatography was performed on 230-400 mesh silica gel. Visualization of the developed chromatogram was performed by staining with L or potassium permanganate solution.JH and13C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCh solutions.JH NMR spectra were referenced to residual CHCI3 (7.26 ppm) and13C NMR spectra were referenced to the central line of the CDCh triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the 5 scale. Multiplicities are reported as “s” (singlet), “d” (doublet), “f ’ (triplet), “q” (quartet), “m” (multiplet), and further qualified as “app” (apparent) and “br” (broad). Low- and high- resolution mass spectra (m / z) were obtained in the electrospray (ESI) mode.Nor-MC3 control

[0149] The ionizable lipid, nor-MC3, was used in Example 2 as a control. This ionizable lipid has the structure:nor-MC3

[0150] WO 2022 / 246571 describes the synthesis of the foregoing ionizable lipid and is incorporated herein by reference.Example 1: Methods for chemically synthesizing representative ionizable lipids of this disclosureSynthesis of building blocks4-chlorobutane-l,2-diol.

[0151] Triethylamine (31 mL, 222 mmol, 1.5 equiv) added slowly and dropwise to a cold (0°C), well-stirred solution of commercial 2-(2,2-dimethyl-l,3-dioxolan-4-yl)ethan-l-ol (21.6 g, 148 mmol, 1.0 equiv) and MsCl (13.7 mL, 178 mmol, 1.2 equiv) in CH2CI2 (150 mL). The reaction mixture was allowed to warm up to room temperature and stirred for 8 h, then quenched with water. The organic layer was separated, and the aqueous layer was further extracted with CH2CI2 (2 x 50 mL). The combined organic layers were washed with brine (sat. solution), dried (NfeSCL), fdtered, and concentrated in vacuo to afford 2-(2,2- dimethyl-l,3-dioxolan-4-yl)ethyl methanesulfonate (33.2 g, 148 mmol, -100%) as a colorless oil that was used for the next step without purification.XH NMR (400 MHz, CDCh) 84.43-4.31 (m, 2H), 4.22 (m, 1H), 4.11 (dt, 1H, J= 8.1, 4.7 Hz), 3.60 (dd, 1H, J = 8.2, 6.6 Hz), 3.02 (s, 3H), 2.08-1.90 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H).13C NMR (100 MHz, CDCh) 8 109.3, 72.1, 69.1, 67.0, 37.3, 33.5, 27.0, 25.6. A solution of 2-(2,2- dimethyl-l,3-dioxolan-4-yl)ethyl methanesulfonate (21.9 g, 97.6 mmol, 1.0 equiv), anhydrous LiCl (8.27 g, 195 mmol, 2 equiv), and NaHCCh (8.2 g, 97.6 mmol, 1 equiv) in dry N, ^-dimethyl formamide (50 mL) was stirred at 60 °C for 24 h. The reaction mixture was quenched with water (200 mL) and extracted with diethyl ether (3 x 100 mL). The combined organic layers were washed with water, brine (sat. solution), dried (Na2SO4), filtered, and concentrated in vacuo to afford a crude of 4-(2-chloroethyl)-2,2-dimethyl-l,3- dioxolane (14.5 g, 88.1 mmol, 91%) as a colorless oil that was used for the next step without purification. This crude material was dissolved in methanol (20 mL) and treated with IM aqueous HC1 (5 mL). The mixture was spun on the rotary evaporator at atmospheric pressure and at a bath temperature of 50 °C for 2h, then it was evaporated to dryness in vacuo to afford 4-chlorobutane-l,2-diol (11 g, 88.3 mmol, -100%) as a colorless oil that was used for the next step without purification. 'H NMR (400 MHz, CDCh) 8 3.99 (m, 1H), 3.79-3.66 (m, 3H), 3.51 (dd, 1H, J= 11.1, 7.1 Hz), 2.12 (br s, 2H), 2.00-1.79 (m, 2H).13C NMR (100 MHz, CDCh) 8 69.3, 66.7, 41.7, 35.7.4-(( / c / 7-Butykliniethylsilyl)oxy )butan-l -amine.H2N^^OTBS

[0152] Solid TBSC1 (3.72 g, 24.7 mmol, 1.1 eq) was added to solution of 4-aminobutanol (2.0 g, 22.4 mmol, 1 equiv) and imidazole (1.83 g, 26.9 mmol, 1.2 equiv) in CH2CI2 (25 mL) at rt and under N2. The mixture was stirred for 18 h at rt, then poured into waster. The organic layer was separated, and the aq. phase was extracted with CH2CI2 (2 x 30 mL). The combined organic layers were sequentially washed with aqueous IM NaOH and brine, dried (NfeSCL), and concentrated in vacuo to afford the title compound as a colorless oil that was used for the next step without further purification. 'H NMR (400 MHz, CDCh) 8 3.62 (t, J = 6.1 Hz, 2H), 2.71 (t, J= 6.7 Hz, 2H), 1.63 - 1.42 (m, 6H), 0.89 (s, 9H), 0.05 (s, 6H). 5-cyclohexylpentan-l-ol.

[0153] To a solution of 5-cyclohexylpentanoic acid (25 g, 136 mmol) in THF (230 mL) in a RBF under inert atmosphere, was added borane DMS complex (18 mL, 190 mmol) over 30 min via syringe pump at 0°C. The mixture was stirred for 1.5 h at RT then cooled to 0°C and slowly quenched with water (250 mL). The mixture was basified with 7M NaOH (5 mL) and diluted with hexanes (100 mL). The layers were allowed to separate and the organic layer was collected. The aqueous phase was back extracted with hexanes (2x200 mL). The combined organic phases were washed with 1 M NaOH (200 mL), brine (100 mL), dried (Na2SO4), filtered though celite, and evaporated to yield the desired alcohol (97% yield). This was carried forward without further purification.1H NMR (400 MHz, CDCh) 8 3.63 (t, J= 6.6 Hz, 2H), 1.72 - 1.04 (m, 17H), 0.91 - 0.78 (m, 2H).13C NMR (101 MHz, CDCh) 8 63.2, 37.7, 37.6, 33.6, 33.0, 26.9, 26.8, 26.6, 26.2.5-cyclohexylpentyl methanesulfonate.

[0154] To a solution of 5-cyclohexylpentan-l-ol (21.3 g, 125 mmol) and triethylamine (26 mL, 190 mmol) in DCM (125 mL) in an RBF under inert atmosphere, was added methanesulfonyl chloride (11.6 mL, 150 mmol) dropwise via syringe at 0°C. The mixture was stirred for 30 min at 0°C then quenched with water (250 mL). The biphasic mixture was separated, and the organic layer was collected. The organic phase was washed with water (250 mL), dried (Na2SO4), filtered, and evaporated to give a black liquid. This was purified by silica plug (-120 g silica) eluding with 15% EtOAc in hexanes (1000 mL) to yieldenriched 5 -cyclohexylpentyl methanesulfonate as a yellow oil (90% yield). 'H NMR (400 MHz, CDCh) 84.22 (t, J= 6.6 Hz, 2H), 3.00 (s, 3H), 1.79 - 1.06 (m, 17H), 0.92 - 0.79 (m, 2H).(5-bromopentyl)cyclohexane.

[0155] To a solution of 5-cyclohexylpentyl methanesulfonate (27.9 g, 112 mmol) in DMF (112 mL) in an RBF under inert atmosphere was added LiBr (11.7 g, 135 mmol). The mixture was stirred for 1 h at 70°C then diluted with water (120 mL) and hexanes (100 mL). The biphasic mixture was separated and the organic layer was collected. The aqueous phase was back-extracted with hexanes (2x100 mL). The combined organic phases were dried (NfeSCL), filtered through silica (-100 g silica) eluding with hexanes, then evaporated to give (5-bromopentyl)cyclohexane as a colorless liquid. 'H NMR (400 MHz, CDCh) 8 3.41 (t, J= 6.9 Hz, 2H), 1.90 - 1.80 (m, 2H), 1.73 - 1.60 (m, 5H), 1.45 - 1.07 (m, 10H), 0.91 - 0.79 (m, 2H).13C NMR (101 MHz, CDCh) 837.7, 37.4, 34.2, 33.5, 33.0, 28.6, 26.9, 26.6, 26.1.But-3-en-l-ylcyclohexane.

[0156] Commercial (4-bromobutyl)cyclo-hexane (116 mmol, 25.4 g) was added to a RBF under inert atmosphere containing 1.0 M tBuOK (139 mmol) in THF (139 mL). The reaction was then stirred at RT for 30 min. The reaction was diluted with hexanes (200 ml) and fdtered through celite, then evaporated to yield a dark crude oil. The crude oil was fdtered through a silica plug eluding with hexanes (200 mL), then evaporated (Note 1), yielding the desired alkene (>95% yield). This was carried forward without further purification.XH NMR (400 MHz, CDCh) 85.81 (ddt, J= 16.9, 10.2, 6.7 Hz, 1H), 5.03-4.96 (m, 1H), 4.95-4.89 (m, 1H), 2.09-2.01 (m, 2H), 1.78-1.00 (m, 11H), 0.94-0.81 (m, 2H).

[0157] The following compound was prepared by the same method: Pent-4-en-l-ylcyclohexane.

[0158] >95 % yield from (5-bromopentyl)cyclohexane (27 g, 116 mmol). 'H NMR (400 MHz, CDCh) 8 5.82 (ddt, J= 16.9, 10.2, 6.7 Hz, 1H), 5.02 - 4.96 (m, 1H), 4.95 - 4.89 (m, 1H), 2.06 - 1.98 (m, 2H), 1.79 - 1.00 (m, 13H), 0.93 - 0.79 (m, 2H).General Procedure for epoxidation.

[0159] Aqueous 30% H2O2 (2 eq.) was added via syringe pump over 5 h to a 0.3 M solution of an alkene (33 mmol, 1 equiv) and formic acid (100 mmol, 3 eqiv) in CH2CI2, under inert atmosphere. The mixture was stirred for 16 h then evaporated. The residue liquid was diluted with hexanes (100 mL) and water (100 mL). The layers were separated and the organic phase was collected. The aqueous phase was back extracted with hexanes (2x50 mL). The combined organic phases were washed with sat NaHCCti (2x100 mL), dried (Na2SO-i). filtered, and evaporated to yield a crude epoxide product. This was further purified by Kugelrohr distillation yielding the desired epoxide.

[0160] The following compounds were thus prepared:2-Cyclohexyloxirane.

[0161] From vinylcyclohexane (16.5g, 150 mmol) to yield (15 g, 119 mmol, 79%) of desired epoxide.XH NMR (400 MHz, CDCh) 82.75 - 2.67 (m, 2H), 2.52 (dd, J= 4.5, 3.3 Hz, 1H), 1.90 - 1.83 (m, 1H), 1.80 - 1.62 (m, 4H), 1.31 - 1.02 (m, 6H).13C NMR (101 MHz, CDCh) 8 56.8, 46.2, 40.5, 29.9, 29.0, 26.5, 25.8, 25.7.2-(2-cyclohexylethyl)oxirane.

[0162] From but-3-enylcyclohexane (19.1 g, 138 mmol) to yield (17.7 g, 115 mmol, 83%) of desired epoxide. 'H NMR (400 MHz, CDCh) 82.91 (tdd, J= 5.6, 3.9, 2.7 Hz, 1H), 2.76 (dd, J= 5.0, 4.0 Hz, 1H), 2.48 (dd, J= 5.0, 2.7 Hz, 1H), 1.80 - 1.09 (m, 13H), 0.95 - 0.84 (m, 2H).2-(3-Cyclohexylpropyl)oxirane.

[0163] From pent-4-enylcyclohexane (22.4 g, 147 mmol) to yield (11.3 g, 67.2 mmol,2.74 (dd, J= 5.0, 4.0 Hz, 1H), 2.46 (dd, J= 5.1, 2.7 Hz, 1H), 1.75 - 1.10 (m, 13H), 0.93 - 0.80 (m, 4H).Procedure for thioacetate formation: methyl 6-(acetylthio)hexanoate (5.2, m = 5).

[0164] To an RBF under inert atmosphere was added DMF (120 mL) and methyl-6- bromohexanoate (25.0 g, 0.12 mol, 1 eq.). The solution was degassed for 10 min, then triethylamine (1.5 eq., 0.18 mol, 18 g, 25 mL) followed by thioacetic acid (1.2 eq., 0.14 mol, 10 mL) were carefully added at RT. The mixture was stirred 1.5 h at 60°C, then diluted with water (100 mL) and extracted with hexanes (100 mL). The aqueous phase was back extracted with hexanes (2x50 mL). The combined organic phases were dried (Na2SO4), decolorized with carbon, filtered through celite and evaporated to yield methyl 6- (acetylthio)hexanoate (24.0 g, 0.12 mol, >95% yield). The product was carried forward without further purification. 'H NMR (400 MHz, CDCh) 5 3.66 (s, 3H), 2.85 (t, J= 7.4 Hz, 2H), 2.32 (s, 3H), 2.28 (t, J= 7.7 Hz, 2H), 1.68-1.23 (m, 6H).

[0165] The following compound was prepared by the same method:Methyl 7-(acetylthio)heptanoate (5.2, m = 6).AcS\^\^^^^COOMe

[0166] >95% yield from ethyl 7-bromoheptanoate (50 g, 0.21 mol). 'H NMR (400 MHz, CDCh) 54.11 (q, J= 7.2 Hz, 2H), 2.85 (t, J= 7.3 Hz, 2H), 2.31 (s, 3H), 2.28 (t, J= 7.5 Hz, 2H), 1.66 - 1.50 (m, 4H), 1.41 - 1.30 (m, 4H), 1.25 (t, J= 7.1 Hz, 3H).Procedure for epoxide opening with a thiolate.

[0167] To a round bottom flask (RBF) under inert atmosphere containing degassed (20 min N2 sparge) methanol (19.4 mL) was added a thioacetate (1 eq., 19.4 mmol). Then NaOMe 25 wt% in MeOH (2 eq., 8.6 mL, 38.7 mmol) was added via syringe. The mixture was stirred for 5 min at RT, then an epoxide (1 eq., 19.4 mmol) was added, then stirred for 15 min at RT. The reaction was then quenched with sat. NH4CI (20 mL), diluted with water (30 mL), and extracted with hexanes (40 mL). The aqueous phase was back extracted with hexanes (2 x 40 mL). The combined organic phases were dried (Na2SO4), filtered and evaporated to yield the crude product as an amber oil (typically -90% yield). The product was carried forward without further purification.

[0168] The following compounds were thus prepared:Methyl 6-((2-cyclohexyl-2-hydroxyethyl)thio)-hexanoate (5.3, m = 5, n = 0).

[0169] 91% yield from methyl 6-(acetylthio)hexanoate (16.8 g, 82.2 mmol) and 2-(2- cyclohexylethyl)oxirane (12.7 g, 82.2 mmol). 'H NMR (400 MHz, CDCh) 5 3.67 (s, 3H), 3.41 - 3.34 (m, 1H), 2.77 (dd, J= 13.5, 2.9 Hz, 1H), 2.54 - 2.43 (m, 3H), 2.31 (t, J= 7.4 Hz, 2H), 1.91 - 0.97 (m, 17H).Methyl 6-((4-cyclohexyl-2-hydroxybutyl)thio)hexanoate (5.3, m = 5, n = 2).

[0170] 91% yield from methyl 6-(acetylthio)hexanoate (16.8 g, 82.2 mmol) and 2-(2- cyclohexylethyl)oxirane (12.7 g, 82.2 mmol. 'H NMR (400 MHz, CDCh) 5 3.66 (s, 3H), 3.63 - 3.55 (m, 1H), 2.72 (dd, J= 13.6, 3.3 Hz, 1H), 2.52 (t, J= 7.4 Hz, 2H), 2.42 (dd, J =13.6, 9.0 Hz, 1H), 2.31 (t, J= 7.4 Hz, 2H), 1.75 - 1.09 (m, 19H), 0.93 - 0.81 (m, 2H).Methyl 7-((2-cyclohexyl-2-hydroxyethyl)thio)heptanoate (5.3, m = 6, n = 0). OHCOOMe

[0171] >95% yield from ethyl 7-acetylsulfanylheptanoate (1.17 g, 5.0 mmol) and 2- cyclohexyloxirane (0.64 g, 5.0 mmol).XH NMR (400 MHz, CDCh) 8 3.66 (s, 3H), 3.37 (ddt, J= 9.2, 6.0, 3.0 Hz, 1H), 2.77 (dd, J= 13.5, 2.9 Hz, 1H), 2.56 (d, J = 3.0 Hz, 1H), 2.52 - 2.41 (m, 3H), 2.30 (t, J= 7.5 Hz, 2H), 1.91 - 1.82 (m, 1H), 1.79 - 1.53 (m, 8H), 1.46 - 0.97 (m, 10H).13C NMR (101 MHz, CDCh) 8 174.3, 73.1, 51.6, 42.9, 37.9, 34.1, 32.1,29.6, 29.2, 28.8, 28.5, 28.5, 26.6, 26.3, 26.2, 24.9.Methyl 6-((5-cyclohexyl-2-hydroxypentyl)thio)hexanoate (5.3, m = 5, n = 3).5.3 m = 5, n = 3

[0172] >95% yield from methyl 6-(acetylthio)hexanoate (11.3 g, 55.3 mmol) and 2-(3- cyclohexylpropyl)oxirane (10.0 g, 55.3 mmol). 'H NMR (400 MHz, CDCh) 8 3.69 (s, 3H), 3.68 - 3.61 (m, 1H), 2.75 (dd, J= 13.6, 3.3 Hz, 1H), 2.59 - 2.52 (m, 3H), 2.45 (dd, J= 13.6, 9.1 Hz, 1H), 2.34 (t, J= 7.4 Hz, 2H), 1.77 - 1.12 (m, 21H), 0.95 - 0.81 (m, 2H).Procedure for O-TMS protection: methyl 6-((2-cyclohexyl-2- ((trimethylsilyl)oxy)ethyl)-thio)hexanoate (5.4, m = 5, n = 0).

[0173] To an RBF under inert atmosphere at 0°C was added methyl 6-((2-cyclohexyl-2- hydroxyethyl)thio)hexanoate (19.3 g, 67 mmol), DCM (67 mL), TEA (14 mL, 100 mmol), and TMSC1 (10.2 mL, 80 mmol). The mixture was stirred for 1 h at RT, then quenched with sat. NH4CI (50 mL) and extracted with DCM (50 mL). The aqueous phase was back- extracted with DCM (2x50 mL). The combined organic phases were dried (Na2SO4), fdtered and evaporated to yield crude product. This was passed though a silica plug eluding with 10% EtOAc in hexanes (1000 mL), and evaporated to yield pure the TMS protected alcohol (24 g, 66 mmol, >95% yield). 'H NMR (400 MHz, CDCh) 8 3.66 (s, 3H), 3.53 (q, J= 5.5 Hz, 1H), 2.64 (dd, J= 13.2, 5.4 Hz, 1H), 2.58 - 2.49 (m, 3H), 2.31 (t, J= 7.5 Hz, 2H), 1.79 - 0.82 (m, 17H), 0.12 (s, 9H).

[0174] The following compounds were prepared by the same method:Methyl 6-((4-cyclohexyl-2-((trimethylsilyl)oxy)butyl)thio)hexanoate (5.4, m = 5, n = 2).

[0175] 82% yield from methyl 6-((4-cyclohexyl-2-hydroxybutyl)thio)hexanoate (23.7 g, 75 mmol). 'H NMR (400 MHz, CDCh) 8 3.74 - 3.67 (m, 1H), 3.66 (s, 3H), 2.58 - 2.49 (m, 4H), 2.35 - 2.28 (m, 2H), 1.78 - 0.99 (m, 19H), 0.93 - 0.80 (m, 2H), 0.12 (s, 9H).Methyl 7-((2-cyclohexyl-2-((trimethylsilyl)oxy)ethyl)thio)heptanoate (5.4, m = 6, n = 0).

[0176] 63% yield from methyl 7-(2-cyclohexyl-2-hydroxy-ethyl)sulfanylheptanoate (1.9 g, 6.3 mmol).XH NMR (400 MHz, CDCh) 8 3.66 (s, 3H), 3.53 (q, J= 5.3 Hz, 1H), 2.64 (dd, J = 13.1, 5.4 Hz, 1H), 2.57 - 2.48 (m, 3H), 2.30 (t, J= 7.5 Hz, 2H), 1.79 - 0.90 (m, 19H), 0.12 (s, 9H).13C NMR (101 MHz, CDCh) 8 174.3, 76.6, 51.6, 42.4, 37.1, 34.1, 33.3, 29.8, 29.7, 28.9, 28.7, 27.7, 26.7, 26.5, 26.4, 25.0, 0.7.Methyl 6-((5-cyclohexyl-2-((trimethylsilyl)oxy)pentyl)thio)hexanoate (5.4, m = 5, n = 3).

[0178] Imidazole (5 g, 74.1 mmol) was added over 20 min to 7-bromoheptan-l-01 (4.8 g, 24.7 mmol) and tert- butyldimethylsilyl chloride (4.47 g, 29.64 mmol) in dimethylformamide (30 ml) at 0 °C. The reaction was warmed to room temperature and stirred for 6 hours, diluted with water and extracted with ether (3 x 20.0 mL). The combined extracts were dried (Na2SO4) and concentrated gave a pale-yellow oil (7.2 g). Distillation via kugelrohr at 140 °C, 0.02-0.04 mm Hg afforded the desired product (6.2 g, 82%) as a colorless oil.XH NMR (400 MHz, CDCI3) 8 3.56-3.53 (t, J= 6.54 Hz, 2H), 3.50-3.46 (t, J = 6.54 Hz, 1H), 3.37-3.36 (t, J = 6.87 Hz, 1H), 1.82-1.70 (m, 2H), 1.48-1.26 (m, 8H), 0.84 (s, 9H), 0.001 (s, 6 H).6-(Benzyl(7-((te / 7-butyldimethyl-silyl)oxy)heptyl)amino)hexan-l-ol

[0179] A solution of 6-(benzylamino)hexan-l-ol (3 g , 14.5 mmol) and ((7- bromoheptyl)oxy)( / c / 7-butyl)dimethyl-silane (4.5 g, 14.5 mmol) in dry acetonitrile (10 mL) containing suspended K2CO3 (3 g, 21.7 mmol, 1.5 equiv) was heated to 75 °C under argon and stirred for 16 hours, diluted with water and extracted with EtOAc (3 x 50 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-50% EtOAc in hexanes) to yield the desired product (4.2 g, 67%).XH NMR (400 MHz, CDCh) 8 7.28-7.15 (m, 5H,), 3.57-3.52 (m, 4H,), 3.48 (s, 2H), 2.35-2.31 (m, 4H,), 1.52-1.40 (m, 9H), 1.26 -1.21 (m, 10H), 0.84 (s, 9H), 0.001 (s, 6 H). / (77-Butyl (7-((ter / -butyldimethyIsilyl)oxy)heptyl)(6-hydroxyhexyI)carbamate (12.1).

[0180] Palladium hydroxide (581 mg, 4.2 mmol) was added to a solution of 6-(benzyl(7- ((ferEbutyl-dimethylsilyl)oxy)heptyl)amino)hexan-l-ol (3.6 g, 8.3 mmol) and di- / c / 7-butyl dicarbonate (2.1 g, 9.9 mmol) in methanol (30 mL) and the mixture was stirred under hydrogen at rt for 12 hours. The solution was filtered through Celite and the filtrate was concentrated under reduced pressure. The residue was purified by silica chromatography (0- 30% EtOAc in hexanes) to yield 12.1 (3.2 g, 89%).XH NMR (400 MHz, CDC13) 8 3.58- 3.53 (m, 4H,), 3.08 (brs, 4H), 1.55-1.22 (m, 30H,), 0.82 (s, 9H), 0.001 (s, 6 H).Procedure for the synthesis of ketones 4: l,ll-bis((2-cyclohexyl-2-hydroxyethyl)thio)- undecan-6-one (4.1).

[0181] A solution of methyl 6-((2-cyclohexyl-2-((trimethylsilyl)oxy)ethyl)-thio)hexanoate (23.9 g, 66 mmol), and tri butylamine (1.8 eq.) in toluene (0.6 M) in a 2 L RBF maintained under N2 was cooled to -20° C. Then a 50% v / v solution of TiCU (1.5 eq.) in toluene was added via syringe pump over 1.5 hours under vigorous stirring. The reaction was found to be complete after the TiCU addition. The reaction was quenched by slowly pouring it into a rapidly stirred mixture of sat. NH4CI (500 mL) and hexanes (200 mL). Stirring was continued until the dark-red color faded away (~10 min). Additional water was added to dissolve white precipitates. The layers were separated and the organic phase was collected. Any remaining dark-red liquid (titanium complex) appearing as a third phase was recovered and quenched with additional sat. NH4CI. The aqueous phase was back extracted with hexanes (2x300 mL). The combined organic phases were sequentially washed with 20% aq. EDTA @ pH 9 (2x250 mL; removal of remaining titanium) and brine (200 mL), dried (Na2SO4), filtered, and evaporated to yield crude beta-ketoester as an amber oil. This crude product was diluted with 1,4-dioxane (0.8 M) under inert atmosphere. Then 7.5 N aq. NaOH (6 eq.) was added. The mixture was heated at 65°C for 0.5 h, then acidified with cone. aq. HC1 (7 eq). The mixture was heated at 65°C for 2 h. The mixture was then diluted with EtOAc (100 mL) and water (100 mL). The mixture was allowed to separate and the organic phase was collected. The aqueous phase was back-extracted EtOAc (IxlOOmL). The combined organic phases were washed with sat. NaHCOs (150mL), dried (Na2SO4), filtered, and rotary evaporated. The solid residue was rerystallized from hot EtOAc (150 mL) gave pure product (20% yield from methyl 6-((2-cyclohexyl-2-((trimethylsilyl)oxy)-ethyl)thio)hexanoate). 'H NMR (400 MHz, CDCh) 8 3.41 - 3.34 (m, 2H), 2.77 (dd, J = 13.5, 2.9 Hz, 2H), 2.55 - 2.42 (m, 8H), 2.39 (t, J= 7.3 Hz, 4H), 1.92 - 0.96 (m, 32H).13C NMR (101 MHz, CDCh) 8211.0, 73.1, 43.0, 42.7, 38.0, 32.0, 29.6, 29.2, 28.5, 28.5, 26.6, 26.3, 26.2, 23.4.

[0182] The following compounds were prepared by the same method: l,ll-bis((4-Cyclohexyl-2-hydroxybutyl)thio)undecan-6-one (4.2).

[0183] 75% yield from methyl 7-((4-cyclohexyl-2-((trimethylsilyl)oxy)butyl)thio)- heptanoate (26.9 g, 67 mmol). The crude product was purified by column chromatography (0-40% EtOAc in hexanes over 12 CV) instead of precipitation. 'H NMR (400 MHz, CDCh) 8 3.63 - 3.54 (m, 2H), 2.73 (dd, J= 13.6, 3.3 Hz, 2H), 2.56 - 2.49 (m, 6H), 2.46 - 2.37 (m, 6H), 1.75 - 1.09 (m, 38H), 0.94 - 0.81 (m, 4H).13C NMR (101 MHz, CDCh) 8 211.0, 69.7, 40.5, 37.9, 33.8, 33.5, 33.5, 33.4, 32.2, 29.7, 28.5, 26.8, 26.5, 26.5, 23.4. l,13-bis((2-Cyclohexyl-2-hydroxyethyl)thio)tridecan-7-one (4.3).

[0184] 39% yield from methyl 7- ((2-cyclohexyl-2-((trimethyl-silyl)oxy)ethyl)thio)- heptanoate (0.4 g, 0.78 mmol).XH NMR (400 MHz, CDCh) 8 3.38 (ddd, J= 9.3, 6.0, 2.9 Hz, 2H), 2.77 (dd, J= 13.5, 2.9 Hz, 2H), 2.57 (br.s, 2H), 2.53 - 2.43 (m, 6H), 2.38 (t, J = 7.4 Hz, 4H), 1.90 - 1.83 (m, 2H), 1.80 - 0.96 (m, 36H).13C NMR (101 MHz, CDCh) 8 211.3, 73.1, 42.9, 42.8, 38.0, 32.1, 29.6, 29.2, 28.9, 28.7, 28.5, 26.6, 26.3, 26.2, 23.8. l,ll-bis((5-Cyclohexyl-2-hydroxypentyl)thio)undecan-6-one (4.4).4.4

[0185] 23% yield from methyl 6- ((5-cyclohexyl-2-((trimethylsilyl)oxy)pentyl)thio)- hexanoate (19.1 g, 47.4 mmol). The crude product was purified by column chromatography (0-40% EtOAc in hexanes over 12 CV) instead of precipitation. 'H NMR (400 MHz, CDCh) 8 3.66 - 3.59 (m, 2H), 2.72 (dd, J= 13.6, 3.3 Hz, 2H), 2.57 - 2.48 (m, 6H), 2.46 - 2.36 (m, 6H), 1.73 - 1.06 (m, 42H), 0.91 - 0.79 (m, 4H).13C NMR (101 MHz, CDCh) 8 210.8, 69.2, 42.6, 40.4, 37.6, 37.6, 37.5, 36.6, 33.4, 33.4, 32.0, 29.5, 28.4, 26.4, 23.3, 23.1.Procedure for Claisen condensation of a lactone: l,ll-dihydroxyundecan-6-one (4.5).4.5

[0186] Neat TiCU (25.0 mL, 227.8 mmol, 1.3 equiv) was slowly added (syringe pump) over 30 minutes to a cold (-78 °C) solution of caprolactone (20.0 g, 19.4 mL, 175.2 mmol,I.0 equiv.) and EhN (36.6 mL, 262.8 mmol, 1.5 equiv) in CH2CI2 (150 mL) under N2 (balloon). The mixture was warmed to room temperature and stirred for 5 h, then it was poured into cold water (100 mL). The organic layer was removed, and the aqueous layer was extracted with 95:5 CFbCkMeOH (5x100 mL). The combined extracts were evaporated in vacuo, and the residue of crude 10 was taken up in CH2CI2 (50 mL) and IM aq. HC1 (50 mL). The flask containing the mixture was placed back on the rotary evaporator, immersed in a water bath at 60 °C and spun at atmospheric pressure for 5 h. The residual aqueous solution was extracted with 95:5 CLEC^MeOH (5x100 mL). The combined extracts were washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by crystallization from 2: 1 diethyl ether: / 7-hexane (cooling to -20 °C). The precipitate was recovered by filtration and dried to afford 4.5 (16.2 g, 79.7 mmol, 91%) as an off white solid, m.p. 57 °C (Momenteau, M.; Loock, B.; Huel, C.; Lhoste,J.-M. J. Chem. Soc., Perkin Trans. 11988, 283-296; m.p. 58.5 °C).XH NMR (300 MHz, CDCh) 8 3.66 (4 H, t, J 6.5, 2xCH2OH), 2.44 (4 H, t, J 7.2, 2xCH2CO), 1.59 (8 H, m, 4XCH2), 1.38 (4 H, m, 2xCH2).13C NMR (100 MHz, CDCh) 8211.7 (CO), 62.6 (2XOCH2), 42.8 (2XCH2), 32.5 (2xCH2), 25.5 (2xCH2), 23.6 (2 x CH2).Procedure for bis-esterification of a dihydroxyketone: ((6-oxoundecane-l,ll-diyl)bis- (sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (3.1).To an RBF containing l,l l-bis((2-cyclohexyl-2-hydroxyethyl)thio)undecan-6-one (1 eq. 6.6 mmol, 3.2 g) under inert atmosphere was added DCM (1 M), alkyl carboxylic acid (2.4 eq.), EDCI-HC1 (2.4 eq.), and DMAP (3 eq.). The mixture was stirred for 18 h then diluted with water (20 mL) and hexanes (20 mL). The layers were separated and the organic phase was collected. The aq. phase was back-extracted with hexanes (2x20mL). The combined organics were washed with IN HC1 (50 mL). sat. NaHCOs (50 mL), dried (Na2SO4),fdtered, and evaporated to yield a crude product. This was then purified by automated silica gel chromatography (0-10% EtOAc in hexanes over 10 CV) to yield ((6-oxoundecane-l,ll- diyl)bis-(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (85% yield).XH NMR (400 MHz, CDCI3) 84.85 - 4.79 (m, 2H), 2.71 (dd, J= 13.8, 4.9 Hz, 2H), 2.61 (dd, J= 13.8, 7.4 Hz, 2H), 2.55 - 2.47 (m, 4H), 2.38 (t, J= 7.4 Hz, 4H), 2.36 - 2.30 (m, 4H), 1.80 - 0.81 (m, 60H).

[0187] The following compounds were prepared by the same method:((6-Oxoundecane-l,ll-diyl)bis(sulfanediyl))bis(4-cyclohexylbutane-l,2-diyl) bis(3- cyclohexylpropanoate) (3.2).

[0188] 70% yield from 1 J l-bis((4-cy cl ohexyl-2 -hydroxy butyl)thio)- undecan-6-one (15.0 g, 18.3 mmol).XH NMR (400 MHz, CDC13) 5 5.00 - 4.91 (m, 2H), 2.70 - 2.59 (m, 4H), 2.55 (tt, J= 7.4, 3.8 Hz, 4H), 2.41 (t, J= 7.4 Hz, 4H), 2.36 - 2.30 (m, 4H), 1.78 - 1.08 (m, 60H), 0.90 (q, J= 12.8 Hz, 8H).[l-cyclohexyl-2-[13-[2-cyclohexyl-2-(3-cyclohexylpropanoyloxy)ethyl]sulfanyl-7-oxo- tridecyl]sulfanyl-ethyl] 3-cyclohexylpropanoate (3.3).

[0189] 44% yield from l,13-bis[(2-cyclohexyl-2-hydroxy-ethyl)sulfanyl]tridecan-7-one (0.40 g, 0.78 mmol).XH NMR (400 MHz, CDCh) 84.83 (dt, J= 7.2, 5.1 Hz, 2H), 2.72 (dd, J= 13.8, 4.9 Hz, 2H), 2.61 (dd, J= 13.8, 7.3 Hz, 2H), 2.55 - 2.47 (m, 4H), 2.35 (m, 8H), 1.79 - 0.82 (m, 64H).((6-Oxoundecane-l,ll-diyl)bis(sulfanediyl))bis(5-cyclohexylpentane-l,2-diyl) bis(3- cyclohexylpropanoate) (3.6).

[0190] 85% yield from 1,1 l-bis((5-cyclohexyl-2-hydroxypentyl)thio)-undecan-6-one (3.0 g, 5.3 mmol). 'H NMR (400 MHz, CDCh) 8 5.00 - 4.91 (m, 2H), 2.70 - 2.59 (m, 4H), 2.55 (tt, J= 7.4, 3.8 Hz, 4H), 2.41 (t, J= 7.4 Hz, 4H), 2.36 - 2.30 (m, 4H), 1.78 - 1.08 (m, 60H), 0.90 (q, J= 12.8 Hz, 8H).

[0191] The following compounds were also prepared by the same method, but using heptanoic acid instead of 3-cyclohexylpropanoic acid:((6-Oxoundecane-l,ll-diyl)bis(sulfanediyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate (3.5).

[0192] 87% yield from l,ll-bis[(4-cyclohexyl-2-hydroxy-butyl)sulfanyl]undecan-6-one (0.50 g, 0.92 mmol).XH NMR (400 MHz, CDCh) 84.95 - 4.86 (m, 2H), 2.67 - 2.56 (m, 4H), 2.56 - 2.50 (m, 4H), 2.39 (t, J= 7.4 Hz, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.76 - 1.06 (m, 54H), 0.88 (h, J = 4.6 Hz, 10H).((6-Oxoundecane-l,ll-diyl)bis(sulfanediyl))bis(5-cyclohexylpentane-l,2-diyl) diheptanoate (3.7).

[0194] To an RBF containing l,l l-dihydroxy-undecan-6-one (1 eq. 6.7 mmol, 1.4 g) under inert atmosphere was added DCM (0.2 M), 4-pentylundecanoic acid (1 eq.), EDCI- HC1 (1.5 eq.), and DMAP (0.5 eq.). The mixture was stirred for 18 h then diluted with water (20 mL) and hexanes (20 mL). The layers were separated and the organic phase wascollected. The aq. phase was back-extracted with hexanes (2x20mL). The combined organics were washed with IN HC1 (50 mL), sat. NaHCCL (50 mL), dried (Na2SO4), fdtered, and evaporated to yield a crude product. This was then purified by automated silica gel chromatography (0-30% EtOAc in hexanes over 15 CV) to yield ll-hydroxy-6- oxoundecyl 4-pentylundecanoate (25% yield). 'H NMR (400 MHz, CDCh) 54.04 (t, J = 6.7 Hz, 2H), 3.67 - 3.62 (m, 2H), 2.45 - 2.36 (m, 4H), 2.28 - 2.22 (m, 2H), 1.67 - 1.14 (m, 35H), 0.90 - 0.84 (m, 6H). ll-((methylsulfonyl)oxy)-6-oxoundecyl 4-pentylundecanoate (8.2).

[0195] To a solution of 11 -hydroxy-6-oxoundecyl 4-pentylundecanoate (0.75 g, 1.7 mmol) and triethylamine (0.36 mL, 2.6 mmol) in DCM (1.7 mL) in a reaction vial under inert atmosphere, was added methanesulfonyl chloride dropwise via syringe at 0°C. The mixture was stirred for 30 min at 0°C then diluted with DCM (5 mL) and quenched with water (10 mL). The biphasic mixture was separated and the organic layer was collected. The organic phase was washed with water (10 mL), dried (Na2SO4), fdtered, and evaporated to yield crude l l-((methylsulfonyl)oxy)-6-oxoundecyl 4-pentylundecanoate (>95% yield). This carried forward without further purification. 'H NMR (400 MHz, CDCh) 54.22 (t, J= 6.4 Hz, 2H), 4.04 (t, J= 6.7 Hz, 2H), 3.00 (s, 3H), 2.45 - 2.37 (m, 4H), 2.29 - 2.22 (m, 2H), 1.81 - 1.70 (m, 2H), 1.67 - 1.52 (m, 8H), 1.46 - 1.14 (m, 25H), 0.91 - 0.84 (m, 6H).Procedure for mesylate displacement: ll-(acetylthio)-6-oxoundecyl 4-pentylundecanoate (8.3).

[0196] To a reaction vial under inert atmosphere was added DMF (1.6 mL) and 11- ((methyl-sulfonyl)oxy)-6-oxoundecyl 4-pentylundecanoate (0.84 g, 1.6 mmol). The solution was degassed via needle sparging for 10 min, then tri ethylamine (0.30 mL, 2.4 mmol) and thioacetic acid (0.14 mL, 1.9 mmol) were added at RT. The mixture was stirred 2 h at 60°C, then diluted with water (5 mL) and extracted with hexanes (5 mL). The aqueous phase was back-extracted with hexanes (2x5 mL). The combined organic phases were dried (Na2SO4), decolorized with carbon, fdtered through celite and evaporated to yield crude 11 -(acetylthio)-6-oxoundecyl 4-pentylundecanoate (>95% yield). This carried forward without further purification. 'H NMR (400 MHz, CDC13) 84.04 (t, J= 6.7 Hz, 2H), 2.89 - 2.81 (m, 2H), 2.42 - 2.37 (m, 4H), 2.32 (s, 3H), 2.29 - 2.22 (m, 2H), 1.70 - 1.16 (m, 35H), 0.91 - 0.84 (m, 6H).Procedure for epoxide ring opening: ll-((5-cyclohexyl-2-hydroxypentyl)thio)-6- oxonndecyl 4-pentylundecanoate (8.4).

[0197] To an RBF under inert atmosphere containing degassed methanol (N2, 20 min needle sparge) (5.3 mL) was added 11 -(acetylthio)-6-oxoundecyl 4-pentylundecanoate (0.38 g, 0.75 mmol) and 2-(3-cyclohexylpropyl)-oxirane (0.13 g, 0.75 mmol). Then NaOMe 1 wt% in MeOH (2.9 mL, 0.52 mmol) was added via syringe pump over 45 min. The reaction was then immediately quenched with sat. NH4CI (10 mL) and extracted with hexanes (10 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexanes (2x1 OmL). The combined organic phases were dried (NfeSCL) and evaporated to yield the crude product as a yellow oil. This was then purified by automated silica gel chromatography (0-20% EtOAc in hexanes over 15 CV) to yield 11- ((5-cyclohexyl-2-hydroxypentyl)thio)-6-oxoundecyl 4-pentylundecanoate (40 % yield).1H NMR (400 MHz, CDCh) 84.04 (t, J= 6.7 Hz, 2H), 3.66 - 3.58 (m, 1H), 2.72 (dd, J= 13.6, 3.3 Hz, 1H), 2.52 (t, J= 7.4 Hz, 2H), 2.46 - 2.37 (m, 5H), 2.29 - 2.23 (m, 2H), 1.75 - 1.07 (m, 50H), 0.93 - 0.79 (m, 8H).13C NMR (101 MHz, CDCI3) 8210.8, 174.4, 69.3, 64.2, 42.8, 42.7, 40.5, 37.7, 37.6, 37.2, 36.7, 33.5, 33.5, 33.4, 33.4, 32.4, 32.2, 32.1, 31.9, 30.2, 29.7, 29.5, 28.9, 28.7, 28.5, 26.9, 26.7, 26.6, 26.4, 25.8, 23.5, 23.4, 23.2, 22.8, 14.3.Procedure for esterification: ll-((5-cyclohexyl-2-((3-cyclohexylpropanoyl)oxy)pentyl)- thio)-6-oxoundecyl 4-butyldecanoate (3.4).

[0198] To an RBF containing l l-((5-cyclohexyl-2-hydroxy-pentyl)thio)-6-oxoundecyl 4- pentylundecanoate (0.19 g, 0.30 mmol) under N2 atmosphere was added DCM (1.5 mL), 3- cyclohexylpropanoic acid (0.060 mL, 0.36 mmol), EDCI-HC1 (85 mg, 0.44 mmol), andDMAP (54 mg, 0.44 mmol). The mixture was stirred for 18 h then diluted with water (5 mL) and hexanes (5 mL). The layers were separated and the organic phase was collected. The aq. phase was back-extracted with hexanes (2x5mL). The combined organics were washed with IN HC1 (10 mL), sat. NaHCCL (10 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. This was then purified by automated silica gel chromatography (0-10% EtOAc in hexanes over 10 CV) to yield 1 l-((5-cyclohexyl-2-((3- cyclohexylpropanoyl)oxy)pentyl)thio)-6-oxoundecyl 4-pentylundecanoate (83% yield).1H NMR (400 MHz, CDCh) 84.98 - 4.89 (m, 1H), 4.05 (t, J= 6.7 Hz, 2H), 2.68 - 2.58 (m, 2H), 2.57 - 2.49 (m, 2H), 2.43 - 2.36 (m, 4H), 2.35 - 2.29 (m, 2H), 2.29 - 2.23 (m, 2H), 1.75 - 1.09 (m, 61H), 0.94 - 0.79 (m, 10H).Synthesis of lipids T-01-T-10Procedure for ketone reduction: ((6-hydroxyundecane-l,ll-diyl)bis(sulfanediyl))-bis(l- cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (9.1).

[0199] Solid NaBH4 (92 mg, 2.4 mmol, 2.0 equiv.) was added portion-wise to a stirred solution of ketone 3.1 (1.0 g, 1.2 mmol, 1.0 equiv.) in 95% ethanol (2 mL) at 0 °C. The resulting mixture was stirred at room temperature for 2 hours. The reaction was quenched by careful addition of aqueous saturated NH4CI solution and concentrated on the rotary evaporator to remove the ethanol. The aqueous residue was extracted with hexanes (3 x 5 mL). The combined extracts were dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (8% EtOAc in Hexane) to afford 9.1 (0.95 g, 1.2 mmol, 95%) as a colorless oil.XH NMR (400 MHz, CDCh) 8 5.01 - 4.75 (m, 2H), 3.57 (p, J= 7.48 Hz, 1H), 2.67 - 2.40 (m, 8H), 2.31 (t, J= 7.76 Hz, 4H), 1.81 - 0.97 (m, 63H), 0.97 - 0.71 (m, 9H).

[0200] The following compound was prepared by the same method:((6-Hydroxyundecane-l,ll-diyl)bis(sulfanediyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate.

[0201] Colorless oil, 90% from 3.5 after purification by silica gel chromatography (8% EtOAc in hexanes).XH NMR (400 MHz, CDCI3) 84.92 - 4.71 (m, 2H), 3.70 - 3.39 (p, J = 7.43 Hz, 1H), 2.84 - 2.39 (m, 8H), 2.33 (t, J= 7.62 Hz, 4H), 1.87 - 0.63 (m, 68H).Procedure for esterification: ((6-((4-(dimethylamino)butanoyl)oxy)undecane-l,ll- diyl)bis-(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (T-01)

[0202] A solution of 4-dimethylaminobutyric acid hydrochloride (0.25 g, 1.4 mmol, 1.2 equiv), EDCI (0.33 g, 1.73 mmol, 1.5 equiv), and DMAP (0.21 g, 1.73 mmol, 1.5 equiv) in dry CH2CI2 (5.0 mL) was stirred at room temperature for 5 minutes prior to the addition of alcohol 9.1 (0.95 g, 1.2 mmol, 1.0 equiv). The mixture was stirred at room temperature for 18 hours, under nitrogen atmosphere. The solution was diluted with more CH2CI2 (5 mL) and sequentially washed with aqueous saturated NaHCCh (3 x 5 mL) and water (5 mL), then dried (NfeSCL) and evaporated. The residue was purified by silica gel column chromatography (5% MeOH in DCM) to afford T-01 (0.67 g, 0.72 mmol, 62%) as a colorless oil.XH NMR (400 MHz, CDCI3) 84.96 - 4.75 (m, 3H), 2.69 - 2.57 (m, 4H), 2.51 (m, 4H), 2.42 - 2.08 (m, 14H), 1.84 - 1.04 (m, 65H), 0.94 - 0.75 (m, 9H).

[0203] The following compound was prepared by the same method:((6-((4-(Dimethylamino)butanoyl)oxy)undecane-l,ll-diyl)bis(sulfanediyl))bis(4- cyclohexyl-butane-l,2-diyl) diheptanoate (T-02).

[0204] Crude product was purified by silica gel chromatography (5% MeOH in DCM) to afford T-02 (0.62 g, 0.70 mmol, 60%) as a colorless oil. 'H NMR (400 MHz, CDCh) 84.83 (m, 3H), 2.91 - 2.10 (m, 22H), 1.98 - 0.61 (m, 70H).Procedure for ketalization: (((4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(pentane-5,l- diyl))bis(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (10.1)

[0205] A solution of ketone 3.1 (1.0 g, 1.31 mmol, 1 eq), 4-chloro-l,2-butanediol (0.49 g, 3.93 mmol, 3 eq), and PPTS (33 mg, 0.13 mmol, 0.1 eq) in toluene (50 mL) was refluxed under nitrogen for 5 h with continuous removal of water (Dean-Stark trap). After completion of the reaction (checked by NMR), the mixture was cooled to room temperature, treated with solid NaHCCh (200 mg), and evaporated to dryness in vacuo. The residue was taken up with n-hexanes (100 mL) and aqueous saturated NaHCCh solution (100 mL). The organic phase was separated and sequentially washed with water, brine, dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by neutral alumina eluting with EtOAc;hexanes (0-5%) afford the title compound (1.07 g, 1.23 mmol, 94 %) as a colorless oil.XH NMR (400 MHz, CDCh) 84.83 (dt, J= 7.4, 5.1 Hz, 2H), 4.22 (td, J = 7.1, 4.7 Hz, 1H), 4.09 (dd, J= 7.9, 6.1 Hz, 1H), 3.68 - 3.61 (m, 2H), 3.51 (t, J= 7.7 Hz, 1H), 2.72 (dd, J= 13.8, 5.0 Hz, 2H), 2.61 (dd, J= 13.7, 7.4 Hz, 2H), 2.51 (t, J= 7.4 Hz, 4H), 2.33 (t, 4H), 2.10 - 1.89 (m, 2H), 1.79 - 0.81 (m, 64H).

[0206] The following compound was prepared by the same method: (((4-(2-Chloroethyl)-l,3-dioxolane-2,2-diyl)bis(pentane-5,l-diyl))bis(sulfanediyl))bis-(4- cyclohexylbutane- 1,2-diyl) diheptanoate.

[0207] From 3.2.XH NMR (400 MHz, CDCh) 84.91 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 4.28 - 4.19 (m, 1H), 4.09 (dd, J= 7.9, 6.1 Hz, 1H), 3.69 - 3.60 (m, 2H), 3.51 (t, J= 7.7 Hz, 1H),2.63 (dd, J= 6.2, 1.7 Hz, 4H), 2.53 (td, J= 7.2, 1.9 Hz, 4H), 2.31 (t, 4H), 2.11 - 1.84 (m, 2H), 1.76 - 1.46 (m, 32H), 1.41 - 1.03 (m, 30H), 0.88 (tdt, J= 12.8, 8.9, 4.2 Hz, 10H).Procedure for chloride displacement: (((4-(2-(dimethylamino)ethyl)-l,3-dioxolane-2,2- diyl)bis(pentane-5,l-diyl))bis(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3- cyclohexylpropanoate) (T-03).

[0208] To a solution of 11.1 (1.14 g, 1.31 mmol, 1 eq) in THF at 0 °C, was added dimethylamine (2 M in THF, 2.0 mL, 3.93 mmol, 3 eq). The resulting mixture was heated in a sealed tube at 125 °C for 24 h. The reaction mixture was concentrated in vacuo. The resulting residue was dissolved in hexanes, washed with NaHCCh (sat solution), brine (sat. solution), dried over (Na2SO4), filtered and concentrated in vacuo. The resulting residue was purified by automated column chromatography eluting with EtOAc:DCM (0-10%) to afford the title compound as colorless oil.1H NMR (400 MHz, CDCh) 54.86 (dt, J= 7.3, 5.1 Hz, 2H), 4.09 (dq, J= 12.9, 6.2 Hz, 2H), 3.49 (t, J= 7.3 Hz, 1H), 2.75 (dd, J= 13.7, 5.0 Hz, 2H), 2.64 (dd, J= 13.7, 7.3 Hz, 2H), 2.54 (t, J= 7.3 Hz, 4H), 2.46 - 2.27 (m, 6H), 2.24 (s, 6H), 1.97 - 1.49 (m, 40H), 1.45 - 0.80 (m, 26H).

[0209] The following compound was prepared by the same method: (((4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(pentane-5,l- diyl))bis(sulfanediyl))-bis(4-cyclohexylbutane-l,2-diyl) diheptanoate (T-04).

[0210] From (((4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(pentane-5,l-diyl))bis(sulfane- diyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate. 'H NMR (400 MHz, CDCI3) 54.92 (ddt, J= 10.7, 8.1, 5.2 Hz, 2H), 4.12 - 4.00 (m, 2H), 3.46 (t, J= 7.2 Hz, 1H), 2.69 - 2.58 (m, 4H), 2.53 (dd, J= 8.2, 6.5 Hz, 4H), 2.44 - 2.25 (m, 6H), 2.22 (s, 6H), 1.90 - 1.48 (m, 38H), 1.43 - 1.06 (m, 28H), 0.88 (tq, J= 11.0, 3.7 Hz, 8H).Procedure for reductive amination: 6-((4-((ter / -butyldimethylsilyl)oxy)butyl)amino)- ll-((5-cyclohexyl-2-((3-cyclohexylpropanoyl)oxy)pentyl)thio)undecyl 4- pentylundecanoate.

[0211] To an RBF was added l l-((5-cyclohexyl-2-((3-cyclohexylpropanoyl)oxy)pentyl)- thio)-6-oxoundecyl 4-pentylundecanoate (0.18 g, 0.23 mmol), 4-[tert-butyl(dimethyl)silyl]- oxybutan-1 -amine (96 mg, 0.48 mmol), and DCE (0.47 mL), then flushed with N2. The mixture was stirred for 15 min, then STAB-H (75 mg, 0.35 mmol) was added. The mixture was stirred overnight then was quenched with sat. NaHCCL (5 mL) followed by dilution with hexanes (5 mL). The layers were separated and the organic phase was collected. The aqueous phase was back extracted with hexanes (2x5 mL). The combined organic phases were washed with satNaHCCL (2x10 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. This was purified by automated chromatography [0-10% MeOH in DCM over 12 CV] to yield 6-((4-((tert-butyldimethylsilyl)oxy)butyl)amino)-ll-((5-cyclohexyl-2- ((3-cyclohexyl-propanoyl)oxy)pentyl)thio)undecyl 4-pentylundecanoate (87% yield).XH NMR (400 MHz, CDCh) 84.99 - 4.90 (m, 1H), 4.05 (t, J= 6.7 Hz, 2H), 3.62 (t, J = 6.0 Hz, 2H), 2.68 - 2.59 (m, 2H), 2.59 - 2.50 (m, 4H), 2.49 - 2.42 (m, 1H), 2.35 - 2.29 (m, 2H), 2.29 - 2.24 (m, 2H), 1.75 - 1.09 (m, 74H), 0.95 - 0.82 (m, 19H), 0.04 (s, 6H).

[0212] The following compounds were prepared by the same method:((7-((4-(ter / -butyldimethyIsilyloxy)butyl)amino)-tridecane-l,13-diyl)bis(sulfanediyl))- bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexyl-propanoate) (11.1).

[0213] >95% yield from [l-cyclohexyl-2-[13-[2-cyclohexyl-2-(3-cyclohexylpropanoyl- oxy)ethyl]sulfanyl-7-oxo-tridecyl]sulfanyl-ethyl] 3-cyclohexylpropanoate (0.269 g, 0.34 mmol).XH NMR (400 MHz, CDCI3) 84.87 - 4.79 (m, 2H), 3.66 (t, J= 6.0 Hz, 2H), 2.72 (dd, J= 13.8, 5.0 Hz, 2H), 2.62 (dd, J= 13.8, 7.3 Hz, 2H), 2.57 - 2.48 (m, 6H), 2.44 - 2.38 (m, 1H), 2.36 - 2.30 (m, 4H), 1.79 - 1.09 (m, 64H), 0.94 - 0.82 (m, 17H), 0.04 (s, 6H).((6-(((4-ter / -Butyldimethylsilyloxy)butyl)amino)undecane-l,ll-diyl)bis(sulfanediyl))- bis(4-cyclohexylbutane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0214] >95 % yield from ((6-oxoundecane- 1,11 -diyl)bis(sulfanediyl))bis(4- cyclohexylbutane-l,2-diyl) bis(3-cyclohexylpropanoate) (0.77 g, 0.94 mmol). 'H NMR (400 MHz, CDCh) 54.96 - 4.87 (m, 2H), 3.69 - 3.62 (m, 2H), 2.67 - 2.58 (m, 4H), 2.58 - 2.49 (m, 6H), 2.46 - 2.38 (m, 1H), 2.35 - 2.27 (m, 4H), 1.77 - 1.09 (m, 67H), 0.93 - 0.80 (m, 17H), 0.04 (s, 6H).((6-((4-(ter / -butyldimethyIsilyloxy)butyl)amino)undecane-l,ll-diyl)bis(sulfanediyl))- bis(4-cyclohexylbutane-l,2-diyl) diheptanoate.

[0215] >95% yield from [3-cyclohexyl-l-[[l l-(4-cyclohexyl-2-heptanoyloxy- butyl)sulfanyl-6-oxo-undecyl]-sulfanylmethyl]propyl] heptanoate (0.61 g, 0.80 mmol). 1H NMR (400 MHz, CDCh) 84.97 - 4.87 (m, 2H), 3.69 - 3.63 (m, 2H), 2.69 - 2.58 (m, 4H), 2.58 - 2.49 (m, 6H), 2.46 - 2.38 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 1.78 - 1.10 (m, 66H), 0.92 - 0.80 (m, 19H), 0.04 (s, 6H).((6-((4-(ter / -Butyldimethylsilyloxy)butyl)amino)undecane-l,ll-diyl)bis(sulfanediyl))- bis(5-cyclohexylpentane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0216] >95% yield from [4-cyclohexyl-l-[[l l-[5-cyclohexyl-2-(3-cyclohexylpropanoyl- oxy)-pentyl]sulfanyl-6-oxo-undecyl] sulfanylmethyl] butyl] 3-cyclohexylpropanoate (0.26 g, 0.30 mmol). 'H NMR (400 MHz, CDCh) 5 5.00 - 4.90 (m, 2H), 3.68 - 3.63 (m, 2H), 2.69 - 2.58 (m, 4H), 2.58 - 2.50 (m, 6H), 2.41 (dd, J= 7.4, 4.4 Hz, 1H), 2.35 - 2.27 (m, 4H), 1.78 - 1.08 (m, 70H), 0.93 - 0.79 (m, 17H), 0.04 (s, 6H).((6-((4-( / c / 7-Biitykliinethylsilyloxy )butyl)amino)undecane- 1 ,1 l-diyl)bis(sulfanediyl))- bis(5-cyclohexylpentane-l,2-diyl) diheptanoate.

[0217] >95% yield from [4-cyclohexyl-l-[[l l-(5-cyclohexyl-2-heptanoyloxy- pentyl)sulfanyl-6-oxoundecyl] -sulfanylmethyl] butyl] heptanoate (0.536 g, 0.67 mmol). 'H NMR (400 MHz, CDCh) 8 5.02 - 4.92 (m, 2H), 3.73 - 3.64 (m, 2H), 2.68 - 2.62 (m, 4H), 2.56 (dd, J= 8.0, 6.1 Hz, 6H), 2.47 - 2.40 (m, 1H), 2.33 (t, J= 7.5 Hz, 4H), 1.76 - 1.11 (m, 66H), 0.95 - 0.82 (m, 19H), 0.04 (s, 6H).Procedure for reductive methylation: ((7-((4-( / c / 7-butykliinethylsilyloxy)butyl)- (methyl)-amino)tridecane-l,13-diyl)bis(sulfanediyl))bis-(l-cyclohexylethane-2,l-diyl) bis(3-cyclo-hexylpropanoate) (11.2).

[0218] To a reaction vial containing ((7-((4-(to7-butyldimethylsilyloxy)- butyl)amino)tridecane-l,13-diyl)bis(sulfanediyl))bis(l-cyclohexyl-ethane-2,l-diyl)bis(3- cyclohexylpropanoate) (0.40 g, 0.36 mmol) was added THF (0.36 mL), and 37% aqueous formaldehyde (0.14 mL, 1.8 mmol). The vessel was flushed with N2 then stirred at RT for 20 min. Then NaBH(OAcs) (0.23 g, 1.1 mmol) was added, the reaction was then stirred for 18 h. The reaction was quenched with sat. NaHCCh (5 mL) followed by dilution with hexanes (5 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexanes (2x5 mL). The combined organic phases were dried (Na2SO4), fdtered, and evaporated to yield a crude product. This was purified by column chromatography [0-40% EtOAc in hexanes over 12 CV] to yield ((7-((4-(fert- butyldimethy lsilyl-oxy)butyl)(methyl)amino)tri decane- 1,13 -diyl)bis(sulfanediyl))bis(l- cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (>95% yield). 'H NMR (400 MHz, CDCh) 84.88 - 4.79 (m, 2H), 3.66 (t, J= 6.2 Hz, 2H), 2.72 (dd, J= 13.8, 4.9 Hz, 2H), 2.61 (dd, J= 13.8, 7.3 Hz, 2H), 2.51 (t, J= 7.4 Hz, 4H), 2.36 - 2.27 (m, 7H), 2.13 (s, 3H), 1.75 - 0.98 (m, 64H), 0.94 - 0.82 (m, 17H), 0.04 (s, 6H).

[0219] The following compounds were nrenared by the same method:6-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-ll-((5-cyclohexyl-2-((3- cyclohexylpropanoyl)oxy)pentyl)thio)undecyl 4-pentylundecanoate.

[0220] 53% yield from 6-((4-((tert-butyldimethylsilyl)oxy)butyl)amino)-l l-((5- cyclohexyl-2-((3-cyclohexyl-propanoyl)oxy)-pentyl)thio)undecyl 4-pentylundecanoate (195 mg, 0.20 mmol). 'H NMR (400 MHz, CDCh) 84.99 - 4.89 (m, 1H), 4.04 (t, J= 6.8 Hz, 2H), 3.61 (t, J= 6.3 Hz, 2H), 2.68 - 2.58 (m, 2H), 2.56 - 2.49 (m, 2H), 2.38 - 2.23 (m, 7H), 2.13 (s, 3H), 1.76 - 1.06 (m, 69H), 0.94 - 0.79 (m, 19H), 0.04 (s, 6H).((6-(((4-ter / -Butylmethylsilyloxy)butyl)(methyl)amino)undecane-l,ll-diyl)bis(sulfane- diyl))-bis(4-cyclohexylbutane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0221] 59% yield from ((6-(((4-ferf-Butyldimethylsilyloxy)butyl)amino)undecane-l,l 1- diyl)bis(sulfanediyl))-bis(4-cyclohexyl-butane-l,2-diyl) bis(3-cyclohexylpropanoate) (1.0 g, 0.94 mmol).XH NMR (400 MHz, CDCh) 84.95 - 4.87 (m, 2H), 3.66 (t, J= 6.1 Hz, 2H), 2.68 - 2.57 (m, 4H), 2.56 - 2.47 (m, 4H), 2.36 - 2.27 (m, 7H), 2.12 (s, 3H), 1.79 - 1.08 (m, 68H), 0.95 - 0.79 (m, 17H), 0.04 (s, 6H).((6-((4-(ter / -Butyldimethylsilyloxy)butyl)(methyl)amino)undecane-l,ll-diyl)bis- (sulfanediyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate.

[0222] >95% yield from ((6-((4-(terf-butyl-dimethylsilyloxy)butyl)amino)undecane-l,l 1- diyl)bis(sulfanediyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate (0.76 g, 0.8 mmol). 'H NMR (400 MHz, CDCh) 84.98 - 4.87 (m, 2H), 3.66 (t, J= 6.2 Hz, 2H), 2.66 - 2.61 (m, 4H), 2.56 - 2.50 (m, 4H), 2.35 - 2.27 (m, 7H), 2.12 (s, 3H), 1.78 - 1.10 (m, 62H), 0.93 - 0.80 (m, 19H), 0.04 (s, 6H).((6-((4-(ter / -Butyldimethylsilyloxy )butyl)(methyl)amino)undecane-l ,1 l-diyl)bis- (sulfanediyl))bis(5-cyclohexylpentane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0223] >95% yield from ((6-((4-(ferLButyldimethylsilyloxy)butyl)amino)undecane-l,ll- diyl)bis(sulfanediyl))bis(5-cyclohexylpentane-l,2-diyl) bis(3-cyclohexylpropanoate) (0.69 g, 0.67 mmol). 'H NMR (400 MHz, CDC13) 84.98 - 4.91 (m, 2H), 3.68 - 3.60 (m, 2H), 2.69 - 2.59 (m, 4H), 2.57 - 2.49 (m, 4H), 2.35 - 2.27 (m, 7H), 2.12 (s, 3H), 1.78 - 1.09 (m, 66H), 0.92 - 0.79 (m, 19H), 0.04 (s, 6H).((6-((4-(ter / -Butyldimethylsilyloxy)butyl)(methyl)amino)undecane-l,ll-diyl)bis- (sulfanediyl))bis(5-cyclohexylpentane- 1,2-diyl) diheptanoate.

[0224] >95% yield from ((6-((4-(ferLbutyldimethylsilyloxy)butyl)amino)undecane-l,ll- diyl)bis(sulfanediyl))bis(5-cyclohexyl-pentane-l,2-diyl) diheptanoate (0.667g, 0.67 mmol).XH NMR (400 MHz, CDCI3) 84.98 - 4.91 (m, 2H), 3.68 - 3.60 (m, 2H), 2.69 - 2.59 (m, 4H), 2.57 - 2.49 (m, 4H), 2.35 - 2.27 (m, 7H), 2.12 (s, 3H), 1.78 - 1.09 (m, 66H), 0.92 - 0.79 (m, 19H), 0.04 (s, 6H).Procedure for desilylation: ((7-((4-Hydroxybutyl)(methyI)amino)tridecane-l,13-diyl)- bis(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexylpropanoate) (T-05).

[0225] To an RBF containing ((7-((4-( / c / -butyldimethylsilyloxy)butyl)(methyl)amino)- tridecane-l,13-diyl)bis(sulfanediyl))bis(l-cyclohexylethane-2,l-diyl) bis(3-cyclohexyl- propanoate) (0.36 g, 0.36 mmol) under N2 atmosphere was added DCM (0.36 mL). The solution was cooled to 0°C then HF pyridine (0.16 mL) was added. The mixture was stirred at 0°C until completion after 15 min (TLC). The reaction mixture was quenched by slowly pouring into a rapidly stirred mixture of sat. NaHCCL (5 mL) and hexanes (5 mL). Thelayers were separated, and the organic phase was collected. The aqueous phase was back extracted with hexanes (2x5 mL). The combined organic phases were washed with sat. NaHCCh (20 mL), dried (NfeSCL), fdtered and evaporated to yield the crude product. The crude product was purified by automated silica gel chromatography (0-10% MeOH in DCM over 12 CV) to yield the final lipid (light yellow oil). The purified lipid was diluted with 5% EtOAc in hexanes (2 mL) then filtered through basic alumina (1 g, Brockmann activity 1), eluding with 5% EtOAc in hexanes (10 mL), then evaporated to give a near-colourless clear oil. This solution was diluted with HPLC grade hexanes (2 mL) then filtered through a 0.2 pm sub-micron PTFE syringe filter on a glass syringe, then evaporated, yielding pure T-05 (27% yield).XH NMR (400 MHz, CDCh) 84.83 (dt, J= 7.2, 5.1 Hz, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.72 (dd, J= 13.8, 4.9 Hz, 2H), 2.62 (dd, J= 13.8, 7.3 Hz, 2H), 2.51 (t, J= 7.4 Hz, 4H), 2.44 (t, J= 5.3 Hz, 2H), 2.39 (t, J= 5.9 Hz, 1H), 2.37 - 2.30 (m, 4H), 2.14 (s, 3H), 1.78 - 0.76 (m, 72H).13C NMR (101 MHz, CDCh) 8 174.0, 76.1, 63.7, 62.9, 54.6, 40.3, 37.3, 35.6, 33.7, 33.2, 33.1, 32.7, 32.6, 32.5, 32.2, 30.2, 29.8, 29.8, 29.4, 29.0, 27.8, 27.7, 26.7, 26.5, 26.4, 26.3, 26.2, 26.1. ll-((5-Vyclohexyl-2-((3-cyclohexylpropanoyl)oxy)pentyl)thio)-6-((4-hydroxybutyl)- (methyl)amino)undecyl 4-pentylundecanoate (T-06).

[0226] 44% yield from 6-((4-(( / c / 7-butyldimethylsilyl)-oxy)butyl)(methyl)amino)- 1 1 -((5- cyclohexyl-2-((3-cyclohexylpropanoyl)oxy)pentyl)thio)-undecyl 4-pentylundecanoate (0.104g, 0.11 mmol). 'H NMR (400 MHz, CDCh) 84.98 - 4.90 (m, 1H), 4.05 (t, J= 6.8 Hz, 2H), 3.58 - 3.53 (m, 2H), 2.68 - 2.58 (m, 2H), 2.56 - 2.51 (m, 2H), 2.47 - 2.36 (m, 3H), 2.34 - 2.29 (m, 2H), 2.29 - 2.24 (m, 2H), 2.14 (s, 3H), 1.76 - 1.09 (m, 69H), 0.94 - 0.79 (m, 10H).((6-((4-Hydroxybutyl)(methyI)amino)undecane-l,ll-diyl)bis(sulfanediyl))bis(4- cyclohexyl-butane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-07).

[0227] 64% yield from ((6-(((4-ferf-butylmethylsilyloxy)butyl)(methyl)amino)undecane- 1,11 -diy l)bis(sulfane-diy l))-bis(4-cy clohexy Ibutane- 1 ,2-diyl) bis(3 -cy clohexylpropanoate) (0.61 g, 0.60 mmol). 1H NMR (400 MHz, CDC13) 84.96 - 4.88 (m, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.70 - 2.58 (m, 4H), 2.58 - 2.49 (m, 4H), 2.47 - 2.36 (m, 3H), 2.35 - 2.28 (m, 4H), 2.14 (s, 3H), 1.79 - 1.06 (m, 68H), 0.94 - 0.80 (m, 8H).13C NMR (101 MHz, CDCh) 8 174.0, 73.3, 63.6, 62.9, 54.5, 37.7, 37.3, 36.1, 35.5, 33.5, 33.3, 33.1, 33.0, 32.9, 32.6, 32.4, 32.3, 30.6, 30.1, 29.7, 29.4, 27.4, 26.8, 26.7, 26.5, 26.5, 26.4, 26.2.((6-((4-Hydroxybutyl)(methyI)amino)undecane-l,ll-diyl)bis(sulfanediyl))bis(4- cyclohexylbutane- 1,2-diyl) diheptanoate (T-08).

[0228] 34% yield from ((6-((4-(ferf-butyldimethylsilyloxy)butyl)(methyl)amino)undecane- l,ll-diyl)bis(sulfane-diyl))bis(4-cyclohexylbutane-l,2-diyl) diheptanoate (0.77 g, 0.80 mmol).XH NMR (400 MHz, CDC13) 84.92 (dtd, J = 8.0, 6.1, 4.4 Hz, 2H), 3.56 (t, J = 4.9 Hz, 2H), 2.69 - 2.58 (m, 4H), 2.57 - 2.49 (m, 4H), 2.47 - 2.36 (m, 3H), 2.30 (t, J = 7.5 Hz, 4H), 2.14 (s, 3H), 1.78 - 1.09 (m, 62H), 0.93 - 0.79 (m, 10H). 13C NMR (101 MHz, CDC13) 8 173.7, 73.3, 63.6, 62.9, 54.6, 37.7, 36.1, 35.6, 34.8, 33.5, 33.3, 33.0, 32.9, 32.4, 31.6, 30.6, 30.1, 29.7, 29.4, 29.0, 27.4, 26.8, 26.5, 26.5, 26.2, 25.2, 22.7, 14.2.((6-((4-Hydroxybutyl)(methyl)amino)undecane-l,ll-diyl)bis(sulfanediyl))bis(5- cyclohexyl-pentane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-09).

[0229] 23% yield from ((6-((4-(ferf-butyldimethylsilyloxy)butyl)(methyl)amino)undecane- l,ll-diyl)bis(sulfane-diyl))bis(5-cyclohexylpentane-l,2-diyl) bis(3-cyclohexyl-propanoate) (0.14 g, 0.13 mmol).XH NMR (400 MHz, CDCh) 84.99 - 4.91 (m, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.70 - 2.59 (m, 4H), 2.58 - 2.49 (m, 4H), 2.47 - 2.36 (m, 3H), 2.36 - 2.27 (m, 4H), 2.14 (s, 3H), 1.77 - 1.06 (m, 72H), 0.94 - 0.78 (m, 8H).13C NMR (101 MHz, CDCh) 8 174.0, 72.9, 63.6, 62.9, 54.6, 37.7, 37.4, 37.3, 36.2, 35.5, 33.6, 33.5, 33.4, 33.1, 32.9, 32.6, 32.4, 32.3, 30.1, 29.7, 29.4, 27.4, 26.8, 26.7, 26.5, 26.5, 26.4, 26.2, 22.7.((6-((4-Hydroxybutyl)(methyl)amino)undecane-l,ll-diyl)bis(sulfanediyl))bis(5- cyclohexylpentane-l,2-diyl) diheptanoate (T-10).

[0230] 35% yield from ((6-((4-(ferLbutyldimethylsilyloxy)butyl)(methyl)amino)undecane- l,ll-diyl)bis(sulfane-diyl))bis(5-cyclohexylpentane-l,2-diyl) diheptanoate (0.67 g, 0.67 mmol).XH NMR (400 MHz, CDC13) 84.99 - 4.90 (m, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.69 - 2.59 (m, 4H), 2.58 - 2.50 (m, 4H), 2.47 - 2.36 (m, 3H), 2.30 (t, J= 7.5 Hz, 4H), 2.14 (s, 3H), 1.74 - 1.05 (m, 66H), 0.94 - 0.77 (m, 10H).13C NMR (101 MHz, CDCI3) 8 173.5, 72.8, 63.5, 62.8, 54.4, 37.5, 37.2, 36.0, 35.4, 34.6, 33.5, 33.4, 33.3, 32.8, 32.3, 31.5, 29.9, 29.6, 29.3, 28.9, 27.2, 26.7, 26.4, 26.4, 26.1, 25.1, 22.6, 22.5, 14.1.Synthesis of lipids T-ll-T-12Procedure for epoxide opening with thioacetic acid: N-(2-hydroxyoctyl) ethanethioate.

[0231] To a solution of 2-hexyloxirane (10 g, 78 mmol, 1 eq.) and AcSH (6.7 mL, 93.6 mmol, 1.2 eq) in DMF at 0 °C, was added NaHCCL (0.66 g, 7.8 mmol, 0.1 eq). The resulting mixture was allowed to warm to RT and stirred for 3 hrs, then poured into water (100 mL). The aq. phase was extracted with n-hexanes (3 x 50 mL), the combined organic layers were dried over Na2SC>4 and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:hexane (0-10%) to afford the title compound (12.1 g, 59.2 mmol, 76%) as yellow oil.XH NMR (400 MHz, CDCI3) 8 3.73 (tq, J= 6.1, 4.2 Hz, 1H), 3.15 (dd, J= 14.0, 3.7 Hz, 1H), 2.88 (dd, J= 14.0, 7.5 Hz, 1H), 2.37 (s, 3H), 1.99 (td, J = 4.8, 2.3 Hz, 1H), 1.63 - 1.18 (m, 10H), 0.93 - 0.82 (m, 3H).

[0232] The following compound was prepared by the same method: N-(4-cyclohexyl-2-hydroxybutyl) ethanethioate.

[0233] From 2-(2-cyclohexylethyl)oxirane.XH NMR (400 MHz, CDCh) 8 3.72 (tdd, J = 7.4, 5.4, 3.7 Hz, 1H), 3.17 (dd, J= 14.0, 3.8 Hz, 1H), 2.90 (dd, J= 14.0, 7.5 Hz, 1H), 2.39 (s, 3H), 2.00 (s, 1H), 1.77 - 1.08 (m, 13H), 0.91 (qt, J= 12.6, 6.1 Hz, 2H).Procedure for esterification: l-(acetylthio)-4-cyclohexylbutan-2-yl 3-cyclohexyl- propanoate (12.7).

[0234] To a solution of EDC.HC1 (6.3 g, 32.8 mmol, 1.3 eq) and 4-DMAP (3.4 g, 27.8 mmol, 1.1 eq) in CH2CI2 (40 mL), was added 3-cylohexylpropanoic acid (5.13 g, 32.8 mmol, 1.5 eq) and stirred for 10 min, then followed by stow addition a solution of thioacetate alcohol (5.82 g, 25.3 mmol, 1 eq) in CH2CI2 (10 mL). The resulting mixture was stirred at room temp for 18 hr, diluted with CH2CI2 (50 mL) and poured into water (100 mL). The organic layer was separated, and the aq. phase was further extracted with CH2CI2 (2 x 30 mL). The combined organic layers were dried (NfeSCL), filtered, and concentrated in vacuo. The resulting residue was purified by automated flash column chromatography eluting with EtOAcm-hexanes (0-10-%) to afford the title compound (14.3 g, 47.3 mmol, 80%) as a colorless oil. 'H NMR (400 MHz, CDCh) 84.96 (qd, J= 6.7, 4.3 Hz, 1H), 3.26 (dd, J= 14.0, 4.4 Hz, 1H), 3.00 (dd, J= 14.0, 6.9 Hz, 1H), 2.36 (s, 3H), 2.31 (t, 2H), 1.77 - 1.49 (m, 14H), 1.22 (tdd, J= 14.6, 11.2, 6.0 Hz, 10H), 0.98 - 0.81 (m, 2H).

[0235] The following compound was prepared by the same method: l-(Acetylthio)octan-2-yl hexanoate (12.3).

[0236] From S-(2 -hydroxy octyl) ethanethioate and hexanoic acid.1H NMR (400 MHz, CDCh) 84.97 (tt, J= 7.0, 4.6 Hz, 1H), 3.24 (dd, J= 14.0, 4.4 Hz, 1H), 2.97 (dd, J= 14.0,6.9 Hz, 1H), 2.33 (s, 3H), 2.27 (t, J= 7.5 Hz, 2H), 1.60 (dp, J= 12.2, 6.8 Hz, 4H), 1.41 -1.10 (m, 12H), 0.88 (dt, J= 9.9, 6.7 Hz, 6H).6-((te / 't-Butoxycarbonyl)(7-((te / 't-butyldimethylsilyl)oxy)heptyl)-amino)hexyl 4-methyl- benzenesulfonate (12.2).Boc12.2

[0237] Solid TsCl (2 g, 11 mmol) was added to a solution of 12.1 (3.2 g, 7.2 mmol), TEA (2.1 mL, 14 mmol) and DMAP (439 mg, 3.6 mmol) in DCM (20.0 mL) at 0 °C undernitrogen. The reaction was warmed to room temperature and stirred for 2 hours, diluted with water and extracted with DCM (3 x 20.0 mL). The combined extracts were dried (NfeSC ) and concentrated. The residue was purified by silica chromatography (0-20% EtOAc in hexanes) to yield 12.2 (3.3 g, 77%).XH NMR (400 MHz, CDC13) 8 7.81-7.78 (d, J= 8.32 Hz, 2H), 7.37-7.35 (d, J= 8.32 Hz, 2H), 4.03-4.00 (t, J = 6.53 Hz, 2H), 3.62-3.59 (t, J = 6.53 Hz, 2H), 3.12 (brs, 4H,), 2.46 (s, 3H), 1.70-1.61 (m, 2H), 1.50-1.43 (m, 15H), 1.34- 1.20 (m, 10H), 0.90 (s, 9H), 0.05 (s, 6 H).Procedure for tosylate displacement with a thiolate: 12-( / c / -But oxy carbonyl )-2.2,3,3- tetramethyl-4-oxa-19-thia-12-aza-3-silaheptacosan-21-yl hexanoate (12.4).

[0238] NaOMe 25 wt% in MeOH (1.67 mL, 7.31 mmol, 1.3 equiv) was added dropwise to a cold (-20 °C) solution of thioester 12.3 (2.04 g, 6.74 mmol, 1.2 equiv) and tosylate 12.2 (3.37 g, 5.62 mmol, 1.0 equiv), in degassed DMF (6 mL). The mixture was stirred for 45 min, then quenched with sat. NH4CI (10 mL) and extracted with hexanes (3 x 10 mL). The combined extracts were washed with brine, dried (ISfeSCL), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5 % EtOAc in Hexane) to afford 12.4 (2.25 g, 3.27 mmol, 58%) as a colorless oil.XH NMR (400 MHz, CDCI3) 85.02 - 4.83 (m, 1H), 3.58 (t, J = 6.56 Hz, 2H), 3.12 (t, J = 7.41 Hz, 4H), 2.66 - 2.47 (m, 4H), 2.29 (t, J = 7.52 Hz, 2H), 1.77 - 1.19 (m, 43H), 0.96 - 0.78 (m, 15H), 0.03 (s, 6H). l-((6-((te7 -ButoxycarbonyI)(7-hydroxyheptyl)amino)hexyI)thio)octan-2-yI hexanoate (12.5).

[0239] HF - pyridine (0.45 mL, 3.49 mmol, 1.2 equiv) was added dropwise to a solution of 12.4 (2.0 g, 2.91 mmol, 1.0 equiv) in CH2CI2 (5 mL) in an ice bath. The mixture was stirred for 10 min, then quenched with sat. NaHCCh (5 mL) and extracted with CH2CI2 (3 10 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (15% EtOAc inHexane) to afford 12.5 (1.54 g, 2.68 mmol, 92%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 5.07 - 4.78 (m, 1H), 3.64 (t, J= 6.49 Hz, 2H), 3.32 - 3.03 (m, 4H), 2.76 - 2.41 (m, 4H), 2.30 (t, J= 7.45 Hz, 2H), 1.86 - 1.04 (m, 43H), 0.88 (q, J= 7.02 Hz, 6H). l-((6-((ter / -ButoxycarbonyI)(7-(tosyloxy )heptyl)amino)hexyl)thio)octan-2-yl hexanoate (12.6).Boc

[0240] p-Toluenesulfonyl chloride (0.75 g, 3.92 mmol, 1.5 equiv) in CH2CI2 (2.0 mL) was added to a solution of l-|6-| / c / 7-butoxycarbonyl(7-hydroxyheptyl)amino|hexylsulfanyl- methyl]heptyl hexanoate 12.5 (1.5 g, 2.61 mmol, 1.0 equiv), triethylamine (0.47 mL, 3.40 mmol, 1.3 equiv) and A i methyl aminopyridine (0.032 g, 0.26 mmol, 0.1 equiv ) in CH2CI2 (3.0 mL) cooled in an ice bath. The mixture was stirred at room temperature for 4 h. The solution was diluted with more CH2CI2 (5 mL) and sequentially washed with aqueous saturated NaHCCL (2 x 5 mL) and water (5 mL), then dried (NfeSCL) and evaporated. The residue was purified by silica gel column chromatography (9 % EtOAc in Hexane) to afford 12.6 (1.43 g, 1.98 mmol, 75%) as a pale yellowish oil. 'H NMR (400 MHz, CDCI3) 87.79 (d, J= 8.11 Hz, 2H), 7.34 (d, J= 7.97 Hz, 2H), 4.95 (m, 1H), 4.01 (t, J= 6.46 Hz, 2H), 3.25 - 2.90 (m, 4H), 2.71- 2.46 (m, 4H), 2.45 (s, 3H), 2.30 (t, J= 7.47 Hz, 2H), 1.93 - 1.07 (m,43H), 0.88 (t, J= 7.07 Hz, 6H).15-(te / t-Butoxy carbonyl)- l-cyclohexyl-5-(2-cyclohexylethyl)-3-oxo-4-oxa-7,22-dithia- 15-azatriacontan-24-yl hexanoate (12.8).

[0241] NaOMe, 25 wt% in MeOH (0.61 mL, 2.68 mmol, 1.3 equiv) was added dropwise to a cold (-20 °C) solution of thioester 8.3 (0.91 g, 2.47 mmol, 1.2 equiv) and tosylate 13.6 (1.5 g, 2.06 mmol, 1.0 equiv), in degassed DMF (5 mL). The mixture was stirred for 45 min, then quenched with sat. NH4CI (10 mL) and extracted with hexanes (3 10 mL). The combined extracts were washed with brine, dried (Na2SO-i). and concentrated in vacuo. Theresidue was purified by silica gel column chromatography (4% EtOAc in Hexane) to afford 12.8 (1.2 g, 1.36 mmol, 66%) as a colorless oil. 'H NMR (400 MHz, CDCh) 85.04 - 4.82 (m, 2H), 3.22 - 3.00 (m, 4H), 2.75 - 2.44 (m, 8H), 2.38 - 2.22 (m, 4H), 1.81 - 1.04 (m, 67H), 0.99 - 0.77 (m, 10H). LRMS m / z 882.6 [M+H]+.Procedure for Boc release: l-cyclohexyl-5-(2-cyclohexylethyl)-3-oxo-4-oxa-7,22-dithia- 15-azatriacontan-24-yl hexanoate (12.9).

[0242] A solution of 12.8 (1.2 g, 1.36 mmol, 1.0 equiv) and trifluoroacetic acid (TFA) (0.52 mL, 6.8 mmol, 5.0 equiv) in CH2CI2 (15 mL) was stirred at rt for 5 hours then concentrated. The mixture was diluted with CH2CI2 (15 mL), neutralized with sat. NaHCCh and the organic phase was collected. The aqueous phase was back extracted with CH2CI2 (3 x 10 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5% MeOH in DCM) to afford 12.9 (0.5 g, 0.64 mmol, 47%) as a pale yellowish oil. 'H NMR (400 MHz, CDCh) 8 5.04 - 4.77 (m, 2H), 2.76 - 2.36 (m, 12H), 2.36 - 2.22 (m, 4H), 2.16 (br, 1H), 1.85 - 1.00 (m, 58H), 0.99 - 0.68 (m, 10H).15-(4-((ter / -ButyldimethyIsilyl)oxy)butyl)-l-cyclohexyI-5-(2-cyclohexyIethyI)-3-oxo-4- oxa-7,22-dithia-15-azatriacontan-24-yl hexanoate (13.2).

[0243] Sodium triacetoxyborohydride (0.12 g, 0.58 mmol, 1.5 equiv.) was added portion wise to a solution of 12.9 (0.3 g, 0.38 mmol, 1.0 equiv) and 4-[tert- butyl(dimethyl)silyl] oxy butanal (0.12 g, 0.58 mmol, 1.5 equiv), in DCE (3.00 mL). The mixture was stirred at room temperature under nitrogen for 18 hours. The reaction was quenched with sat. NaHC'CL (5 mL) and extracted with CH2CI2 (3 5 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residuewas purified by silica gel column chromatography (5% MeOH in DCM) to afford 13.2 (0.23 g, 0.24 mmol, 62%) as a colorless oil. 'H NMR (400 MHz, CDC13) 85.04 - 4.78 (m, 2H), 3.61 (t, J= 5.85 Hz, 2H), 2.80 - 2.38 (m, 14H), 2.36 - 2.25 (m, 4H), 1.89 - 1.04 (m, 64H), 1.02 - 0.71 (m, 17H), 0.04 (s, 6H). l-Cyclohexyl-5-(2-cyclohexylethyl)-15-(4-hydroxybutyl)-3-oxo-4-oxa-7,22-dithia-15- azatriacontan-24-yl hexanoate (T-ll).

[0244] To a solution of 13.2 (0.20 g, 0.21 mmol, 1.0 equiv.) in CH2CI2 (0.8 mL) was added HF-pyridine (0.04 mL, 0.31 mmol, 1.5 equiv.) at 0°C under nitrogen. The mixture was stirred at 0°C until completion after 15 min. The reaction mixture was quenched by sat. NaHCCL (3.0 mL) and CH2CI2 (3.0 mL). The layers were separated, and the organic phase was collected. The aqueous phase was back extracted with CH2CI2 (2 x 5 mL). The combined organic phases were dried (ISfeSCL) and concentrated in vacuo. The residue was purified by silica gel column chromatography (7% MeOH in DCM) to afford T-ll (0.1 g, 0.12 mmol, 56%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 5.02 - 4.82 (m, 2H), 3.70 (t, J= 5.34 Hz, 2H), 3.08 - 2.90 (m, 6H), 2.70 - 2.48 (m, 8H), 2.38-2.25 (m, 4H), 1.91- 1.02 (m, 62H), 1.01 - 0.70 (m, 10H). LRMS m / z 854.6 [M+H]+.Procedure for / V-alkylation: l-cyclohexyl-5-(2-cyclohexylethyl)-15-(2-(2-hydroxy- ethoxy)-ethyl)-3-oxo-4-oxa-7,22-dithia-15-azatriacontan-24-yl hexanoate (T-12).

[0245] A solution of 12.9 (0.30 g, 0.38 mmol, 1.0 equiv.) and 2-(2-bromoethoxy)ethan-l- ol (71 mg, 0.42 mmol, 1.1 equiv) in dry acetonitrile (3 mL) containing suspended anhydrous K2CO3 (63 mg, 0.46 mmol, 1.2 equiv) was heated to 75 °C in a sealed reactor, under N2 atmosphere. After 20 hours, the solvent was evaporated, and the residue was taken up with water (5 mL) and CH2CI2 (5 mL). The aqueous phase was back extracted with CH2CI2 (3 x5 mL). The combined extracts were washed with water (2 x 5 mL), dried (NazSCh) and evaporated. The residue was purified by silica gel column chromatography (6% MeOH in DCM) to afford T-12 (100 mg, 0.11 mmol, 30%) as a pale yellowish oil.1!! NMR (400 MHz, CDCh) 8 5.01 - 4.77 (m, 2H), 3.74 (t, J= 6.21 Hz, 2H), 3.54- 3.39 (m, 4H), 2.89 - 2.18 (m, 14H), 1.91 - 1.07 (m, 62H), 1.00 - 0.65 (m, 10H)Example 2: mRNA-containing LNPs comprising ionizable lipids with cyclic moieties described herein exhibit in vivo delivery of mRNA to the liver and / or spleen that is superior to the nor-MC3 benchmark

[0246] LNP formulations containing 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG with a nitrogen-to-phosphorous ratio (N / P) of 6 and mRNA encoding luciferase were prepared as described in the Materials and Methods. The poly dispersity index (PDI), encapsulation efficiency and size of the LNP formulations are shown in Figure 1.

[0247] The LNP formulations were subsequently tested for in vivo transfection efficiency in the liver and spleen after injection to CD-I mice. The mRNA dose was 1 mg / kg. Luminescence intensity in the liver and spleen was measured at 4 hours post-injection.

[0248] The results in Figure 2A show luminescence intensity per mg liver for each formulation relative to the nor-MC3 benchmark. Results for luminescence intensity per mg spleen for the formulations tested relative to the MC3 benchmark are shown in Figure 2B.

Claims

1. WE CLAIM:

1. An ionizable, cationic amino lipid or a pharmaceutically acceptable salt thereof having a structure of FORMULA A:FORMULA A wherein R1is H or a substituent selected from,C1-C10 alkyl,Cs-Cs cycloalkyl,C1-C10 alkoxy,C1-C10 alkylthio, or an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C10 alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl, and the dash represents a bond to a ring atom of the ring structure having the n at its center, the ring structure having the n at its center comprising a total of n atoms, wherein n is 3 to 20, or 4 to 10, said ring structure being carbocyclic or heterocyclic, wherein m is 0 to 6,G is an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C14 alkyl, a C3-C16 cycloalkyl, or a C3-C16 alkylcycloalkyl, and the dash represents a bond to the CH group, wherein n is 1 to 4,A1is absent, a heteroatom that is optionally O or S, or an ester moiety, -C(O)-O-, wherein the C of the ester moiety is bonded to the (CH2)nand the O is bonded to the (CH2)P, or the C of the ester moiety is bonded to the (CH2)Pand the O is bonded to the (CH2)n, p is 2 to 10, and q is 2 to 10,A2is absent, a heteroatom optionally selected from O or S, or an ester moiety, C(O)-O, wherein the C of the ester moiety is bonded to the (CH2)qand O is bonded to an atom of the R2, or the C is bonded to (CH2)qand O is bonded to an atom of the R2,R2is a linear, branched or cyclic, optionally substituted, Ci to C10 alkyl, or wherein R2is a moiety of FORMULA A.l:FORMULA A.l wherein t is 0 to 6, and u is 1 to 4, wherein 1-3 carbon atoms of an alkyl of at least one R1, R2or R3optionally shares a double bond of E or Z configuration with an adjacent atom, or shares a triple bond with an adjacent atom,G is the ester -COO-R’ or R’-COO- as defined above, the circle of Formula A.l with the n in its center represents the ring structure as defined for Formula A comprising a total of n atoms, with n ranging from 3 to 20, or 4 to 10, R3is H or a substituent independently selected from a structure defined in R1above, A3is either C or N, and if A3is C, thenW1and Y are either bonded to each other or not bonded to each other as indicated by the dashed bond; and if W1and Y are bonded to each other thenW1is O or S;W2is O or S;X is CH;Y is (CH2)m, wherein m is 1 or 2; andZ is a group selected from one of structures a-c below, wherein the wavy line represents the bond to X, / a. - / '(CH2)n_N\ type 2 ionizable head, wherein n of the type 2 ionizable head is 1 to 5;— (CH2)m-O^ / (CH2)n^ / b. U | type 3 ionizable head, wherein m and n of the type 3 ionizable head are independently 1 to 5; andR c. •~v(CH2)m— N— (CH2)— OH tyPe4 ionizable head, wherein m and n of the type 4 ionizable head are independently 2 to 5, and wherein R of the type 4 ionizable head is Ci-Ce alkyl or cycloalkyl, if W1and Y are not bonded to each other, then:W1is H;W2is O, S, NH or NR3, wherein R3is a Ci to C4 alkyl optionally substituted with an OH group; and group is selected from one of structures d to 1 below, whereinthe wavy line represents the bond to W2: d-lf w2is O, type 1 ionizable head, wherein n of the type1 ionizable head is 1 to 5; -O, type 5 ionizable head, wherein m and n of the type 5 ionizable head are independently 1 to 5;O R f.jjJ^(CH2)^N^ (CH2)n-OH if W2is O, type 6 ionizable head, wherein m of the type 6 ionizable head is 1 to 5, and independently n of the type 6 ionizable head is 2 to 5, wherein R is Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl; g. (CH2)n_OH if W2is NH or NR3, type 7 ionizable head, wherein n of the type 7 ionizable head is 1 to 5; h. (CH2)n— is if W2is NH or NR3, type 8 ionizable head, wherein n of the type 8 ionizable head is 1 to 5; i.if W2is O, type 9 ionizable head, whereinO m and n of the type 9 ionizable head are independently 1 to 5;o j if W2is O, type 10 ionizable head, whereinthe ring structure of the type 10 ionizable head has 2 to 8 C atoms, and wherein j of the type 10 ionizable head is 0 to 5; k if W2is NH or NR3, type 11 ionizable head,wherein the circle of the type 11 ionizable head represents a homocyclic or heterocyclic ring comprising from 3 to 8 atoms, and wherein j of the type 11 ionizable head is 0 to 5; and crR1lf w2is O, type 12 ionizable head, and wherein j of the type 12 ionizable head is 1 to 5 and wherein R = Ci-Ce alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, if A3is N, thenW1and Y are absent;W2and X together form a group of structure (CRaRb)P, wherein Raand Rbare independently selected from H, C1-C5 alkyl, cycloalkyl, and wherein p is 2 to 6, 3 to 6 or 4 to 6; andZ is OH, SO2NH2 or NR’R”, wherein R’ and R” are independently C1-C5 alkyl, or cycloalkyl, or wherein R’ and R” form a heterocyclic group that incorporates the N atom to which R’ and R” are bound.

2. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of claim 1, wherein the ring structure with the n of Formula A or Formula A. 1 at its center is the heterocyclic and wherein the heterocyclic comprises carbon atoms and at least one nitrogen, oxygen, and sulfur atom.

3. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of claim 1, wherein the ring structure with the n of Formula A or Formula A. 1 at its center is the carbocyclic and wherein the carbocyclic is a 4 to 7 membered ring or a 5 to 6 membered ring.

4. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of claim 1, 2 or 3, wherein the ring structure of Formula A or Formula A. 1 having the n in itscenter comprises at least one substituent bonded to a ring atom thereof, the substituent selected from Ci to Cio alkyl, aryl, alkoxy, alkylthio or an ester having a structure as defined by -COO-R’ or R’-COO-or an amide having a structure as defined by -CONR’R” or R’- CON(R”)-, wherein R’ and R” are, independently, optionally substituted Ci to Ci6 alkyl or cycloalkyl and the dash represents the bond to an atom of the ring structure and optionally R’ and R” share a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom.

5. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 4, wherein the A3is N, and Z is NR’N”, wherein R’ and R” form the heterocyclic group that incorporates the N atom to which R’ and R” are bound and wherein the heterocyclic group is pyrrolidine, piperidine or morpholine.

6. An ionizable, cationic amino lipid or a pharmaceutically acceptable salt thereof comprising: a protonatable amino head group; two lipophilic chains, wherein the protonatable amino head group has a central carbon or nitrogen atom to which each of the two lipophilic chains are directly bonded; at least one of the two lipophilic chains has a structure of FORMULA A.2:FORMULA A.2 wherein R1is H or a substituent selected from,Ci-Cio, alkyl,Cs-Cs cycloalkyl,C1-C10 alkoxy,C1-C10 alkylthio, or an ester that is -COO-R’ or R’-COO-, wherein R’ is a C1-C10 alkyl, C3-C8 cycloalkyl, phenyl, substituted phenyl, and the dash represents a bond to a ring atom of the ring structure having the n at its center, the ring structure having the n at its center comprising a total of n atoms, wherein n is 3 to 20, or 4 to 10, said ring structure being carbocyclic or heterocyclic, wherein m is 0 to 6,G is an ester that is -COO-R’ or R’-COO-, wherein R’ is an optionally substituted alkyl selected from a linear or branched C1-C14 alkyl, C3-C16 or C4-C10 cycloalkyl, or C3-C16 alkylcycloalkyl, and the dash represents a bond to the CH group, wherein n of (CH)2 is 1 to 4,A1is absent, a heteroatom that is optionally O or S, or an ester moiety, -C(O)-O-, wherein the C of the ester moiety is bonded to the (CH2)nand the O is bonded to the (CH2)P, or the C of the ester moiety is bonded to the (CH2)Pand the O is bonded to the (CH2)n, p is 2 to 10, each lipophilic chain has between 15 and 40 carbon atoms in total; and wherein the ionizable, cationic amino lipid (i) imparts an apparent pKaof between 6 and 7.5 to a lipid nanoparticle when formulated therein; and (ii) has a ClogP of at least 11.

7. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of claim 6, wherein a second one of the two lipophilic chains bonded to the central carbon or nitrogen atom of the head group has a structure as defined by FORMULA A.3:R2— A2-(CH2)q~wFORMULA A.3 q is 2 to 10; wherein A2is absent, a heteroatom that is optionally O or S, or an ester moiety, C(O)-O, wherein the C of the ester moiety is bonded to the (CH2)qand the O of the ester moiety is bonded to an atom of the R2, or the C is bonded to (CH2)qand the O of the ester moiety is bonded to an atom of the R2, wherein R2is a linear, branched or cyclic, optionally substituted, Ci to C10 alkyl, optionally wherein 1 to 3 carbon atoms form a double bond of E or Z configuration with an adjacent atom, or share a triple bond with an adjacent atom; or wherein R2is a moiety ofFORMULA A.1:FORMULA A.1 wherein the wavy line represents a bond to A2, t is 0 to 6, u is 1 to 4,wherein the alkyl of at least one R1, R2or R3optionally shares a double bond of E or Z configuration with an adjacent atom, or shares a triple bond with an adjacent atom, G is the ester -COO-R’ or R’-COO- as defined above, the circle of Formula A.l with the n in its center represents the ring structure as defined above for Formula A comprising a total of n atoms, with n ranging from 3 to 20 or 4 to 10.

7. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 6, wherein the R’ of the G that is an ester (-COO-R’ or R’-COO-) is a C2-C14 alkyl or cycloalkyl, C3-C14 alkyl or cycloalkyl, C3-C10 alkyl or cycloalkyl or C4-C10 alkyl or cycloalkyl.

8. The ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of claim 1, having a structure selected from any one of compounds T-01-T-09, T-ll and T-12 as defined below or a pharmaceutically acceptable salt thereof:

9. A lipid nanoparticle comprising the ionizable, cationic amino lipid of any one of claims 1 to 8, a nucleic acid and a helper lipid.

10. The lipid nanoparticle of claim 9, wherein the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

11. A method for administering a nucleic acid to a subject in need thereof, the method comprising administering the lipid nanoparticle of claim 9 or 10 comprising the nucleic acid to the subject.

12. A method for delivering a nucleic acid molecule to a cell, the method comprising contacting the lipid nanoparticle of claim 9 or 10 with the cell in vivo or in vitro.

13. Use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 8 or the lipid nanoparticle of claim 9 or 10 in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

14. Use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 8 or the lipid nanoparticle of claim 9 or 10 to deliver a nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

15. The use as defined in claim 13 or 14, wherein the nucleic acid is an mRNA, DNA vector, siRNA or antisense oligonucleotide.

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