Ionizable lipids comprising sulfur atoms and cyclic moieties for the delivery of therapeutic agents

Abstract

Provided are novel sulfur-containing lipids and nanoparticles containing such lipids and a cargo molecule, such as a nucleic acid, methods to formulate the lipids with nucleic acids to produce lipid nanoparticles and chemical routes for making the lipids. The lipids may have the structure of Formula A as defined herein (Formula A)

Description

IONIZABLE LIPIDS COMPRISING SULFUR ATOMS AND CYCLIC MOIETIES FOR THE DELIVERY OF THERAPEUTIC AGENTS TECHNICAL FIELD

[0001] Provided herein are sulfur-containing lipids that may be formulated in a delivery vehicle so as to facilitate the encapsulation of cargo, 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 (Scheme 1), by investigators. Furthermore, MC3 represents an evolution of a structurally related ionizable lipid, referred to by investigators as “KC2”, 2 (Scheme 1). MC3 is considered a state-of-the art ionizable lipid for the delivery of siRNA, requiring about 3 times less siRNA than KC2, although KC2 is superior in other applications, and it remains a valuable research tool.

[0005] While the foregoing ionizable lipids are especially efficacious for the delivery of siRNAcontaining LNPs to hepatic cells, they are much less effective for the hepatic delivery of mRNA-containing LNPs. To illustrate, mRNA vaccines, including the COVID-19 Pfizer / BioNTech and Moderna vaccines, rely on lipid nanoparticles to deliver mRNA to the cytoplasm of liver cells. After entry into the host cell, the mRNA is transcribed to produce antigenic proteins. In the case of the COVID-19 vaccines, the mRNA encodes the highly immunogenic Sars-Cov-2 spike protein. Such vaccines, however, incorporate other types of ionizable lipids besides MC3 or KC2. In particular, the Pfizer / BioNTech vaccine comprises an ionizable lipid referred to as “ALC-0315”, 3 (Scheme 1), and the Moderna vaccine comprises an ionizable lipid referred to as “SM-102”, 4.

[0006] Furthermore, 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 diseaseconditions 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 relationship between lipid structure and nucleic acid delivery efficiency is complex and not readily predictable from structural considerations alone. Minor structural changes in lipophilic chains can affect nanoparticle formation, biodistribution, and endosomal escape. Branched ionizable lipids incorporating ester groups and having sulfur atoms distal to the branch point have been described in co-owned WO 2024 / 065042, and such architectures have been found to improve biodistribution, notably to the liver and spleen. However, there remains a need to identify lipid architectures that lead to further improvements in nucleic acid delivery and / or that can be more readily synthesized. This is often challenging because modification of distal hydrophobic substituents within ionizable lipid structures can significantly influence lipid nanoparticle performance. For example, modification of an ionizable lipid to adjust chain length may impact physicochemical properties such as CLogP. As a result, the identification of ionizable lipid architecture suitable for nucleic acid delivery often involves empirical investigation and screening of candidate lipids.

[0008] 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

[0009] As used herein, “type 1 ionizable head” refers to a head group that includes a moiety of Formula I below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, n ranging from 1 to 5:X(CH2)n-N Formula I

[0010] As used herein, “MC-type ionizable head” refers to a moiety that has a head group of the lipid of Formula la below, or equivalents thereof, with n ranging from 1 to 5:o[lipophilic chain(s)]— O (CH2)^Formula la

[0011] As used herein, “type 2 ionizable head” refers to a head group that includes a moiety of Formula II below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, with n ranging from 1 to 5:— (CH2)n-N\Formula II

[0012] As used herein, “KC-type ionizable head” refers to a moiety that has a head group of the lipid of Formula Ila below, or equivalents thereof, with n ranging from 1 to 5:_ / [lipophilic chain(s)] O-^ ^(CH2)nN[lipophilic chain(s)] X O JFormula Ila

[0013] As used herein, “type 3 ionizable head” refers to a head group that includes a moiety of Formula III below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, m and n independently ranging from 1 to 5:(CH2)m-O (CH2)n\NOFormula III

[0014] As used herein, “type 4 ionizable head” refers to a head group that includes a moiety of Formula IV below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, R = C₁-C₆ alkyl or cycloalkyl, and with m and n independently ranging from 2 to 5:R— (CH2)m-N-(CH2)n— OHFormula IV

[0015] As used herein, “type 5 ionizable head” refers to a head group that includes a moiety of Formula V below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, with m and n independently ranging from 1 to 5:O O— i X(CH2)A^< CH2,"-fFormula V

[0016] As used herein, “type 6 ionizable head” refers to a head group that includes a moiety of Formula VI below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, R = C₁-C₆ alkyl or cycloalkyl and with m ranging from 1 to 5, and n, independently, ranging from 2 to 5:0^\cH2) / N^(CH2)n-OHFormula VI

[0017] As used herein, “type 7 ionizable head” refers to a head group that includes a moiety of Formula VII below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, and with n ranging from 1 to 5:(CH2)n— OHFormula VII

[0018] As used herein, “type 8 ionizable head” refers to a head group that includes a moiety of Formula VIII below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, and with n ranging from 1 to 5:— (CH2)n-NFormula VIII

[0019] As used herein, “type 9 ionizable head” refers to a head group that includes a moiety of Formula IX below, or equivalents thereof, wherein the wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, with m and n independently ranging from 1 to 5:O / Ji O-(CH2)n-NoFormula IX

[0020] 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 wavy line connects to W selected from O, S, NH and NRa, with Rabeing C₁-C₆ alkyl or cycloalkyl, in which X = O or S, m independently ranging from 2 to 4 in each [(CH2)m-X] unit, n ranging from 1 to 3 and p ranging from 2 to 4:[(CH2)m-X]n— (CH2)p-OHFormula X

[0021] 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.

[0022] As used herein, the term “lipophilic chain” refers to an alkyl group bonded to a nitrogen or carbon atom of the lipid, the alkyl group comprising at least 6 C atoms and optionally comprising C=C double bonds, and / or ring structures, and / or carbonyl groups, and / or heteroatoms such as N, O, S, and such that the parent compound of the alkyl group has a CLogP of at least 6.

[0023] For example, lipid MC3, 1, and lipid KC2, 2, have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene, which has a CLogP of 9.25:parent compound of lipiphilic chain(s):(6Z,9Z)-octadeca-6,9-diene: CLogP: 9.25

[0024] Lipid ALC-0315, 3, has a pair of lipophilic chains derived from hexyl 2-hexyldecanoate, which has a CLogP of 10.01:lipiphilic hexyl 2-hexyldecanoate chain(s) CLogP: 10.013

[0025] 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:undecyl hexanoate CLogP: 7.588 heptadecan-9-yl octanoate CLogP: 11.6

[0026] As used herein, the term “alkyl” or “alkyl group” is a carbon-containing chain that is linear or branched and that optionally comprises C=C double bonds and / or ring structures, and that is optionally substituted.

[0027] As used herein, the term “Cm to Cn alkyl” or “Cm to Cn alkyl 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.

[0028] The term “optionally substituted” with reference to an alkyl 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 by one or more substituents comprising heteroatoms selected from O, S and NR’, wherein R’ is as defined below. Non-limiting examples of groups that may replace a hydrogen atom include halogen; alkyl groups; cycloalkyl groups; oxo groups (=0); hydroxyl groups (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; —OR'; — S(O)XR'; — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=0)R'; — C(=0)NR'R'; — NR'C(=0)NR'R'; — 0C(=0)NR'R'; — NR'C(=0)0R'; — 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.

[0029] 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), phosphatidyl serine (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. 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.

[0030] 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 polymerlipid hybrids, including particles in which the lipid is attached to a polymer.

[0031] 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.

[0032] 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.

[0033] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.SUMMARY

[0034] The present disclosure is based, at least in part, on the surprising discovery that LNP formulations of nucleic acids comprising ionizable lipids that incorporate at least one lipophilic chain substituted with a sulfur atom and an ester moiety are more potent than a nor-MC3 benchmark LNP (Example 2) for liver delivery of therapeutic RNA. As further described herein, such lipids may exhibit a different organ selectivity relative to known lipids. In particular, nonlimiting examples described herein demonstrate that such lipids promote the delivery of mRNA selectively to the spleen more efficiently than other known lipids. In addition, the chemical synthesis of the lipids of certain embodiments herein is more straightforward and / or economical than that of known lipids.

[0035] In certain embodiments, the hydrophobic substituent distal to the sulfur atom of the lipid backbone comprises a cycloalkyl group. Suitable cycloalkyl groups may include, for example, cyclohexyl, cycloheptyl or cyclooctyl. In some embodiments, the cyclohexyl, cycloheptyl or cyclooctyl group is bound to the sulfur atom through a methylene spacer or an ethylene spacer. In alternative embodiments, the cycloalkyl group is cycloheptyl or cyclooctyl and lacks such a spacer group.

[0036] According to one aspect of the disclosure, there is provided a lipid having a structure of Formula A:r2“s- — (CH2)m\ W2X^Z2 A IR3-S"G"'^(CH2)n / W1- YE2R4Formula Aor a pharmaceutically acceptable salt thereof;whereinindices m and n are independently 4 to 8; andR1and R4are independently, optionally substituted, linear, branched or cyclic C3 to C20 alkyl groups, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising C₃ to C₈ cycloalkyl substituents;R2and R3are independently groups of Formula B:^(CH^(CH2)pFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is at least 1,E1and E2are ester groups, that is -C(O)-O- or -O-C(O)-;G1and G2are, independently, optionally substituted, linear, branched or cyclic Ci to Ce alkyl groups;A is C or N, andif A is C, then W1and Y are either bonded to each other or not bonded to each other, andif W1and Y are bonded to each other, thenW1is O or S;W2is O or S;X is CH;Y is (CH2)q, wherein q is 1 or 2; andZ is selected from one of structures a-c below, wherein the wavy line represents the bond to X:a. ^v'(CH2)n- N type 2 ionizable head group, wherein the n of the type 2 ionizable head group is 1 to 5;— (CH2)m-O^ / (CH2)n\N / b. | type 3 ionizable head group, wherein the m and n of the type 3 ionizable head group are independently 1 to 5;Rc. ■~v(CH2)m-N— (CH2)— OH type 4 ionizable head group, wherein the m and n of the type 4 ionizable head group are independently 2 to 5 and wherein R = Ci-Ce alkyl or cycloalkyl;if W1and Y are not bonded to each other, thenW1is H;W2is O, S, NH or NR2a, wherein R2ais a Ci to Ce alkyl optionally substituted with an OH group, and optionally a cycloalkyl; andthe moiety | of Formula A is a group selected from one of Ystructures d-j below, wherein the wavy line represents the bond to W2:d. J0L N I type 1 ionizable head group, wherein the n of (CH2)the type 1 ionizable head group is 1 to 5;Oe. > — (CH2)n— type 5 ionizable head group,(CH2)mO |wherein the m and n of the type 5 ionizable head group are independently 1 to 5;O Rif.(CH2)mN (CH2)n— OH type 6 ionizable head group, wherein the m of the type 6 ionizable head group is 1 to 5 and the n of the type 6 ionizable head group is independently 2 to 5 and wherein the R = Ci-Ce alkyl or cycloalkyl;g. (CH2)n“OH type 7 ionizable head group, wherein the n of the type 7 ionizable head group is 1 to 5, optionally wherein W2is NR2a;h. "w (CH2)n“N\ type 8 ionizable head group, wherein the n of the type 7 ionizable head group is 1 to 5, optionally wherein W2is NR2a;o / i. x > / ° (CH2)n N\ type 9 ionizable head group,Owherein m and n of the type 9 ionizable head group are independently 1 to 5;j. ■~v'[(CH2)m-X]n— (CH2)p-OH type 10 ionizable head group, wherein X = O or S, m of the type 10 ionizable head group is 2 to 4, n of the type 10 ionizable head group is 1 to 3, and p of the type 10 ionizable head group is 2 to 4, optionally wherein W2is NR2a,if A is N, thenW1and Y are absent;W2and X together form a group of structure (CRaRb)P, wherein Raand Rbare independently H, C1-C5alkyl or cycloalkyl, and wherein p is 2 to 6; andZ is OH or NR’R”, wherein R’ and R” are independently optionally substituted C1-C5 alkyl or cycloalkyl, or wherein R’ and R” together with the N atom of NR’R”, form an optionally substituted heterocyclic ring that incorporates the N atom to which the R’ and R” are each bound, and optionally wherein the heterocyclic group that incorporates the N atom to which R’ and R” are bound is pyrrolidine, piperidine or morpholine.

[0037] In certain embodiments of the foregoing aspect, the A is C. In some preferred embodiments, W1and Y are not bonded to each other. In some non-limiting embodiments, the W2is O.~xIz

[0038] In some embodiments the moietyYof Formula A is the structure d. In other • xIzembodiments, the moietyYof Formula A is the structure j.

[0039] In certain non-limiting embodiments, the W1 and Y of Formula A are bonded to each other. In some embodiments, W1 and W2 are each O. In certain non-limiting embodiments, the Z in Formula A is the structure a.

[0040] In certain other embodiments, the A is N. In some preferred embodiments, the Z is OH.

[0041] In some non-limiting embodiments of the lipid, at least one of the ester groups selected from El and E2 has a carbonyl carbon (C(O)) bonded to R1 or R4, respectively. In some embodiments, El and E2 have carbonyl carbon (C(O)) bonded to R1 or R4, respectively.

[0042] In some embodiments, G1 and G2 are, independently, optionally substituted, linear or branched Cl to C6 alkyl or C3 to C6 cycloalkyl groups. In some preferred embodiments, G1 and G2 are, independently, optionally substituted, linear Cl to C6 alkyl groups, optionally a methylene.

[0043] In some embodiments, G1 and G2 are, independently, optionally substituted, linear or branched Cl to C6 alkyl or C3 to C6 cycloalkyl groups. In some preferred embodiments, G1 and G2 are, independently, optionally substituted, linear Cl to C6 alkyl groups, optionally a methylene.

[0044] In another aspect of the foregoing disclosure, there is presented a lipid or a pharmaceutically acceptable salt thereof comprising:a protonatable amino head group;at least two lipophilic chains, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic chains are directly bound;at least one of the lipophilic chains has the formula:R2-S^) - (CH2)nJUW>R1— Ewherein (CH2)n- of the formula is bound to the central nitrogen atom or carbon atom; wherein R1is a linear, branched or cyclic optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation, optionally comprising C₃ to C₈ cycloalkyl substituents; wherein R2is a structure of Formula B:r^(CH2)q'AA~'(CH2)pFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is 1 or greater,n is 4 to 8;E is an ester;each lipophilic chain has between 15 and 40 carbon atoms in total; andwherein the lipid is ionizable and has a CLogP of at least 11.

[0045] In yet another aspect of the foregoing disclosure, there is presented an ionizable amino lipid with at least two lipophilic chains directly bound to a central nitrogen or carbon atom in which at least one of the lipophilic chains has the formula:R5*y~(CH2)nJVVV'R6wherein the wavy line represents a bond to the central nitrogen atom or carbon atom; n is 4 to 8;wherein the * represent a carbon branch point;wherein R5is a linear, cyclic or branched C3-C30 alkyl group, wherein a carbon atom of the C3-C30 alkyl group is substituted with an ester E;wherein R6is a C3-C20 alkyl group with a sulfur atom at an alpha (Ci), beta (C2) or gamma (C3) position with respect to the carbon branch point and wherein the sulfur is bound to a distal R2, the R2having a structure of Formula B:r^(CH2)q—(CH2)PFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is 1 or greater.

[0046] In certain embodiments of the foregoing aspect, the ester E is substituted at an alpha (Ci), beta (C2) or gamma (C3) position with respect to the carbon branch point.

[0047] In certain non-limiting embodiments, the q of Formula B is 0. In certain other embodiments, the index p of Formula B is 5 or 6 and the index q is 0 or 1.

[0048] In certain non-limiting embodiments, the index p of Formula B is 4 and the index q is 1 or 2. In certain other embodiments, the index p of Formula B is 5 or 6 and the index q is 0 or 1.

[0049] In yet other embodiments, the index p of Formula B is 5 and the index q is 0 or 1. In some non-limiting embodiments, the index p of Formula B is 6 and the index q is 0.

[0050] In certain embodiments of the foregoing aspect of the disclosure, the R1 and R4 are, independently, C6 to C11 linear saturated alkyl or independently have a structure of Formula B.l:^(CH2)q^(CH2)pwherein p of Formula B.l is 4 or 5 and q of Formula B.l is 1, 2, 3 or 4.

[0051] In certain non-limiting embodiments, R1 and R4 are independently C8 or C9 linear saturated alkyl and wherein q of Formula B.l is 1, 2 or 3.

[0052] In certain non-limiting embodiments of the foregoing disclosure, if A is C thenW1and Y are bonded to each other, and wherein W1is O; W2is O; X is CH; Y is (CH2)q, wherein q is 1 or 2; and Z is structure a type 2 ionizable head group; or W1and Y are not bonded to each other, and wherein W1is H; W2is O, S, NH or NR2a, wherein R2ais a Ci to Ce alkyl optionally substituted with an OH group, and optionally a cycloalkyl; and the moiety 'UV'X'Zof Formula A is structure d type 1 ionizable head ygroup, structure g type 7 ionizable head group or structure j type 10 ionizable head group; andwherein if A is N thenW1and Y are absent; W2and X together form a group of structure (CRaRb)P, wherein Raand Rbare H, and wherein p is 4; and Z is OH.

[0053] In certain non-limiting embodiments of the foregoing aspect, the lipid is selected from the following group:

[0054] According to a further embodiment of any of the foregoing aspects of the disclosure, the lipid of Formula A, when formulated in a lipid nanoparticle comprising an mRNA, results in an increase in biodistribution of the lipid nanoparticle of at least about 10% in the liver and / or one or more extrahepatic tissues, such as the spleen, relative to a lipid nanoparticle containing DLin-nor-MC3-DMA (also referred to as n-MC3 or nor-MC3) as measured by luminescence of the mRNA in vivo in the liver and / or the one or more extrahepatic tissues. The assay and the formulations used to determine the biodistribution are as described in Example 2.

[0055] According to yet another embodiment of any of the foregoing aspects of the disclosure, the lipid of Formula A, when formulated in a lipid nanoparticle comprising an mRNA, results in an increase in biodistribution of the lipid nanoparticle in the liver and / or one or more extrahepatic tissues, such as the spleen, relative to a lipid nanoparticle containing nor-MC3, Benchmark-1 or Benchmark-2 (lipid details have been provided in Table 1) as measured by luminescence of the mRNA in vivo in the liver and / or the one or more extrahepatic tissues. The assay and the formulations used to determine the biodistribution are as described in Examples 3 and 4.

[0056] In certain embodiments, the novel lipid comprises a cycloalkyl group connected through a methylene or an ethylene spacer positioned distal to the sulfur atom of the lipid backbone. In some non-limiting embodiments, the introduction of the alkylene spacer between the sulfur atom and the cycloalkyl ring in the ionizable lipid contributes to an increase in in vivo protein expression when formulated in an mRNA LNP compared to a lipid lacking the methylene or ethylene spacer. In a particularly preferred embodiment, the lipid comprises a cyclohexyl group connected through a methylene or an ethylene spacer distal to the branching point of the hydrophobic tail.

[0057] In certain embodiments, the novel lipid comprises an additional carbon atom in the cyloalkyl positioned distal to the sulfur atom of the lipid backbone. In some embodiment, the cyclic ring is a cycloheptyl moiety. In some other embodiments, the cycloalkyl ring is a cyclooctyl moiety. In some non-limiting embodiments, the introduction of the carbon atom contributes to an increase in in vivo protein expression when formulated in an mRNA LNP compared to a lipid bearing a cyclohexyl ring. In a particularly preferred embodiment, the lipid comprises a cycloheptyl moiety distal to the branching point of the hydrophobic tail. In yet another embodiment, the lipid comprises a cyclooctyl moiety distal to the branching point of the tail.

[0058] In another aspect, there is provided a lipid nanoparticle comprising the lipid of any one of the foregoing aspects or embodiments thereof and a nucleic acid.

[0059] The lipid nanoparticle may comprise a helper lipid and a hydrophilic polymer-lipid conjugate. The helper lipid may be selected from cholesterol, a diacylglycerol and a sphingolipid.

[0060] In a further aspect, there is provided a method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle as described in any of the foregoing aspects or embodiments comprising the nucleic acid and administering the lipid nanoparticle to the subject.

[0061] According to another aspect of the disclosure, there is provided a method for delivering a cargo molecule to a cell, the method comprising contacting the lipid nanoparticle as described in any aspect or embodiment as described above with the cell in vivo or in vitro. In one embodiment, the cargo molecule is a nucleic acid.

[0062] According to a further aspect of the disclosure, there is provided a use of the lipid or a pharmaceutically acceptable salt thereof as defined above or the lipid nanoparticle as defined in any aspect or embodiment described above, 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.

[0063] According to a further aspect of the disclosure, there is provided a use of the lipid or the pharmaceutically acceptable salt thereof as defined above or the lipid nanoparticle of any one of the aspects or embodiments described above to deliver a nucleic acid to a patient to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid. In one embodiment, the nucleic acid is an mRNA.

[0064] 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

[0065] FIGURE 1 is a bar graph showing entrapment (%), particle size and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising the ionizable lipids nor-MC3, T-l, T-3, T-5, T-10, T-17, T-19, T-22, T-26, and T-27 described hereinafter. 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. The details of the lipids are found in Table 1.

[0066] FIGURE 2A shows luminescence intensity / mg in the liver tissue for the mRNA-containing LNPs comprising the ionizable lipids nor-MC3, Tl, T3, T5, T10, T17, T19, T22,T26, and T27 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).

[0067] FIGURE 2B shows luminescence intensity / mg in the spleen tissue for the mRNA-containing LNPs comprising the ionizable lipids nor-MC3, Tl, T3, T5, T10, T17, T19, T22, T26, and T27 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).

[0068] FIGURE 3A shows luminescence intensity / mg in the CD-I mice liver tissue 4 hours post-intravenous administration of mRNA-containing LNPs comprising the following ionizable lipids: nor-MC3, Benchmark-1, T-03 and T-05. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0069] FIGURE 3B shows luminescence intensity / mg in the CD-I mice liver tissue 4 hours post-intravenous administration of mRNA-containing LNPs comprising the following ionizable lipids: nor-MC3, Benchmark-2, T-19, T-20, T-22 and T-26. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0070] FIGURE 4A shows luminescence intensity / mg in the CD-I mice liver tissue 4 hours post-intravenous administration of mRNA-containing LNPs comprising the following ionizable lipids: nor-MC3, Benchmark-1 and T-01. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0071] FIGURE 4B shows luminescence intensity / mg in the CD-I mice liver tissue 4 hours post-intravenous administration of mRNA-containing LNPs comprising the following ionizable lipids: nor-MC3, Benchmark-2, T17 and T-25. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).DETAILED DESCRIPTION

[0072] Various aspects and embodiments of the disclosure are directed to ionizable lipids having structures of Formula A and pharmaceutically acceptable salts thereof.R,'E>R2“S^ J - (CH2)m\W2_X^Zl A1IR3-S"Gx1— (CH2)nZ W"YE2R4Formula A

[0073] 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.Methods to produce lipids of Formula A

[0074] Lipids of Formula A or pharmaceutically acceptable salts thereof can be prepared using any suitable method known to those of skill in the art. Particularly suitable methods are shown below.

[0075] As described in co-owned and co-pending applications WO 2022 / 246555, WO 2023 / 147657, WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 130421, WO 2026 / 000083, WO 2026 / 000082, each of which is incorporated herein by reference, a lipid of Formula A wherein A is a carbon atom can be prepared from ketone 2.1 (Scheme 2). Ketone carbonyl group in 2.1 can subsequently be transformed into a suitable ionizable head group through appropriate synthesis steps.Scheme 2

[0076] Ketone 2.1 wherein R1and R4, R2and R3, E1and E2, and m and n all differ, can be made starting with a selective mono-alkylation of tosylmethyl isocyanide (TosMIC, 3.1) with alkyl halide or sulfonate 3.2 (L = Cl, Br, I, or OSO2Ph, OTs, OMs, OTf) in the presence of a bulky, moderately strong organic base such as DBU in an appropriate solvent such as DMF (Scheme 3).This produces 3.3, which can be alkylated again with 3.4 (X = as for 3.2) under basic conditions in an appropriate solvent, for example, with NaH in DMF, to give 3.5. Treatment of 3.5 with an aqueous solution of a strong mineral acid in an organic cosolvent, for example, concentrated aqueous HCl in diethyl ether, converts it into 2.1.R2-S.1^(CH2)m-L o 3.2 DBU, DMFScheme 3

[0077] Alkylating agents 3.2 and 3.4 can be represented with the general structure 4.1 (Scheme 4). Compounds of this type can be prepared by methods that are well known to those skilled in the art. Without intending to be limiting, representative synthetic routes are set forth in Schemes 5-13Rp= R1or R4RpRq= R2or R3E E = E1or E2Rq-S / ^(CH2)r-L, zG^ G = G1or G2R3-S ^(CH2)n— L r = m or n4.1E2L = halide or sulfonateR43.4Scheme 4

[0078] A compound 4.1 wherein the moiety Rp-E is an ester of structure RP-O-C(O)- can be represented with structure 5.4 (Scheme 5). A route to 5.4 starts with the displacement of a leaving group L (L = halide or sulfonate) from 5.1 (P = protecting group) with a thiol Rq-SH under basic conditions. Subsequent release of protecting group P and conversion of the OH group in the resulting alcohol 5.3 into a leaving group L produces 5.4.RP-0Rq-SH Rp-Oconditions(CH2)r— O-P Rq-S (CH2)r— O-P -baseL effective5.1 L = leaving grp. 5.2P = prot. grp.RP = R1or R4RP-O conditions Rp-O^,0 Rq= R2or R3G = G1or G2Rq-S (CH2)r— OH effective Rq-S (CH2)r-L r = m or nG = C1to C6alkyl5.3 5.4L = halide or sulfonate Scheme 5

[0079] In certain embodiments, group G in 5.4 may be a moiety of structure (Ru)(Rv)C, with Ruand Rvindependently equal to H or small alkyl; namely, 5.4 can be represented as structure 6.3 (Scheme 6). In such a case, it may be advantageous to prepare 6.3 by 1,4-addition of Rq-SH to a conjugated ester of structure 6.1. As before, this would be followed by release of protecting group P and conversion of the OH group in 6.2 into leaving group L, resulting in formation of 6.3.RP-0^,0 Rq-SH Scheme 5 Rux^ (CH2)r— O-P base (CH2)r— OP (CH2) -L□v 6.1 6.2 6.3Scheme 6

[0080] A compound 4.1 wherein the moiety Rp-E is an ester of structure RP-C(O)-O- can be represented with structure 7.4 (Scheme 7). A route to 7.4 entails the displacement of a leaving group L (L = halide or sulfonate) from 7.1 (P = protecting group) with a thiol Rq-SH under basic conditions. Subsequent release of protecting group P and conversion of the OH group in the resulting alcohol 7.3 into a leaving group L produces 7.4.oRP-O^O Rq-SH conditions / L(CH2)r-O-Pi base effectiveL 7.1 L = leaving grp.P = prot. grp.RP = R1or R4conditions Rq= R2or R3G = G1or G2RUrJ - (CH2)r— OHeffective r = m or n7.3 7.4 G = C1to C6alkylL = halide or sulfonate Scheme 7

[0081] In certain embodiments, group G in 7.4 may be a methylene (CH2); namely, 7.4 can be represented as structure 8.5 (Scheme 8). In such a case, it may be advantageous to prepare 8.5 starting with the reaction of thiol Rq-SH with epoxide 8.1, followed by esterification of the OH group in the resulting 8.2 with an appropriate carboxylic acid. As before, this would be followed by release of protecting group P and conversion of the OH group in 8.4 into leaving group L, resulting in formation of 8.5.ORq-SH OH RP-COOH (CH2)r-O-P RP O8.1 r = m or n base 8.2 conditions P = prot. grp. 8.3effectiveO ORPreleas RpO conditions Oe Peffective8.5 L = halide or sulfonateScheme 8

[0082] In some embodiments, ketone 2.1 may be such that one or more of R1and R4, R2and R3, G1and G2, E1and E2, m and n, may be identical. In such cases, alternative synthetic routes may be advantageous.

[0083] For example, a ketone 2.1 wherein R1= R4, R2= R3, E1= E2= -C(O)-O-, G1= G2= CH2, and m = n, namely, 9.6 (Scheme 9), can be prepared as described in detail in co-owned and copending US 2024 / 0294462, incorporated herein by reference. Accordingly, TosMIC, 3.1 is doubly alkylated with alkyl halide or sulfonate 9.1 (L = Cl, Br, I, or OSO2Ph, OTs, OMs, OTf) under basicconditions in an appropriate solvent, for example, in the presence of NaH in DMF, followed by treatment of the resulting 9.2 with an aqueous solution of a strong mineral acid in an organic cosolvent, for example, concentrated aqueous HCl in diethyl ether. This produces 9.3, which is then doubly epoxidized with an appropriate reagent, for example, a preformed peroxycarboxylic acid like meta-chloroperoxybenzoic acid, peracetic acid, magnesium monoperoxyphthalate, and the like, or a mixture of formic acid and aqueous hydrogen peroxide solution. Bis-epoxide 9.4 is then caused to react with a thiol in the presence of base, leading to the formation of 9.5. The OH groups in the latter are esterified with a carboxylic acid in the presence of a suitable condensing agent, for example, a carbodiimide such as EDCI, to produce the desired ketone 9.6., Tscone.9.1 (CH2)m, Ts aq. HCI (CH2)mepoxidation ©Nbase7m© ether 3.19.20O9.3O OH R1(CH2), R2-SH R1-COOH R2-SOzO / (CH2)mbase R2-S coupling R2-S 'm O 9.4 OH agent R! JD9.5OScheme 9

[0084] Alternatively, ketone 9.3 can be prepared starting with the Claisen condensation of an ester such as 10.1 to give 10.2, for example, under Mukaiyama conditions, as described in co-owned and co-pending WO 2022 / 246555, incorporated herein by reference (Scheme 10). Saponification of 10.2 and acidification results in decarboxylation of transient ketoacid 10.3 to give 9.3.(CH2)m oaq. NaOH ^X'(CH2)m-COOMeEt3N’TlC'410.1 CH2CI2^< CH2U10,—.2 CoMe,he"aq-HCI^(CH2)m<5^(CH2)m-1- (COOH10.3 9.3Scheme 10

[0085] As another example, a ketone 9.6 wherein R2= R3, but R1and R4differ, namely, 11.2 (Scheme 11), can be prepared starting with the mono-esterification of a compound such as 9.5 with about one molar equivalent of carboxylic acid R'-COOH in the presence of a suitable condensing agent, for example, a carbodiimide such as EDCI, to produce monoester 11.1, as described in detail in co-owned and co-pending WO 2023 / 147657, WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 130421 and WO 2024 / 065043, each of which is incorporated herein by reference. The free OH group in monoester 11.1 can then be esterified with carboxylic acid, R4-COOH, also in the presence of a suitable condensing agent, for example, a carbodiimide such as EDCI, to produce diester 11.2.Oabout 1 equiv of R1-COOH R4-COOHcoupling coupling agent ~ OH agentaO 11.1Scheme 11

[0086] As yet another example, a ketone 2.1 wherein R1= R4, R2= R3, E1= E2= -C(O)-O-, G1= G2= CH2, but m and n differ, namely, 12.7 (Scheme 12), can be prepared starting with the selective mono-alkylation of TosMIC with alkyl halide or sulfonate 12.1 in the presence of a bulky, moderately strong organic base such as DBU in an appropriate solvent such as DMF. This produces 12.1, which can be alkylated again with alkyl halide or sulfonate 12.2 (L = Cl, Br, I, or OSO2Ph, OTs, OMs, OTf) under basic conditions in an appropriate solvent, for example, in the presence of NaH in DMF. Treatment of the resulting 12.3 with an aqueous solution of a strong mineral acid in an organic cosolvent, for example, concentrated aqueous HCl in diethyl ether, produces 12.4, which can be transformed into the desired 12.7 by the method outlined in Scheme 11 above.<^(CH2)m-L ^ / (CH2)n-L^(CH2)mTs “ 9.1 ^(CH2)m“C|12.2 DBU, DMF 12.1 NaH, DMF12.3Scheme 12

[0087] As another example still, a ketone of the type 2.1 wherein R1= R4, E1= E2= -C(O)-O-, G1= G2= CH2, m = n, but R2and R3differ, namely, 13.3 (Scheme 13), can be prepared starting with R2-SH OH(CH2)m(about R3-SH (CH2) / 1 equiv) base9.4 base13.1 oOHR1-COOH couplingagentScheme 13the reaction of bis-epoxide 9.4 with about one molar equivalent of thiol R2-SH in the presence of an appropriate base, whereupon selective opening of one of the oxirane rings gives 13.1. Opening of the remaining epoxide ring with a different thiol, R3-SH, in the presence of an appropriate base, transforms 13.1 into 13.2, bis-esterification of which produces 13.3.

[0088] As a further example, a ketone of the type 2.1 wherein R1= R4, E1= E2= -O-C(O)- and m = n, namely, 14.8 (Scheme 14), can be synthesized starting from dihydroxyketone 14.1, available as described in the aforementioned co-pending and co-owned PCT application WO 2023 / 147657. Conversion of the OH groups into leaving groups, L (cf. 14.2, L= halide or sulfonate), and selectivedisplacement of one L with a malonate derivative such as 14.3 produces 14.4, which can be transformed into 14.6 by displacement of the second L with a malonate derivative such as 14.5.Saponification and decarboxylation of 14.6 gives diacid 14.7, which can be esterified with RLOH in the presence of a condensing agent, for example a carbodiimide like EDCI, to produce 14.8COOMe MeOOC COOMez-(CH2) R2_S R2-S ^G2COOMe " G1COOMe G1R3-S 14.3014.5 COOMe L- (CH2Z-(CH2) )mbase base 14.4OH - 1 conditionsL (halide, sulfonate)' effectiveMeOOC COOMe COOH2Gaq. NaOHR -S.1^(CH2U R1-OH thenG2 / CR3_S (CH2)maq. acid EDCI MeOOC COOMe 14.6 heat, - CO2R1— OR2-S1^- (CH2)m(J, „G?R3-S "plCH^R1-OA)14 8Scheme 14

[0089] A lipid of Formula A wherein A is a nitrogen atom can be represented with the general formula 15.1 (Scheme 15).A = NR2~SG1 I >. N-[(CH2)rX]u— (CH2)V-OH o ^Gt / R “sV- (CH2)n X = O or S. t = independently 2 to 4 in eachR‘ [(CH2)t-X] unit, u = 0 to 2, v = 2 to 6Scheme 15

[0090] Lipid 15.1 can be prepared by methods described in co-owned and co-pending applications WO 2023 / 173203, WO 2024 / 065043, WO 2024 / 065042 and WO 2026 / 000081, incorporatedherein by reference. In one such method, an appropriate aminoalcohol or an O-protected variant thereof, represented in Scheme 16 with the generic formula 16.1 (G = H or protecting group), is selectively mono N-alkylated with about one molar equivalent of 3.2 in a polar solvent, for example, DMF, in the presence of an inorganic base, such as K₂CO₃, and at a temperature between 0 and 50 °C, whereupon secondary amine 16.2 forms. The latter can then be N-alkylated a second time with 3.4 by heating in acetonitrile in the presence of Na2COs. If G in 16.1 and 16.2 is H, this results in direct formation of 15.1. If G in 16.1 and 16.2 is a protecting group, a final deprotection step can be used to transform the product 16.3 into 15.1.H2N-[(CH2)t-X]u— (CH2)V-O-G R2-S. ^(CH^-L 16.1 3.2 G = H or prot. grp., X = O or S t = independently 2 to 4 in each (about 1 equiv) [(CH2)t-X] unit, u = 0 to 2, v = 2 to 6 DMF, K2CO3, ^G R3_S2^(CH2)n— L 3.4 HN-[(CH2)t-X]u— (CH2)V-O-GR,MeCN, Na2CO3, heat -E’ 16.2R2-S'G’ 16.3 G = pKrot. g — [(CH2)t-X]u— (CH2),-O-Garpr. — N cond..it..ions, ^G? effective R3_S ^(CH2)n15.1 G = HScheme 16

[0091] In some embodiments, lipid 15.1 may be such that one or more of R1and R4, R2and R3, G1and G2, E1and E2, m and n, may be identical. In such cases, alternative synthetic routes may be advantageous.

[0092] For example, a lipid 15.1 wherein R1= R4, R2= R3, G1= G2, E1= E2, and m = n, can be represented with structure 17.2 (Scheme 17). In such a case, amine 16.1 may be more conveniently doubly N-alkylated by reaction with at least two molar equivalents of 3.2 in a polar solvent, for example, acetonitrile, in the presence of an inorganic base, preferably Na2COs, and at a temperature between 40 and 100 °C. If G in 16.1 is H, this results in direct formation of 17.2. If G in 16.1 is a protecting group, a final deprotection step can be used to transform 17.1 into 17.2.H2N-[(CH2)t-X]u— (CH2)V-O-G(about 2 equiv) MeCN, Na2CO3, heatR 16.1E1R2-C J - fCH-9G1 m\ 17.1 G = prot. grp. —conditions N-[(CH2)t-X]u— (CH2)V-O-Geffective R2-S"G^(CH2)m'Z 17‘2 G-H / E1R1Scheme 17

[0093] A lipid of the type 15.1 wherein R1= R4, R2= R3, G1= G2= CH2, E1= E2= -C(O)-O-, but m and n differ, namely, compound 18.7 (Scheme 18) can be made starting with tertiary amine 18.2, which can be prepared by sequential N-alkylation of 16.1 with halides or sulfonates 9.1 and 12.2, according to the method outlined earlier in Scheme 16. Compound 18.2 can then be subjected to synthetic steps described in co-owned and co-pending WO 2026 / 000081, incorporated herein by reference. Accordingly, reaction with an appropriate oxidant, for example, a peroxycarboxylic acid like MCPBA, transforms 18.2 into 18.3. The reaction of 18.3 with a thiol under basic conditions results in epoxide opening and formation of 18.4. The A-oxide in the latter can be reduced back to a tertiary amine by reaction with triphenylphosphine, optionally in the presence of an acid such as acetic acid, and 13.5 thus obtained can be esterified to produce 18.6. Release of the TBS group with fluoride ion, provided, e.g., by pyridine-HF complex, transforms 18.6 into 18.7^(CH2)m-L9.1 (CH2)m xH2N-[(CH2)rX]u— (CH2)V-O-G N-[(CH2)t-X]u-(CH2)v-OTBSH16.1, G = TBS DMF, rt, K2CO318.1<^ / (CH2)n-L.^(CH^12.2N-[(CH2)t-X]u-(CH2)v-OTBS<5^(CH2)n iR 9MeCN, 80 °C MCPBA Na2COsO O R2-SHN-[(CH2)t-X]u-(CH2)v-OTBSbase18.3OHR2-S^ X(CH2)m\ PPh3N-[(CH2)t-X]u-(CH2)v-OTBSacidR2-s y" (CH2)n ®18.4OH OHR2-S^(CH2)m\ R1-COOHN-[(CH2)t-X]u-(CH2)v-OTBScouplingO R2-s y" (ch2) / 18.5agentR1O OHR2-S^(CH2)m\ pyr-HF / N-[(CH2)t-X]u-(CH2)v-OTBS(CH2)R2-s yn18.6R! OO R1O'm\N-[(CH2)t-X]u-(CH2)v-OHR2-S'xyn18.7R!OScheme 18

[0094] Lipid 18.7 can also be prepared by a method described in detail in co-owned and copending WO 2026 / 000081, incorporated herein by reference. The synthesis of 18.7 by this alternative route starts with amine 19.2 (Scheme 19). Amines of the type 19.2 have been described in the chemical literature and can be prepared by the reported procedure (Curran, D. P. et al., J.Chem. Soc., Perkin Trans. 7, 1994, 1377-1393, which is incorporated herein by reference). Alternatively, they can be made by mono-N-alkylation of 19.1 with halide or sulfonate 12.2. N-Boc protection of 19.2 gives 19.3. Epoxidation of the alkenes as described earlier and epoxide opening with a thiol produces 19.5, which can be esterified and N-deprotected to 19.7. The latter can then be converted into 18.7 by N-alkylation with alkyl halide or sulfonate 19.8 in an appropriate solvent and at a suitable temperature in the presence of an inorganic base; for example, in MeCN at 80 °C in the presence of Na2COs.^(CH2)n-L12.2 ^(CH2)mxBOC2O ^(CH2)m(CH2)m-NH2- ► NH - ► N— Boc19-1DMF, rt, K2CO3^(CH2)nZ^(CH2) / 1 19.3OHR2-S (X(CH2)m yR2-SH ^(CH2)mepoxidationN-Boc N— Boc■(CHz) / _ conditionsR2-S 19.5OH effective OO OR1O R1OR1-COOH R2-S^ X R2-S.(CH2)m xTFA X (CH2)mN-Boc NHcouplingR2-S y (CH2) / c'19.6B0CR2-S y (CH2) / ,19.7agentR! R1O O OR1OR2-S^ A L-[(CH2)t-X]u-(CH2)v-OH (CH2)m x19.8N-[(CH2)t-X]u-(CH2)v-OH(CHzVR2-S y 18.7 MeCN, Na2CO3, 80 °CR1 L = halide or sulfonateScheme 19

[0095] A modification of the procedures of Schemes 18 and 19 can be used to prepare a lipid of the type 15.1 wherein R2= R3, G1= G2= CH2, E1= E2= -C(O)-O-, m = n, but R1and R4differ, namely, compound 20.9 (Scheme 20). Thus, compounds 20.1 and 20.2 are prepared according to Schemes 18 and 19 by the use of appropriate amine and halide or sulfonate building blocks, andsubsequently converted into 20.3 and 20.4. As seen earlier in Scheme 11, 20.3 and 20.4 can be selectively mono-esterified with about one molar equivalent of carboxylic acid R1-COOH in the presence of a suitable condensing agent, for example, a carbodiimide such as EDCI. The free OH group in the resulting monoesters 20.5 or 20.6 can be further esterified with carboxylic acid R4-COOH, also in the presence of a suitable condensing agent, for example, a carbodiimide such as EDCI, to produce diesters 20.7 or 20.8 which can be converted into 20.9 by the procedures outlined in Schemes 18 and 19 above.— (CH2)m\ Scheme 18N-(CH2-CH2-X)U-(CH2)V-OTBS (CH2) / 20.1Scheme 19 N-Boc20.2 20.3 Z = [(CH2)t-X]u-(CH2)v-OTBS O 20.4 Z = BocR4-COOH R1-COOHcoupling coupling agent agent20.5 Z = [(CH2)t-X]u-(CH2)v-OTBS20.6 Z = Boc20.7 Z = [(CH2)t-X]u-(CH2)v-OTBS20.8 Z = Boc O Scheme 1820.7 - ►N-[(CH2)t-X]u-(CH2)v-OH Scheme 1920.8 - ► 20.9Scheme 20

[0096] Without intending to be limiting, the foregoing synthetic methods are exemplified below with the preparation of compounds T01-T52 set forth below. Those skilled in the art would appreciate that alternative starting materials could be employed in the same sequence, leading to congeners of compound T01-T52 as defined by Formula A. Therefore, the synthetic schemes set forth below are merely illustrative of select embodiments.Ionizable lipids T01-T52

[0097] In accordance with the synthetic schemes above, the ketones required for the synthesis of lipids of the type 2.1, such as T-01-T-28, are compound 21.1-21.7 (Scheme 21).precursor of T-01, T-04, T-08, T-17, T-25precursor of T-02, T-09, T-018precursor of T-03, T-10, T-11, T-12, T-19, T-20, T-21, T-26precursor of T-05, T-06, T-13, T-14, T-22, T-23, T-27oprecursor of T-07OH 21.5 OHprecursor ofT-15, T-24precursor ofT-16, T-28Scheme 21

[0098] Furthermore, ketones 21.1-21.6 can be made from bis-epoxide 22.4, and 21.7 from 22.8 (Scheme 22). The synthesis of 22.4 proceeds as shown earlier in Scheme 9. Accordingly, TosMIC, 3.1, is doubly alkylated with, for example, 6-bromo-1-hexene, 22.1, followed by acid hydrolysis of the resulting 22.2. Ketone 22.3 is then doubly epoxidized to 22.4. The synthesis of 22.8 proceeds as shown earlier in Scheme 12. Accordingly, TosMIC, 3.1, is sequentially alkylated with, for example, 6-bromo-1-hexene, 22.1, and 7-bromo-1-heptene, 22.6. The resulting 22.7 is then transformed into 22.8 by the same method used for the conversion of 22.2 into 22.4.<^(CH2)4-Br cone.22.1 ^^(CH^ Ts aq. HCI MCPBA NaH, DMF <^(CH2) / ©N^ ether ^(CH2)422.3<^(CH2)4-Br ^^(CHzls-Br ^(CH2)5Ts as22 1> / <TS 226DBU, DMF ^<CH2)4 ©N^NaH, DMF A^(CH2) / ©^ above22.5Scheme 22

[0099] In accord with Scheme 9, bis-epoxide 22.4 can be transformed into ketones 21.1-21.5 by double epoxide opening with a thiol under basic conditions. In certain embodiments, it may beexpedient to utilize the thiol in the form of a thioacetate (cf. 23.1, R as defined in Scheme 23). The reaction of 22.4 with 23.2 can then be carried out in methanol in the presence of sodium methoxide. In the same manner, bis-epoxide 22.8 can be transformed into ketone 21.7.OR-SAc 23.1MeONa0H21.1 R = cycloheptyl0HMeOH 21.2 R = cyclooctyl21.3 R = (cyclohexyl)methyl21.4 R = (2-cyclohexyl)-1 -ethyl21.5 R = (cycloheptyl)methylR-SAc23.1 OH OMeONaR' [ ^s'MeOH 21.7 R = (cyclohexyl)methyl OHScheme 23

[0100] In accord with Scheme 13, ketone 21.6 can be synthesized by sequential epoxide opening of 22.4 with two different thiols, as shown in Scheme 24.

[0101] The conversion of dihydroxy ketones 21.1-21.7 into the representative, but by no means limiting, lipids T-01-T-28 of the disclosure starts with the esterification of the OH groups with appropriate carboxylic acids in the presence of a condensing agent, for example a carbodiimide such as EDCI. This operation can be carried out so that both OH groups undergo esterification with the same carboxylic acid, or in such a manner that one OH group is esterified with one carboxylic acid, and the other, with a different carboxylic acid, as shown earlier in Scheme 11.Accordingly, Scheme 25 illustrates how ketones 21.1-21.7 can be transformed into compounds 25.1-25.12, The skilled artisan will appreciate that carboxylic acids and esterification methodsother those of Scheme 21 can be employed to produce congeners of 25.1-25.12 not explicitly contemplated herein.R-COOH 21.1 - * about 2 equiv, EDCI 25.2 R = (CH2)2-cyc / o-C6H11as 21.2 - aboveas 21.3 aboveas 21.5 aboveo o0 IIR' O O. —. as / \... S.....-L,.-x,x-x._.x- < ) 21.7 - \ ) ■ ■v■vY S \ — / above' ' 25.12 R = (CHJJH-CHJ.0Scheme 25

[0102] The synthesis of lipids T-01-T-07 involves the conversion of the carbonyl group in the appropriate ketone of Scheme 25 into a type 1 ionizable head group. In general, the ketone carbonyl can be selectively reduced using a suitable hydride reagent, such as NaBTE, in a protic solvent (for example, an alcohol such as ethanol) to provide the corresponding alcohol intermediate. The resulting alcohol may then undergo esterification with 4-(dimethylamino)butanoic acid or a corresponding salt (e.g., the hydrochloride) in the presence of an appropriate condensing agent, such as a carbodiimide (for example, EDCI), to afford the corresponding lipid product.

[0103] By the method of Scheme 26, 25.1 can be transformed into T-01; 25.3 into T-02; 25.4 into T-03; 25.2 into T-04; 25.8 into T-05; 25.9 into T-06; 25.10 into T-07.o OH•-'L' ^' 's'R3NaBH4, EtOH_R% ^ <r- ' " 'O.. R4R\, O O.... R4II I IQ O O 25.1: R1= R4= (CHf)0-CH3,J= RJ= Cy oheplyl 26.1: R1= R4= (CHJ)B-CH3. R2= R3= Cyclohepl l 25.2: R1= R4= (CH^-cyric-CsHr, R2= Rs= Cytlaheplyl 26.2: R1= R4= (CH;j;j-c dL:‘-CtiH1|. R2= R3= Cytlahaptyl 25.3: R1= R4= (CH2)U-CHJ,J= Ru=■ Cy ooclyl 26.3: R1= R4= (CH?)B-CH3. R2= RJ=■ Cyclooctyl 25.4: R1= R4= (CH;)B-CH3, RJ= RJ= Cyclohexylmethyl 26.4: R1= R4- (CHf)B-CH3. R2- Rs- Cyclohe ylmethyl 25.8: R1= R4= (CH^u-CHj, R2= R3=■ Cyclohexyleihyl 26.8: R1= R4- (CH?)u-CHj. R2- R3- Cyclohexyleihyl 25.9: R1= R4= (CH u-cycJL-UBHv, R2= RJ= Cyclohexyleihyl 26.9: R1= R4= (CH^-cydc-CuHn. R2= RJ= Cyclohexylethyl 25.10: R1= R4= CHjla-CHi. R2=J= Cydohaptylmethyl 26.10: R1= R4= (CH^ - H;, R2= R1= CydohapLylmethyl OX.. N.R?S- 'Y..■- O O-., R4EDCI. DCM 1 Io oT-fll: R1= R4= ■[CH2jw-CH.31R2= R = CydohaptylT-fl4: R1= R4= (CHjk-cycto-CyH, Rf= R ' = CydoheplylT-02: R1= R4= R2= R3= CydooclylT-fl3: R1= R4= ■(CH£jw-CH.31R2= R1= Cydohaxyl methylT-fl5: R1= R4= (CHjJi-CHj., R2= R = CydohaxylelhylT-06: R1= R4= ^CHj^-cycto-CyH, R2= R3= CydahaxytelhylT-fl7: R1= R4= CHV -CH;, R2= R1= CydahaptylmethylScheme 26

[0104] The synthesis of lipids T-08-T-16 entails the conversion of the carbonyl group in the appropriate ketone of Scheme 25 into a type 2 ionizable head group. The ketone is ketalized with 4-chlorobutane-l,2-diol. In all cases, the reaction is carried out in an appropriate solvent that forms an azeotrope with water, for example cyclohexane, 1,2-di chloroethane, toluene, and the like, in the presence of an acid catalyst such as pyridinium / ?-toluenesulfonate (PPTS), quinolinium camphorsulfonate (QCS), / ?-toluenesulfonic acid (TsOH), and the like. The reaction is conducted at a suitably elevated temperature, for example at reflux, with continuous removal of the water formed during the course of the reaction, for example using a Dean-Stark trap or an equivalent device.

[0105] As an example, compound 27.1 is the product of the reaction of 25.1 with 4-chlorobutane-l,2-diol. Displacement of the chloride from 27.1 with dimethylamine results in formation of T-08.. Cl HO OH PP7S. iolune reflux, -H^C 25.1: R1= RJ= (CH2)U-CHJ. R2=■ R3= Cyclahaplyl. m = 1 27.1: R1= R4= (CHJB-CHJ. R3= R3= Cycloheplyl, 25.3: R1= Rfl= (CHf)5-CH5. R2=■ R3= Cyclaoclyl, m = 1 27.3: R1= R4= (CH^-CH;, R2= RJ= Cyclooclyl, m = 1 25.4: R1= R4= (CH2)U-CHJ. R;' = R3= Cytlohaxylmeihyl. m = 1 27.4: R1= R4= (C H?)f.-CH.-,, R3= R3= Cyclohexylmelhyl, m = 1 25.5: R1= R4= (CH^j-cydo-CijH-,. 27.5: R1= R4= (CH^j-cycfo-CtHT, R2= R2= Cydahexylmslhyl. m = 1 R2= RJ= Cyclohexylmethyl, m = 1 25.7: R1= (CHJu-CH j, R4= (CHz)j-cycfc-CuHr, 27.7: R1^tCHjjs-CHs, R4= iCHjivcycta-C^Hn, R‘ - R- = Cyclahsxylmeihyl. m = 1 R2= R3= Cyclohexylmethyl, m = 1 25.3: R1= R4= (CHf)5-CH3. R2= R3= Cyclahaxylelhyl, m = 1 27. B: R1= R4= (C H?>-CH,-.. R2= RJ= Cyclohexylelhyl, m = 1 25.9: R1= R4= (CH^-cycfo-C.jH-,. R3= R3= Cyclahaxylelhyl, m = 1 27.9: R1= R4= (CH?)rcycfo-CcHi.. R3= R3= Cyclohexylelhyl, m = 1 25.11: R1= R4= (CH^u-CHj, 27.11: R1= R4= (CHJu-CH-i R3= Cyclohexylmelhyl, R " = Cyclohexylelhyl, m = 1 R2= Cydohexylmalhyl, R3= Cyclohaxylalhyl, m = 1 25.12: R1= R4= (CHJfCH-j, R?= R3= Cyclohexylmelhyl, m = 2 27.12: R1= R4= (CHf)B-CH3. R3= R3= Cyclohaxyl methyl. m = 2MejNH7-08: R1= R4= (CHJIR-CH.,. R2= R2= Cycloheplyl, m = 1 7-09: R1= R4=; CH£jfc-CH,. R3= R ' = Cycloociyl, m = 1 7-10: R1=■ R4= ■; CH^i-CHi, R3= R ' = Cydohexylmalhyl, m = 1 7-11: R1= R4= (CH ffc-cy 11. R2-RJ= Cydohaxyl methyl. m =■ 1 7-12: R = (CHJ)J-CHJ, R4= (CHf)r<ry<r / n-C^ H.,. R3= R' = Cydohexylmalh yl. m = 1 7-13: R1= R4= ■tCHJk-CHj. R2= R2= Cyclohexylethyl, m = 1 7-14: R1= R4= {CH^-cycfo-GjHii, R2= R2= Cyclohexylethyl, m = 1 7-15: R1= R4= tCHjjii-CHi. R2= Cyclohexylmethyl, R3= Cyclohaxylethyl, m = 17-16: R1= R4= ^CHj Ji-CH;, R2= R2= Cyclohexylmalhyl, m = 2Scheme 27

[0106] By the method of Scheme 27, 25.3 can be transformed into T-09, 25.4 into T-10; 25.5 into T-11; 25.7 into T-12; 25.8 into T-13; 25.9 into T-14; 25.11 into T-15; 25.12 into T-16.

[0107] The synthesis of lipids T-17- T-25 and T-28 entail the conversion of the carbonyl group in the appropriate ketone of Scheme 21 into a type 7 ionizable head group. In all cases, the reductive amination of the ketone with primary amine, 4-amino-l -butanol or a suitably O-protected derivative thereof, for example, methyl amine in THF or 4-amino- l-( / c / 7-butyldimethylsilyoxy)butane, in an appropriate solvent, for example, 1,2-di chloroethane (DCE), at a temperature between 0 and 100°C, in the presence of a reducing agent, for example, NaBH(OAc)3, and optionally in of an acid catalyst such as acetic acid, results in formation of secondary amine Scheme 28.25.1: R = R4= (CHj)x-CH.v R2= R2= Cyclohepiyl, m = 125.3: R' = R4= (CHj)x-CH.v Rf= R3= Cyclooctyl, m = 1 o 25.4: R' = R4= (C HvL.-CH-,., Rf= R3= Cyclohexylmelhyl, m = 125.5: R = R4= (CHjJ-.-cyclo-CxHn, R2= R2= Cyclohexylmethyl, m = 1 25.7: R' = (CHE)e-CH3, R4= (CHjJj-cyclo-CjzH,., R2- R3- Cyclohexylmelhyl, m = 1 R1.. Om° yRl25. B: R' = R4= (C Rf= R3= Cyclohexyleihyl, m = 125.9: R1= R4- (CHf)2-cydo-CyHn R2- Ra- Cyclohexyleihyl. m =■ 1 O O 25.11: R' = R4= (CHjfc-CH.-, R2= Cyclohexylmelhyl, R " = Cyclohexyleihyl, m = I R9= Me: (CHfJd-CIBS; RS-NH? 25.12: R1= R4=■ (CH?)U-CH3. R2=■ R9= Cyclo hexylmelhyl, m = 2 (CH?)?-O-(CH?)a-O1BS NaBH|OAc.:i,28.1: R4= R4= (CHE)B-CH3, R2= R3= Cycloheplyl. m = 1. Rs= (CH^-OTBS 25.3: R1= R’ = R2= R2= Cyclooctyl, m =■ 1. Ra= Ma 254: R1= R4= (CHE)B-CH3, R2= R2= Cyclohexylmelhyl. m = 1, F? = (CH^-OTBS 25.5: R4= R4= (CH^Jj-cyclo-CuHn R2=■ Ft3= Cyclohexylmelhyl, m = 1. Rs= (CH^-CUBS 25.7: R1=■ R4=■ [CH?)a-cyclo-C4H |. Ft2= Fl2= Cyclohexylmelhyl. m =■ 1. Ra=■ Me 268: R1= R’ = (CHE)B-CH3, R2= R2= Cyclohexyleihyl. m =■ 1 Ra=■ (CHjJx-OTBS 25.9: R4= R4= (CH2)a-cyclo-CbH1| R2= R3=■ Cyclohexyleihyl. m =■ 1. Rs=■ Me 25.11: R1= R4= R2= Cyclohexylmelhyl, Ra= Cyclohexyleihyl. m = 1. Ra= Me CH2O (aq) or 26 12: R4= (CH3)f-CH.-., Rf= R ’ = Cyclohexylmelhyl, m = 2. R3= Me 25.13: R4= R4= (CHJu-CH?, Rf= R3= Cycloheplyl, m = 1. R9= (CH?]?-O-(CH;!]?-O1SS NaHH(OAc)y 29.1: R1= R4= R2= R2= Cycloheplyl, m = 1, R9= R ° = Me 29.3: R4= R4= (CHjJ^CH.i, Rf= R •'= Cyclooctyl, m = 1, R9- Me, R43- (CH,)lj-OTBS 29.4: R4= R4= (CHaj,.-CH,, Rf= R'= Cyclohexylmelhyl. m = 1, R’ = iCH?t-O1BS, R,n= Me 29.5: R1= R4= (CH3J.1-cyclo-CF. Hn, R2= R2= Cyclohexylmethyl, m = 1, R9= R ’= Me 29.7: R4= (CH2)f-CH.i, R4= (C H2)vcyclo-CcH,(, R3= R3- Cyclohexylmelhyl, m = 1, R9= Me, Rlu= (CH?ji-O1BS 29.5: R1= R4= (CHjJa-CHi, R2= Ra= Cyclohexylethyl, m = 1, R" = (CH3).i-CHBS. RIL= Me 29.9: R4= R4= (CH^^-cyclo-C^H Rf= R ' = Cyclohexyleihyl, m = 1, R9= Me, R43- (CH?)q-O1BS 29.11: R1= R4= (CHab-CH, R* = Cyclohexylmelhyl, R3= Cyclohexyleihyl, m = 1, Ra= Ma, Rlu= HF-Pyr 29.12: R4= (CH^-CH?, R2= R3= Cyclohexylmelhyl, m = 2, R9= Me. R13= (CHa)q-OTBS 29.13: R1= Rs- R2-Ra= Cycloheptyl, m - 1 Ra- (CH?)a-O-(CHa)a-OTBS, R1J= Me T17: R1= R4= R2= R3=■ Cycloheplyl, m = 1, X = CH?, n = 1 118: R1= R4= R2= RJ= Cyclooctyl, m = 1. X = CH£, n = 1 119: R1= R4= R2= R3=■ Cyclohexylmelhyl, m = 1, X = CH?, n = 1 120: R1= R4= (CRJj-cyclo-CuHn. R2= R4= Cyclohexylmelhyl, m = 1, X = CH?, n = 1 121: R1= R4= (CHf)3-cydo-C^H i. R2= R1= Cyclohexylmelhyl. m = 1. X = CH£, n = 1 122: R1= R4= R2= R3=■ Cyclohexyleihyl. m =■ 1. X = CH?, n = 1 123: R1= R4= (CRJa-cyclo-CiiHu. R2= Ra=■ Cyclohexyleihyl. m = 1. X = CH?, n = 1 124: R1= R4= (CHf)B-CH3R2= Cyclohexylmelhyl, RJ= Cyclohexyleihyl, m = 1, X = CH?, n = 1 128: R1= R2= R3= Cyclohexylmelhyl, m = 2, X= CH?, n = 1125: R1= R4= (CH? Ju-CHj. R2= RJ=■ Cycloheplyl, m = 1, X = O, n = 2Scheme 28

[0108] Further reductive alkylation of secondary amine, for example by treatment with aqueous formaldehyde or 4-(tert-butyldimethylsilyloxy)butanal in a solvent such as THF, at a temperature comprised between 0 and 100°C, and in the presence of a reducing agent such as NaBH(OAc)3, converts secondary amine like 28.1 into 29.1, which can be transformed into T-17 by release of the silyl protecting group with a source of fluoride ion, for example, HF-pyridine complex.

[0109] By the method of Scheme 28, 25.1 can be transformed into T-17 and T25; 25.3 into T-18; 25.4 into T-19; 25.5 into T-20; 25.7 into T-21; 25.8 into T-22; 25.9 into T-23; 25.11 into T- 24; 25.12 into T-28.

[0110] The synthesis of lipids T-26 and T-27 illustrates an alternative method for the introduction of a type 10 head group. As demonstrated in Scheme 29 with the synthesis of T-26 and T-27, said alternative route starts with the reductive amination of ketone 25.4 and 25.8 with methylamine in the presence of a reducing agent, for example, a boron hydride such as sodium triacetoxyborohydride, and optionally in the presence of an acid such as acetic acid, to produce compounds 28.14 and 28.15. The latter compounds can then be A- alkylated with 2-(2-bromoethoxy)ethan-l-ol in an appropriate solvent, for example, DMF, and in the presence of a base, for example, K₂CO₃, at a temperature comprised between 20 and 100°C, resulting in formation of corresponding lipids T-26 and T-27.QMeNH; \ - / NaBH(OAcl, V..-Xx.--s... O25 4 n=1O..✓■xx.--x X X- - -| 25.0 n=2 - O O NH / s-. -k ■Y"'S'’ VUK2C°3 20.14 n 20.15 n...oT'26 n“10. ' 1 T-27 n=-2 KO O Scheme 29

[0111] Lipids of the type 15.1, such as T-29-T-32, can be prepared, for example, by the methods outlined earlier in Schemes 16-20. Accordingly, and without intending to be limiting, lipids T-29-T-32 can be made starting with epoxidation of compounds 30.1 and 30.2, for example with a peroxy carboxylic acid such as MCPBA, followed by double epoxide opening with an appropriate sulfur nucleophile (Scheme 30).B i oc / NIn (' MCPBA30.1 n = 4 (precursor ofT-29-T31)30.2 n = 5 (precursorof T-32)BocHO i OH / nN30.5 n = 430.6 n = 5Scheme 30

[0112] Furthermore, a bis-epoxide such as 30.3 can be subjected to selective mono-epoxide opening by reaction with about one equivalent of a sulfur nucleophile, followed by opening of the second epoxide with a different sulfur nucleophile. Without intending to be limiting, this is exemplified in Scheme 31 with the conversion of 30.3 into 31.1 and 31.2.Scheme 31

[0113] The synthesis of T-29, T-31, and T-32 continues with the esterification of the OH groups in 30.5, 31.2 and 30.6 with decanoic acid (Scheme 32).Scheme 32

[0114] Furthermore, a diol such as 30.5 can be selectively mono esterified with about one equivalent of a carboxylic acid, followed by esterification of the second OH group with a different carboxylic acid. Without intending to be limiting, this is exemplified in Scheme 33 with the conversion of 30.5 into 33.1 and 33.2.Scheme 33

[0115] Release of Boc group in compounds 32.1-32.3 and 33.2, for example by treatment with trifluoroacetic acid in CH2CI2, produces secondary amines 34.1, 34.2, 34.3 and 34.4, respectively. Further reductive amination of 34.1, for example by treatment with 4-((tert-butyldimethylsilyl)oxy)butanal in a solvent such as DCE, at a temperature comprised between 0 and 100 °C, and in the presence of a reducing agent such as NaBH(OAc)3, converts 34.1 into 35.1, which can be transformed into T-29 by release of the silyl protecting group with a source of fluoride ion, for example, HF-pyridine complex.

[0116] By the method of Scheme 34, 35.2 can be transformed into T-32; 35.3 into T-31; 35.4 into T-30.Boo, r! H *.. OTBS «5zxz|J^ N^r s. R3TFA. DCM31 ' ’ 1s- - R4.0 0.. R4- R!, O O..., R«'I p NaBH(OAc)3. DCE T n 0 a O O 32.1: R1= R4= iCHJj-CH-i, R2= RJ= Cyclohexylmethyl, m 34.1: R' = Rfl= (CH jg-CHj, RL= R3= Cyclohexylmelhyl. m = 1 32.2: R1= R* = iCH2)s-CH.-., Rf= R3= Cyclohexylmelhyl, m 34.2: R' = Rd= (CH ju-CHj, R3= Rs= Cyclohexylmelhyl. m = 2 32.3: R1= R4= iCHJj-CH-i, R4= Cyclohexylmethyl, 34.3: R' = Rfl= (CHjn-CHj, RL= Cyclohexylmethyl, R;= Cyclohexylethyl, m = 1 RJ= Cyclohexylethyl. m = 1 33.2: R1= {CHJa-CH.-., R'1= (CH?j3-cycfo-CfiHi-. 34.4: R’ = (CHja-CHj. Rd= (CH2)3-c^-CuH| Rf = R' = Cyclohexylmethyl, m = 1 R* = RJ= Cyclahexylmetriyl. m = 1■-■■""■■■ ■■■■■""■■OTBS HF-Pyr, DCM R\.. O,rO... R11 r 0 0 35.1: R1= R4= {CHjfe-CH,, Rf= R' = Cyclohexylmethyl, m T-29: R1= R- = R4= Ri =Cyclohexylmelhyl, 35.2: R1= R- = {CHjii-CHi, R4= R1= Cyclohexylmethyl, m T-32: R1= R4= {CH£)S-CH,, R' = R '= Cyclohexylmelhyl,T-31: R1= R- = {CH^-GH,, R4= Cyclohexylmelhyl, =5 =5 35.3: R1= Ri=; CHsj(s-CH.i. R4= Cyclohexylmelhyl, R1= Cyclohexylethyl, m = 1 RJ= Cyclohexylethyl, m = 1 35.4: R1= {CHjfe-CHj, R* = (CH Jj-cycto-CuH,- T-30: R1= \CHjJu-CHj., R4= (CHJ-j-cycto-CtH,-R4= R* = Cyclohexylmelhyl, m = 1 RJ= RJ= Cyclohexylmethyl, m = 1Scheme 34Formulation of the above lipids in a delivery vehicle

[0117] 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.

[0118] 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 helper lipids, such as vesicle forming lipids and optionally an aggregation inhibiting lipid, such as a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid).

[0119] As set forth previously, a helper lipid includes a sterol, a diacylglycerol, a ceramide or derivatives thereof.

[0120] Examples of sterols include cholesterol, or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl -2 '-hydroxy ethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, and the like.

[0121] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoylol eoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (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.

[0122] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[0123] 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 aremixed using a rapid-mixing device as described in Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni et al., 2017, Nanoscale, 36:133347, each of which is incorporated herein by reference in its entirety.

[0124] 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 without lipids. 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.

[0125] 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 filled core stabilized by an emulsifying component such as a monolayer or bilayer of lipids.

[0126] Lipids described herein may be incorporated into a micelle. Micelles are self-assembling 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

[0127] 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.

[0128] 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.

[0129] 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, smallactivating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in WO 2022 / 251953 Al, which is incorporated herein by reference.

[0130] 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.

[0131] 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 or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C 5 -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).

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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 location in 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.

[0138] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5’ and 3’ ends and thus may be more stable in vivo due to its resistance to degradation by exonucleases. The circular mRNA may be prepared by any known method, including any one of the methods described in Deviatkin et al., 2023, “Cap-Independent Circular mRNA Translation Efficiency”, Vaccines, 11(2), 238, which is incorporated herein by reference. Translation of the circular mRNA is carried out by a cap-independent initiation mechanism.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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 targetingspecific 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.

[0143] 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.

[0144] In another embodiment, the cargo is a DNA vector as described in co-owned and copending 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.

[0145] 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 double-stranded 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 selfreplicating systems such as vector DNA.

[0146] 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 linkagessubstituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0147] 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 azasugars 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] While pharmaceutical compositions are described above, the lipids described herein can be a component of any nutritional, cosmetic, cleaning or foodstuff product.Pharmaceutical formulations

[0156] 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 nontoxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and the like.

[0157] 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.

[0158] In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, thepharmaceutical 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.

[0159] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0160] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

[0161] The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.EXAMPLESMaterials

[0162] The lipid 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000 (PEG-DMG) were 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.

[0163] An mRNA encoding firefly luciferase synthesized by NanoVation Therapeutics Inc. (Vancouver, BC) was used to analyse luciferase activity.MethodsPreparation of lipid nanoparticles (LNP) containing mRNA

[0164] Lipids described herein, DSPC, cholesterol, and PEG-DMG, were dissolved in ethanol at the appropriate ratios to a final concentration of 10 - 20 mM total lipid. Nucleic acid (mRNA) was dissolved in an appropriate buffer such as 25 mM sodium acetate 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 Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni 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 ofLNP

[0165] 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 Tris-EDTA (TE) containing 2% Triton Tx-100, and free RNA vector in solution (external to LNP) was measured based on the RiboGreen™ fluorescence in a TE solution without Triton. Total siRNA or mRNA content in the formulation was determined using a modified Bligh-Dyer extraction procedure. Briefly, LNP formulations containing siRNA or mRNA 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

[0166] 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 tissues isolated. A small piece of each 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

[0167] Unless otherwise specified, reagents and solvents were commercial products and were used without purification. All reactions were performed under inert atmosphere (nitrogen or argon). Reaction mixture from aqueous workups were dried by passing over a plug of anhydrous Na2SC>4 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). Visualization of the developed chromatogram was performed by staining with I2 or potassium permanganate solution. Chromatographic purifications were performed on a BiotageISCO system. 'H and13C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCl₃ solutions.1H NMR spectra were referenced to residual CHCl₃ (7.26 ppm) and13C NMR spectra were referenced to the central line of the CDCl₃ triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the 6 scale. Multiplicities are reported as “s” (singlet), “d” (doublet), “t” (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.Example 1: Representative procedures for chemically synthesizing ionizable lipids(A) Preparation of building blocksProcedure for the preparation of thioacetates: 5-cycloheptyl ethanethioate.

[0168] To a solution of cycloheptanol (5 g, 43.8 mmol, 1 equiv) and Et3N (9.15 mL, 65.7 mmol, 1.5 equiv) in dry CH₂Cl₂ (50 mL) at 0 °C, was added mesyl chloride (4.1 mL, 52.5 mmol, 1.2 equiv) slowly. The reaction mixture was then allowed to warm up to room temperature and stirred for 8h. The reaction mixture was then poured into NaHCO₃ (sat. solution), the organic layer was separated, washed with brine (sat. solution), dried (Na2SC>4), filtered and concentrated in vacuo to afford cycloheptyl methanesulfonate (8.4 g, 43.8 mmol, -100%) as a colorless oil and used for the next step without further purification.JH NMR (400 MHz, CDCl₃) δ 4.87 (1H, tt, J 8.0, 4.6, CH), 2.99 (3H, s, Me), 2.05 (2H, dddd, J 12.0, 8.8, 4.7, 2.8, CH2), 1.88 (2H, dddd, J 14.4, 9.3, 8.0, 2.7, CH2), 1.75 - 1.63 (2H, m, CH2), 1.63 - 1.47 (4H, m, 2 x CH2), 1.50 - 1.38 (2H, m, CH2). To a solution of the above cycloheptyl methanesulfonate (8.4 g, 43.8 mmol, 1 equiv) and thioacetic acid (4.1 mL, 56.8 mmol, 1.3 equiv) in DMF at 0 °C, was added Et3N (7.9 mL, 56.8 mmol, 1.3 equiv) dropwise. The resulting mixture was then stirred for 2 hr at 60 °C, then poured into water (100 mL). The aq. phase was extracted with w-hexanes (3 x 50 mL), the combined extracts were dried (Na2SC>4) and concentrated in vacuo to afford the title compound (7.53 g, 43.7 mmol, 100%) as yellow liquid. 'H NMR (400 MHz, CDCl₃) δ 3.64 (1H, tt, J 8.9, 4.4, CH), 2.28 (3H, s, Me), 1.96 (2H, ddd, J 13.8, 7.2, 4.2, CH2), 1.69 - 1.48 (10H, m, 5 x CH2).The following compounds were prepared by the same method:S-Cyclooctyl ethanethioate.

[0169] To a solution of cyclooctanol (10 g, 78.0 mmol, 1 equiv) and Et3N (16.3 mL, 117.0 mmol, 1.5 equiv) in dry CH2CI2 (150 mL) at 0 °C, was added mesyl chloride (22.3 g, 117.0 mmol, 1.5 equiv) slowly. The reaction mixture was then allowed to warm up to room temperature and stirred for 8h. The reaction mixture was then poured into NaHCO₃ (sat. solution), the organic layer was separated, washed with brine (sat. solution), dried (Na2SC>4), filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography eluting with EtOAc:hexane (0-5%) to afford cyclooctyl 4-methylbenzenesulfonate (21 g, 74.4 mmol, 95%) as a colorless oil.. S-(Cyclohexylmethyl) ethanethioate.

[0170] Yellow liquid (5.84 g, 33.9 mmol, quant.), from bromomethyl cyclohexane (6 g, 33.9 mmol, 1 equiv) and thioacetic acid (2.91 mL, 40.7 mmol, 1.2 equiv). 'H NMR (400 MHz, CDCl3) δ 2.78 (2H, d, J 6.8, CH2), 2.32 (3H, s, Me), 1.82 - 1.54 (6H, m, 3 x CH2), 1.50 - 1.35 (1H, m, CH), 1.29 - 1.06 (4H, m, 2 x CH2), 1.01 - 0.87 (2H, m, CH2).S-(2-Cyclohexylethyl) ethanethioate.

[0171] Yellow liquid (6.94 g, 37.2 mmol, quant.), from bromoethyl cyclohexane (7.12 g, 37.2 mmol, 1 equiv) and thioacetic acid (2.80 mL, 39.1 mmol, 1.05 equiv).1H NMR (400 MHz, CDCl3) δ 2.89 (2H, t, J8.02, CH2), 2.34 (3H, s, Me), 1.79 - 1.61 (5H, m, 2 x CH2, CH), 1.47 (2H, q, J7.7, CH2), 1.39 - 1.11 (4H, m, 2 x CH2), 1.02 - 0.84 (2H, m, CH2).S-(Cycloheptylmethyl) ethanethioate.

[0172] Yellow liquid (4.24 g, 22.8 mmol, 76%) from cycloheptylmethanol (3.84 g, 30.0 mmol) via the mesylate. 'H NMR (400 MHz, CDCl3) δ 2.79 (2H, d, J 6.7, CH2), 2.34 (3H, s, Me), 1.77- 0.90 (13H, m).Procedure for the bis-alkylation of TosMIC: l-((7-isocyanotrideca-l,12-dien-7-yl)sulfonyl)- 4-methylbenzene (22.2).^YCH2)4, Ts^ / (CH2)4^N22.2 ©

[0173] To a cold (0° C), well stirred solution of TosMIC (5 g, 25.6 mmol, 1 equiv) and 6-bromo- 1-hexene (8.77 g, 36.8 mmol, 2.1 equiv.) in dry DMF (15 mL) was carefully added sodium hydride (60% wt. in oil, 2.25 g, 56.3 mmol, 2.2 equiv.) in small portions. The mixture was allowed to warm up to room temperature and stirred for 2 hr, whereupon the reaction was complete. The mixture was diluted with diethyl ether (75 mL) and carefully poured into a vigorously stirred, ice-cold, saturated aqueous NH4Cl solution (100 mL). The organic layer was separated and the aqueous layer was extracted with diethyl ether (2 × 40 mL). The combined extracts were washed with brine, dried (Na2SC>4), filtered, and concentrated in vacuo. The residue was passed through a plug of basic alumina, which was further washed with ethyl acetate-hexanes (1:9). The filtrate was concentrated in vacuo to give crude 22.2 as a colorless oil, which was used for the next step without purification. 'H NMR (400 MHz, CDCl3) δ 7.88-7.83 (m, 2H), 7.44-7.38 (m, 2H), 5.76 (ddt, J = 16.9, 10.2, 6.6 Hz, 2H), 5.04-4.93 (m, 4H), 2.48 (s, 3H), 2.10-1.86 (m, 8H), 1.64-1.35 (m, 8H).Procedure for the mono-alkylation of TosMIC: l-((l-isocyanohex-5-en-l-yl)sulfonyl)-4- methylbenzene (22.5).Ts<% / (CH2)4e22.5

[0174] To a solution of TosMIC (8.23 g, 42.2 mmol, 1.1 equiv) and 6-bromo-l -hexene (6.25 g, 38.3 mmol, 1 equiv) in DMF (5.1 mL) was added DBU (5.8 g, 38 mmol, 1.4 equiv.). The mixture was stirred for 18 hr. The mixture was diluted with ether (75 mL) and carefully poured into a vigorously stirred, ice-cold, saturated aqueous NH4CI solution (100 mL). The organic layer was separated and the aqueous layer was extracted with ether (2 x 40 mL). The combined extracts were washed with water (200 mL), brine (100 ml), dried (Na2SC>4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography by eluting with 0-15% EtOAc in hexanes to give 22.5 (4.2 g, 15 mmol, 40%). 'H NMR (400 MHz, CDCl3) δ 7.89 - 7.84 (m, 2H), 7.46 - 7.40 (m, 2H), 5.77 (ddt, J= 16.9, 10.2, 6.7 Hz, 1H), 5.05 - 4.95 (m, 2H), 4.44 (dd, J= 10.9, 3.5 Hz, 1H), 2.49 (s, 3H), 2.24 - 2.14 (m, 1H), 2.12 - 2.05 (m, 2H), 1.91 - 1.80 (m, 1H), 1.72 -1.58 (m, 1H), 1.57 - 1.40 (m, 3H).13C NMR (101 MHz, CDCl3) δ 165.1, 146.7, 138.0, 131.3, 130.3, 130.2, 115.3, 73.1, 33.3, 28.4, 27.9, 24.9, 22.0.Procedure for the alkylation of a mono-alkylated TosMIC derivative: l-((6-isocyanododeca-l,ll-dien-6-yl)sulfonyl)-4-methylbenzene (22.7).^(CH2)5, Ts^ / (CH2)4^N22.7 °

[0175] To a cold (0° C), well stirred solution of 22.5 (3.98 g, 14.3 mmol, 1 equiv.) and 7-bromo-1 -heptene (3.8 g, 22 mmol, 1.5 equiv in dry DMF (14 mL) was carefully added sodium hydride (60% wt. in oil, 670 mg, 17 mmol, 1.2 equiv) in small portions. The mixture was allowed to warm up to room temperature and stirred for 2 hr, whereupon the reaction was complete. The mixture was diluted with hexanes (75 mL) and carefully poured into a vigorously stirred, ice-cold, saturated aqueous NH4Cl solution (100 mL). The organic layer was separated and the aqueous layer was extracted with hexanes (2 × 40 mL). The combined extracts were washed with water (200 mL), brine (100 ml), dried (Na2SC>4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography by eluting with 0-10% EtOAc in hexanes to give 22.7 (3.63 g, 9.7 mmol, 68%). 'H NMR (400 MHz, CDCl3) δ 7.88 - 7.84 (m, 2H), 7.43 - 7.39 (m, 2H), 5.85 -5.70 (m, 2H), 5.05 - 4.91 (m, 4H), 2.49 (s, 3H), 2.13 - 1.85 (m, 8H), 1.64 - 1.23 (m, 10H).13C NMR (101 MHz, CDCl3) δ 163.9, 146.4, 138.7, 138.2, 131.3, 130.4, 130.0, 115.2, 114.8, 81.9, 33.6, 33.3, 33.2, 33.1, 29.0, 28.7, 28.5, 23.6, 23.2, 21.9.Procedure for the conversion of a bis-alkylated TosMIC derivative into a ketone: trideca- l,12-dien-7-one (22.3).^(CH2)4^ / (CH2) / 22.3

[0176] Aqueous concentrated (12M) HC1 solution (12.8 mL, 154 mmol, 6 equiv) was added dropwise to a cold (0 °C) solution of the above crude 18.2 in ether (10 mL). The mixture was stirred for 15 min at 0 °C, then it was diluted with hexanes (50 mL) and carefully poured into cold (0 °C), well stirred, saturated aqueous NaHCO3solution (100 mL) containing additional undissolved NaHCOs (20 g). The organic layer was separated, and the aqueous layer was extracted with more hexanes (2 x 30 mL). The combined extracts were washed with brine, dried (Na2SC>4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography using ethyl acetate-hexanes (1:9) to afford 22.3 (3.57 g, 18.4 mmol, -100%) as a colorless oil.1H NMR (300 MHz, CDCl3) δ 5.73 (2 H, ddt, J 16.9, 10.1, 6.7), 4.92 (4 H, m), 2.35 (4 H, t, J7.3 ), 2.01 (4 H, q, J7.1), 1.54 (4 H, p, J7.4), 1.36-1.28 (4 H, m).13C NMR (75 MHz, CDCl3) δ 210.6, 138.3, 114.5, 42.5, 33.5, 28.4, 23.2 (2 x CH2).

[0177] The following compound was prepared by the same method:Tetradeca-l,13-dien-7-one(CH2)4

[0178] 83% colourless liquid from l-((6-isocyanododeca-l,l l-dien-6-yl)sulfonyl)-4-methylbenzene (3.63 g, 9.7 mmol).1H NMR (400 MHz, CDCl3) δ 5.85 - 5.73 (m, 2H), 5.04 - 4.90 (m, 4H), 2.42 -2.35 (m, 4H), 2.10 -2.00 (m, 4H), 1.63 - 1.52 (m, 4H), 1.43 - 1.24 (m, 6H).13C NMR (101 MHz, CDCl3) δ 211.5, 139.0, 138.7, 114.8, 114.5, 42.9, 42.7, 33.7, 33.7, 28.8, 28.8, 28.6, 23.8, 23.5.(B) Preparation of epoxides and ring opening thereofProcedure for epoxidation: l,9-di(oxiran-2-yl)nonan-5-one (22.4).OL^(CH2)4.022.4

[0179] Commercial mCPBA (75% stated purity, 11.8 g, 51.5 mmol, 2 equiv) was added portionwise to a cold (0 °C), well-stirred solution of 22.3 (5 g, 25.7 mmol) in CH2CI2 (25 mL). The mixture was allowed to warm to room temperature and stirred for 2 hr, then it was concentrated in vacuo. The residue was taken up with hexanes (100 mL) and the solution was filtered through cotton. Triethylamine (5.3g, 7.3 mL, 52 mmol) was added to the filtrate with vigorous stirring and stirring was continued for 10 min. The solution was washed with water, dried (Na2SC>4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (1:3 ethyl acetate-hexanes) to afford 22.4 (5.82 g, 25.7 mmol, -100%) as a colorless oil.1H NMR (400 MHz, CDCl3) δ 2.89 (m, 2H), 2.74 (dd, J= 5.0, 4.0 Hz, 2H), 2.45 (dd, J= 5.0, 2.7 Hz, 2H), 2.41 (t, J= 7.3 Hz, 4H), 1.68 - 1.37 (m, 12H).

[0180] The following compounds were prepared by the same method:l,8-di(Oxiran-2-yl)octan-4-one (22.8).CL^(CH2)5.[> / (CH2)4 / 022.8

[0181] White solid (1.56 g, 6.5 mmol, 80%) from tetradeca-1,13-dien-7-one (1.7 g, 8.1mmol).1H NMR (400 MHz, CDCl3) δ 2.93 - 2.86 (m, 2H), 2.76 - 2.71 (m, 2H), 2.47 - 2.44 (m, 2H), 2.43 -2.37 (m, 4H), 1.69 - 1.27 (m, 14H).13C NMR (101 MHz, CDCl3) δ 211.0, 52.4, 52.2, 47.2, 42.8, 42.7, 32.4, 29.1, 25.9, 25.7, 23.8, 23.6.tert- Butyl bis(4-(oxiran-2-yl)butyl)carbamate (30.3).

[0182] 86% from tert-butyl di(hex-5-en-l-yl)carbamate 30.1 (2.2 g, 7.82 mmol, prepared from the litrature J. Am. Chem. Soc. 2011, 133, 19087). 'H NMR (400 MHz, CDCl3) δ 3.16 (brs, 4H), 2.89 (m, 2H), 2.77 - 2.70 (m, 2H), 2.45 (dd, J= 5.1, 2.7 Hz, 2H), 1.67 - 1.32 (m, 21H).tert- Butyl (4-(oxiran-2-yl)butyl)(5-(oxiran-2-yl)pentyl)carbamate (30.4).

[0183] 81% from tert-butyl tert-butyl hept-6-en-l-yl(hex-5-en-l-yl)carbamate 30.2 (2.0 g, 6.78 mmol). 'H NMR (400 MHz, CDCl3) δ 3.15 (brs, 4H), 2.93 - 2.84 (m, 2H), 2.73 (t, J = 4.5 Hz, 2H), 2.45 (dt, J= 4.9, 2.2 Hz, 2H), 1.68 - 1.17 (m, 23H).Procedure for bis-epoxide opening: l,13-bis(cycloheptylthio)-2,12-dihydroxytridecan-7-one

[0184] To a solution of 5-cycloheptyl ethanethioate (4.11g, 23.9 mmol, 3.6 equiv) in dry MeOH at RT, was slowly added NaOMe 25 wt% in MeOH (4.55 mL, 19.9 mmol, 3 equiv). The resulting mixture was stirred for 20 min at the same temperature, then transferred into a solution of 22.4 (1.5 g, 6.63 mmol, 1 equiv) in MeOH and stirred for further 15 min at the same temperature. Then, the reaction mixture was poured into sat. NH4Q solution (100 mL). The aqueous layer was extracted with / / -hexanes (3 x 50 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with DCM:EtOAc(0-15%) to afford 21.1 (3.575 g, 18.4 mmol, -100%) as a white solid. 'H NMR (400 MHz, CDCl3) δ 3.61 (ddq, J= 9.7, 6.7, 3.6 Hz, 2H), 2.84 (tt, J= 9.0, 4.3 Hz, 2H), 2.75 (dd, J= 13.5, 3.4 Hz, 2H), 2.64 (d, J= 3.1 Hz, 2H), 2.46 - 2.37 (m, 6H), 1.99 (ddt, J= 14.2, 7.7, 2.7 Hz, 4H), 1.70 (ttd, J= 12.9, 6.1, 3.6 Hz, 4H), 1.64 - 1.31 (m, 26H).

[0185] The following compounds were prepared by the same method:l,13-bis(Cyclooctylthio)-2,12-dihydroxytridecan-7-one (21.2).

[0186] 68% yield from l,9-di(oxiran-2-yl)nonan-5-one (3.0 g, 13 mmol) and commercial cyclooctanethiol (4.7 g, 29 mmol). 'H NMR (400 MHz, CDCl3) δ 3.65 - 3.56 (m, 2H), 2.89 (tt, J = 9.0, 3.9 Hz, 2H), 2.74 (dd, J= 13.4, 3.4 Hz, 2H), 2.64 (d, J= 3.2 Hz, 2H), 2.45 - 2.37 (m, 6H), 1.99 - 1.87 (m, 4H), 1.80 - 1.30 (m, 34H).13C NMR (101 MHz, CDCl3) δ 211.2, 69.2, 44.9, 42.8, 39.2, 36.2, 32.8, 32.3, 27.3, 27.3, 25.8, 25.5, 25.2, 23.8.l,13-bis((Cyclohexylmethyl)thio)-2,12-dihydroxytridecan-7-one (21.3).

[0187] 90% yield from l,9-bis(oxiran-2-yl)nonan-5-one (3.0 g, 13.3 mmol). *H NMR (400 MHz, CDCl3) δ 3.68 - 3.55 (m, 2H), 2.70 (dd, J= 13.57, 3.31 Hz, 2H), 2.61 (d, J= 2.99 Hz, 2H), 2.47 - 2.33 (m, 10H), 1.91 - 1.76 (m, 4H), 1.77 - 1.07 (m, 24H), 1.02 - 0.85 (m, 4H).l,13-bis((2-Cyclohexylethyl)thio)-2,12-dihydroxytridecan-7-one (21.4).

[0188] 88% yield from l,9-bis(oxiran-2-yl)nonan-5-one (1.25 g, 5.52 mmol). 'H NMR (400 MHz, CDCl3) δ 3.68 - 3.57 (m, 2H), 2.72 (dd, J= 13.61, 3.30 Hz, 2H), 2.60 - 2.47 (m, 4H), 2.47 - 2.36 (m, 6H), 2.06 - 1.83 (m, 6H), 1.74 - 1.06 (m, 28H), 0.95 - 0.82 (m, 4H).l,13-bis((Cycloheptylmethyl)thio)-2,12-dihydroxytridecan-7-one (21.5).

[0189] 35% yield from from l,9-di(oxiran-2-yl)nonan-5-one (1.0 g, 4.4 mmol) and S-(Cycloheptylmethyl) ethanethioate (1.8 g, 9.7 mmol). 'H NMR (400 MHz, CDCl3) δ 3.66 - 3.57 (m, 2H), 2.69 (dd, J= 13.5, 3.3 Hz, 2H), 2.62 (d, J= 3.1 Hz, 2H), 2.46 - 2.37 (m, 10H), 1.87 -1.76 (m, 4H), 1.71 - 1.31 (m, 28H), 1.29 - 1.17 (m, 4H).13C NMR (101 MHz, CDCl3) δ 211.2, 68.9, 42.8, 41.1, 40.8, 39.7, 36.1, 34.3, 34.1, 28.5, 28.5, 26.5, 26.4, 25.6, 23.8.l,14-bis((Cyclohexylmethyl)thio)-2,13-dihydroxytetradecan-7-one (21.7).

[0190] 81% yield from l-((6-isocyanododeca-l,l l-dien-6-yl)sulfonyl)-4-methylbenzene (1.56 g, 6.51 mmol) and S-(Cyclohexylmethyl) ethanethioate (2.47 g, 14.3 mmol).1H NMR (400 MHz, CDCl3) δ 3.65 - 3.56 (m, 2H), 2.73 - 2.59 (m, 4H), 2.46 - 2.34 (m, 10H), 1.89 - 1.77 (m, 4H), 1.76- 1.07 (m, 26H), 1.01 -0.85 (m, 4H).13C NMR(101 MHz, CDCl3) δ 211.3, 69.1, 68.9, 42.8, 42.7, 41.2, 41.1, 40.0, 38.2, 36.1, 36.1, 33.0, 32.8, 29.3, 26.5, 26.2, 26.2, 25.7, 25.6, 23.8.tert-Butyl bis(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)carbamate (30.5).Boc1

[0191] 96% from tert-butyl bis(4-(oxiran-2-yl)butyl)carbamate 30.3 (7.5 g, 23.9 mmol) and S- (Cyclohexylmethyl) ethanethioate (9.9 g, 57.5 mmol). ’H NMR (400 MHz, CDCl3) δ 3.62-3.56 (m, 2H), 3.31 -2.97 (m, 4H), 2.81 -2.54 (m, 4H), 2.54 -2.28 (m, 6H), 1.89 - 1.75 (m, 4H), 1.73 - 1.56 (m, 4H), 1.56 - 1.02 (m, 31H), 0.99 - 0.76 (m, 4H).tert-Butyl (6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)(7-((cyclohexylmethyl)thio)-6- hydroxyheptyl)carbamate (30.6).

[0192] 90% from tert-butyl (4-(oxiran-2-yl)butyl)(5-(oxiran-2-yl)pentyl)carbamate 30.4 (1.8 g, 5.5 mmol) and S-(Cyclohexylmethyl) ethanethioate (2.0 g, 11.5 mmol).1H NMR (400 MHz, CDCl3) δ 3.61 (brs, 2H), 3.14 (brs, 4H), 2.68 (tt, J= 15.0, 7.5 Hz, 4H), 2.47 - 2.36 (m, 6H), 1.91 - 1.77 (m, 4H), 1.76 - 0.77 (m, 41H).Procedure for sequential epoxide opening: l-((2-Cyclohexylethyl)thio)-13-((cyclohexyl- methyl)thio)-2,12-dihydroxytridecan-7-one (21.6).

[0193] To a solution of 5-cyclohexylmethyl ethanethioate (514 mg, 2.9 mmol, 0.9 equiv) in dry MeOH at RT, was slowly added NaOMe 25 wt% in MeOH (0.9 mL, 3.9 mmol, 1.5 equiv). The resulting mixture was stirred for 20 min at the same temperature, then transferred into a solution of 22.4 (1.5 g, 6.63 mmol, 1 equiv) in MeOH and stirred for further 15 min at the same temp. Then, the reaction mixture was poured into sat. NH4CI solution (50 mL). The aqueous layer was extracted with w-hexanes (3 x 50 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography eluting with DCM: EtOAc (0-15-%) to afford 24.1 (733 mg, 2.06 mmol, 71%). To a solution of S- cyclohexylethyl ethanethioate (460 mg, 2.5 mmol, 1.2 equiv) in dry MeOH at RT, was slowly added NaOMe 25 wt% in MeOH (0.8 mL, 3.8 mmol, 1.5 equiv). The resulting mixture was stirred for 20 min at the same temperature, then transferred into a solution of 24.1 (733 mg, 2.06 mmol, 1 equiv) in MeOH and stirred for further 15 min at the same temp. Then, the reaction mixture was poured into sat. NH4Q solution (50 mL). The aqueous layer was extracted with w-hexanes (3 x 50 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography eluting with DCM: EtOAc (0-15-%) to afford 21.6 (1.01 g, 2.0 mmol, 97%) as a white solid. 'll NMR (400 MHz, CDCl3) δ 3.61 (ddq, J = 9.7, 6.7, 3.6 Hz, 2H), 2.84 (tt, J= 9.0, 4.3 Hz, 2H), 2.75 (dd, J= 13.5, 3.4 Hz, 2H), 2.64 (d, J= 3.1 Hz, 2H), 2.46-2.37 (m, 6H), 1.99 (ddt, J= 14.2, 7.7, 2.7 Hz, 4H), 1.70 (ttd, J= 12.9, 6.1, 3.6 Hz, 6H), 1.64 - 1.31 (m, 26H).

[0194] The following compound was prepared by the same method:tcrt-butyl (6-((2-cyclohexylethyl)thio)-5-hydroxyhexyl)(4-(oxiran-2-yl)butyl)carbamate (31.1)

[0195] To a solution of tert-butyl bis(4-(oxiran-2-yl)butyl)carbamate 30.3 (2.5 g, 7.99 mmol, 1.0 equiv) and S-(2-cyclohexylethyl) ethanethioate (1.78 g, 9.59 mmol, 1.2 equiv) in MeOH (25 mL) at 0 °C, was slowly added NaOMe 25 wt% in MeOH (2 mL, 1.2 equiv). The resulting mixture was allowed to proceed at RT for 30 min, at which point starting material was consumed, the reaction was quenched with water (25 mL) followed by dilution with hexanes (50 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexanes (2 x 30 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. The crude product was purified by flash column chromatography with 15-25% EtOAc in hexanes to afford 31.1 (950 mg, 2.1 mmol, 26% yield). 'H NMR (400 MHz, CDCl3) δ 3.71 - 3.50 (m, 1H), 3.16 (s, 4H), 2.92 - 2.84 (m, 1H), 2.77 - 2.57 (m, 3H), 2.56 - 2.48 (m, 2H), 2.47 - 2.35 (m, 2H), 1.77 -1.06 (m, 32H), 0.93 - 0.81 (m, 2H).te / 7- Butyl (6-((2-cyclohexylethyl)thio)-5-hydroxyhexyl)(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)carbamate (31.2).Boc1bks Y y sOH OH

[0196] To a solution of tert-butyl (6-((2-cyclohexylethyl)thio)-5-hydroxyhexyl)(4-(oxiran-2-yl)butyl)carbamate 31.1 (650 mg, 1.42 mmol, 1.0 equiv) and S-(cyclohexylmethyl) ethanethioate (293 mg, 1.71 mmol, 1.2 equiv) in MeOH (25 mL) at 0 °C, was slowly added NaOMe 25 wt% in MeOH (370 pL, 1.70 mmol, 1.2 equiv). dropwise. The resulting mixture was allowed to proceed at RT for 30 min, at which point starting material was consumed, the reaction was quenched with water (15 mL) followed by dilution with hexanes (30 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexanes (2 x 20 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. The crude product was purified by flash column chromatography with 15-25% EtOAc in hexanes to afford 31.2 (790 mg, 1.34 mmol, 79% yield). 'H NMR (400 MHz, CDCl3) δ 3.69 - 3.49 (m, 2H), 3.16 (s, 4H), 2.77 - 2.57 (m, 4H), 2.56-2.47 (m, 2H), 2.48 -2.38 (m, 4H), 1.91 - 1.78 (m, 2H), 1.77- 1.03 (m, 37H), 0.94 - 0.76 (m, 4H).(C) Preparation of compounds 25.1-25.12Procedure for alcohol esterification: l,13-bis(cycloheptylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (25.1).

[0197] To a solution of EDCI*HCl (2.52 g, 13.2 mmol, 2.4 equiv) and 4-DMAP (1.68 g, 13.7 mmol, 2.5 equiv) in CH2CI2 (20 mL), was added decanoic acid (2.08 g, 12.1 mmol, 2.2 equiv) and stirred for 10 min, then followed by addition a solution of diol 21.1 (2.67 g, 5.48 mmol, 1 equiv) in CH2CI2 (10 mL) dropwise. The resulting mixture was stirred at room temp for 2 hrs, diluted with CH₂Cl₂ (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 (Na2SC>4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:hexane (0-10-%) to afford 25.1 (4.1 g, 5.16 mmol, 94%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.92 (dtd, J= 8.1, 6.2, 4.2 Hz, 2H), 2.86 (tt, J= 8.9, 4.3 Hz, 2H), 2.70 - 2.57 (m, 4H), 2.38 (t, J= 7.4 Hz, 4H), 2.29 (t, J= 7.5 Hz, 4H), 2.02 - 1.92 (m, 4H), 1.76 - 1.39 (m, 32H), 1.36 - 1.20 (m, 28H), 0.91 - 0.83 (m, 6H).

[0198] The following compounds were prepared by the same method:l,13-bis(Cycloheptylthio)-7-oxotridecane-2,12-diyl bis(3-cyclohexylpropanoate) (25.2).

[0199] 84 % yield from 21.1 (2.5 g, 5.13 mmol) as a colorless oil. 'H NMR (400 MHz, CDCl3) § 4.97 > 4.87 (m, 2H), 2.86 (tt, J= 8.95, 4.30 Hz, 2H), 2.71 - 2.56 (m, 4H), 2.38 (t, J= 7.42 Hz, 4H), 2.29 (t, J= 7.53 Hz, 4H), 2.03 - 1.92 (m, 4H), 1.77 - 1.37 (m, 20H), 1.37 - 1.17 (m, 30H), 0.92 - 0.81 (m, 8H).l,13-bis(Cyclooctylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (25.3).

[0200] 95% yield from l,13-bis(Cyclooctylthio)-2,12-dihydroxytridecan-7-one (4.65 g, 90.4 mmol) and decanoic acid (3.7 g, 22 mmol).1H NMR (400 MHz, CDCh) 64.97 - 4.88 (m, 2H), 2.91 (tt, J= 9.0, 3.9 Hz, 2H), 2.69 - 2.58 (m, 4H), 2.38 (t, J= 7.4 Hz, 4H), 2.29 (t, J= 7.5 Hz, 4H), 1.98- 1.88 (m, 4H), 1.80 - 1.20 (m, 64H), 0.93 -0.82 (m, 6H).13C NMR (101 MHz, CDCh) 6210.7, 173.6, 73.0, 45.1, 42.7, 34.7, 34.7, 33.1, 32.5, 32.4, 32.0, 31.7, 29.6, 29.4, 29.4, 29.3, 27.4, 27.3, 25.9, 25.2, 25.2, 25.2, 25.1, 23.7, 22.8, 22.8, 14.3.l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (25.4).

[0201] 67% yield from l,13-bis(cyclohexylmethylsulfanyl)-2,12-dihydroxy-tridecan-7-one (2.0 g, 4.11 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.99 – 4.87 (m, 2H), 2.68 – 2.54 (m, 4H), 2.49 – 2.34 (m, 9H), 2.29 (t, J= 7.53 Hz, 4H), 1.85 – 1.03 (m, 57H), 0.95 – 0.79 (m, 10H).l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(4-cyclohexylbutanoate) (25.5).

[0202] 84% yield from l,13-bis(cyclohexylmethylsulfanyl)-2,12-dihydroxy-tridecan-7-one (1.14 g, 2.3 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.93 (dtd, J= 8.2, 6.1, 4.2 Hz, 2H), 2.67 – 2.55 (m, 4H), 2.49 – 2.30 (m, 8H), 2.27 (t, J= 7.5 Hz, 4H), 1.88 – 1.77 (m, 4H), 1.76 – 1.05 (m, 52H), 0.91 (ddt, J= 25.0, 13.1, 6.4 Hz, 8H).l,13-bis((Cyclohexylmethyl)thio)-12-hydroxy-7-oxotridecan-2-yl decanoate (25.6).O

[0203] To a solution of EDC. HC1 (1.9 g, 9.9 mmol, 1.2 equiv) and 4-DMAP (1.2 g, 9.9 mmol, 1.2 equiv) in CH2CI2 (10 mL), was added decanoic acid (1.42 g, 8.22 mmol, 1 equiv) and stirred for 10 min, then followed by addition a solution of l,13-bis(cyclohexylmethylsulfanyl)-2,12-dihydroxy-tridecan-7-one (4.0 g, 8.22 mmol, 1 equiv) in CH2CI2 (10 mL) dropwise. The resulting mixture was stirred at room temp for 18 h, diluted with CH₂Cl₂ (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 (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:hexane (15- 20%) to afford the title compound, 25.6 (2.8 g, 4.4 mmol, 53%). ¹H NMR (400 MHz, CDCl₃) δ 4.98 – 4.85 (m, 1H), 3.68 – 3.54 (m, 1H), 2.74 – 2.53 (m, 4H), 2.49 – 2.34 (m, 9H), 2.29 (t, J = 7.52 Hz, 2H), 1.91 – 1.04 (m, 43H), 0.99 – 0.80 (m, 7H).12-((4-Cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7-oxotridecan-2-yl decanoate (25.7).

[0204] To a solution of EDC. HC1 (0.72 g, 3.74 mmol, 1.2 equiv) and 4-DMAP (0.46 g, 3.74 mmol, 1.2 equiv) in CH₂Cl₂ (5 mL), was added 4-cyclohexylbutanoic acid (0.53 g, 3.12 mmol, 1 equiv) and stirred for 10 min, then followed by addition a solution of 25.6 (2.0 g, 3.12 mmol, 1 equiv) in CH₂Cl₂ (5 mL) dropwise. The resulting mixture was stirred at room temp for 18 h, diluted with CH2CI2 (30 mL) and poured into water (50 mL). The organic layer was separated, and the aq. phase was further extracted with CH2CI2 (2 x 20 mL). The combined organic layers were dried (Na2SC>4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:hexane (7%) to afford the title compound, 25.7 (1.85 g, 2.3mmol, 75%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.98 – 4.88 (m, 2H), 2.68 – 2.54 (m, 4H), 2.49 – 2.33 (m, 8H), 1.88 – 1.05 (m, 60H), 0.98 – 0.79 (m, 10H).l,13-bis((2-Cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (25.8).

[0205] 86% yield from l,13-bis(2-cyclohexylethylsulfanyl)-2,12-dihydroxy-tridecan-7-one, 25.7 (1.5 g, 3.0 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.99 – 4.88 (m, 2H), 2.67 – 2.47 (m, 8H), 2.38 (t, J= 7.37 Hz, 4H), 2.29 (t, J= 6.80 Hz, 4H), 1.77 – 1.49 (m, 18H), 1.50 – 1.04 (m, 44H), 0.95 – 0.80 (m, 10H).l,13-bis((2-Cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(3-cyclohexylpropanoate) (25.9).

[0206] 65% yield from l,13-bis(2-cyclohexylethylsulfanyl)-2,12-dihydroxy-tridecan-7-one (1.2 g, 2.3 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.99 – 4.87 (m, 2H), 2.67 – 2.48 (m, 8H), 2.38 (t, J = 7.40 Hz, 4H), 2.30 (t, J= 6.80 Hz, 4H), 1.82 – 1.03 (m, 56H), 0.96 – 0.77 (m, 8H). l,13-bis((Cycloheptylmethyl)thio)-7-oxotridecane-2,12-diyl didecanoate (25.10).

[0207] 95% yield l,13-bis((Cycloheptylmethyl)thio)-2,12-dihydroxytridecan-7-one (0.803 g, 1.56 mmol) and decanoic acid (0.65 g, 3.7 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.98 – 4.87 (m,2H), 2.66 - 2.55 (m, 4H), 2.50 - 2.41 (m, 4H), 2.38 (t, J= 7.4 Hz, 4H), 2.29 (t, J= 7.5 Hz, 4H), 1.88 - 1.17 (m, 66H), 0.92 - 0.84 (m, 6H).l-((2-Cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (25.11).

[0208] 87% yield from l-(2-cyclohexylethylsulfanyl)-13-(cyclohexylmethylsulfanyl)-2,12-dihydroxy-tridecan-7-one (3.0 g, 6.0 mmol) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.97 – 4.86 (m, 2H), 2.65 – 2.49 (m, 6H), 2.45 – 2.32 (m, 6H), 2.27 (t, J = 7.50 Hz, 4H), 1.86 – 1.76 (m, 2H), 1.75 – 1.02 (m, 58H), 0.96 – 0.78 (m, 10H).l,14-bis((Cyclohexylmethyl)thio)-7-oxotetradecane-2,13-diyl bis(decanoate) (25.12).OO

[0209] 94% yield from 1, 14-bis((Cyclohexylmethyl)thio)-2, 13-dihydroxytetradecan-7-one (2.63 g, 5.25 mmol) and decanoic acid (2.2 g, 13 mmol.1H NMR (400 MHz, CDCl₃) 64.97 - 4.89 (m, 2H), 2.67 - 2.55 (m, 4H), 2.49 - 2.40 (m, 4H), 2.41 - 2.35 (m, 4H), 2.32 - 2.26 (m, 4H), 1.86 -1.15 (m, 60H), 0.93 - 0.83 (m, 10H).((tert-Butoxycarbonyl)azanediyl)bis(1-((cyclohexylmethyl)thio)hexane-6,2-diyl) bis(decanoate) (32.1).

[0210] 79% from tert-butyl bis(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)carbamate 30.5 (3.2 g, 5.6 mmol) and decanoic acid (2.3 g, 13.4 mmol).1H NMR (400 MHz, CDCh) δ 5.01 – 4.85 (m, 2H), 3.12 (brs, 4H), 2.68 – 2.54 (m, 4H), 2.49 – 2.36 (m, 4H), 2.29 (t, J= 7.5 Hz, 4H), 1.95 – 1.00 (m, 67H), 1.00 – 0.78 (m, 10H).7-((tert-Butoxycarbonyl)(6-((cyclohexylmethyl)thio)-5-(decanoyloxy)hexyl)amino)-l-((cyclohexylmethyl)thio)heptan-2-yl decanoate (32.2).

[0211] 79% from tert-butyl (6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)(7-((cyclohexylmethyl)thio)-6-hydroxyheptyl)carbamate 30.6 (2.9 mg, 4.94 mmol) and decanoic acid (2.12 g, 12.3 mmol). ¹H NMR (400 MHz, CDCl₃) δ 5.02 – 4.81 (m, 2H), 3.12 (brs, 4H), 2.68 – 2.54 (m, 4H), 2.49 – 2.36 (m, 4H), 2.28 (q, J= 7.7 Hz, 4H), 1.88 – 1.05 (m, 69H), 1.00 – 0.75 (m, 10H).6-((tert-butoxycarbonyl)(6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (32.3).

[0212] 84% from tert-butyl (6-((2-cyclohexylethyl)thio)-5-hydroxyhexyl)(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)carbamate 31.2 (780 mg, 1.33 mmol) and decanoic acid (548 mg, 3.18 mmol). ¹H NMR (400 MHz, CDCl₃) δ 5.03 – 4.84 (m, 2H), 3.14 (brs, 4H), 2.76 – 2.35 (m, 7H), 2.29 (t, J= 7.5 Hz, 4H), 1.93 – 0.99 (m, 70H), 0.98 – 0.74 (m, 10H).6-((tert-Butoxycarbonyl)(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)amino)-l-((cyclohexyl-methyl)thio)hexan-2-yl decanoate (33.1, from 0.9 equiv of decanoic acid) (33.1).

[0213] 43% from 6-((ter / -butoxycarbonyl)(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)amino)- l-((cyclohexylmethyl)thio)hexan-2-yl decanoate 30.5 (6.2 g, 10.8 mmol) and decanoic acid (2.04 g, 11.9 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.93 (t, J= 6.6 Hz, 1H), 3.67 – 3.52 (m, 1H), 3.13 (brs, 4H), 2.73 – 2.54 (m, 4H), 2.49 – 2.34 (m, 5H), 2.29 (t, J= 7.6 Hz, 2H), 1.94 – 1.04 (m, 53H), 1.00 – 0.79 (m, 7H).6-((tert-butoxycarbonyl)(5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)- amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (33.2).

[0214] 85% from 6-((ter / -butoxycarbonyl)(6-((cyclohexylmethyl)thio)-5-hydroxyhexyl)amino)- l-((cyclohexylmethyl)thio)hexan-2-yl decanoate 33.1 (3.4 g, 4.7 mmol) and 4-cyclohexylbutanoic acid (959 g, 5.64 mmol). 'H NMR (400 MHz, CDCl3) δ 4.96 – 4.89 (m, 2H), 3.12 (brs, 4H), 2.68 – 2.54 (m, 4H), 2.49 – 2.36 (m, 4H), 2.28 (q, J= 7.7 Hz, 4H), 1.88 – 1.05 (m, 65H), 1.00 – 0.77 (m, 10H).(D) Preparation of lipids T-01 - T-28Procedure for ketone reduction: l,13-bis(cycloheptylthio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (26.1).

[0215] To a solution of ketone 25.1 (200 mg, 0.25 mmol, 1 equiv) in ethanol- THF (1:1, 4 mL) at 0 °C, was added sodium borohydride (143 mg, 0.38 mmol, 1.5 equiv) portion wise. The resulting mixture was allowed to warm to RT and stirred for 2 h, then diluted with / / -hexanes (30 mL) and poured slowly [carefully] into a stirred an ice-cold NH4CI (sat. solution). The organic layer was separated, and the aqueous layer was further extracted with / / -hexanes (2 x 30 mL). The combined organic layers were washed with brine (sat. solution), dried (Na2SO4), filtered, and concentrated in vacuo to afford 26.1 (200 mg, 0.25 mmol, -100%) as a colorless oil and used for the next step without further purification.1H NMR (400 MHz, CDCl3) δ 5.02 – 4.91 (m, 2H), 3.63 – 3.53 (m, 1H), 2.89 (tt, J= 8.9, 4.3 Hz, 2H), 2.73 – 2.61 (m, 4H), 2.32 (t, J= 7.5 Hz, 4H), 2.05 – 1.96 (m, 4H), 1.79 – 1.22 (m, 64H), 0.90 (t, 6H).

[0216] The following compounds were prepared by the same method:l,13-bis(Cyclooctylthio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (26.3).

[0217] 82% yield from l,13-bis(cyclooctylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.50 g, 0.61 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.99 – 4.90 (m, 2H), 3.60 – 3.51 (m, 1H), 2.92 (tt, J= 9.0, 3.9 Hz, 2H), 2.71 – 2.58 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.98 – 1.88 (m, 4H), 1.80 – 1.19 (m, 68H), 0.92 – 0.83 (m, 6H).l,13-bis((Cyclohexylmethyl)thio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (26.4).

[0218] 99 % from l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.30 g, 0.4 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.96 – 4.82 (m, 2H), 3.6 – 3.49 (m, 1H), 2.62 – 2.32 (m, 4H), 2.43 – 2.33 (m, 4H), 2.23 (t, J= 7.5 Hz, 4H), 1.81 – 1.71 (m, 4H), 1.71 – 1.49 (m, 20H), 1.44 – 1.04 (m, 38H), 0.92 – 0.84 (m, 4H), 0.83 – 0.79 (m, 6H).l,13-bis(Cycloheptylthio)-7-hydroxytridecane-2,12-diyl bis(3-cyclohexylpropanoate) (26.2).

[0219] 98 % from l,13-bis(Cycloheptylthio)-7-oxotridecane-2,12-diyl bis(3- cyclohexylpropanoate) (0.30 g, 0.4 mmol). ’H NMR (400 MHz, CDCl₃) δ 4.99 – 4.87 (m, 2H), 3.62 - 3.51 (m, 1H), 2.67 - 2.49 (m, 6H), 2.31 (t, J = 7.45 Hz, 4H), 1.78 - 1.68 (m, 4H), 1.67 - 1.04 (m, 54H), 0.99 - 0.79 (m, 8H).l,13-bis((2-cyclohexylethyl)thio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (26.8).

[0220] 98 % from l,13-bis((2-Cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.30 g, 0.4 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.95 (dq, J = 10.4, 5.2 Hz, 2H), 3.60 – 3.52 (m, 1H), 2.63 (d, J= 6.1 Hz, 4H), 2.55 (td, J= 7.3, 1.3 Hz, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.78 – 1.53 (m, 20H), 1.51 – 1.08 (m, 46H), 0.97 – 0.81 (m, 10H).l,13-bis((2-Cyclohexylethyl)thio)-7-hydroxytridecane-2,12-diyl bis(3-cyclohexylpropan- oate) (26.9).

[0221] 95 % from l,13-bis(2-cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(3- cyclohexylpropanoate) (0.40 g, 0.51 mmol).¹H NMR (400 MHz, CDCl₃) δ 5.01 – 4.87 (m, 2H), 3.62 – 3.51 (m, 1H), 2.67 – 2.49 (m, 8H), 2.31 (t, J= 7.45 Hz, 4H), 1.78 – 1.04 (m, 60H), 0.99 – 0.79 (m, 8H).l,13-bis((Cycloheptylmethyl)thio)-7-hydroxytridecane-2,12-diyl didecanoate (26.10).

[0222] 95% yield from l,13-bis((Cycloheptylmethyl)thio)-7-oxotridecane-2,12-diyl didecanoate (0.40 g, 0.48 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.96 – 4.82 (m, 2H), 3.6 – 3.51 (m, 1H), 2.62 – 2.32 (m, 4H), 2.43 – 2.33 (m, 4H), 2.23 (t, J= 7.5 Hz, 4H), 1.81 – 1.71 (m, 4H), 1.71 – 1.49 (m, 20H), 1.44 – 1.04 (m, 42H), 0.92 – 0.84 (m, 4H), 0.83 – 0.79 (m, 6H)Procedure for alcohol esterification: l,13-bis(cycloheptylthio)-7-((4-(dimethylamino)-butanoyl)oxy)tridecane-2,12-diyl bis(decanoate) (T-01).

[0223] A solution of 4-(dimethylamino)-butyric acid hydrochloride (51 mg, 0.3 mmol, 1.6 equiv), 4-DMAP (37 mg, 0.3 mmol, 1.6 equiv) and EDC. HCI (58 mg, 0.3 mmol, 1.6 equiv) in dry CH₂Cl₂ (5 mL) was stirred at room temperature for 10 min, followed by a slow addition of a solution of alcohol 26.1 (150 mg, 0.19 mmol, 1.0 equiv ) in CH2CI2 (2 mL). The reaction mixture was then stirred at RT for 18 hr, then diluted with CH2CI2 (20 mL) and poured into a stirred solution of NaHCO₃ (sat. solution). The organic layer was separated, and the aqueous layer was further extracted with CH2CI2 (2 × 10 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography eluting with dichloromethane: MeOH (0-5%) to afford T-01 (143 mg, 0.16 mmol, 84%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 4.91 (dt, J= 10.6, 5.5 Hz, 2H), 4.85 (q, J= 6.1 Hz, 1H), 2.87 (tt, J= 8.9, 4.3 Hz, 2H), 2.69 – 2.58 (m, 4H), 2.35 – 2.25 (m, 8H), 2.22 (s, 6H), 2.02 – 1.93 (m, 4H), 1.82 – 1.39 (m, 36H), 1.28 – 1.24 (m, 30H), 0.88 (d, 6H).

[0224] The following compounds were prepared by the same method:l,13-bis(Cyclooctylthio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(decanoate) (T-02).

[0225] 36% yield from l,13-bis(cyclooctylthio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (0.412 g, 0.499 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.96 - 4.88 (m, 2H), 4.84 (p, J= 6.2 Hz, 1H), 2.92 (tt, J= 9.0, 3.9 Hz, 2H), 2.69 - 2.58 (m, 4H), 2.35 - 2.24 (m, 8H), 2.21 (s, 6H), 1.98 -1.88 (m, 4H), 1.82 - 1.19 (m, 70H), 0.91 - 0.84 (m, 6H).13C NMR (101 MHz, CDCh) 6 173.6, 173.5, 74.0, 73.1, 59.1, 45.6, 45.1, 34.7, 34.2, 33.2, 32.5, 32.5, 32.4, 32.0, 29.6, 29.4, 29.4, 29.3, 27.4, 27.3, 25.9, 25.4, 25.3, 25.2, 25.2, 25.2, 23.3, 22.8, 14.3.l,13-bis((Cyclohexylmethyl)thio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(decanoate) (T-03).

[0226] 90% from l,13-bis((Cyclohexylmethyl)thio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (0.30 g, 0.4 mmol). 'H NMR (400 MHz, CDCh) 64.91 (dt, J= 10.5, 5.5 Hz, 2H), 4.84 (p, J= 6.3 Hz, 1H), 2.67 - 2.55 (m, 4H), 2.49 - 2.38 (m, 4H), 2.35 - 2.25 (m, 8H), 2.22 (s, 6H), 1.88 - 1.39 (m, 28H), 1.38 - 1.05 (m, 36H), 0.99 - 0.90 (m, 4H), 0.88 (t, 6H).l,13-bis(Cycloheptylthio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(3-cyclohexylpropanoate) (T-04).

[0227] 65% from l,13-bis(Cycloheptylthio)-7-hydroxytridecane-2,12-diyl bis(3- cyclohexylpropanoate) (0.200 g, 0.26 mmol).1H NMR (400 MHz, CDCl₃) δ 4.99 – 4.87 (m, 2H), 4.82 (p, J= 6.4 Hz, 1H), 2.64 - 2.42 (m, 8H), 2.22 (s, 6H), 2.31 (t, J= 7.45 Hz, 4H), 1.72 - 1.64 (m, 4H), 1.67 - 1.04 (m, 58H), 0.99 - 0.79 (m, 8H).l,13-bis((2-Cyclohexylethyl)thio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(decanoate) (T-05).

[0228] 93% from l,13-bis((2-cyclohexylethyl)thio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (0.30 g, 0.4 mmol). 1H NMR (400 MHz, CDCh) 64.97 - 4.89 (m, 2H), 4.84 (p, J = 6.1 Hz, 1H), 2.68 - 2.57 (m, 4H), 2.57 - 2.52 (m, 4H), 2.35 - 2.25 (m, 8H), 2.22 (s, 6H), 1.84 - 1.06 (m, 68H), 0.88 (td, J= 6.0, 3.4 Hz, 10H).l,13-bis((2-Cyclohexylethyl)thio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(3-cyclohexylpropanoate) (T-06).

[0229] 60% from [12-(2-cyclohexylethylsulfanyl)-l-(2-cyclohexylethylsulfanylmethyl)-l l-(3- cyclohexylpropanoyloxy)-6-hydroxy-dodecyl] 3-cyclohexylpropanoate (0.37 g, 0.47 mmol). 'HNMR (400 MHz, CDCh) 64.97 - 4.88 (m, 2H), 4.84 (p, J= 6.14 Hz, 1H), 2.67 - 2.59 (m, 4H), 2.58 - 2.50 (m, 4H), 2.35 - 2.27 (m, 6H), 2.21 (s, 6H), 1.81 - 1.03 (m, 64H), 0.98 - 0.81 (m, 8H). l,13-bis((Cycloheptylmethyl)thio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl didecanoate (T-07).

[0230] 32% yield from l,13-bis((Cycloheptylmethyl)thio)-7-hydroxytridecane-2,12-diyl didecanoate (0.37 g, 0.45 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.87 (m, 2H), 4.87 - 4.80 (m, 1H), 2.66 - 2.55 (m, 4H), 2.50 - 2.41 (m, 4H), 2.38 (t, J= 7.4 Hz, 4H), 2.29 (t, J= 7.5 Hz, 4H), 2.21 (s, 6H), 1.88 - 1.17 (m, 68H), 0.92 - 0.84 (m, 10H).Procedure for ketalization: (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((cyclohexyl-methyl)thio)hexane-6,2-diyl) bis(decanoate) (27.4).Clo o

[0231] A solution of ketone 25.4 (500 mg, 0.63 mmol, 1 equiv), 4-chloro-l,2-butanediol (235 mg, 1.9 mmol, 3 equiv), and PPTS (16 mg, 0.063 mmol, 0.1 equiv) in toluene (20 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 NaHCO3(100 mg), and evaporated to dryness in vacuo. The residue was taken up with w-hexanes (100 mL) and aqueous saturated NaHCO3solution (100 mL). The organic phase was separated and sequentially washed with water, brine, dried (Na2SC>4), filtered, and concentrated in vacuo to afford the title compound (567 mg, 0.63 mmol, -100%) as a colorless oil that was used for the next step without purification. 'H NMR (400 MHz, CDCl₃) δ 5.02 - 4.89 (m, 2H), 4.25 (ddt, J= 12.0,7.5, 3.7 Hz, 1H), 4.11 (dd, J= 7.9, 6.1 Hz, 1H), 3.69 - 3.64 (m, 2H), 3.52 (t, J= 7.8 Hz, 1H), 2.71 - 2.58 (m, 4H), 2.52 - 2.40 (m, 4H), 2.32 (t, J= 7.5 Hz, 4H), 2.12 - 1.90 (m, 2H), 1.88 - 1.80 (m, 4H), 1.77 - 1.54 (m, 20H), 1.52 - 1.08 (m, 38H), 1.04 - 0.83 (m, 10H).

[0232] The following compounds were prepared by the same method:(4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cycloheptylthio)hexane-6,2-diyl) bis(decanoate) (27.1).

[0233] 68% yield from l,13-bis(cycloheptylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (1.00 g, 1.26 mmol). ¹H NMR (400 MHz, CDCl₃) δ 4.92 (m, 2H), 4.27 - 4.18 (m, 1H), 4.12 - 4.05 (m, 1H), 3.68 - 3.55 (m, 2H), 3.50 (t, J= 7.8 Hz, 1H), 2.87 (tt, J= 8.9, 4.3 Hz, 2H), 2.70 - 2.59 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 2.10 - 1.83 (m, 6H), 1.77 - 1.10 (m, 64H), 0.87 (t, 6H).(4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cyclooctylthio)hexane-6,2-diyl) bis(decanoate) (27.3).

[0234] 65% yield from l,13-bis(cyclooctylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (1.00 g, 1.21 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.89 (m, 2H), 4.28 - 4.17 (m, 1H), 4.12 -4.05 (m, 1H), 3.69 - 3.60 (m, 2H), 3.54 - 3.46 (m, 1H), 2.97 - 2.88 (m, 2H), 2.70 - 2.57 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 2.08 - 1.88 (m, 6H), 1.80 - 1.19 (m, 68H), 0.91 - 0.85 (m, 6H).(4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((cyclohexylmethyl)thio)hexane-6,2-diyl) bis(4-cyclohexylbutanoate) (27.5).

[0235] 68% from l,13-bis((cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(4-cyclohexylbutanoate) (0.78 g, 1.0 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.89 (m, 2H), 4.22 (qd, J= 7.6, 4.9 Hz, 1H), 4.08 (dd, J= 7.9, 6.1 Hz, 1H), 3.68 - 3.60 (m, 2H), 3.49 (t, J= 7.8 Hz, 1H), 2.67 - 2.55 (m, 4H), 2.42 (dt, J= 8.3, 4.3 Hz, 4H), 2.27 (t, J= 7.5 Hz, 4H), 2.20 - 1.89 (m, 2H), 1.87 - 1.77 (m, 4H), 1.74 - 1.05 (m, 56H), 0.99 - 0.79 (m, 8H).6-(4-(2-chloroethyl)-2-(5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (27.7)

[0236] 89% from 12-((4-cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7-oxotridecan-2-yl decanoate (1.0 g, 1.3 mmol).1H NMR (400 MHz, CDCh) 84.99 - 4.85 (m, 2H), 4.28 - 4.16 (m, 1H), 4.08 (dd, J= 7.94, 6.11 Hz, 1H), 3.66 - 3.59 (m, 2H), 3.49 (t, J= 7.79 Hz, 1H), 2.68 - 2.54 (m, 4H), 2.48 - 2.36 (m, 4H), 2.36 - 2.22 (m, 4H), 2.07 - 1.87 (m, 2H), 1.87 -1.78 (m, 4H), 1.76 - 1.05 (m, 57H), 0.97 - 0.79 (m, 9H).(4-(2-Chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2-cyclohexylethyl)thio)hexane-6,2-diyl) bis(decanoate) (27.8).

[0237] 65% yield from l,13-bis((2-cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (1.03 g, 1.25 mmol). 'H NMR (400 MHz, CDCl3) 8 4.98 - 4.89 (m, 2H), 4.27 -4.18 (m, 1H), 4.12 - 4.05 (m, 1H), 3.67 - 3.60 (m, 2H), 3.50 (t, J= 7.8 Hz, 1H), 2.69 - 2.59 (m, 4H), 2.58 - 2.52 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 2.10 - 1.82 (m, 3H), 1.78 - 1.01 (m, 65H), 0.94 - 0.82 (m, 10H).(4-(2-Chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2-cyclohexylethyl)thio)hexane-6,2-diyl) bis(3-cyclohexylpropanoate) (27.9).

[0238] 91% yield from l,13-bis((2-cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(3-cyclohexylpropanoate) (0.861g, 1.09 mmol). 'H NMR (400 MHz, CDCl3) 64.98 - 4.89 (m, 2H), 4.27 - 4.17 (m, 1H), 4.12 - 4.06 (m, 1H), 3.68 - 3.55 (m, 2H), 3.50 (t, J= 7.7 Hz, 1H), 2.70 -2.59 (m, 4H), 2.59 - 2.51 (m, 4H), 2.35 - 2.27 (m, 4H), 2.09 - 1.83 (m, 2H), 1.78 - 1.02 (m, 60H), 0.94 - 0.81 (m, 8H).6-(4-(2-chloroethyl)-2-(6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (27.11).

[0239] 80% from l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7-oxotridecane- 2, 12-diyl bis(decanoate) (1.0 g, 1.24 mmol). 'H NMR 400 MHz, CDCl₃) δ 4.99 - 4.85 (m, 2H), 4.28 - 4.16 (m, 1H), 4.08 (dd, J = 7.94, 6.11 Hz, 1H), 3.66 - 3.59 (m, 2H), 3.54 - 3.46 (m, 1H), 2.97 - 2.88 (m, 2H), 2.70 - 2.57 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 2.08 - 1.88 (m, 6H), 1.80 - 1.19 (m, 66H), 0.91 - 0.84 (m, 6H).7-(4-(2-Chloroethyl)-2-(6-((cyclohexylmethyl)thio)-5-(decanoyloxy)hexyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)heptan-2-yl decanoate (27.12).

[0240] 68% yield from l,14-bis((cyclohexylmethyl)thio)-7-oxotetradecane-2,13-diyl bis(decanoate) (983 mg, 1.21 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.89 (m, 2H), 4.29 -4.18 (m, 1H), 4.11 - 4.05 (m, 1H), 3.72 - 3.58 (m, 2H), 3.54 - 3.47 (m, 1H), 2.68 - 2.56 (m, 4H), 2.47-2.42 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 2.09- 1.89 (m, 2H), 1.87- 1.79 (m, 4H), 1.76- 1.08 (m, 60H), 0.98 - 0.86 (m, 10H).Procedure for nucleophilic displacement: (4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2- diyl)bis(l-((cyclohexylmethyl)thio)hexane-6,2-diyl) bis(decanoate) (T-10).

[0241] Cold (0 °C) commercial dimethylamine solution (2 M in THF, 0.75 mL, 27.5 mmol, 3 equiv) was added to a cold (0 °C) solution of (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l- ((cyclohexyl-methyl)thio)hexane-6,2-diyl) bis(decanoate) (250 mg, 0.28 mmol, 1 equiv) in 1 mL of dry THF in a sealable microwave glass tube. The tube was sealed and heated at 120 °C in an oil bath for 48 h, whereupon the reaction was complete. The cooled mixture was poured into aqueous saturated NaHCO3solution and extracted with / / -hexanes (3 × 15 mL). The combined extracts were washed with brine, dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by automated column chromatography (0→25% EtOAc: CH2C12) to afford T-10 (170 mg, 6.01 mmol, 67%) as a colorless oil. 'H NMR (400 MHz, C6D6) 6 5.24 (dt, J= 10.8, 5.4 Hz, 2H), 4.08 (p, J= 6.6 Hz, 1H), 3.93 (dd, J= 7.8, 5.9 Hz, 1H), 3.41 (t, J= 8.0 Hz, 1H), 2.67 (dd, J= 14.3, 7.0 Hz, 2H), 2.58 (dd, J= 13.5, 5.2 Hz, 2H), 2.48 (dd, J= 12.5, 6.9 Hz, 2H), 2.40 (dd, J= 12.5,6.8 Hz, 2H), 2.34 - 2.16 (m, 6H), 2.05 (s, 6H), 1.87 (d, J= 12.7 Hz, 4H), 1.80 - 1.01 (m, 60H), 0.90 (q, = 8.0 Hz, 10H).

[0242] The following compounds were prepared by the same method:(4-(2-(dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cycloheptylthio)hexane-6,2-diyl) bis(decanoate) (T-08).I< I f )S Y ■■ y' S-.... o T-oa 0.-..-..-.-■ -- K r ■ ■o o

[0243] 67% yield from (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cycloheptylthio)hexane-6,2-diyl) bis(decanoate) (0.770 g, 0.854 mmol). 'H NMR (400 MHz, CDCl3) 54.97 - 4.86 (m, 2H), 4.11 - 4.00 (m, 2H), 3.49 - 3.43 (m, 1H), 2.87 (tt, J= 9.0, 4.3 Hz, 2H), 2.71 - 2.58 (m, 4H), 2.42 - 2.34 (m, 1H), 2.32 - 2.25 (m, 5H), 2.22 (s, 6H), 2.02 - 1.93 (m, 4H), 1.86 - 1.18 (m, 66H), 0.87 (t, J= 6.7 Hz, 6H).(4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cyclooctylthio)hexane-6,2-diyl) bis(decanoate) (T-09).

[0244] 25% yield from (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-(cyclooctylthio)hexane-6,2-diyl) bis(decanoate) (0.733 g, 0.788 mmol). 'H NMR (400 MHz, CDCh) 64.97 - 4.88 (m, 2H), 4.12 - 4.00 (m, 2H), 3.50 - 3.41 (m, 1H), 2.96 - 2.87 (m, 2H), 2.70 - 2.58 (m, 4H), 2.45 -2.34 (m, 1H), 2.33 - 2.25 (m, 5H), 2.22 (s, 6H), 1.98 - 1.87 (m, 4H), 1.85 - 1.19 (m, 70H), 0.91 -0.85 (m, 6H).13C NMR (101 MHz, CDCl3) 6 173.6, 111.8, 75.0, 73.3, 70.2, 56.4, 45.7, 45.1, 37.8, 37.6, 34.7, 34.7, 33.4, 33.3, 32.5, 32.4, 32.0, 32.0, 29.6, 29.4, 29.4, 29.3, 27.4, 27.3, 25.9, 25.8, 25.8, 25.2, 25.2, 25.2, 24.0, 23.7, 22.8, 14.3.(4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(l-((cyclohexylmethyl)thio)hexane-6,2-diyl) bis(4-cyclohexylbutanoate) (T-11).

[0245] 69% yield from (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2-cyclohexylethyl)thio)hexane-6,2-diyl) bis(3-cyclohexylpropanoate) (0.890 g, 1.0 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.89 (m, 2H), 4.11 -4.02 (m, 2H), 3.49 - 3.43 (m, 1H), 2.68 -2.58 (m, 4H), 2.58 -2.51 (m, 4H), 2.43 - 2.34 (m, 1H), 2.34 - 2.25 (m, 5H), 2.22 (s, 6H), 1.86 -1.06 (m, 62H), 0.95 - 0.81 (m, 8H).6-(2-(5-((4-Cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)-4-(2-(dimethyl-amino)ethyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (T-12).

[0246] 85% from 6-(4-(2-chloroethyl)-2-(5-((4-cyclohexylbutanoyl)oxy)-6- ((cyclohexylmethyl)thio)hexyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (1.0 g, 1.11 mmol).1H NMR (400 MHz, CDCl₃) δ 4.98 – 4.85 (m, 2H), 4.12 - 3.97 (m, 2H), 3.51 - 3.40 (m, 1H), 2.66 - 2.56 (m, 4H), 2.49 - 2.33 (m, 5H), 2.32 - 2.17 (m, 11H), 1.89 -1.02 (m, 63H), 0.98 - 0.78 (m, 9H).(4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2-cyclohexylethyl)thio)hexane-6,2-diyl) bis(decanoate) (T-13).

[0247] 76% yield from (4-(2-chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2- cyclohexylethyl)thio)hexane-6,2-diyl) bis(decanoate) (0.730 g, 0.785 mmol). 'H NMR (400 MHz, CDCh) 64.97 - 4.86 (m, 2H), 4.11 - 4.00 (m, 2H), 3.49 - 3.43 (m, 1H), 2.87 (tt, J= 9.0, 4.3 Hz, 2H), 2.71 - 2.58 (m, 4H), 2.42 - 2.34 (m, 1H), 2.32 - 2.25 (m, 5H), 2.22 (s, 6H), 2.02 - 1.93 (m, 4H), 1.86 - 1.18 (m, 70H), 0.87 (t, J= 6.7 Hz, 6H).(4-(2-(Dimethylamino)ethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2-cyclohexylethyl)thio)hexane- 6,2-diyl) bis(3-cyclohexylpropanoate) (T-14).

[0248] 65% from (4-(2-Chloroethyl)-l,3-dioxolane-2,2-diyl)bis(l-((2- cyclohexylethyl)thio)hexane-6,2-diyl) bis(3-cyclohexylpropanoate) (0.89 g, 1.0 mmol).1H NMR (400 MHz, CDCl3) 8 5.02 - 4.89 (m, 2H), 4.15 - 4.03 (m, 2H), 3.52 - 3.43 (m, 1H), 2.71 - 2.51 (m, 8H), 2.46 - 2.26 (m, 6H), 2.25 (s, 6H), 1.91 - 1.07 (m, 62H), 0.99 - 0.81 (m, 8H).6-(2-(6-((2-Cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)-4-(2-(dimethylamino)ethyl)-l,3- dioxolan-2-yl)- l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (T- 15).

[0249] 35% from 6-(4-(2-chloroethyl)-2-(6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)-l,3-dioxolan-2-yl)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (1.0 g, 1.1 mmol).1H NMR (400 MHz, CDCl₃) δ 4.98 – 4.85 (m, 2H), 4.12 - 3.97 (m, 2H), 3.50 - 3.40 (m, 1H), 2.67 - 2.56 (m, 4H), 2.49 - 2.33 (m, 5H), 2.32 - 2.24 (m, 5H), 2.21 (s, 6H), 1.89 - 1.02 (m, 68H), 0.98 - 0.79 (m, 10H).l-(Cyclohexylmethyl)thio)-6-(2-(7-((cyclohexylmethyl)thio)-6-(decanoyloxy)heptyl)-4-(2-(dimethylamino)ethyl)-l,3-dioxolan-2-yl)hexan-2-yl decanoate (T-16).

[0250] 25% yield from 7-(4-(2-chloroethyl)-2-(6-((cyclohexylmethyl)thio)-5-(decanoyloxy)hexyl)- 1,3 -dioxolan-2-yl)- 1 -((cy clohexylmethyl)thio)heptan-2-yl decanoate (0.857g, 0.936 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.89 (m, 2H), 4.12 - 4.01 (m, 2H), 3.50 - 3.41 (m, 1H), 2.67 - 2.56 (m, 4H), 2.49 - 2.34 (m, 5H), 2.33 - 2.25 (m, 5H), 2.22 (s, 6H), 1.88 - 1.07 (m, 66H), 0.99 - 0.84 (m, 10H).Procedure for reductive amination: 7-((4-((tert-Butyldimethylsilyl)oxy)butyl)amino)-l,13-bis(cycloheptylthio)tridecane-2,12-diyl bis(decanoate) (28.1).

[0251] To a solution of ketone 25.1 (500 mg, 0.63 mmol, 1.0 equiv) in DCE (5 mL) at RT was added 4-(tert-butyldimethylsilyl)oxy-1-butanol (256 mg, 1.26 mmol, 2 equiv), and the resulting mixture was stirred for 15 min at the same temperature, then followed by addition STAB-H (266 mg, 1.26 mmol, 2 equiv). The resulting mixture was stirred for further 48 h, then quenched with sat. solution of NaHCO3(75 mL). The aq. phase was extracted with CH2CI2 (3 x 30 mL). The combined organic layers were washed brine (sat. solution), dried (Na2SC>4), filtered, andconcentrated in vacuo. The resulting residue was purified by column chromatography eluting with (0→5% MeOH:CH₂Cl₂) to afford 28.1 (400 mg, 0.47 mmol, 65%) as a colorless oil.1H NMR (400 MHz, CDCl₃) δ 4.92 (dt, J = 10.6, 5.6 Hz, 2H), 3.61 (t, J= 6.1 Hz, 2H), 2.87 (tt, J= 8.9, 4.3 Hz, 2H), 2.71 - 2.59 (m, 4H), 2.55 (t, J= 6.8 Hz, 2H), 2.43 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 2.03 - 1.93 (m, 4H), 1.75 - 1.20 (m, 69 H), 0.93 - 0.83 (m, 15H), 0.04 (s, 6H).Procedure for reductive amination: l,13-bis(cyclooctylthio)-7-(methylamino)tridecane-2, 12-diyl bis(decanoate) (28.3)

[0252] To a 4 mL reaction vial containing l,13-bis(cyclooctylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.500 g, 0.607 mmol) under inert atmosphere was added methylamine (2M in THF) (1.5 mL, 3.0 mmol, 5 equiv.). The solution was stirred at RT for 15 min then STAB-H (0.19 g, 0.91 mmol, 1.5 equiv.) was added. The reaction was quenched with sat. NaHCO3(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 sat. NaHCO3(2 x 20 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. This was passed though a pad of basic alumina (1 g, Brockmann activity 1), eluding with 10% EA in hexanes (20 mL), evaporated, yielding l,13-bis(cyclooctylthio)-7-(methylamino)tridecane-2, 12-diyl bis(decanoate) 28.3 (0.411 g, 0.526 mmol, 87%).1H NMR (400 MHz, CDCl₃) 6 5.00 - 4.88 (m, 2H), 2.92 (tt, J= 9.0, 3.9 Hz, 2H), 2.70 - 2.59 (m, 4H), 2.39 -2.32 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.98 - 1.89 (m, 4H), 1.81 - 1.20 (m, 68H), 0.91 - 0.85 (m, 6H).

[0253] The following compounds were prepared by the same method:7-((4-((tert-Butyldimethylsilyl)oxy)butyl)amino)-l,13-bis((cyclohexylmethyl)thio)t ridecane-2,12-diyl bis(decanoate) (28.4).

[0254] 80 % from l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.50 g, 0.63 mmol). 'H NMR (400 MHz, CDCl₃) δ 5.00 – 4.87 (m, 2H), 3.63 (t, J= 5.8 Hz, 2H), 2.67 - 2.56 (m, 4H), 2.51 - 2.37 (m, 4H), 2.30 (t, J= 7.6 Hz, 4H), 2.21 - 2.14 (m, 1H), 1.82 (d, J = 12.9 Hz, 4H), 1.77 - 1.07 (m, 64H), 0.99 - 0.79 (m, 19H), 0.05 (s, 6H).7-((4-((terMJutyldimethylsilyl)oxy)butyl)amino)-l,13-bis((cyclohexylmethyl)thio)tridecane-2,12-diyl bis(4-cyclohexylbutanoate). (28.5).

[0255] 81% from l,13-bis((cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(4-cyclohexylbutanoate) (0.5 g, 0.63 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.93 (dt, J = 10.8, 5.4 Hz, 2H), 3.62 (dt, J= 8.0, 5.9 Hz, 2H), 3.27 (p, J= 6.3 Hz, 1H), 2.67 - 2.52 (m, 6H), 2.49 - 2.37 (m, 4H), 2.28 (t, J= 7.5 Hz, 4H), 1.88 - 1.77 (m, 4H), 1.74 - 1.06 (m, 60H), 0.99 - 0.80 (m, 17H), 0.05 (s, 6H).12-((4-cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7-(methylamino)tridecan-2-yl decanoate (28.7).

[0256] 68% from 12-((4-cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7-oxotridecan-2-yl decanoate (1.0 g, 1.3 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.99 – 4.87 (m, 2H),2.66 - 2.55 (m, 4H), 2.42 (dt, J= 6.86, 2.03 Hz, 4H), 2.37 - 2.32 (m, 4H), 2.28 (t, J = 1.50 Hz, 4H), 1.89 - 1.76 (m, 4H), 1.73 - 0.99 (m, 57H), 0.99 - 0.77 (m, 9H).7-((4-((terCButyldimethylsilyl)oxy)butyl)amino)-l,13-bis((2-cyclohexylethyl)thio)tridecane-2,12-diyl bis(decanoate) (28.8).

[0257] 68% from l,13-bis((2-cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (0.5 g, 0.61 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.93 (dt, J = 10.7, 5.5 Hz, 2H), 3.63 (t, J= 6.1 Hz, 2H), 2.63 (d, J= 6.1 Hz, 5H), 2.55 (td, J= 7.4, 1.6 Hz, 4H), 2.30 (t, J= 7.6 Hz, 4H), 1.88 - 1.03 (m, 72H), 0.95 - 0.80 (m, 19H), 0.05 (s, 6H).7-((4-((terCButyldimethylsilyl)oxy)butyl)amino)-l,13-bis((2-cyclohexylethyl)thio)t ridecane-2,12-diyl bis(3-cyclohexylpropanoate) (28.9).

[0258] 68% from l,13-bis((2-Cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(3- cyclohexylpropanoate) (0.7 g, 0.9 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.99 – 4.89 (m, 2H), 2.63 (dd, J= 6.09, 1.20 Hz, 4H), 2.60 - 2.51 (m, 4H), 2.38 - 2.27 (m, 7H), 1.78 - 1.05 (m, 62H), 0.96 - 0.81 (m, 8H).l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7-(methylamino)tridecane-2,12-diyl bis(decanoate) (28.11).

[0259] 90 % from l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7-oxotridecane-2, 12-diyl bis(decanoate) (1.2 g, 1.5 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.94 (tt, J = 10.73, 3.87 Hz, 2H), 2.67 - 2.50 (m, 4H), 2.43 (dd, J= 6.85, 2.54 Hz, 2H), 2.35 (s, 3H), 2.29 (t, J= 7.54 Hz, 4H), 1.88 - 1.77 (m, 2H), 1.76 - 1.05 (m, 66H), 0.94 - 0.80 (m, 10H).l,14-bis((cyclohexylmethyl)thio)-7-(methylamino)tetradecane-2,13-diyl bis(decanoate) (28.12).

[0260] 77% yield from l,14-bis((cyclohexylmethyl)thio)-7-oxotetradecane-2,13-diyl bis(decanoate) (0.491g, 0.607 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.99 – 4.89 (m, 2H), 2.68 - 2.55 (m, 4H), 2.49 - 2.38 (m, 4H), 2.38 - 2.33 (m, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.88 - 1.77 (m, 4H), 1.77 - 1.08 (m, 60H), 0.97 - 0.83 (m, 10H).7-((2-(2-((tert-Butyldimethylsilyl)oxy)ethoxy)ethyl)amino)-l,13-bis(cycloheptylthio)-tridecane-2, 12-diyl bis(decanoate) (28.13).

[0261] 65% from bisthiolyl ketone (1.0 g, 1.26 mmol,) and TBS-protected ethoxy amino butanol (141 mg, 1.89 mmol) as colorless oil. 'H NMR (400 MHz, CDCl₃) δ 4.95 (dq, J = 10.6, 5.9 Hz, 2H), 3.78 (t, J= 5.3 Hz, 2H), 3.56 (dt, J= 17.5, 5.3 Hz, 4H), 2.89 (tt, J= 9.0, 4.2 Hz, 2H), 2.79 -2.72 (m, 2H), 2.72 - 2.61 (m, 4H), 2.53 - 2.42 (m, 1H), 2.32 (t, J= 7.5 Hz, 4H), 2.05 - 1.94 (m, 4H), 1.78 - 1.19 (m, 62H), 0.95 - 0.85 (m, 18H), 0.09 (s, 6H).l,13-bis((cyclohexylmethyl)thio)-7-(methylamino)tridecane-2, 12-diyl bis(decanoate) (28.14)

[0262] -100% from l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (814 mg, 1.02 mmol) as a colorless oil and used for the next step without further purification.1H NMR (400 MHz, CDCl₃) δ 5.01 – 4.91 (m, 2H), 2.69 - 2.57 (m, 4H), 2.50 - 2.41 (m, 4H), 2.38 (m,4H), 2.32 (t, J= 7.5 Hz, 4H), 1.78 - 1.09 (m, 62H), 1.01 - 0.85 (m, 10H).l,13-bis((2-cyclohexylethyl)thio)-7-(methylamino)tridecane-2,12-diyl bis(decanoate) (28.15)

[0263] -98% from l,13-bis((2-cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (500 mg, 0.6 mmol) as a colorless oil and used for the next step without further purification. 'H NMR (400 MHz, CDCl₃) δ 5.01 – 4.90 (m, 2H), 2.63 (dd, J = 6.1, 1.0 Hz, 4H), 2.59 - 2.51 (m, 4H), 2.37 (s, 4H), 2.30 (t, J= 7.6 Hz, 4H), 1.75 - 1.07 (m, 66H), 0.88 (t, J= 6.9 Hz, 10H).Procedure for reductive methylation: 7-((4-((tert-butyldimethylsilyl)oxy)butyl)- (methyl)amino)-l,13-bis(cycloheptylthio)tridecane-2,12-diyl bis(decanoate) (29.1).

[0264] To a solution of amine 28.1 (390 mg, 0.4 mmol, 1.0 equiv) in THF (5 mL) at RT was added 37% aq. formaldehyde (0.12 mL mg, 1.6 mmol, 4 equiv), and the resulting mixture was stirred for 20 min at the same temperature, then followed by addition of NaBH(OAc)₃ (252 mg, 1.2 mmol, 3 equiv). The resulting mixture was stirred for 36 h, then quenched with NaHCO3(25 mL). The aq. phase was extracted with n-hexanes (3 x 30 mL). The combined organic layers werebrine (sat. solution), dried (Na2SC>4), filtered, and concentrated in vacuo to afford 29.1 (396 mg, 0.4 mmol, -100%) as a colorless oil and used for the next step without further purification.1H NMR (400 MHz, CDCl₃) δ 4.88 (tt, J = 9.0, 4.6 Hz, 2H), 3.73 – 3.56 (t, J = 6.2 Hz, 2H), 2.82 (tt, J= 8.9, 4.3 Hz, 2H), 2.67 - 2.53 (m, 4H), 2.28 (dt, J= 19.0, 7.4 Hz, 5H), 2.08 (s, 3H), 1.93 (tdd, J = 8.3, 5.1, 2.1 Hz, 4H), 1.83 - 1.78 (m, 1H), 1.70 - 1.06 (m, 69H), 0.87 - 0.78 (m, 15H), 0.00 (s, 6H).Procedure for reductive amination:7-((4-((tert- Butyldimethylsilyl)oxy)butyl)( met hyl )amino)-l,13-bis(cyclooctylthio)t ridecane-2, 12-diyl bis(decanoate) (29.3).

[0265] To a reaction vial containing l,13-bis(cyclooctylthio)-7-(methylamino)tridecane-2, 12-diyl bis(decanoate) (0.441 g, 0.526 mmol) under inert atmosphere was added 4-((tert-butyldimethylsilyl)oxy)butanal (0.12 g, 0.58 mmol, 1.1 equiv) and DCE (1 mL). The solution was stirred at RT for 15 min then STAB-H (0.17 g, 0.79 mmol, 1.5 equiv) was added. The reaction was stirred for 18 h at RT. The reaction was quenched with sat. NaHCO3(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 sat. NaHCOs, (1x20 mL), dried (Na2SO4), filtered, and evaporated to yield a crude product. This was purified by automated chromatography (10-30% EA in hexanes over 12 CV) yielding 7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis(cyclooctylthio)tridecane-2, 12-diyl bis(decanoate) (0.435 g, 0.425 mmol, 81%). 'H NMR (400 MHz, CDCl₃) δ 4.97 – 4.90 (m, 2H), 3.61 (t, J= 6.3 Hz, 2H), 2.92 (tt, J= 9.0, 3.9 Hz, 2H), 2.70 - 2.59 (m, 4H), 2.38 - 2.26 (m, 7H), 2.13 (s, 3H), 1.98 - 1.88 (m, 4H), 1.81 - 1.02 (m, 72H), 0.93 - 0.82 (m, 15H), 0.05 (s, 6H).

[0266] The following compounds were prepared by the same method:7-((4-((tert-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((cyclohexylmethyl)thio)-tridecane-2, 12-diyl bis(decanoate). (29.4).

[0267] 82% from 7-((4-((ter / -Butyldimethylsilyl)oxy)butyl)amino)-l,13-bis((cyclohexylmethyl) thio)tridecane-2,12-diyl bis(decanoate) (0.45 g, 0.46 mmol).1H NMR (400 MHz, CDCl₃) δ 4.98 – 4.87 (m, 2H), 3.74 (t, J = 6.5 Hz, 1H), 3.61 (t, J= 6.2 Hz, 2H), 2.68 -2.57 (m, 4H), 2.50 - 2.40 (m, 4H), 2.35 (t, J= 7.0 Hz, 2H), 2.30 (t, J= 7.5 Hz, 4H), 2.13 (s, 3H), 1.88 - 1.78 (m, 4H), 1.75 - 1.08 (m, 62H), 0.99 - 0.83 (m, 19H), 0.04 (s, 6H).7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((cyclohexylmethyl)-thio)tridecane-2,12-diyl bis(4-cyclohexylbutanoate) (29.5).

[0268] 95% from 7-((4-((tert-butyldimethylsilyl)oxy)butyl)amino)-l,13-bis((cyclohexylmethyl)thio)tridecane-2,12-diyl bis(4-cyclohexylbutanoate) (0.35 g, 0.36 mmol).'H NMR (400 MHz, CDCl₃) δ 4.98 – 4.89 (m, 2H), 3.61 (t, J = 6.3 Hz, 2H), 2.68 – 2.57 (m, 4H), 2.43 (dt, J= 8.4, 4.3 Hz, 4H), 2.31 (dt, J= 25.8, 7.4 Hz, 7H), 2.13 (s, 3H), 1.83 (d, J= 12.9 Hz, 4H), 1.73 - 1.00 (m, 60H), 0.98 - 0.78 (m, 17H), 0.05 (s, 6H).l-Cyclohexyl-9-(5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)- 10,16,16,17,17-pentamethyl-15-oxa-2-thia-10-aza-16-silaoctadecan-4-yl decanoate (29.7).

[0269] 65% from 12-((4-cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7- (methylamino)tridecan-2-yl decanoate (0.7 g, 0.9 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 –4.87 (m, 2H), 3.60 (t, J= 6.26 Hz, 2H), 2.68 - 2.55 (m, 4H), 2.48 - 2.38 (m, 4H), 2.37 - 2.24 (m, 6H), 2.13 (s, 3H), 1.88 - 1.77 (m, 4H), 1.73 - 1.05 (m, 62H), 1.00 - 0.79 (m, 18H), 0.04 (s, 6H).7-((4-((tert-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((2-cyclohexylethyl)thio)-tridecane-2, 12-diyl bis(decanoate) (29.8).

[0270] 82% from 7-((4-((tert-butyldimethylsilyl)oxy)butyl)amino)-l,13-bis((2- cy cl ohexylethyl)thio)tridecane-2, 12-diyl bis(decanoate) (0.44 g, 0.44 mmol).1H NMR (400 MHz, CDCl₃) δ 4.95 – 4.78 (m, 2H), 3.78 - 3.72 (m, 1H), 3.65 (t, J= 6.2 Hz, 2H), 2.71 - 2.60 (m, 6H), 2.60 - 2.51 (m, 4H), 2.30 (t, J= 7.6 Hz, 4H), 2.09 (s, 3H), 1.99 - 1.06 (m, 70H), 0.88 (d, J= 2.5 Hz, 19H), 0.04 (s, 6H).7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((2-cyclohexylethyl)thio)-tridecane-2, 12-diyl bis(3-cyclohexylpropanoate) (29.9).

[0271] 72% from l,13-bis((2-cyclohexylethyl)thio)-7-(methylamino)tridecane-2, 12-diyl bis(3- cyclohexylpropanoate) (0.48 g, 0.6 mmol). 'H NMR (400 MHz, CDCl₃) δ 5.01 – 4.87 (m, 2H), 3.61 (t, J= 6.27 Hz, 2H), 2.64 (d, J= 6.08 Hz, 4H), 2.58 - 2.50 (m, 4H), 2.38 - 2.24 (m, 6H), 2.13 (s, 3H), 1.82 - 1.02 (m, 65H), 0.96 - 0.79 (m, 17H), 0.05 (s, 6H).7-((4-((tert-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l-((2-cyclohexylethyl)thio)-13- ((cyclohexylmethyl)thio)tridecane-2, 12-diyl bis(decanoate). (29.11).

[0272] 65 % from l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7- (methylamino)tridecane-2,12-diyl bis(decanoate) (1.1 g, 1.33 mmol).1H NMR (400 MHz, CDCl₃) δ 4.94 (tt, J = 8.02, 3.87 Hz, 2H), 3.61 (t, J= 6.22 Hz, 2H), 2.71 - 2.50 (m, 5H), 2.47 -2.40 (m, 3H), 2.36 -2.26 (m, 6H), 2.14 (s, 3H), 1.88 - 1.75 (m, 3H), 1.75 - 1.01 (m, 66H), 1.00 - 0.80 (m, 19H), 0.05 (s, 6H).7-((4-((tert-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,14-bis((cyclohexylmethyl)thio)-tetradecane-2, 13-diyl bis(decanoate). (29.12)

[0273] 78% yield from l,14-bis((cyclohexylmethyl)thio)-7-(methylamino)tetradecane-2, 13-diyl bis(decanoate) (0.386 g, 0.468 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 – 4.89 (m, 2H), 3.61 (t, J = 6.2 Hz, 2H), 2.68 – 2.57 (m, 4H), 2.49 – 2.38 (m, 4H), 2.38 – 2.26 (m, 7H), 2.13 (s, 3H), 1.87 - 1.78 (m, 4H), 1.76 - 1.10 (m, 64H), 0.98 - 0.83 (m, 19H), 0.05 (s, 6H).7-((2-(2-((tert-Butyldimethylsilyl)oxy)ethoxy)ethyl)(methyl)amino)-l,13-bis(cycloheptyl-thio)tridecane-2,12-diyl bis(decanoate) (29.13).

[0274] 85% from 7-((2-(2-((ter / -Butyldimethylsilyl)oxy)ethoxy)ethyl)amino)-l,13-bis(cycloheptylthio)-tridecane-2,12-diyl bis(decanoate) (28.13) (1.15 g, 1.15 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.95 (p, J = 6.0 Hz, 2H), 3.77 (t, J= 5.5 Hz, 2H), 3.61 - 3.44 (m, 5H), 2.90(tt, J= 8.9, 4.3 Hz, 2H), 2.73 - 2.62 (m, 4H), 2.58 (t, J= 6.6 Hz, 2H), 2.32 (t, J= 7.6 Hz, 4H), 2.24 (s, 3H), 2.06 - 1.96 (m, 4H), 1.79 - 1.14 (m, 60H), 0.91 (d, J= 7.2 Hz, 19H), 0.09 (s, 6H).Procedure for desilylation: (l,13-bis(cycloheptylthio)-7-((4-hydroxybutyl)(methyl)- amino)tridecane-2,12-diyl bis(decanoate) (T-17).

[0275] To a solution of compound 29.1 (390 mg, 0.4 mmol, 1.0 equiv) in CH₂Cl₂ (5 mL) at 0 °C was added HF-pyridine (0.08 mL, 0.8 mmol, 2 equiv), and the resulting mixture was stirred for 20 min at the same temperature. The resulting mixture was then quenched with NaHCO3(25 mL). The organic layer was separated, and the aq. phase was further extracted with CH2CI2 (2 × 30 mL). The combined organic layers were brine (sat. solution), dried (Na2SC>4), filtered, and concentrated in vacuo. The resulting residue was purified by automated chromatography with (0— >5% MeOH: CH2Cl2) to afford T-17 (250 mg, 0.28 mmol, -72%) as a colorless oil.1H NMR (400 MHz, CDCl₃) 64.96 (ddd, J= 8.2, 6.3, 4.4 Hz, 2H), 3.58 (t, J= 5.0 Hz, 2H), 2.89 (tt, J= 8.8, 4.3 Hz, 2H), 2.74 - 2.59 (m, 4H), 2.46 (t, J= 5.4 Hz, 2H), 2.41 (t, J= 5.9 Hz, 1H), 2.32 (t, J= 7.5 Hz, 4H), 2.16 (s, 3H), 2.00 (dq, J= 13.6, 4.2 Hz, 4H), 1.77 - 1.41 (m, 36H), 1.39 - 1.20 (m, 32), 0.93 - 0.86 (m, 6H).

[0276] The following compounds were prepared by the same method:l,13-bis(Cyclooctylthio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-18).

[0277] 82% yield from 1 7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis(cyclooctylthio)tridecane-2,12-diyl bis(decanoate) (0.441 g, 0.430 mmol). 'H NMR (400 MHz, CDCl₃) δ 5.62 (br. s, 1H), 4.97 - 4.87 (m, 2H), 3.56 (t, J= 5.0 Hz, 2H), 2.92 (tt, J= 9.0, 3.9 Hz,Ill2H), 2.70 - 2.58 (m, 4H), 2.43 (t, J= 5.4 Hz, 2H), 2.42 - 2.35 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 2.13 (s, 3H), 1.98 - 1.88 (m, 4H), 1.80 - 1.17 (m, 72H), 0.90 - 0.84 (m, 6H).l,13-bis((Cyclohexylmethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-19)

[0278] 75% from 7-((4-((ter / -Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((cyclohexylmethyl)thio)-tridecane-2,12-diyl bis(decanoate) (0.47 g, 0.47 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.88 (m, 2H), 3.56 (t, J = 5.0 Hz, 2H), 2.67 - 2.55 (m, 4H), 2.50 -2.36 (m, 8H), 2.30 (t, J = 7.5 Hz, 4H), 2.14 (s, 3H), 1.88 - 1.77 (m, 4H), 1.76 - 1.54 (m, 18H), 1.44 (dtt, J= 14.7, 7.4, 3.5 Hz, 4H), 1.38 - 1.06 (m, 40H), 1.01 - 0.81 (m, 10H).l,13-bis((Cyclohexylmethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(4-cyclohexylbutanoate) (T-20)

[0279] 72% from 7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((cyclohexylmethyl)thio)tridecane-2,12-diyl bis(4-cyclohexylbutanoate) (0.20 g, 0.20 mmol).1!! NMR (400 MHz, CDCl₃) δ 4.93 (dt, J= 10.8, 5.3 Hz, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.67 - 2.56 (m, 4H), 2.50 - 2.35 (m, 7H), 2.28 (t, J= 7.5 Hz, 4H), 2.14 (s, 3H), 1.87 - 1.77 (m, 4H), 1.77 - 1.05 (m, 60H), 0.92 (ddt, = 25.1, 13.1, 6.4 Hz, 8H).12-((4-Cyclohexylbutanoyl)oxy)-l,13-bis((cyclohexylmethyl)thio)-7-((4-hydroxybutyl)- (methyl)amino)tridecan-2-yl decanoate (T-21).

[0280] 52% from 7-((4-((ter / -butyldimethylsilyl)oxy)butyl)(methyl)amino)-l-((2- cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)tridecane-2,12-diyl bis(decanoate) (0.55 g, 0.55 mmol). 'H NMR (400 MHz, CDCl₃) δ 5.61 (s, 1H), 4.98 - 4.88 (m, 2H), 3.55 (t, J= 5.01 Hz, 2H), 2.67 - 2.56 (m, 4H), 2.50 - 2.34 (m, 7H), 2.29 (t, J= 7.65 Hz, 4H), 2.13 (s, 3H), 1.88 - 1.77 (m, 4H), 1.76 - 1.03 (m, 60H), 1.00 - 0.80 (m, 10H).l,13-bis((2-cyclohexylethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-22).

[0281] 70% from 7-((4-((tert-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((2- cyclohexylethyl)thio)-tridecane-2,12-diyl bis(decanoate) (0.45 g, 0.44 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.94 (dtd, J= 8.0, 6.0, 4.4 Hz, 2H), 3.56 (t, J= 4.9 Hz, 2H), 2.63 (d, J= 6.2 Hz, 4H), 2.55 (td, J= 7.4, 1.5 Hz, 4H), 2.48 - 2.36 (m, 3H), 2.30 (t, J= 7.6 Hz, 4H), 2.14 (s, 3H), 1.77 - 1.06 (m, 70H), 0.92- 0.81 (m, 10H).l,13-bis((2-Cyclohexylethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(3-cyclohexylpropanoate) (T-23).

[0282] 58% from 7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((2- cyclohexylethyl)thio)-tridecane-2,12-diyl bis(3-cyclohexylpropanoate) (0.35 g, 0.35 mmol). 'HNMR (400 MHz, CDCl₃) δ 5.00 - 4.87 (m, 2H), 3.56 (t, J= 5.01 Hz, 2H), 2.63 (d, J= 6.11 Hz, 4H), 2.59 - 2.51 (m, 4H), 2.46 - 2.35 (m, 3H), 2.31 (t, = 7.31 Hz, 4H), 2.13 (s, 3H), 1.79 - 1.06 (m, 64H), 0.95 - 0.81 (m, 8H).l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)-tridecane-2, 12-diyl bis(decanoate) (T-24).

[0283] 54% from 7-((4-((ter / -Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l-((2-cyclohexylethyl)thio)-13-((cyclohexylmethyl)thio)tridecane-2,12-diyl bis(decanoate) (0.20 g, 2.0 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.99 - 4.88 (m, 2H), 3.56 (t, J= 5.01 Hz, 2H), 2.69 - 2.50 (m, 4H), 2.47 - 2.35 (m, 5H), 2.30 (t, J= 7.52 Hz, 4H), 2.14 (s, 3H), 1.88 - 1.76 (m, 3H), 1.78 - 1.03 (m, 67H), 0.98 - 0.79 (m, 10H).l,13-bis(Cycloheptylthio)-7-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-25).

[0284] 72% from 7-((2-(2-((tert-Butyldimethylsilyl)oxy)ethoxy)ethyl)(methyl)amino)-l,13-bis (cy cl oheptyl-thio)tridecane-2, 12-diyl bis(decanoate) (950 mg, 0.94 mmol) as a colorless oil. 'H NMR (400 MHz, CDCl₃) δ 4.93 (t, J= 6.6 Hz, 2H), 3.71 - 3.67 (m, 2H), 3.58 (dt, J= 10.7, 5.1 Hz, 4H), 2.87 (dq, J= 8.9, 4.3 Hz, 2H), 2.70 - 2.56 (m, 4H), 2.58 (t, J= 5.7 Hz, 2H), 2.41 - 2.34 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 2.22 (s, 3H), 1.97 (d, J= 11.5 Hz, 4H), 1.76 - 1.15 (m, 64H), 0.90 - 0.84 (m, 6H).l,13-bis((cyclohexylmethyl)thio)-7-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)tridecane-2, 12-diyl bis(decanoate) (T-26).

[0285] A solution of amine (1 mmol), 2-(2-bromoethoxy)ethanol (0.9 mmol) and K₂CO₃ (0.9 mmol) in DMF was heated 70 °C for 36 h. The reaction was diluted with hexanes (50 mL) and water (30 mL). The layers were separated and the organic phase was collected and the aqueous phase was further extracted with hexanes (2 x 30 mL). The combined organic phases were dried (Na₂SO₄), filtered and concentrated in vacuo to yield the crude product. This was purified by automated chromatography [0-5% MeOH in DCM] to yield the desired lipid T-26 (320 mg, 0.35 mmol, 34%). ’H NMR (400 MHz, CDCl₃) δ 4.93 (dt, J= 10.9, 5.4 Hz, 2H), 3.73 - 3.65 (m, 2H), 3.63 - 3.52 (m, 4H), 2.68 - 2.53 (m, 6H), 2.49 - 2.35 (m, 5H), 2.30 (t, J= 7.5 Hz, 4H), 2.22 (s, 1H), 1.87 - 1.78 (m, 4H), 1.76 - 1.06 (m, 60H), 0.99 - 0.83 (m, 10H).l,13-bis((2-cyclohexylethyl)thio)-7-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-27).

[0286] 44% from 7-((4-((te / 7-Butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,13-bis((2-cyclohexylethyl)thio)-tridecane-2,12-diyl bis(decanoate) (320 mg, 0.38 mmol).1!! NMR (400 MHz, CDCl₃) δ 4.94 (dt, J= 11.1, 5.3 Hz, 2H), 3.72 - 3.66 (m, 2H), 3.58 (dt, J= 10.4, 5.1 Hz, 4H), 2.64 (d, J= 6.1 Hz, 4H), 2.61 - 2.51 (m, 6H), 2.42 - 2.33 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 2.22 (s, 3H), 1.74 - 1.05 (m, 66H), 0.88 (t, J= 6.7 Hz, 10H).l,14-bis((cyclohexylmethyl)thio)-7-((4-hydroxybutyl)(methyl)amino)tetradecane-2,13-diyl bis(decanoate) (T-28).

[0287] 55% yield from 7-((4-((tert-butyldimethylsilyl)oxy)butyl)(methyl)amino)-l,14-bis((cyclohexylmethyl)thio)tetradecane-2,13-diyl bis(decanoate) (0.368 g, 0.364 mmol).1H NMR (400 MHz, CDCl₃) δ 5.68 (br. s, 1H), 4.98 - 4.89 (m, 2H), 3.56 (t, J= 5.0 Hz, 2H), 2.68 -2.57 (m, 4H), 2.50-2.36 (m, 7H), 2.30 (t, J= 7.5 Hz, 4H), 2.14 (s, 3H), 1.89 - 1.78 (m, 4H), 1.76 - 1.08 (m, 64H), 0.99 - 0.83 (m, 10H).(E) Preparation of lipids T-29-T32.i. Procedure for BOC group release: azanediylbis(l-((cyclohexylmethyl)thio)hexane-6,2- diyl) bis(decanoate) (34.1).

[0288] To a solution of 32.1 (3.9 g, 4.4 mmol, 1.0 equiv) in CH₂Cl₂ (40 mL) at 0 °C, was added TFA (2.5 g, 22 mmol, 5.0 equiv) dropwise. The resulting mixture was allowed to proceed at RT for 2 h, at which point full deprotection had occurred, the reaction was quenched with sat. NaHCO3(40 mL) followed by dilution with CH₂Cl₂ (50 mL). The layers were separated and the organic phase was collected. The aqueous phase was back -extracted with CH2CI2 (2 x 30 mL), dried (Na2SC>4), filtered, and evaporated to yield a crude product. The crude product was purified by flash column chromatography with 3% MeOH in CH2CI2 to afford 34.1 (2.6 g, 3.32 mmol, 75% yield). 'H NMR (400 MHz, CDCl₃) δ 4.96 - 4.82 (m, 2H), 2.89 (t, J= 8.1 Hz, 4H), 2.73 - 2.52 (m, 4H), 2.42 (dd, J= 6.8, 2.8 Hz, 4H), 2.35 - 2.21 (m, 4H), 1.89 - 1.52 (m, 19H), 1.48 - 1.01 (m, 39H), 1.01 - 0.74 (m, 10H).

[0289] The following compounds were prepared by the same method:l-((Cyclohexylmethyl)thio)-6-((7-((cyclohexylmethyl)thio)-6-(decanoyloxy)heptyl)amino)- hexan-2-yl decanoate (34.2).OO

[0290] Synthesized from 7-((tert-butoxycarbonyl)(6-((cyclohexylmethyl)thio)-5- (decanoyloxy)hexyl)amino)-l-((cyclohexylmethyl)thio)heptan-2-yl decanoate 32.2 (3.4 g, 3.8 mmol), yielding desired amine 34.2 (2.4 g, 3.0 mmol, 80%). 'H NMR (400 MHz, CDCl₃) δ 5.05 - 4.82 (m, 2H), 2.94 - 2.80 (m, 4H), 2.67 - 2.52 (m, 4H), 2.49 - 2.36 (m, 4H), 2.29 (t, J= 7.6 Hz, 4H), 1.94 - 1.05 (m, 61H), 1.01 - 0.73 (m, 10H).6-((6-((2-Cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)amino)-l-((cyclohexylmethyl)thio)- hexan-2-yl decanoate (34.3).

[0291] 58% from 6-((tert-butoxycarbonyl)(5-((4-cyclohexylbutanoyl)oxy)-6- ((cyclohexylmethyl)thio)hexyl)-amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (1.0 g, 1.1 mmol). 'H NMR (400 MHz, CDCl₃) δ 5.02 - 4.85 (m, 2H), 2.70 - 2.49 (m, 10H), 2.47 - 2.36 (m, 2H), 2.30 (t, J= 7.5 Hz, 4H), 1.94 - 1.77 (m, 2H), 1.75 - 1.07 (m, 59H), 0.99 - 0.79 (m, 10H).6-((5-((4-Cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)amino)-l-((cyclohexyl- methyl)thio)hexan-2-yl decanoate (34.4).

[0292] 66% from 6-((tert-butoxycarbonyl)(5-((4-cyclohexylbutanoyl)oxy)-6- ((cyclohexylmethyl)thio)hexyl)amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate 33.2 (3.6 g, 4.1 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.98 - 4.80 (m, 2H), 2.89 (t, J= 8.0 Hz, 4H), 2.73 -2.53 (m, 4H), 2.49 -2.36 (m, 4H), 2.28 (q, J= 7.7 Hz, 4H), 1.91 - 1.50 (m, 28H), 1.48 - 1.02 (m, 29H), 0.99 - 0.68 (m, 9H).Procedure for reductive amination:l-cyclohexyl-9-(6-((cyclohexylmethyl)thio)-5-(decanoyloxy)hexyl)-15, 15,16, 16-tetramethyl- 14-oxa-2-thia-9-aza-15-silaheptadecan-4-yl decanoate (35.1).

[0293] To a solution of amine 34.1 (2.1 g, 2.7 mmol, 1.0 equiv) in DCE (5 mL) at RT was added 4-((tert-butyldimethylsilyl)oxy)butanal (546 mg, 2.7 mmol, 1 equiv), and the resulting mixture was stirred for 15 min at the same temperature, then followed by addition STAB-H (858 mg, 4.05 mmol, 1.5 equiv). The resulting mixture was stirred for further 24 h, then quenched with sat. solution of NaHCO3(75 mL). The aq. phase was extracted with CH2CI2 (3 x 30 mL). The combined organic layers were washed brine (sat. solution), dried (Na2SC>4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with (0→25% EtOAc: hexanes) to afford 35.1 (1.7 g, 1.76 mmol, 65%) as a colorless oil. 'H NMR (400 MHz, CDCl₃) δ 5.02 - 4.82 (m, 2H), 3.69 - 3.51 (m, 2H), 2.68 - 2.54 (m, 4H), 2.50 - 2.36 (m, 10H), 2.33 - 2.17 (m, 4H), 1.88 - 1.77 (m, 4H), 1.76 - 1.00 (m, 56H), 1.00 - 0.80 (m, 21H), 0.04 (s, 6H).l-cyclohexyl-10-(6-((cyclohexylmethyl)thio)-5-(decanoyloxy)hexyl)-16, 16,17, 17-tetramethyl- 15-oxa-2-thia-10-aza-16-silaoctadecan-4-yl decanoate (35.2)

[0294] 58% from l-((cyclohexylmethyl)thio)-6-((7-((cyclohexylmethyl)thio)-6-(decanoyloxy)heptyl)amino)hexan-2-yl decanoate (1.6 g, 2.01 mmol) as a colorless oil.1H NMR (400 MHz, CDCl₃) δ 4.99 - 4.88 (m, 2H), 3.60 (t, J= 6.0 Hz, 2H), 2.68 - 2.55 (m, 4H), 2.50 -2.25 (m, 14H), 1.88 - 1.77 (m, 4H), 1.75 - 1.06 (m, 58H), 1.00 - 0.80 (m, 21H), 0.04 (s, 6H). l-cyclohexyl-9-(6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)-15,15,16,16-tetramethyl- 14-oxa-2-thia-9-aza-15-silaheptadecan-4-yl decanoate (35.3)

[0295] 74% from 6-((6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (500 mg, 0.62 mmol.1H NMR (400 MHz, CDCl₃) 65.08 - 4.84 (m, 2H), 3.68 - 3.52 (m, 2H), 2.70 - 2.50 (m, 6H), 2.47 - 2.23 (m, 12H), 1.87 - 1.77 (m, 2H), 1.76 - 0.76 (m, 81H), 0.04 (s, 6H).l-cyclohexyl-9-(5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)- 15,15,16,16-tetramethyl-14-oxa-2-thia-9-aza-15-silaheptadecan-4-yl decanoate (35.4)

[0296] 65% from 6-((5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (1.6 g, 2.05 mmol). 'H NMR (400 MHz, CDCl₃) δ 4.93 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 3.60 (t, J= 6.0 Hz, 2H), 2.68 - 2.55 (m, 4H), 2.50 - 2.33 (m, 11H), 2.28 (q, J= 7.6 Hz, 4H), 1.90 - 1.77 (m, 5H), 1.76 - 0.78 (m, 73H), 0.04 (s, 6H).Procedure for desilylation: ((4-hydroxybutyl)azanediyl)bis(l- ((cyclohexylmethyl)thio)hexane-6,2-diyl) bis(decanoate) (T-29).O O

[0297] To a solution of compound 35.1 (1 g, 1.03 mmol, 1.0 equiv) in CH₂Cl₂ (7 mL) at 0 °C, was added 70% HF-pyridine (220 μL, 7.65 mmol, 7.4 equiv), and the resulting mixture was stirred for 60 min at the same temperature. The resulting mixture was then quenched with NaHCO3(25 mL). The organic layer was separated, and the aq. phase was further extracted with CH2CI2 (2 x 30 mL). The combined organic layers were brine (sat. solution), dried (Na2SC>4), filtered, and concentrated in vacuo. The resulting residue was purified by automated chromatography with (0→7% MeOH: CH2Cl2) to afford T-29 (510 mg, 0.6 mmol, 58%) as a colorless oil.1H NMR (400 MHz, CDCl₃) 65.12 - 4.80 (m, 2H), 3.53 (t, J= 4.7 Hz, 2H), 2.68 - 2.54 (m, 4H), 2.49 - 2.35 (m, 10H), 2.29 (t, J= 7.5 Hz, 4H), 1.88 - 1.77 (m, 5H), 1.76 - 1.05 (m, 58H), 0.99 - 0.81 (m, 10H).6-((5-((4-cyclohexylbutanoyl)oxy)-6-((cyclohexylmethyl)thio)hexyl)(4-hydroxybutyl)amino)-l-((cyclohexylmethyl)thio)hexan-2-yl decanoate (T-30)

[0298] 47% from l-cyclohexyl-9-(5-((4-cyclohexylbutanoyl)oxy)-6- ((cyclohexylmethyl)thio)hexyl)-15,15,16,16-tetramethyl-14-oxa-2-thia-9-aza-15-silaheptadecan-4-yl decanoate (550 mg, 0.6 mmol) as a colorless oil. 'H NMR (400 MHz, CDCl₃) δ 6.21 (brs, 1H), 5.06-4.81 (m, 2H), 3.53 (t,. / = 4,6 Hz, 2H), 2.68 - 2.53 (m, 4H), 2.50 - 2.34 (m, 10H), 2.33 - 2.19 (m, 4H), 1.90 - 1.05 (m, 61H), 1.01 - 0.77 (m, 9H).6-((6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)(4-hydroxybutyl)amino)-l- ((cyclohexylmethyl)thio)hexan-2-yl decanoate (T-31)

[0299] 43% from l-cyclohexyl-9-(6-((2-cyclohexylethyl)thio)-5-(decanoyloxy)hexyl)- 15,15,16,16-tetramethyl-14-oxa-2-thia-9-aza-15-silaheptadecan-4-yl decanoate (410 mg, 0.42 mmol) as a colorless oil. 'H NMR (400 MHz, CDCl3) δ 5.09 – 4.82 (m, 2H), 3.54 (t, J= 4.6 Hz, 2H), 2.68 -2.51 (m, 6H), 2.49 - 2.35 (m, 9H), 2.30 (t, J= 7.5 Hz, 4H), 1.88 - 1.05 (m, 63H), 0.99 - 0.81 (m, 10H).l-((cyclohexylmethyl)thio)-6-((7-((cyclohexylmethyl)thio)-6-(decanoyloxy)heptyl)(4- hydroxybutyl)amino)hexan-2-yl decanoate (T-32)

[0300] 52% from 1 -cyclohexyl- 10-(6-((cy clohexylmethyljthi o)-5-(decanoyloxy)hexyl)- 16,16,17,17-tetramethyl-15-oxa-2-thia-10-aza-16-silaoctadecan-4-yl decanoate (390 mg, 0.39 mmol) as a colorless oil. 'H NMR (400 MHz, CDCl3) δ 5.01 – 4.85 (m, 2H), 3.53 (t, J= 4.6 Hz, 2H), 2.61 (dd, J= 6.2, 2.8 Hz, 4H), 2.52 - 2.35 (m, 11H), 2.34 - 2.25 (m, 4H), 1.88 - 1.77 (m, 4H), 1.75 - 1.06 (m, 60H), 0.99 - 0.81 (m, 10H).Example 2: mRNA-containing LNPs comprising ionizable lipids of the disclosure exhibit in vivo delivery of mRNA to the liver and spleen that is superior to the MC3 benchmark

[0301] 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 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 postinjection. The ionizable lipids were T-01, T-03, T-05, T-10, T-17, T-19, T-22, T-26, and T-27 set forth above. The ionizable lipid used as a control was nor-MC3 (described in WO 2022 / 246571, which is incorporated herein by reference) having the following structure:

[0302] The results in Figure 2A show that luminescence intensity per mg liver was higher for lipids T-01, T-03, T-05, T-10, T-17, T-19, T-22, T-26, and T-27 than the nor-MC3 benchmark. Results for luminescence intensity per mg spleen were found to be higher for lipids T-17, T-19, T-22, T-26, and T-27 than the nor-MC3 benchmark (Figure 2B).Example 3: Introduction of methylene and ethylene groups between sulfur atom and cycloalkyl moiety in ionizable lipids used in mRNA LNP contributes to increased in vivo protein expression

[0303] To understand the effect of distal chain modifications in the ionizable lipids used in mRNA LNPs, novel lipids bearing alkylene introductions in the distal chain were tested.

[0304] 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 tested for in vivo expression efficiency in the liver at 4 hours post-injection to CD-I mice. The mRNA dose was 1 mg / kg. nor-MC3, Benchmark-1 and Benchmark-2 ionizable lipids were used as positive controls. Benchmark-1 has a type 1 ionizable head group and Benchmark-2 has a type 7 ionizable head group. The details of the ionizable lipids tested are listed in Table 1.Table 1: Ionizable lipids used in mRNA LNPs for in vivo protein expressionIonizable Lipid Head group CLogP Distal chain Reference Modificationsnor-MC3 Type 1 16.629 Not applicable Disclosed in WO 2022 / ionizable head 246571 Algroup (incorporated herein by reference)Benchmark -1 Type 1 17.334 Sulfur atom Lipid #6 in WO ionizable head directly attached to 2024 / 065042 Al group cyclohexyl ring (incorporated herein by reference)T-01 Type 1 18.452 Sulfur atom N / Aionizable head directly attached togroup cycloheptyl ringT-03 Type 1 18.572 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring bya methylene groupT-05 Type 1 19.63 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring byan ethylene groupBenchmark-2 Type 7 16.153 Sulfur atom Lipid #32 with one less ionizable head directly attached to C atom in inner alkyl group cyclohexyl ring chain and an additional C atom in the acyl chain in WO 2024 / 065042 Al (incorporated herein by reference)T-17 Type 7 17.271 Sulfur atom N / Aionizable head directly attached togroup cycloheptyl ringT-19 Type 7 17.391 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring bya methylene groupT-20 Type 7 16.343 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring bya methylene groupT-22 Type 7 18.449 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring byan ethylene groupT-25 Type 10 17.319 Sulfur atom N / Aionizable head directly attached togroup cycloheptyl ringT-26 Type 10 17.44 Sulfur atom N / Aionizable head separated from agroup cyclohexyl ring bya methylene group

[0305] Novel lipids T-03 and T-05 bearing type 1 ionizable head groups and having methylene or ethylene groups, respectively, between the sulfur atom and the cyclohexyl moiety in the lipid were compared against Benchmark-1 lipid lacking the alkylene introduction therein. Figure 3A shows the comparison in vivo liver luminescence intensity per mg.

[0306] Novel lipids T-19 and T-20 bearing a type 7 ionizable head group and having a methylene group introduced between the sulfur atom and the cyclohexyl ring in the distal chain were compared with Benchmark-2 lipid lacking the alkylene introduction therein. Novel lipids T-22 bearing an ethylene introduction in the distal chain and T-26 having a type 10 head group and amethylene introduction in the distal chain were also tested. Figure 3B shows the comparison data. Surprisingly, the novel lipids T-03, T-05, T-19, T-20, T-22 and T-26 bearing the alkylene introductions in the distal chain showed increased in vivo liver luminescence intensity.

[0307] Thus, in some embodiments, the introduction of the methylene and ethylene groups between the sulfur atom and the cyclohexyl moiety in the distal chain contributes to increased transfection and / or protein expression in the mRNA LNPs.Example 4: mRNA LNPs with ionizable lipids bearing cycloheptyl rings in the distal chain show increased in vivo protein expression compared to lipids with cyclohexyl rings

[0308] Further, to understand the effect of cyclic ring modifications in the distal chain of ionizable lipids used in mRNA LNPs, novel lipids bearing cycloheptyl rings were studied.

[0309] 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 tested for in vivo expression efficiency in the liver at 4 hours post-injection to CD-I mice.

[0310] Novel lipid T-01 (see Table 1 for details on the novel lipids used) bearing a cycloheptyl ring in the distal chain was compared with Benchmark-1 lipid having a cyclohexyl ring therein. Both lipids have a type 1 ionizable head group. Figure 4A shows the comparison in vivo liver luminescence intensity data.

[0311] Novel lipids T-17 and T-25 bearing type 7 and type 10 ionizable head groups, respectively, and having cycloheptyl rings in the distal chain were compared with Benchmark-2 lipid bearing a distal cyclohexyl ring. Figure 4B shows the comparison data. Surprisingly, the novel lipids T-01, T-17 and T-25 bearing cycloheptyl rings distal chain showed increased in vivo liver luminescence intensity.

[0312] Thus, in some embodiments, the cycloheptyl rings in the distal chain of ionizable lipids contributes to higher transfection and / or protein expression in the mRNA LNPs compared to distal cyclohexyl rings.

[0313] The examples are intended to illustrate the novel ionizable lipids, lipid nanoparticle comprising the lipids, use of the lipids or the lipid nanoparticle comprising the lipids, and methods of administering and / or delivering nucleic acids using the lipids and / or lipid nanoparticles comprising the novel lipids and are in no way intended to limit the scope of the invention.

[0314] The article "a" or "an" as used herein is meant to include both singular and plural, unless otherwise indicated.

[0315] The detailed description set forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

[0316] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent that any document incorporated by reference is inconsistent with the express content of this disclosure, the express content controls. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

Claims

1. WE CLAIM:

1. A lipid having a structure of Formula A:R,E’r2“S- (CH2)m\ W2X^Z2 A IW1-YE2R4Formula Aor a pharmaceutically acceptable salt thereof;whereinindices m and n are independently 4 to 8; andR1and R4are independently, optionally substituted, linear, branched or cyclic C3 to C20 alkyl groups, optionally comprising 0-2 carbon-carbon double bonds, optionally comprising C₃ to C₈ cycloalkyl substituents;R2and R3are independently groups of Formula B:(CH2)q—(CH2)pFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is at least 1,E1and E2are ester groups, selected from -C(O)-O- or -O-C(O)-;G1and G2are, independently, optionally substituted, linear, branched or cyclic Ci to Ce alkyl groups;A is C or N, andif A is C, then W1and Y are either bonded to each other or not bonded to each other, andif W1and Y are bonded to each other, thenW1is O or S;W2is O or S;X is CH;Y is (CH2)q, wherein q is 1 or 2; andZ is selected from any one of structures a-c below, wherein the wavy line represents the bond to X:a. ' / v’(CH2)n_N\ type 2 ionizable head group, wherein the n of the type 2 ionizable head group is 1 to 5;— (CH2)m-O^(CH2)n\N / b. | type 3 ionizable head group,wherein the m and n of the type 3 ionizable head group are independently 1 to 5;Rc. — (CH2)m-N— (CH2)ri—OH type 4 ionizable head group, wherein the m and n of the type 4 ionizable head group are independently 2 to 5 and wherein R = Ci-Ce alkyl or cycloalkyl;if W1and Y are not bonded to each other, thenW1is H;W2is O, S, NH or NR2a, wherein R2ais a Ci to Ce alkyl optionally substituted with an OH group, and optionally a cycloalkyl; and'vv' V 'the moiety | of Formula A is a group selected from one of Ystructures d-j below, wherein the wavy line represents the bond to W2:d.0N I type 1 ionizable head group, wherein the n of(CH2Xthe type 1 ionizable head group is 1 to 5;Oe- Xo / ^(CH2)n-Nztype 5 ionizable head group,wherein the m and n of the type 5 ionizable head group are independently 1 to 5;O RA i(CH2)^fN(CH2)n-OH6ionizable head group, wherein the m of the type 6 ionizable head group is 1 to 5 and the n of the type 6 ionizable head group is independently 2 to 5 and wherein the R = Ci-Ce alkyl or cycloalkyl;g. (CH2)n-OH type 7 ionizable head group, wherein the n of the type 7 ionizable head group is 1 to 5, optionally wherein W2is NR2a;h. (CH2)n- type 8 ionizable head group, wherein the n of the type 7 ionizable head group is 1 to 5, optionally wherein W2is NR2a;0 / i.. / ° <CH2>n N\ type 9 ionizable head group,(CH2)mOwherein m and n of the type 9 ionizable head group are independently 1 to 5;j. ■ / vv'[(CH2)m-X]n— (CH2)p-OH type 10 ionizable head group, wherein X = O or S, m of the type 10 ionizable head group independently is 2 to 4 in each [(CH2)m-X] unit, n of the type 10 ionizable head group is1 to 3 and p of the type 10 ionizable head group is 2 to 4, optionally wherein W2is NR2a,if A is N, thenW1and Y are absent;W2and X together form a group of structure (CRaRb)P, wherein Raand Rbare independently H, C1-C5alkyl or cycloalkyl, and wherein p is 2 to 6; andZ is OH or NR’R”, wherein R’ and R” are independently optionally substituted C1-C5 alkyl or cycloalkyl, or wherein R’ and R” together with the N atom of NR’R”, form an optionally substituted heterocyclic ring that incorporates the N atom to which the R’ and R” are each bound, and optionally wherein the heterocyclic group that incorporates the N atom to which R’ and R” are bound is pyrrolidine, piperidine or morpholine.

2. The lipid or the pharmaceutically acceptable salt of claim 1, wherein the A is C.

3. The lipid or the pharmaceutically acceptable salt of claim 2, wherein W1and Y are not bonded to each other.

4. The lipid or the pharmaceutically acceptable salt of claim 3, wherein W2is O.

5. The lipid or the pharmaceutically acceptable salt of claim 3, wherein the moiety —X'Zof YFormula A is the structure d.

6. The lipid or the pharmaceutically acceptable salt of claim 3, wherein the moiety^X^ of y Formula A is the structure j.

7. The lipid or the pharmaceutically acceptable salt of claim 2, wherein W1and Y are bonded to each other.

8. The lipid or the pharmaceutically acceptable salt of claim 7, wherein W1and W2are each O.

9. The lipid or the pharmaceutically acceptable salt of claim 8, wherein the Z is the structure a.

10. The lipid or the pharmaceutically acceptable salt of claim 1, wherein the A is N.

11. The lipid or the pharmaceutically acceptable salt of claim 10, wherein the Z is OH.

12. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 11, wherein at least one of the ester groups selected from E1and E2has a carbonyl carbon (C(O)) bonded to R1or R4, respectively.

13. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 12, wherein the G1and G2are, independently, optionally substituted, linear or branched Ci to Ce alkyl or C3 to Ce cycloalkyl groups.

14. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 13, wherein the G1and G2are, independently, optionally substituted, linear Ci to Ce alkyl groups.

15. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 14, wherein the G1and G2are, independently, methylene.

16. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 13, wherein the G1and G2are, independently, optionally substituted, branched Ci to Ce alkyl groups.

17. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 11, wherein at least one of the ester groups selected from E1and E2has a non-carbonyl oxygen atom bonded to R1or R4, respectively, and wherein G1and G2are, independently, optionally substituted, linear or branched Ci to Ce alkyl or C3 to Ce cycloalkyl groups.

18. A lipid or a pharmaceutically acceptable salt thereof comprising:a protonatable amino head group;at least two lipophilic chains, wherein the amino head group has a central nitrogen atom or carbon atom to which each of the two lipophilic chains are directly bound;at least one of the lipophilic chains has the formula:R2—) - (CH2)n^R1— Ewherein (CH2)n- of the formula is bound to the central nitrogen atom or carbon atom;wherein R1is a linear, branched or cyclic optionally substituted C3-C20 alkyl and optionally with varying degrees of unsaturation, optionally comprising C₃ to C₈ cycloalkyl substituents; wherein R2is a structure of Formula B:r^(CH2)q—(CH2)PFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is 1 or greater,n is 4 to 8;E is an ester;each lipophilic chain has between 15 and 40 carbon atoms in total; andwherein the lipid is ionizable and has a ClogP of at least 11.

19. An ionizable amino lipid with at least two lipophilic chains directly bound to a central nitrogen or carbon atom in which at least one of the lipophilic chains has the formula:R5rvwherein the wavy line represents a bond to the central nitrogen atom or carbon atom; n is 4 to 8;wherein the * represent a carbon branch point;wherein R5is a linear, cyclic or branched C3-C30 alkyl group, wherein a carbon atom of the C3-C30 alkyl group is substituted with an ester E;wherein R6is a C3-C20 alkyl group with a sulfur atom at an alpha (Ci), beta (C2) or gamma (C3) position with respect to the carbon branch point and wherein the sulfur is bound to a distal R2, the R2having a structure of Formula B:r^(CH2)q'~'~'(CH2)PFormula Bwherein index p is 1 to 6 and index q is 0 to 4, andwherein if p = 4, then q is 1 or greater.

20. The ionizable amino lipid of claim 19, wherein the ester E is substituted at an alpha (Ci), beta (C2) or gamma (C3) position with respect to the carbon branch point.

21. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 19, wherein q of Formula B is 0.

22. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 19, wherein index p of Formula B is 5 or 6 and wherein index q is 0 or 1.

23. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 18, wherein the lipid, when formulated in a lipid nanoparticle comprising an mRNA, results in an increase in biodistribution of the lipid nanoparticle of at least about 10% in the liver and / or one or more extrahepatic tissues relative to an otherwise identical lipid nanoparticle containing nor-MC3 as measured by luminescence of the mRNA in vivo in the liver and / or one or more extrahepatic tissues as measured at 4 hours post-injection at 1 mg / kg in a mouse model as a proxy.

24. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 16, wherein index p of Formula B is 4 and wherein index q is 1 or 2 or wherein index p of Formula B is 5 or 6 and wherein index q is 0 or 1.

25. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 16 and 24, wherein index p of Formula B is 5 and wherein index q is 0 or 1.

26. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 16 and 24, wherein index p of Formula B is 6 and wherein index q is 0.

27. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 16, 24, 25 and 26, wherein R1 and R4 are, independently, C6 to C11 linear saturated alkyl or independently have a structure of Formula B.l:r^(CH2)q—(CH2)PFormula B.lwherein p of Formula B.l is 4 or 5 and wherein q of Formula B.l is 1, 2, 3 or 4.

28. The lipid or the pharmaceutically acceptable salt of claim 27, wherein R1 and R4 are independently C8 or C9 linear saturated alkyl and wherein q of Formula B.l is 1, 2 or 3.

29. The lipid or the pharmaceutically acceptable salt of any one of claims 1 to 16, 24, 27 and 28,wherein if A is C thenW1and Y are bonded to each other, and wherein W1is O; W2is O; X is CH; Y is (CH2)q, wherein q is 1 or 2; and Z is structure a type 2 ionizable head group; or W1and Y are not bonded to each other, and wherein W1is H; W2is O, S, NH or NR2a, wherein R2ais a Ci to Ce alkyl optionally substituted with an OH group, and optionallyWV. ZXIa cycloalkyl; and the moiety of Formula A is structure d type 1 ionizable head group, structure g type 7 ionizable head group or structure j type 10 ionizable head group; andwherein if A is N thenW1and Y are absent, W2and X together form a group of structure (CRaRb)P, wherein Raand Rbare H, and wherein p is 4; and Z is OH.

30. The lipid or the pharmaceutically acceptable salt of claim 29, wherein the lipid is selected from the group consisting of:

31. A lipid nanoparticle or the pharmaceutically acceptable salt comprising the lipid of any one of claims 1 to 30 and a nucleic acid.

32. The lipid nanoparticle of claim 31, comprising a helper lipid and optionally a hydrophilic polymer-lipid conjugate.

33. The lipid nanoparticle of claim 32, wherein the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate and a sphingolipid.

34. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle of any one of claims 31 to 33 comprising the nucleic acid and administering the lipid nanoparticle to the subject.

35. A method for delivering a cargo molecule to a cell, the method comprising contacting the lipid nanoparticle of any one of claims 31 to 33 with the cell in vivo or in vitro.

36. The method of claim 35, wherein the cargo molecule is a nucleic acid.

37. Use of the lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 30 or the lipid nanoparticle of any one of claims 31 to 33 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.

38. Use of the lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 30 or the lipid nanoparticle of any one of claims 31 to 33 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.

39. The use as defined in claim 37 or 38, wherein the nucleic acid is an mRNA.

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