Sulfur-containing ionizable lipids for delivery of nucleic acids and other therapeutic agents

Abstract

Provided herein is ionized cationic aminolipid or its pharmaceutically acceptable salt.Ionized cationic aminolipid has protonated amino head group;Two lipophilic chains, and said protonated amino head group has a central carbon atom to which each of said two lipophilic chains is directly bonded;At least one of said two lipophilic chains has the structure of formula C;E is ester in either orientation.This compound can be incorporated into lipid nanoparticles for use in delivering charged cargo such as nucleic acid. JPEG2026501183000257.jpg28170

Description

[Technical Field]

[0001] (Incorporation by reference to priority application) This application claims priority to U.S. Provisional Patent Application No. 63 / 434,506, filed December 22, 2022, which is expressly incorporated herein by reference in its entirety.

[0002] Provided herein are sulfur-containing lipids that can be incorporated into delivery vehicles to facilitate encapsulation of a wide range of therapeutic agents or their prodrugs, including, but not limited to, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceuticals, and salts thereof. [Background technology]

[0003] (background) Nucleic acid-based therapeutics hold great promise in medicine. However, to realize this potential, nucleic acids must be delivered to target sites in patients. This poses a challenge because, upon administration, nucleic acids are rapidly degraded by enzymes in plasma. Even if nucleic acids are delivered to the disease site, intracellular delivery remains a challenge. To address these issues, lipid nanoparticles have been developed that protect nucleic acids from such degradation and facilitate their passage across cell membranes to access intracellular compartments where the associated translation machinery resides.

[0004] The main components of lipid nanoparticles (LNPs) are ionized lipids. Ionized lipids are positively charged at low pH, which facilitates their association with negatively charged nucleic acids. However, ionized lipids are neutral at physiological pH, making them more biocompatible in biological systems. Furthermore, it has been suggested that after LNPs are taken up by cells by endocytosis, the ionization of these lipids at low pH allows them to escape from endosomes, allowing the nucleic acid to be released into intracellular compartments.

[0005] An early example of an LNP product approved for clinical use that relies on ionizable lipids is Onpattro®. Onpattro® is a lipid nanoparticle-based short interfering RNA (siRNA) drug intended for the treatment of polyneuropathy caused by hereditary transthyretin amyloidosis. Onpattro® relies on an ionizable lipid designated by researchers as "DLin-MC3-DMA" or more commonly "MC3" (Scheme 1). MC3 is an evolved form of a structurally related ionizable lipid, designated "KC2" (Scheme 1). MC3 is considered a cutting-edge ionizable lipid, capable of delivering siRNA with approximately threefold less siRNA than KC2. As a result, MC3 is now considered a benchmark for evaluating the efficacy of new lipids. Nevertheless, KC2 remains a valuable research tool.

[0006] Ionizable lipids are also key components of certain COVID-19 vaccines. For example, the Pfizer / BioNTech and Moderna vaccines utilize LNPs to deliver mRNA into the cytoplasm of hepatocytes. This mRNA encodes the highly immunogenic Sars-Cov-2 spike protein. Once inside the host cell, the mRNA is transcribed to produce antigenic proteins. The Pfizer / BioNTech vaccine contains an ionizable lipid called "ALC-0315," 3 (Scheme 1), while the Moderna vaccine contains an ionizable lipid called "SM-102," 4. [ka]

[0007] All of the above ionizable lipids have been optimized for the delivery of therapeutic nucleic acids to the liver. However, there is still a need to develop new ionizable lipids for delivering charged cargoes, such as nucleic acids, to other organs, such as the spleen, lungs, bone marrow, and skin. Delivering therapeutic agents to organs other than the liver will expand the clinical utility of LNPs and enable them to target a wider range of disease states. There is also a continuing need to develop LNPs with improved capabilities for delivering nucleic acids or other charged cargoes to the liver.

[0008] The present disclosure aims to address one or more of the problems identified above and / or to provide useful alternatives to known products and / or compositions for delivery of nucleic acids or other charged cargoes. definition

[0009] As used herein, a "type 1 ionization head" or "MC-type ionization head" refers to a moiety having a lipid head group of formula I below, or its equivalent, where n is in the range of 1 to 5. [ka]

[0010] As used herein, a "type 2 ionization head" or "KC-type ionization head" refers to a moiety having a lipid head group of formula II below, or an equivalent thereof, where n is in the range of 1 to 5. [ka]

[0011] As used herein, a "Type 3 ionizing head" refers to a moiety that is a head group of the structure defined in Formula III below, or an equivalent thereof, where m and n independently range from 1 to 5. [ka]

[0012] As used herein, a "Type 4 ionizing head" refers to a moiety that is a head group of the structure defined in Formula IV below, or an equivalent thereof, where R=C1-C6 alkyl or cycloalkyl, and m and n independently range from 2 to 5. [ka]

[0013] As used herein, a "Type 5 ionizing head" refers to a moiety that is a head group of the structure defined in Formula V below, or an equivalent thereof, where m and n independently range from 1 to 5. [ka]

[0014] As used herein, a "Type 6 ionizing head" refers to a moiety that is a head group of the structure defined in Formula VI below, or an equivalent thereof, where R=C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, m ranges from 1 to 5, and n independently ranges from 2 to 5. [ka]

[0015] As used herein, a "Type 7 ionizing head" refers to a moiety that is a head group of the structure defined in Formula VII below, or its equivalent, where R=C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, and n ranges from 1 to 5, (CH2) n The methylene is optionally substituted with a sulfur atom or an oxygen atom. [ka]

[0016] As used herein, a "Type 8 ionizing head" refers to a moiety that is a head group of the structure defined in Formula VIII below, or an equivalent thereof, where R=C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, and n ranges from 1 to 5. [ka]

[0017] As used herein, a "Type 9 ionizable head" refers to a moiety that is a head group of the structure defined in Formula IX below, or an equivalent thereof, where m and n independently range from 1 to 5. [ka]

[0018] As used herein, a "Type 10 ionizing head" refers to a moiety that is a head group of a structure defined by formula X below, or an equivalent thereof, where the curved lines represent the atoms of the ring structure that includes the N atom, the ring structure has 2 to 8 C atoms, and j ranges from 0 to 5. [ka]

[0019] As used herein, a "Type 11 ionizing head" refers to a moiety that is a head group of the structure defined in Formula XI below, or an equivalent thereof, where R=C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, the circle represents a homocyclic or heterocyclic ring containing 3 to 8 atoms, and j ranges from 0 to 5. [ka]

[0020] As used herein, a "Type 12 ionizing head" refers to a moiety that is a head group of the structure defined in Formula XII below, or an equivalent thereof, where R=C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, and j ranges from 1 to 5. [ka]

[0021] As used herein, the term "ionizable lipid" refers to a lipid that is in an electrostatically neutral form at a particular pH, can accept or donate a proton to become electrostatically charged, and whose electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) greater than 8.

[0022] As used herein, the term "ionizable cationic amino lipid" refers to a lipid that is electrostatically neutral at physiological pH and contains a nitrogen atom in its head group that accepts a proton, thereby becoming electrostatically positively charged at pHs below its pKa.

[0023] The terms "protonated amino head group," "ionizable head group," or "head group" are used interchangeably herein to refer to the portion of an ionizable cationic amino lipid that contains a nitrogen atom in its head group that accepts a proton, thereby making it electrostatically positively charged at pH levels below its pKa. A protonated amino head group has a central carbon atom to which each of the two lipophilic chains is directly attached.

[0024] For example, a Type 2 ionizable head group has a central carbon atom to which each lipophilic chain is directly attached, as indicated by the * symbol. [ka]

[0025] In another example, the central carbon atom of a type 12 ionizable head group is the carbon atom indicated with the * symbol in the following structure: [ka]

[0026] As used herein, the term "lipophilic chain" refers to an optionally substituted alkyl group attached to the central carbon atom of a lipid head group, the alkyl group containing at least 6 carbon atoms and optionally containing a C=C double bond, and the parent compound of said alkyl group has a CLogP of at least 6.

[0027] For example, the known lipids MC3,1 and KC2,2 have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene with a CLogP of 9.25. [ka]

[0028] The lipid ALC-0315, 3, has a pair of lipophilic chains derived from hexyl 2-hexyldecanoate with a CLogP of 10.01: [ka]

[0029] The lipid SM-102,4 has one lipophilic chain derived from undecylhexanoate with a CLogP of 7.59 and one lipophilic chain derived from heptadecan-9-yloctanoate with a CLogP of 11.6: [ka]

[0030] As used herein, the term "alkyl" or "alkyl group" refers to a C1-C alkyl group that is linear, cyclic (monocyclic or polycyclic), and / or branched, optionally containing a C=C double bond and / or one or more substituent ring structures, and that is optionally substituted. 40 It is a carbon-containing chain.

[0031] As used herein, the term "Cm ~C n Alkyl" or "C m ~C n An "alkyl group" refers to an optionally unsaturated, optionally substituted straight, cyclic, and / or branched carbon chain having a minimum of m carbon atoms and a maximum of n carbon atoms in total. For example, a "C1-C3 alkyl" or a "C1-C3 alkyl group" is an alkyl having 1 to 3 carbon atoms.

[0032] The term "ring structure" refers to an optionally substituted, optionally unsaturated, 3- to 22-membered monocyclic or polycyclic alkyl ring. In some non-limiting examples, the ring structure is an optionally substituted 3- to 16-membered monocyclic or polycyclic alkyl ring. In further examples, the ring structure is an optionally substituted 3- to 8-membered monocyclic or polycyclic alkyl ring.

[0033] The term "monocyclic" refers to an optionally substituted alkyl group that is a single ring or contains a single ring substituent.

[0034] The term "polycyclic" refers to an optionally substituted alkyl group that is or includes as a substituent(s) two or more ring structures chemically bonded together or two or more separate ring structures.

[0035] The term "optionally substituted," with respect to an alkyl or alkyl group, means that at least one hydrogen atom of the alkyl group can be replaced with a non-hydrogen atom or group of atoms (i.e., a "substituent") and / or the alkyl is interrupted (i.e., a -(CH)2- group is replaced) with a non-carbon atom or one or more substituents (including, but not limited to, substituents containing a heteroatom selected from O, S, and NR', where R' is as defined below). Non-limiting examples of atoms or substituents that can replace a hydrogen atom include halogen, deuterium, alkyl groups, cycloalkyl groups (monocyclic or polycyclic), oxo groups (=O), hydroxyl groups (-OH); -(C=O)OR'; -O(C=O)R'; -C(=O)R'; O(C=O)OR'-; -OR'; -S(O) x R';-SR',-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x and NR'R', where R' is independently at each occurrence H, C1-C 15 alkyl or cycloalkyl, where x is 0, 1, or 2. Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) include cycloalkyl groups (monocyclic or polycyclic); -O-; -(C=O)O-; -O(C=O)-; -C(=O); -O(C=O)O-; -S(O) x -;-S-;-SS-;-C(=O)S-;-SC(=O)-;-NR'-;-NR'C(=O)-;-C(=O)NR'-;-NR'C(=O)NR'-;-OC(=O)NR'-;-NR'C(=O)OR'-;-NR'S(O) x NR'-;-NR'S(O) x R'-; and -S(O) x and NR'-, where R' is independently at each occurrence H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0036] The term "helper lipid" used herein refers to a compound selected from sterols such as cholesterol or its derivatives; diacylglycerol or its derivatives, such as glycerophospholipids containing phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), etc.; and sphingolipids such as ceramide, sphingomyelin, cerebroside, ganglioside, or their reduced analogues, which lack double bonds in the sphingosine unit.An example of a diacylglycerol derivative is a glycerophospholipid-cholesterol conjugate, in which one of the acyl chains is replaced with a moiety containing cholesterol.The term includes naturally occurring lipids or synthetic lipids.

[0037] As used herein, the term "delivery vehicle" includes any preparation into which the lipids described herein can be formulated, including, but not limited to, delivery vehicles containing one or a combination of the aforementioned helper lipids.

[0038] The term "nanoparticle" as used herein refers to any suitable particle that can be formulated with ionizable lipids and can contain one or more helper lipids.Ionizable lipids are formulated with additional lipid components, such as one or more helper lipids.The term includes, but is not limited to, one or more bilayers, continuous or discontinuous monolayers (including multilayer vesicles, unilamellar vesicles, and particles with a core having an electron-dense region).The term also includes polymer-lipid hybrids, which include particles in which lipids are attached to polymers.

[0039] As used herein, the term "encapsulation," with respect to incorporating a cargo molecule (e.g., a nucleic acid such as mRNA) within a delivery vehicle, refers to any association of the cargo with any component or compartment of the delivery vehicle, such as a nanoparticle.

[0040] The term "pharmaceutically acceptable salts," when referring to lipid forms of the present disclosure in their protonated form (i.e., charged form) and / or as part of a pharmaceutical formulation in which the LNPs are formulated, refers to salts prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. (overview)

[0041] The present disclosure is based, at least in part, on the surprising discovery that LNP formulations of nucleic acids containing ionizable cationic lipids incorporating at least one lipophilic chain substituted with a sulfur atom and an ester moiety are more potent than the benchmark nor-MC3 in terms of nucleic acid delivery. As further described herein, such lipids may exhibit improved delivery to specific organs compared to known lipids. Non-limiting examples described herein demonstrate that such lipids promote nucleic acid delivery to the spleen and / or liver more efficiently than other known lipids. Furthermore, chemical synthesis of the lipids of certain embodiments herein is simpler and / or more economical than chemical synthesis of known lipids.

[0042] According to an aspect of the present disclosure, there is provided an ionized cationic amino lipid, or a pharmaceutically acceptable salt thereof, comprising: with a protonated amino head group; two lipophilic chains, the protonated amino head group having a central carbon atom to which each of the two lipophilic chains is directly attached; At least one of the two lipophilic chains has the structure of formula C; [ka] The wavy line represents the bond to the central carbon atom; m and n are independently 2 to 8; E is an ester group that is —(C═O)O— or —O(C═O)—; R 1 is a linear, branched, monocyclic or polycyclic, optionally substituted, C3-C 20 is an alkyl group; R 2 is a linear or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds; 10 is an alkyl group or R 2 is R 3 to form a ring structure shown by the dashed curve; R 3 is H or a linear or branched, optionally substituted C1-C 10 alkyl group or R 3 is R 2 to form a ring structure shown by the dashed curve; R 4 and R 5 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 4 and R 5 are bonded together to form a ring structure; each lipophilic chain having a total of 15 to 40 carbon atoms; Lipids have (i) a pK between 6 and 7.5 a (ii) has a log P of at least 11;

[0043] According to an embodiment of the aforementioned second aspect of the present disclosure, the second of the two lipophilic chains attached to the central carbon atom of the head group has a structure defined by Formula D: [ka] where the wavy line represents the bond to the central carbon atom of the head group; R 6 and R 7 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 6 and R 7 are bonded together to form a ring structure; A is O, S or carbonyl (C=O); If A is O, then R 8 is an acyl group [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; When A is carbonyl (C=O), R 8 teeth [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; If A is S, then R 8 is a group of formula E: [ka] where E' is an ester group that is -(C=O)O- or -O(C=O)-; The wavy line represents the bond with A, and R 9 is R 1 is as defined above, and R 10 is R 2 is as defined above, and R 11 is R 3 is as defined above.

[0044] Some non-limiting examples are: 2 and R 3 , R 4 and R 5 , R 6 and R 7 , and R 10 and R 11 The ring structures formed by the dotted curves between are independently optionally substituted 3- to 8-membered monocyclic or polycyclic alkyl rings.

[0045] According to one embodiment of the foregoing aspect, the head group has the structure of any one of the above types 1 to 12 head groups as defined herein.

[0046] According to an alternative embodiment of the present disclosure, there is provided an ionizable cationic amino lipid having the structure of Formula A: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, m, n, p, and q in Formula A are independently 2 to 8; R 1 is a linear, branched, monocyclic or polycyclic, optionally substituted C3-C ring containing 0-2 carbon-carbon double bonds; 20 is an alkyl group; E is an ester group that is —(C═O)O— or —O(C═O)—; R 2 is a linear or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds; 10 is an alkyl group or R 2 is R 3 to form a ring structure shown by the dashed curve; R 3 is H or a linear or branched, optionally substituted C1-C 10 alkyl group or R 3 is R 2 to form a ring structure shown by the dashed curve; R 4 and R 5 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 4 and R 5 are bonded together to form a ring structure; R 6 and R 7 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 6 and R 7 are bonded together to form a ring structure; A is O, S or carbonyl (C=O); If A is O, then R 8 is an acyl group [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; When A is carbonyl (C=O), R 8 teeth [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; If A is S, then R 8 is a radical with the structure: [ka] where E is an ester group, which is -(C=O)O- or -O(C=O)-, the wavy line represents the bond to A, and R 9 is R 1 is as defined for R 10 is R 2 is as defined for R 11 is R 3 as defined for W 1 and Y are bonded to each other or not bonded to each other, W 1 When and Y are bonded to each other, W 1 is O or S; W 2 is O or S; X is CH; Y is (CH2) t where t is 1 or 2; Z is selected from any of the following structures a to c, and the wavy line represents represents a bond with X: a. [ka] a Type 2 ionizable head group, wherein n in the Type 2 ionizable head group is 1 to 5; b. [ka] Type 3 ionizable head groups, wherein m and n in the Type 3 ionizable head groups are independently 1 to 5; c. [ka] Type 4 ionizable head groups, wherein m and n in the Type 4 ionizable head group are independently 2 to 5; W 1 If and Y are not bonded to each other, W 1 is H; W 2 is O, S, NH or NR 12 and R 12 is a C1-C4 alkyl optionally substituted with an OH group; Part of formula A [ka] is a group selected from any one of the following structures d to l, and the wavy line is W 2 represents a bond to: d. [ka] Type 1 ionizable head group, where n in the Type 1 ionizable head group is 1 to 5; e. [ka] Type 5 ionizable head groups, wherein m and n in the Type 5 ionizable head group are independently 1 to 5; f. [ka] Type 6 ionizable head groups, where m is 1 to 5, n is independently 2 to 5, and R is C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl; g. [ka] Type 7 ionizable head group, n of Type 7 ionizable head group is 1 to 5, and (CH) of Type 7 ionizable head group n wherein the methylene (CH2) is optionally replaced with a sulfur atom or an oxygen atom; h. [ka] an ionizable head group of type 8, wherein n in the ionizable head group of type 8 is 1 to 5; i. [ka] Ionizable head groups of type 9, wherein m and n in the type 9 ionizable head group are independently 1 to 5; j. [ka] Type 10 ionizable head groups, where the curved lines represent atoms of a ring structure containing an N atom, the ring structure has 2 to 8 carbon atoms, and j of the Type 10 ionizable head group is 0 to 5; k. [ka] Type 11 ionizable head groups, where the circle represents a homocyclic or heterocyclic ring containing 3 to 8 atoms, and j in the Type 11 ionizable head group is 0 to 5; l. [ka] Ionizable head groups of type 12, where R=C-C alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, and j ranges from 1 to 5.

[0047] According to the foregoing aspect or any embodiment thereof, the ionizable cationic amino lipid of formula A has (i) a pKa of 6 to 7.5, and / or (ii) a logP of at least 11.

[0048] According to the foregoing embodiment, the ionizable cationic amino lipid can have the structure of any one of compounds 5-35, or a pharmaceutically acceptable salt thereof, as defined in Table 1 below. In another embodiment, the ionizable cationic amino lipid or a pharmaceutically acceptable salt thereof has the structure of compounds 5-13, 16-19, 22-31, or 33-35.

[0049] According to further examples of any one of the foregoing aspects or embodiments thereof, an ionized cationic amino lipid or a pharmaceutically acceptable salt, when incorporated into lipid nanoparticles containing mRNA, increases the biodistribution of the lipid nanoparticles in the spleen by at least about 10-fold compared to otherwise identical lipid nanoparticles containing norDLin-MC3-DMA (nor-MC3), as measured by in vivo mRNA luminescence in the spleen.

[0050] According to further examples of any one of the foregoing aspects or embodiments thereof, the ionized cationic amino lipid or a pharmaceutically acceptable salt, when formulated into lipid nanoparticles comprising mRNA, increases the biodistribution of the lipid nanoparticles in the liver by at least about two-fold compared to lipid nanoparticles comprising norDLin-MC3-DMA (nor-MC3), as measured by in vivo mRNA luminescence in the liver.

[0051] According to a further aspect of the present disclosure, there is provided a lipid nanoparticle comprising an ionizable cationic amino lipid and a nucleic acid according to any of the preceding aspects or embodiments.

[0052] In one embodiment, the lipid nanoparticles comprise a helper lipid, hi one example, the helper lipid is selected from cholesterol, diacylglycerol, glycerophospholipid-cholesterol conjugates, sphingolipids, and mixtures thereof.

[0053] According to another aspect of the present disclosure, there is provided a method of treating a subject in need of nucleic acid therapy, the method comprising preparing or providing lipid nanoparticles of any one of the foregoing aspects or embodiments thereof comprising a nucleic acid, and administering the lipid nanoparticles to the subject.

[0054] According to another aspect of the present disclosure, there is provided a method of delivering a nucleic acid molecule to a cell, the method comprising contacting the described lipid nanoparticles with the cell in vivo or in vitro.

[0055] According to another aspect of the present disclosure, there is provided use of an ionized cationic amino lipid or a pharmaceutically acceptable salt thereof, or a lipid nanoparticle of any of the preceding aspects or embodiments, in the manufacture of a medicament for treating or preventing a disease, disorder, or condition treatable and / or preventable by a nucleic acid.

[0056] According to another aspect of the present disclosure, there is provided the use of an ionized cationic amino lipid or a pharmaceutically acceptable salt thereof, or the lipid nanoparticle of any one of the preceding aspects or embodiments, to deliver a nucleic acid to a subject for treating or preventing a disease, disorder, or condition treatable or preventable by a nucleic acid.

[0057] In one embodiment, the nucleic acid is mRNA.

[0058] Other objects, features, and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description and drawings. [Brief explanation of the drawings]

[0059] [Figure 1]1 is a bar graph showing the encapsulation efficiency (%), particle size (nm), and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) containing ionizable lipids 1, 5-13, and 16-35 described in Table 1 and Example 2. The LNPs were composed of 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG2000-DMG, with an amine-to-phosphate charge ratio (N / P) of 6.

[0060] [Figure 2A] Figure shows the luminescence intensity / mg in the liver of mRNA-containing LNPs containing ionizable lipids 1, 5–19, 22–31, 33, 34, and 35, 4 h after intravenous administration to CD-1 mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% ionizable lipid / DSPC / chol / PEG2000-DMG (N / P = 6).

[0061] [Figure 2B] Figure shows the luminescence intensity / mg in the spleen of mRNA-containing LNPs containing ionizable lipids 1, 5–19, 22–31, 33, 34, and 35, 4 h after intravenous administration to CD-1 mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% ionizable lipid / DSPC / chol / PEG2000-DMG (N / P = 6). DETAILED DESCRIPTION OF THE INVENTION

[0062] (Detailed explanation) Various aspects and embodiments of the present disclosure relate to ionized cationic amino lipids having the structure of formula A and their pharmaceutically acceptable salts.Preparations containing such lipids are used to deliver nucleic acids to any target site.In some embodiments, such lipids have been found to be particularly effective in delivering mRNA when formulated in a suitable delivery vehicle.In further embodiments, such lipids can be easily synthesized and prepared by a process that is more economical than known methods for producing ionized lipids.

[0063] Embodiments disclosed herein include ionizable cationic amino lipids having the structure of Formula A: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, m, n, p, and q in Formula A are independently 2 to 8; R 1 is a linear, branched, monocyclic or polycyclic, optionally substituted C3-C ring containing 0-2 carbon-carbon double bonds; 20 is an alkyl group; E is an ester group that is —(C═O)O— or —O(C═O)—; R 2 is a linear or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds; 10 is an alkyl group or R 2 is R 3 to form a ring structure shown by the dashed curve; R 3 is H or a linear or branched, optionally substituted C1-C 10 alkyl group or R 3 is R 2 to form a ring structure shown by the dashed curve; R 4 and R 5 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 4 and R 5 are bonded together to form a ring structure; R 6 and R 7 are independently H or a straight or branched, optionally substituted C1-C alkyl group containing 0-2 carbon-carbon double bonds. 10 alkyl group or R 6 and R 7 are bonded together to form a ring structure; A is O, S or carbonyl (C=O); If A is O, then R 8 is an acyl group [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; When A is carbonyl (C=O), R 8 teeth [ka] where the wavy line represents the bond to A and R' is R 1 is as defined above; If A is S, then R 8 is a radical with the structure: [ka] where E is an ester group, which is -(C=O)O- or -O(C=O)-, the wavy line represents the bond to A, and R 9 is R 1 is as defined for R 10 is R 2 is as defined for R 11 is R 3 as defined for W 1 and Y are bonded to each other or not bonded to each other, W 1 When and Y are bonded to each other, W 1 is O or S; W 2 is O or S; X is CH; Y is (CH2) t where t is 1 or 2; Z is selected from any of the following structures a to c, and the wavy line represents represents a bond with X: a. [ka] a Type 2 ionizable head group, wherein n in the Type 2 ionizable head group is 1 to 5; b. [ka] Type 3 ionizable head groups, wherein m and n in the Type 3 ionizable head groups are independently 1 to 5; c. [ka] Type 4 ionizable head groups, wherein m and n in the Type 4 ionizable head group are independently 2 to 5; W 1 If and Y are not bonded to each other, W 1 is H; W 2 is O, S, NH or NR 12 and R 12 is a C1-C4 alkyl optionally substituted with an OH group; Part of formula A [ka] is a group selected from any one of the following structures d to l, and the wavy line is W 2 represents a bond to: d. [ka] Type 1 ionizable head group, where n in the Type 1 ionizable head group is 1 to 5; e. [ka] Type 5 ionizable head groups, wherein m and n in the Type 5 ionizable head group are independently 1 to 5; f. [ka] Type 6 ionizable head groups, where m is 1 to 5, n is independently 2 to 5, and R is C1-C6 alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl; g. [ka] Type 7 ionizable head group, n of Type 7 ionizable head group is 1 to 5, and (CH) of Type 7 ionizable head group n wherein the methylene (CH2) is optionally replaced with a sulfur atom or an oxygen atom; h. [ka] an ionizable head group of type 8, wherein n in the ionizable head group of type 8 is 1 to 5; i. [ka] Ionizable head groups of type 9, wherein m and n in the type 9 ionizable head group are independently 1 to 5; j. [ka] Type 10 ionizable head groups, where the curved lines represent atoms of a ring structure containing an N atom, the ring structure has 2 to 8 carbon atoms, and j of the Type 10 ionizable head group is 0 to 5; k. [ka] Type 11 ionizable head groups, where the circle represents a homocyclic or heterocyclic ring containing 3 to 8 atoms, and j in the Type 11 ionizable head group is 0 to 5; l. [ka] Ionizable head groups of type 12, where R=C-C alkyl, cycloalkyl, deuterated alkyl, or deuterated cycloalkyl, and j ranges from 1 to 5. Method for producing lipids of formula A

[0064] The lipid of formula A or its pharmaceutically acceptable salt can be prepared by any suitable method known to those skilled in the art.The particularly suitable method is described below.Those skilled in the art will understand that alternative starting materials can be used in the same order to obtain the homologue of the compound envisaged herein.Therefore, the synthetic scheme shown below is merely illustrative of selected embodiments.

[0065] Ester group E is R 1 A lipid of formula A, or a pharmaceutically acceptable salt thereof, in which is oriented so that is attached to the carbon atom of the ester carbonyl, can be represented by the general structure of formula B: [ka]

[0066] Without intending to be limiting, the synthesis of lipids of Formula B is exemplified by the synthesis of compounds 5-35 in Table 1. Those skilled in the art will appreciate that alternative starting materials can be used in the same order to obtain analogs of compounds 5-35 defined by Formula B. Thus, the synthetic schemes shown below are merely illustrative of selected embodiments. JPEG2026501183000060.jpg211170JPEG2026501183000061.jpg214170JPEG2026501183 000062.jpg221170JPEG2026501183000063.jpg224170JPEG2026501183000064.jpg30170

[0067] Lipids of formula B can be prepared from the appropriate ketone of general structure 36 (Scheme 2) by converting the keto group to an ionizable head group of type 1-12, where A is either O, S, or carbonyl (C=O), as described above. When A is O, compound 36 can be more precisely designated as 37. When A is S, compound 36 can be more precisely designated as 38. When A is carbonyl, (C=), compound 36 can be more precisely designated as 39. Thus, the synthesis of lipids of formula B where A=O begins with the preparation of a ketone of general structure 37, followed by conversion of the keto group to an ionizable head group; the synthesis of lipids of formula B where A=S begins with the preparation of a ketone of general structure 38, followed by conversion of the keto group to an ionizable head group; and the synthesis of lipids of formula B where A=carbonyl begins with the preparation of a ketone of general structure 39, followed by conversion of the keto group to an ionizable head group. [ka]

[0068] Ketones of general structure 37 (m=p, n=q, R 4 =R 6 , and R 5 =R 7Specific steps in the synthesis of ketones of general structure 46 in Scheme 3 are described in detail in co-pending and commonly owned WO 2023 / 147657, which is incorporated herein by reference. As described in the foregoing disclosure, one such step is the Claisen condensation of an appropriate lactone 40 under Mukaiyama conditions to form compound 41. The OH group of 41 is then converted to a leaving group, e.g., a sulfonate ester such as tosylate, to generate 42. The tosylate in 42 is then displaced with a sulfur nucleophile, which can serve as a precursor to the SH group of, for example, thioacetic acid. The resulting 43 is then treated with a suitable base, e.g., NaOH, in the presence of a suitable epoxide, resulting in the release of the acetyl group from 43, opening the lactone moiety, and decarboxylation of the resulting beta-keto acid. The anion of the mercaptan formed upon release of the acetyl group then reacts with the epoxide to generate compound 44. The primary OH group of 44 is selectively esterified with a carboxylic acid R'-COOH or a chloroformate R'-OCOCl or equivalent reagent in the presence of a condensing agent to produce compound 45. The secondary OH group of 45 is selectively esterified with a carboxylic acid R'-COOH or a chloroformate R'-OCOCl or equivalent reagent in the presence of a condensing agent to produce compound 45. 1 Further esterification with —COOH or with the chloroformate R′—OCOCl or equivalent reagents converts 45 to the ketone 46. [ka]

[0069] As described in the aforementioned application, lactones such as 40 can be converted directly to ketodiols of general formula 48 (Scheme 4), optionally under conditions that avoid the isolation of the Claisen product 41. [ka]

[0070] Thus, adding water to the reaction mixture in which 41 is formed releases acidity through hydrolysis of TiCl. If the resulting mixture is stirred for a sufficient period of time, the strongly acidic aqueous medium opens the lactone to give keto acid 47, which is then decarboxylated to give 48. The aforementioned application teaches that ketodiols such as 48 can be converted to monosulfonic acid esters, such as monomesylate 49, which can be converted to thioester 50 in the presence of a suitable base by reaction with a sulfur nucleophile, which can serve as a precursor to the SH group of, for example, thioacetic acid. According to Scheme 3, treatment of 50 with a suitable base, such as NaOH, in the presence of a suitable epoxide releases the acetyl group, and the anion of the mercaptan thus formed reacts with the epoxide to give compound 44. The latter can be converted to ketone 46 by the method shown in Scheme 3 above.

[0071] In some cases, the lactone required for the preparation of ketodiols of type 48 may be expensive and / or not readily available. In such cases, it is advantageous to carry out the synthesis of the desired 48 starting from the specific procedures described in co-pending and commonly owned WO 2023 / 147657, incorporated herein by reference. Thus, Claisen condensation under Mukaiyama conditions of an appropriately O-protected derivative of a hydroxyester of general structure 51, followed by hydrolysis of the resulting β-ketoester, release of the O-protecting group, and decarboxylation, forms ketodiol 48 (Scheme 5). In certain embodiments, these steps of β-ketoester hydrolysis, release of the O-protecting group, and decarboxylation are most advantageously carried out as a "one-pot operation," meaning that synthetic intermediates 52 and 53 do not need to be isolated, although they can be. [ka]

[0072] Without intending to be limiting, the process of Scheme 3 above is exemplified below with reference to the synthesis of specific ketones that are precursors to lipids 5-35. Lipids 5-11 can be made from ketones 54-59 of Scheme 6. [ka]

[0073] The lactone required for the synthesis of ketones 54–59 is caprolactone 60 (Scheme 7), which can be converted to 61 by Claisen condensation under Mukaiyama conditions, for example, in the presence of titanium tetrachloride and triethylamine in an inert solvent, such as dichloromethane, initially at a suitable low temperature, e.g., −20°C, between −80°C and 0°C, and then gradually increasing to ambient temperature. The OH group of 61 is converted to a sulfonate ester, e.g., tosylate 62, which can then be displaced with a sulfur nucleophile, such as thioacetic acid, which can serve as a precursor to an SH group, in the presence of a suitable base, e.g., a tertiary amine such as triethylamine, in a solvent that promotes nucleophilic substitution, e.g., N,N-dimethylformamide (DMF). This results in the formation of thioacetate 63, which, when treated with a base, e.g., NaOH, which can release the acetyl group from 63 and thus liberate the anion of the corresponding mercaptan, generates compound 64 in the presence of an epoxide, such as 1-epoxyoctane, in a suitable solvent, e.g., an alcohol, e.g., ethanol. [ka]

[0074] Selective acylation of the primary alcohol of 64 can be achieved by treatment with a carboxylic acid in the presence of a coupling agent, for example, a carbodiimide (e.g., EDCI), optionally in the presence of a nucleophilic catalyst such as 4-(dimethylamino)-pyridine (DMAP), or by treatment with an alkyl chloroformate or equivalent reagent such as an alkoxycarbonylimidazolide or alkyl 4-nitrocarbonate, in the presence of a base such as triethylamine, optionally in the presence of DMAP. Without intending to be limiting, Scheme 8 illustrates the selective esterification of 64 with cyclopentadecanecarboxylic acid to produce 65, which is further esterified with 3-cyclohexylpropanoic acid to give 54 in the presence of a coupling agent (e.g., a carbodiimide such as EDCI), optionally in the presence of a nucleophilic catalyst such as DMAP. The advantages of ionizable lipids having macrocyclic moieties such as cyclopentadecyl groups are described in detail in commonly owned and co-pending U.S. Provisional Patent Application No. 63 / 517,628, filed August 4, 2023, and incorporated herein by reference. [ka]

[0075] Similarly, compound 64 can be converted to ketones 55–59 by starting with esterification of the primary alcohol with 2-hexyldecanoic acid and then esterifying the secondary OH group of the resulting 66 with the appropriate carboxylic acid (Scheme 9). [ka]

[0076] Lipids 12–15 can be prepared from ketones 67–70 in Scheme 10. The lactone required for the synthesis of ketones 67–70 is 71 (Scheme 11), which can be prepared by Baeyer-Villiger oxidation of commercially available 4-pentylcyclohexanone with a peracid such as MCPBA. Claisen condensation of 71 under Mukaiyama conditions, e.g., titanium tetrachloride and triethylamine, in an inert solvent such as dichloromethane, initially at a suitable low temperature (e.g., −20°C) between −80°C and 0°C, followed by gradual warming to ambient temperature, produces 72. The OH group of 72 is converted to a sulfonate ester, e.g., tosylate 73, which can then be displaced with a sulfur nucleophile, e.g., thioacetic acid, which can serve as a precursor to an SH group, in a solvent that favors nucleophilic substitution, e.g., N,N-dimethylformamide (DMF), in the presence of a suitable base, e.g., a tertiary amine such as triethylamine. This results in the formation of thioacetate 74. Treatment of 74 with a base, such as NaOH, in the presence of an epoxide, such as 1-hexene oxide, in a suitable solvent, such as an alcohol, such as ethanol, which is capable of releasing the acetyl group from 74 and thus liberating the anion of the corresponding mercaptan, yields compound 75. The same treatment, carried out in the presence of cyclohexene oxide, converts 74 to 76. Compounds 75 and 76 can then be O-acylated as described above. [ka] [ka]

[0077] The synthesis of ketones 67–70 requires acylation of the primary and secondary OH groups of 75–76 with the same groups. Thus, 74 can be esterified with at least two equivalents of octanoic acid or 3-cyclohexylpropanoic acid in the presence of a coupling agent, e.g., a carbodiimide such as EDCI, and optionally a nucleophilic catalyst such as DMAP, to form ketones 67 and 68, respectively. Esterification of 75 in a similar manner yields ketones 69 and 70. [ka]

[0078] Lipids 16-35 can be prepared from ketones 77-83 in Scheme 13. Specific steps in the synthesis of ketones 77-83 are described in detail in co-pending and commonly owned WO 2022 / 246555. As described in the foregoing disclosure, one such step is to subject an appropriate ester of general structure 84 to a Claisen condensation under Mukaiyama conditions to form a β-ketoester product of general structure 85 (Scheme 14). Compound 85 is then converted to β-keto acid 86, which is not isolated (as indicated by the square brackets) but is decarboxylated directly to produce ketone 87. [ka] [ka]

[0079] While not intended to be limiting, the following Scheme 15 illustrates a process encompassing a route to ketones 77-83. The esters of type 84 required for the preparation of said ketones are compounds 90 and 91. The synthesis of these materials begins with the displacement of a leaving group, such as a halide or sulfonate, from an appropriate haloester or sulfonyloxyester (e.g., bromine ester 88) with a suitable sulfur nucleophile (e.g., thioacetic acid) that can serve as a precursor to an SH group in the presence of a base (e.g., triethylamine) in a suitable solvent (e.g., dimethylformamide (DMF)). Treatment of the resulting 69 with a nucleophilic metal alkoxide, e.g., sodium methoxide, in the presence of an epoxide, e.g., 1-hexene oxide, in a suitable solvent, e.g., an alcohol such as methanol, results in the formation of ester 90. The same reaction of 89 with 1-octene oxide instead yields compound 91. Conversion of 90–91 to ketones of type 87 continues with the protection of the OH group as a trialkylsilyl ether, such as trimethylsilyl, triethylsilyl, or tert-butyldimethylsilyl. For example, treatment of 90–91 with tert-butyldimethylsilyl chloride (TBS-Cl) and imidazole generates silyl ethers 92 and 93, respectively. The latter are then treated with, for example, TiCl4 and a tertiary amine base, such as triethylamine or tributylamine, in a solvent such as toluene or dichloromethane at a suitable temperature, e.g., −20°C to room temperature, resulting in the formation of β-ketoesters 94 and 95. Hydrolysis and acidification of the ester decarboxylates the transient keto acid of type 86, releasing the silyl group and directly generating ketodiols 96 and 97. The use of a more labile silyl protecting group, such as trimethylsilyl (TMS), can be advantageous in that the protecting group is more easily released during hydrolysis and decarboxylation. In cases where a stronger silyl protecting group is advantageous, a separate desilylation step can be applied to provide the desired ketodiol. [ka]

[0080] An alternative route to ketones such as 96 and 97 begins with the preparation of esters of type 90-91 by halide or sulfonate displacement with a mercapto alcohol, such as 98, from a sulfonyloxy ester, such as a haloester of 88, in the presence of a base, such as an amine, such as triethylamine, in a suitable solvent, such as DMF (Scheme 16). 91, obtained in exemplary, but not limited to, Scheme 16, can then be treated to generate ketone 97 as shown in Scheme 15 above. [ka]

[0081] Alternatively, ketones such as 96-97 can be prepared by the synthetic procedures detailed in the aforementioned co-pending and commonly owned WO 2023 / 147657. One such step involves the Claisen condensation of a lactone under Mukaiyama conditions, followed by hydrolysis and decarboxylation of the resulting product to form a ketodiol. In certain embodiments, these steps are most advantageously performed as a "one-pot operation," meaning that various synthetic intermediates can be, but do not need to be, isolated. While not intended to be limiting, the process is exemplified in Scheme 17 for the preparation of ketodiol 100 and its conversion to ketones 96-97, where the required lactone is caprolactone 60, the synthetic intermediates, which optionally do not need to be isolated, are compounds 61 and 99, and the product of the reaction is ketodiol 100. Direct formation of 100 is achieved by adding water to the reaction mixture of the Claisen step, as shown previously in Scheme 4, where the acidity generated by the reaction of TiCl4 with HO opens lactone 61 to 99, which undergoes decarboxylation in the process, allowing 100 to be isolated. [ka]

[0082] Finally, ketodiols of type 100 can also be made by an alternative method, also described in the aforementioned co-pending and commonly owned WO 2023 / 147657. This synthetic method is particularly advantageous when lactones suitable for preparing ketodiols are not readily available. Thus, Claisen condensation of an appropriately O-protected derivative of a hydroxyester under Mukaiyama conditions, followed by hydrolysis of the resulting β-ketoester, release of the O-protecting group, and decarboxylation, forms the ketodiol. In certain embodiments, these steps of β-ketoester hydrolysis, release of the O-protecting group, and decarboxylation are most advantageously carried out as a "one-pot operation," meaning that various synthetic intermediates can be, but do not need to be, isolated. Without intending to be limiting, the synthesis of ketodiol 106 is exemplified in Scheme 18, where the O-protected derivative of the hydroxyester is 103, and the synthetic intermediates that optionally do not need to be isolated are compounds 104 and 105. [ka]

[0083] The above synthetic methods yield ketones of general structure 37 and 38 (see Scheme 2), where m=p, n=q, R 4 =R 6 , and R 5 =R 7 m and p, n and q, R 4 and R 6 , and R 5 and R 7 Ketones 37-38, which vary by one or more of the following, can be prepared by the general schemes shown below. These schemes are merely illustrative of representative embodiments and should not be construed as limiting in any way.

[0084] m and p, n and q, R 4 and R 6 , and R 5 and R 7Ketones 37-38, differing in one or more of the following, can be advantageously prepared starting from compounds of general structure 107 as shown in Scheme 19, where P 1 and P 2 is an orthogonal O protecting group, i.e., P 1 and P 2 You can release one of them while leaving the other intact. So, for example, P 1 Release of P gives 108, which can be converted to 109 by obvious modifications of the methods shown in Schemes 3, 4, and 7 above. Compound 109 can then be converted to P 2 Material 110 is then converted to ketone 37 by obvious modifications of the methods shown above in Schemes 8 and 9, or to ketone 38 by obvious modifications of the methods shown above in Schemes 3, 4, and 7. Thus, when m and p, n and q, R 4 and R 6 , and R 5 and R 7 The synthesis of ketones 37-38, which differ in one or more of m and p, n and q, and R 4 and R 6 , and R 5 and R 7 can be achieved by starting with a compound of general structure 107 where one or more of the

[0085] Compounds of general structure 107 are composed of m and p, n and q, and R 4 and R 6 , and R 5 and R 7These compounds may be advantageously prepared by certain synthetic procedures detailed in commonly owned and co-pending U.S. Provisional Patent Application No. 63 / 445,854, filed February 15, 2023, which is incorporated herein by reference. Thus, tosylmethylisonitrile (TosMIC) can be mono-alkylated with an alkyl halide (X = Cl, Br, I) or sulfonate (X = OTs, OTs, etc.) of structure 111 in the presence of base to form product 112. Subsequent alkylation of 112 with an alkyl halide (X = Cl, Br, I) or sulfonate (X = OTs, OTs, etc.) of structure 113 provides 114, which can be hydrolyzed to ketone 107 upon treatment with aqueous acid. The P amine group can be modified to include a protecting group that can be released under the acidic conditions required to convert 114 to 107. 1 or P 2 It is appropriate to use it as one of the following. For example, P 1 If P is a protecting group that can be released under the acidic conditions required for the hydrolysis of 114, 114 can be advantageously converted directly to 108. Otherwise, a suitable deprotection procedure can be used to obtain P 1 or P 2 can emit either [ka] [ka]

[0086] m and p, n and q, R 4 and R 6 , R 5 and R 7An alternative method for the synthesis of compounds of general structure 106, where one or more of the groups are different, involves converting an O-protected hydroxy acid of general structure 115 to a β-ketoester 117, for example, by the method of Oikawa (Oikawa, T., et al., Org. Syn. 1985, 63, 198, incorporated herein by reference). Subsequent alkylation of 117 with an alkyl halide (X=Cl, Br, I) or sulfonate (X=OTs, OMs, etc.) of structure 118 provides 119, which upon ester saponification and decarboxylation generates ketone 107 (Scheme 21). [ka]

[0087] Representative ketones 77-83 in Scheme 13 can be prepared from ketodiols 96-97 in Scheme 15 by acylation of the OH group with the appropriate carboxylic acid or chloroformate (or equivalent reagent). Furthermore, the acylation step can be carried out to yield either diacyl or monoacyl derivatives of the starting ketodiol. Thus, reaction of a ketodiol with slightly more than two equivalents of a carboxylic acid in the presence of a condensing agent, such as a carbodiimide (e.g., EDCI), and optionally in the presence of DMAP, or reaction of a ketodiol with a chloroformate or equivalent reagent, such as an alkoxycarbonylimidazolide or alkyl 4-nitrophenyl carbonate, in the presence of triethylamine, optionally in the presence of DMAP, yields diesters or decarboxylation products containing two identical acyl groups. While not intended to be limiting, this is exemplified in Scheme 22, which converts compound 96 to 79, and Scheme 23, which converts compound 97 to 77, 78, 80, 82, and 83. [ka]

[0088] Conversely, reaction of a ketodiol with about one equivalent of a carboxylic acid in the presence of a condensing agent, such as a carbodiimide (e.g., EDCI), and optionally in the presence of DMAP, or reaction of a ketodiol with a chloroformate or equivalent reagent, such as an alkoxycarbonylimidazolide or alkyl 4-nitrophenylcarbonate, in the presence of triethylamine, optionally in the presence of DMAP, provides a monoacyl derivative, which can be converted to a ketone containing two different acyl groups by further acylation with a different carboxylic acid or chloroformate (or equivalent reagent), as described above. While not intended to be limiting, this is exemplified in Scheme 24 by the preparation of ketone 81 via monoester 120. [ka] [ka]

[0089] Below are procedures for preparing representative lipids 5-35 from the ketones of Schemes 6, 10, and 13. These procedures are merely illustrative of representative embodiments and should not be construed as limiting in any way.

[0090] Lipid 5 can be produced from ketone 54 by a synthetic procedure that converts the keto functionality to a Type 1 ionizable head group. Thus, the ketone is selectively reduced with a hydride reagent, such as sodium borohydride, and the resulting alcohol 121 is esterified with 4-(dimethylamino)butanoic acid hydrochloride in the presence of a condensing agent, such as a carbodiimide such as EDCI, to produce lipid 5 (Scheme 25). The same synthetic procedure can be used to convert any of the ketones in Schemes 6, 10, and 13 to the corresponding lipids containing a Type 1 ionizable head group. [ka]

[0091] Lipid 6 can be generated from ketone 55 by a synthetic procedure that converts the keto functionality to an ionizable head group of type 7. Thus, the ketone is reductively aminated with an O-protected derivative of 4-aminobutanol, such as the O-tert-butyldiphenylsilyl derivative 122, in the presence of a hydride reagent, such as sodium triacetoxyborohydride, and an acid catalyst, such as acetic acid, to give 123. A second reductive alkylation, for example with formaldehyde in the presence of sodium triacetoxyborohydride, converts 123 to 124, which can be converted to lipid 6 by releasing the silyl protecting group with a fluoride ion source, such as HF-pyridine complex (Scheme 26). Using the same synthetic procedure, any of the representative ketones from Schemes 6, 10, and 13 can be converted to the corresponding lipid containing an ionizable head group of type 7. Thus, lipids 7, 8, 9, 10, 11, 12, 13, 14, and 15, all containing ionizable head groups of type 7, can be prepared by the method of Scheme 26 from ketones 56, 57, 58, 59, 54, 67, 68, 69, and 70, respectively. [ka]

[0092] Lipids 16, 17, 18, and 19 contain ionizable head groups of Type 1. Therefore, they can be prepared from ketones 77, 78, 79, and 80, respectively, by the method of Scheme 25 above.

[0093] Lipid 20 can be generated from ketone 80 by a synthetic procedure that converts the keto functionality into an ionizable head group of type 10. Thus, the ketone is selectively reduced with a hydride reagent, e.g., sodium borohydride, and the resulting alcohol 125 is esterified with N-methylazetidine-3-carboxylic acid hydrochloride in the presence of a condensing agent, e.g., a carbodiimide such as EDCI, to generate 20 (Scheme 27).

[0094] Lipid 21 can be prepared from alcohol 125 in Scheme 27 by synthetic steps that introduce an ionizable head group of type 12. [ka]

[0095] Thus, reaction of 125 with 1-bromo-2-ethoxyethene in the E- or Z-configuration in the presence of a suitable catalyst (e.g., a Brønsted acid such as pyridinium paratoluenesulfonate (PPTS)) produces bromoketal 126, which can be converted to lipid 21 by reaction with methylamine, optionally under microwave irradiation conditions (Scheme 28). [ka]

[0096] Lipid 22 can be generated from ketone 80 by a synthetic procedure that converts the keto functionality into an ionizable head group of type 2. Thus, ketone 80 is converted to ketal 127 by reaction with 1,2,4-butanetriol in the presence of a suitable catalyst, e.g., a Brønsted acid such as PPTS. The OH group of 127 is converted to a good leaving group, e.g., a sulfonate ester such as tosylate 128. Reaction of 128 with dimethylamine (optionally under microwave irradiation conditions) generates lipid 22 (Scheme 29). [ka]

[0097] Lipids 23-35 contain ionizable head groups of type 7. Thus, they can be prepared from the appropriate ketones of Scheme 13 by appropriate modification of the method of Scheme 26.

[0098] Lipids 23 and 24 can be obtained from ketones 78 and 77, respectively, as shown in Scheme 26 above.

[0099] Lipids 25 and 26 can be obtained from ketones 78 and 81, respectively, by a modification of the method of Scheme 26 above, in which acetaldehyde is used instead of formaldehyde in the second reductive alkylation step.

[0100] Lipid 27 can be obtained from ketone 80 by the method of Scheme 26 above.

[0101] Lipid 28 can be prepared starting from the reductive amination of ketone 80 with compound 122 (Scheme 30). The resulting product 129 can be converted to 130 by reaction with CD3-I. Removal of the silyl group affords 28. The deuterated methyl group serves to favorably change the pKa of the lipid. [ka]

[0102] Lipid 29 can be obtained from ketone 80 by the same method used to synthesize lipids 25 and 26, i.e., a modification of the method of Scheme 26 above, using acetaldehyde instead of formaldehyde in the second reductive amination step.

[0103] Lipid 30 can be obtained from ketone 80 by a modification of the method of Scheme 30 above, in which compound 129 is N-alkylated with CD3-CD2-I instead of CD3-I prior to release of the silyl group.

[0104] Lipids 31 and 32 can be obtained from ketone 80 by a modification of the method of Scheme 26 above, where the first reductive amination step is carried out using compounds 131 and 132, respectively, instead of compound 122 (Scheme 31). [ka]

[0105] Lipids 33 and 34 can be obtained from ketones 82 and 83, respectively, by the methodology of Scheme 26 above.

[0106] Lipid 35 can be obtained from ketone 80 by a modification of the method of Scheme 26 above, where the first reductive amination step is carried out using compound 133 instead of compound 122 (Scheme 32). [ka]

[0107] Those skilled in the art will appreciate that a variety of lipids of types 5-35 can be prepared by using alternative starting materials in the above synthetic scheme.

[0108] Ketones of general structure 39 in Scheme 2 can be prepared by appropriately modifying the methods outlined in Schemes 20 and 21 above. For example, a route similar to Scheme 20 begins with alkylation of TosMIC with a halide or tosylate of general formula 134 (THP = tetrahydropyranyl). This affords 135, which can be re-alkylated with a halide or tosylate of general formula 136 to generate compounds of general structure 137. Acidic hydrolysis of the latter occurs with concomitant loss of the THP group, converting it to compound 138. The OH group of the latter can be converted to an excellent leaving group, such as a tosylate, as in 139. Displacement of the tosylate with thioacetic acid in the presence of triethylamine converts 139 to 140, which, when reacted with methanolic NaOH and an epoxide, generates 141 via the release of the acetyl group and liberation of the thiolate, which nucleophilically opens the epoxide and simultaneously saponifies the methyl ester. The free acid is esterified with an alcohol R'-OH, and the secondary alcohol is converted to the carboxylic acid R, for example, in the presence of EDCI. 1 Acylation with -COOH gives 39 (Scheme 33).

[0109] Scheme 34 depicts an alternative synthetic route that mimics Scheme 21 and leads to compound 148, the ethyl ester analog of 138, which can be converted to 39 in the same manner as shown in Scheme 33. Thus, dicarboxylic acid monoester 143 is converted to β-ketoester 145 by the method of Oikawa, and 145 is alkylated with a halide or sulfonate of general formula 146. Compound 147 thus produced undergoes Clapcho decarboxylation and release of the THP group to give 148, which is converted to 39 by the method described in Scheme 33. [ka]

[0110] The keto group of 39 can then be converted to any ionizable head group of types 1-12 by the methods described in the scheme above, thus producing lipids of formula B where A is a carbonyl group. [ka]

[0111] Ester group E is R 1 A lipid of formula A, or a pharmaceutically acceptable salt thereof, in which is oriented so that is attached to the oxygen atom of the ester carbonyl, can be represented by the general structure of formula C: [ka]

[0112] Lipids of formula C can be prepared from appropriate ketones of general structure 149 (Scheme 35), where A is either O or S or carbonyl (C=O), by converting the keto group to an ionizable head group of type 1-12. Ketone 148 can then be advantageously prepared from thioacetates of general formula 150. Methods for the synthesis of various thioacetates, where A is either O or S or carbonyl (C=O) in general structure 150, are described in detail in the paragraphs above. [ka]

[0113] Conversion of the thioacetate of general formula 150 to a compound of general structure 149 can be achieved as shown in Scheme 36. Treatment of 150 with, for example, methanol-soluble K2CO2 in the presence of the conjugate ester of general formula 152 results in very rapid acetyl group removal. The normal ester groups present in 150 and 153 undergo transesterification to methyl esters at a rate negligible compared to the thioacetate ester. Thus, the above treatment rapidly liberates the thiolate of general structure 151. The latter undergoes 1,4-addition to 152 to produce 149. The keto group of 149 can then be converted to an ionizable group of type 1-12 by the methods detailed in the above paragraphs and schemes, thus converting ketone 149 to an ionizable lipid of formula C. [ka] Formulating the lipids into a delivery vehicle

[0114] The lipids of the present disclosure can be formulated into a variety of drug delivery vehicles (also referred to herein as "delivery vehicles") known to those skilled in the art. Examples of delivery vehicles include lipid nanoparticles, including liposomes, lipoplexes, lipid-containing polymer nanoparticles, polymer-based nanoparticles, emulsions, and micelles.

[0115] In one embodiment, a lipid having the structure of Formula A of the present disclosure is formulated into a delivery vehicle by mixing it with additional lipids, including helper lipids, such as vesicle-forming lipids, and optionally aggregation-inhibiting lipids, such as hydrophilic polymer-lipid conjugates (e.g., PEG lipids).

[0116] As mentioned above, helper lipids include sterols, diacylglycerols, ceramides, or derivatives thereof.

[0117] Examples of sterols include cholesterol or cholesterol derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol.

[0118] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-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, DSPC-cholesterol conjugate, or a mixture thereof. These lipids can be synthesized or obtained from natural sources such as eggs. DSPC-cholesterol conjugate is a lipid in which one of the acyl chains is replaced with a cholesterol moiety linked to the head group by a succinate linker.

[0119] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[0120] The lipid-incorporated delivery vehicles of the present disclosure can be prepared using a variety of well-described formulation methods known to those skilled 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 an aqueous solution and an organic solution are mixed using a rapid mixer, 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.

[0121] The delivery vehicle can also be a nanoparticle, which is a lipoplex containing a lipid core stabilized by a surfactant. Vesicle-forming lipids can be used as stabilizers. In another embodiment, the lipid nanoparticle is a polymer-lipid hybrid system containing a polymer nanoparticle core surrounded by a stabilizing lipid. The lipid-containing nanoparticles of the present invention can alternatively be prepared from lipid-free polymers. Such nanoparticles can contain a concentrated core of a therapeutic agent surrounded by a polymer shell, or they can contain a solid or liquid dispersed throughout a polymer matrix.

[0122] The lipids described herein can be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or oil cores.Emulsions can be lipid-stabilized.For example, emulsions can contain an oil-filled core stabilized by emulsifying components such as lipid monolayers or bilayers.

[0123] The lipids described herein can be incorporated into micelles, which are self-assembled particles composed of amphiphilic lipid or polymer components that reside within a hydrophobic core and are utilized for drug delivery. Delivery of nucleic acids, genetic material, proteins, peptides, or other charged materials

[0124] The lipids disclosed herein can facilitate the incorporation of compounds or molecules (also referred to herein as "cargo" or "cargo molecules") having a net negative or positive charge into a delivery vehicle and subsequent delivery to target cells in vitro or in vivo.

[0125] In one embodiment, the cargo molecule is genetic material such as a nucleic acid. Nucleic acids include, but are not limited to, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), messenger RNA (mRNA), or DNA, such as vector DNA or linear DNA. The length of the nucleic acid can vary and can include nucleic acids from 5 to 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 acids include antisense oligonucleotides.

[0126] In one embodiment, cargo is mRNA, which comprises the polynucleotide that encodes at least one peptide, polypeptide or protein.MRNA includes but is not limited to small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in WO2022 / 251953A1, which is incorporated herein by reference.

[0127] As used herein, mRNA includes both modified and unmodified mRNA.In one embodiment, mRNA comprises one or more coding regions and non-coding regions.MRNA can be purified from natural sources, or can be produced using recombinant expression systems and optionally purified, or can be chemically synthesized.

[0128] In those embodiments in which mRNA is a chemically synthesized molecule, the mRNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the mRNA may include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deoxyuridine ... zaadenosine, 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); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0129] The mRNA of the present invention can be synthesized according to any of various known methods.For example, in certain embodiments, the mRNA can be synthesized by in vitro transcription (IVT).Briefly, IVT is usually carried out using a linear or circular DNA template, which contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that can contain DTT and magnesium ions, and a suitable RNA polymerase (for example, T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0130] In some embodiments, in vitro synthesized mRNA may be purified prior to encapsulation to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0131] The present disclosure can be used to encapsulate mRNAs of various lengths, in some embodiments, the present disclosure can be used to encapsulate in vitro synthesized mRNAs ranging in length from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0132] Typically, mRNA synthesis involves the addition of a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important for conferring resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from degradation by exonucleases.

[0133] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation (e.g., an iron-responsive element). In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0134] In some embodiments, the 3' untranslated region includes a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides or more in length.

[0135] In a further embodiment, the mRNA is circular. Advantageously, such mRNA lacks 5' and 3' ends, making it less susceptible to exonuclease degradation and therefore more stable in vivo. Circular mRNA can 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. Circular mRNA is translated by a cap-independent translation initiation mechanism.

[0136] In certain embodiments, mRNA provided from an in vitro transcription reaction may be desirable, although other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0137] The mRNA sequence may include a reporter gene sequence, although including a reporter gene sequence in the pharmaceutical formulation for administration is optional. Such sequences may be incorporated into the mRNA for in vitro studies to assess biodistribution or in vivo studies in animal models.

[0138] In another embodiment, cargo is siRNA.SiRNA is incorporated into endogenous cellular machinery, causing mRNA degradation and preventing transcription.Because RNA is easily degraded, its incorporation into delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to target site.

[0139] The siRNAs included in the embodiments of the present disclosure can be used to specifically inhibit the expression of a wide variety of target polynucleotides. siRNA molecules targeting specific polynucleotides can be readily prepared according to procedures known in the art. An siRNA target site can be selected, and the corresponding siRNA can be chemically synthesized, generated by in vitro transcription, or expressed from a vector or PCR product. A wide variety of siRNA molecules can be used to target specific genes or transcripts. siRNAs can be double-stranded RNA or hybrid molecules containing both RNA and DNA (e.g., one RNA strand and one DNA strand). siRNAs can be of various lengths, such as 15-30 nucleotides or 20-25 nucleotides. In certain embodiments, the siRNA is double-stranded and has a 3' or 5' overhang. In certain embodiments, the overhang is UU or dTdT3'. In certain embodiments, the siRNA comprises a stem-loop structure.

[0140] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. MicroRNA (miRNA) is a short non-coding RNA molecule that is transcribed from genomic DNA but is not translated into protein. These RNA molecules are thought to play a role in regulating gene expression by binding to regions of target mRNA. When miRNA binds to target mRNA, gene expression can be downregulated, such as by inducing translational repression, deadenylation, or degradation of the target mRNA. Small nuclear RNA (snRNA) is a longer non-coding RNA molecule that is usually involved in gene splicing. snRNA molecules may play an important role in the treatment of diseases caused by splicing defects.

[0141] In another embodiment, cargo is a DNA vector as described in co-owned and co-pending WO2022 / 251959, which is incorporated herein by reference.DNA vector can be administered to subject for the purpose of repairing, enhancing, inhibiting or reducing the expression of cellular protein or peptide.Therefore, nucleotide polymer can be the nucleotide sequence comprising genomic DNA, cDNA or RNA.

[0142] As those skilled in the art will understand, vectors can encode promoter regions, operator regions, or structural regions.DNA vectors can comprise double-stranded DNA or can be composed of DNA-RNA hybrids.Non-limiting examples of double-stranded DNA include structural genes, genes containing operator control and termination regions, and self-replicating systems such as vector DNA.

[0143] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triplex-forming oligonucleotides. To enhance activity, single-stranded nucleic acids preferably have some or all of the nucleotide bonds replaced with stable non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkylphosphotriester linkages.

[0144] DNA vectors may contain nucleic acids in which one or more sugar moieties and / or one or more pyrimidine or purine bases have been modified. Such sugar modifications may include replacing one or more hydroxyl groups with halogens, alkyl groups, amines, azides, or functionalizing them as ethers or esters. In another embodiment, the entire sugar may be replaced with a sterically and electronically similar structure, including azasugars and carbocyclic sugar analogs. Modifications of purine or pyrimidine base moieties include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substituents known to those skilled in the art.

[0145] In certain embodiments, DNA vectors can be modified with modifying molecules such as peptides, proteins, steroids, or sugar moieties. Modifying DNA vectors with such molecules can facilitate delivery to the desired target site. In some embodiments, such modifications enable the DNA vector to translocate beyond the nucleus of the target cell. As an example, modifiers can be attached to specific portions of the DNA vector (usually portions that do not encode the gene of interest), but they can also contain peptides or other modifiers with nuclear homing effects, such as nuclear localization signals. A non-limiting example of a modifier is the steroid-peptide nucleic acid conjugate described in Rebuffat et al., 2002, Faseb J. 16(11):1426-8, which is incorporated herein by reference. DNA vectors can contain sequences encoding various proteins or peptides. Promoters, enhancers, stress- or chemically-regulated promoters, antibiotic- or nutrient-sensitive regions, and sequences encoding therapeutic proteins can be included as needed. Non-encoding sequences can also be present in DNA vectors.

[0146] The nucleic acids used in the present method can be isolated from natural sources, obtained from sources such 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. Solid-phase synthesis is generally preferred. Detailed descriptions of procedures for solid-phase synthesis of nucleic acids using phosphite triester, phosphate triester, and H-phosphonate chemistries are widely available.

[0147] In one embodiment, the DNA vector is double-stranded DNA and comprises at least 700 base pairs, at least 800 base pairs, at least 900 base pairs, or at least 1000 base pairs.

[0148] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0149] Gene editing systems can also be incorporated into delivery vehicles containing charged lipids. These include Cas9-CRISPR, TALEN, and zinc finger nuclease gene editing systems. In the case of Cas9-CRISPR, guide RNA (gRNA) can be incorporated into the lipid-containing delivery vehicle described herein along with a plasmid or mRNA encoding the Cas9 protein. Optionally, a ribonucleoprotein complex can be incorporated into the lipid-containing delivery vehicle described herein. Similarly, the present disclosure includes embodiments in which genetic material encoding the DNA binding and cleavage domains of zinc finger nuclease or TALEN systems is incorporated into the delivery vehicle along with the lipids disclosed herein.

[0150] While various nucleic acid cargo molecules are described above, it is understood that the above examples are not limiting and that the present disclosure is not to be considered limiting with respect to the particular cargo molecules encapsulated in the delivery vehicles.

[0151] For example, the lipids described herein can also facilitate the incorporation of proteins and peptides into delivery vehicles that include ribonucleoproteins, including both linear and non-linear peptides, proteins, or ribonucleoproteins.

[0152] Although pharmaceutical compositions are described above, the lipids described herein can be ingredients in any nutritional, cosmetic, cleaning, or food product. Pharmaceutical preparations

[0153] The ionizable lipid of the present invention may exist in the form of a salt.The salt is typically a pharmaceutically acceptable salt.Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron (III), ferrous (II), lithium, magnesium, manganese salts, manganese, potassium, sodium, and zinc.In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium, and sodium.Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, etc.

[0154] In some embodiments, the delivery vehicle containing the cargo molecule is part of a pharmaceutical composition and is administered to treat and / or prevent a disease state. Treatment can provide a prophylactic, ameliorative, or therapeutic effect. The pharmaceutical composition is administered in any suitable dose.

[0155] In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intraarterially, intravenously, subcutaneously, or intramuscularly. In yet another embodiment, the pharmaceutical composition is for intratumoral or intrafetal administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinal, intrathecal, or via other topical routes.

[0156] The pharmaceutical composition comprises a pharmaceutically acceptable salt and / or excipient.

[0157] The compositions described herein can be administered to individuals, including patients. As used herein, the term "patient" is not limited and includes a human or non-human subject, and the treatment may be a prophylactic, diagnostic, or therapeutic treatment.

[0158] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Example material

[0159] The lipids 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and 10x phosphate buffered saline (pH 7.4) were purchased from Sigma-Aldrich (St Louis, MO). Ionized amino lipids were synthesized as previously described in WO2022 / 246555, which is incorporated herein by reference.

[0160] Luciferase activity was assayed using mRNA encoding firefly luciferase purchased from APExBIO Technology LLC (Houston, TX). method Preparation of mRNA-containing lipid nanoparticles (LNPs)

[0161] Lipids 1 and 5-35, DSPC, cholesterol, and PEG-DMG described herein were dissolved in ethanol in the appropriate ratios to a final concentration of 10 mM total lipid. Nucleic acids (siRNA or mRNA) were dissolved in an appropriate buffer, such as 25 mM sodium acetate pH 4 or sodium citrate pH 4, to the concentration required to achieve the appropriate amine-to-phosphate ratio. The aqueous and organic solutions were mixed at a flow ratio of 3:1 (v / v; respectively) and a total flow rate of 20 mL / min using the rapid mixing device described in Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni et al., 2017, Nanoscale, 36:133347 (each incorporated herein by reference). The resulting mixture was directly dialyzed against 1000 volumes of PBS pH 7.4. All formulations were concentrated using Amicon™ centrifugal filter units and analyzed using the methods described below. Analysis of LNPs

[0162] Particle size analysis of LNPs in PBS was performed using dynamic light backscattering measurements with a Malvern Zetasizer™ (Worcestershire, UK). Reported particle sizes correspond to number-weighted mean diameters (nm). Total lipid concentration was determined by extrapolation from cholesterol content measured using a Cholesterol E-Total Cholesterol Assay (Wako Diagnostics, Richmond, VA) according to the manufacturer's recommendations. The encapsulation efficiency of the formulations was measured using the Quant-iT RiboGreen™ Assay Kit (Invitrogen, Waltham, MA). Briefly, the total siRNA or mRNA content in solution was measured by dissolving the lipid nanoparticles in a solution of TE containing 2% Triton Tx-100, and the free DNA vector in solution (outside the LNPs) was measured based on RiboGreen™ fluorescence in a Triton-free TE solution. The total siRNA or mRNA content in the formulations was measured using a modified Bligh-Dyer extraction procedure. Briefly, LNP formulations containing siRNA or mRNA were dissolved in a mixture of chloroform, methanol, and PBS to form a single phase, and the absorbance at 260 nm was measured using a spectrophotometer. In vivo analysis in CD-1 mice

[0163] LNP-mRNA encoding firefly luciferase was injected intravenously (tail vein) into 6-8 week-old CD-1 mice. Four hours after injection, the animals were euthanized, and the liver and spleen were removed. The tissues were homogenized in Glo Lysis buffer, and luciferase assays were performed using the Steady Glo Luciferase Assay Kit (according to the manufacturer's recommendations). Organic synthesis of lipids 5-35.

[0164] Unless otherwise specified, all reagents and solvents were commercially available and used without further purification, except for THF (freshly distilled from Na / benzophenone under Ar) and CHCl (freshly distilled from CaH under Ar). "Dry methanol" was freshly distilled from magnesium turnings. All reactions were carried out under an argon atmosphere. The reaction mixture from the aqueous workup was dried by passing it through a plug of anhydrous NaSO held in a filter tube and concentrated under reduced pressure on a rotary evaporator. Thin-layer chromatography was performed on silica gel-coated plates (Merck 60 F254 plates), and column chromatography was performed on 230-400 mesh silica gel. Visualization of the developed chromatograms was performed by staining with I or potassium permanganate solution. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCl3 solution. 1 H NMR spectra were referenced to residual CHCl (7.26 ppm). 13 C NMR spectra were referenced to the CDCl3 triplet centerline (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the δ scale. Multiplicities are reported as "s" (singlet), "d" (doublet), "t" (triplet), "q" (quartet), and "m" (multiplet), and are further modified as "app" (apparent) and "br" (broad). Low- and high-resolution mass spectra (m / z) were acquired in electrospray (ESI) and field desorption / field ionization (FD / FI) modes.

[0165] Lipids 5-15 were synthesized from caprolactone as follows. As previously mentioned, the synthesis of these lipids involves the Claisen condensation of specific esters or lactones under Mukaiyama conditions. This technique is described in co-owned and co-pending WO 2023 / 147657 (incorporated herein by reference). The products of such Claisen reactions are then converted to the final products as described in the scheme above and below. Example 1: Chemical synthesis of ionizable lipids (A) Preparation of building blocks

[0166] (i) 3-(6-hydroxyhexanoyl)oxepan-2-one (61). [ka]

[0167] Titanium tetrachloride (TiCl4) (1.25 mL, 11.39 mmol, 1.3 equiv) was added over 30 min via syringe pump to a cold (-78 °C) well-stirred solution of caprolactone (0.97 mL, 8.77 mmol, 1.0 equiv) and triethylamine (1.8 mL, 13.14 mmol, 1.5 equiv) in dichloromethane (20 mL) under argon. The resulting mixture was warmed to room temperature and stirred for 2 h. The reaction was then quenched by the addition of water (25 mL) at 0 °C. The organic layer was removed, and the aqueous layer was further extracted with dichloromethane (5 × 25 mL). The combined organic layers were washed with brine (saturated solution), dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluting with dichloromethane:acetone (9:1) to give 61 (922 mg, 4.03 mmol, 92%) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 4.39-4.13 (2H, m), 3.67-3.55 (3H, m), 2.61 (1H, dt, J 17.4, 7.4), 2.44 (1H, dt, J 17.4, 7.2), 2.17-1.85 (3H, m), 1.85-1.65 (2H, m), 1.65-1.46 (6H, m, 3 × CH2), 1.36 (2H, m). 13 C NMR (75 MHz, CDCl3) δ 205.0, 173.4, 69.7, 62.8, 56.4, 41.6, 32.6, 28.9, 27.4, 25.3, 25.1, 23.4.

[0168] (ii) 6-oxo-6-(2-oxoxoxepan-3-yl)hexyl 4-methylbenzenesulfonate (62). [ka]

[0169] To a solution of 3-(6-hydroxyhexanoyl)oxepan-2-one (1.0 gm, 4.38 mmol, 1.0 equiv.), pyridine (0.46 mL, 5.69 mmol, 1.3 equiv.), and N,N-dimethylaminopyridine (spatula tip) in dichloromethane (10 mL) was added p-toluenesulfonyl chloride (1.25 g, 6.57 mmol, 1.5 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 5 h, and then the reaction was quenched with water (25 mL). The organic layer was removed, and the aqueous layer was further extracted with dichloromethane (3 × 25 mL). The combined organic layers were washed with brine (saturated solution), dried (NaSO), filtered, and concentrated in vacuo to give 62 (1.40 g, 3.67 mmol, 84%) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 7.75 (2H, d, J=8.3), 7.32 (2 H, d, J =8.1), 4.33 (1H, dd, J = 12.5, 4.1), 4.20 (1H, dd, J = 12.5, 10), 3.98 (2H, t, J = 6.4), 3.60 (1H, dd, J = 11, 2), 2.57 (1H, dd, J = 17.4, 7.3), 2.42 (3H, s), 2.36 (1H, dd, J 17.3, 7.4), 2.13-1.91 (2H, m), 1.80-1.15 (10H, m), 1.36-1.23 (2H, m). 13 C NMR (75 MHz, CDCl3) δ 204.4, 173.2, 144.8, 133.1, 129.9, 127.9, 70.4, 69.5, 56.1, 41.2, 28.7, 28.6, 27.3, 24.9, 24.8, 22.8, 21.6.

[0170] (iii) S-(6-oxo-6-(2-oxoxoxepan-3-yl)hexyl)ethanethioate (63). [ka]

[0171] To a solution of 6-oxo-6-(2-oxoxoxepan-3-yl)hexyl 4-methylbenzenesulfonate (crude product, 1.40 g, nominally 3.67 mmol) and TEA (1.3 mL, 964 mg, 9.5 mmol, 2.6 equiv.) in DMF (10.0 mL) was added thioacetic acid (668 μL, 722 mg, 9.5 mmol, 2.6 equiv.). The mixture was stirred at 60 °C for 16 h, diluted with water (50.0 mL), and extracted with hexanes (3 × 40.0 mL). The combined extracts were washed (brine), dried (NaSO), and concentrated to give 63 (745 mg, 71% over two steps). 1 H NMR (400 MHz, CDCl3) δ 4.33 (dd, J 12.5, 4.1, 1H), 4.20 (dd, J 12.5, 10, 1H), 3.60 (dd, J 11, 2, 1H), 2.84 (t, J = 7.3 Hz, 2H), 2.56 (dd, J 17.4, 7.3, 1H), 2.42 (s, 3H), 2.36 (1 H, dd, J 17.3, 7.4, CH2CO), 2.31 (s, 3H), 2.13-1.91 (2 H, m, CH2), 1.80-1.151 (10 H, m, 4 × CH2), 1.36-1.23 (2 H, m, CH2).

[0172] (iv) 1-hydroxy-11-((2-hydroxyoctyl)thio)undecan-6-one (64). [ka]

[0173] A solution of S-(6-oxo-6-(2-oxoxoxepan-3-yl)hexyl)ethanethioate (23, 760 mg, 2.65 mmol), 2-hexyloxirane (0.486 mL, 3.18 mmol), and NaOH (318 mg, 7.95 mmol) in EtOH (8.00 mL) was stirred at reflux under nitrogen for 4 h. It was then cooled to room temperature, diluted with water (15.0 mL), acidified to pH 2 with concentrated HCl, and extracted with DCM (3 × 20.0 mL). The combined organics were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–75% EtOAc in hexanes) to give 64 (829 mg, 90%). 1 H NMR (400 MHz, CDCl3) δ 3.62 (m, 2H), 3.58 (m, 1H), 2.71 (m, 1H), 2.47-2.32 (m, 7H), 1.68-1.18 (m, 22H), 0.87 (t, J= 7.2 Hz, 3H).

[0174] (v) 11-((2-hydroxyoctyl)thio)-6-oxoundecyl cyclopentadecanecarboxylate (65). [ka]

[0175] To a reaction vial containing the starting cyclopentadecanecarboxylic acid (520 mg, 2.04 mmol) was added CHCl (3 mL), EDCI-HCl (361 mg, 1.88 mmol), and DMAP (192 mg, 1.57 mmol) under an inert atmosphere. Alcohol 64 (500 mg, 1.57 mmol) was then added to the mixture. The mixture was stirred for 18 h and then washed with NaOH (0.1 M, 10 mL). The aqueous phase was back-extracted with CHCl (2 × 10 mL), collected, dried over NaSO, filtered, and evaporated to give the crude product. The product, 0.55 g, 60%, was purified by column chromatography (20% EtOAc / hexanes). 1H NMR (400 MHz, CDCl3) δ 4.04 (td, J = 6.63, 1.77 Hz, 2H), 3.69-3.52 (m, 1H), 2.72 (dt, J = 13.59, 2.77 Hz, 1H), 2.59-2.48 (m, 3H), 2.44-2.31 (m, 6H), 1.76-1.17 (m, 50H), 0.85 (q, J = 9.69 Hz, 3H). LRMS m / z 605 [M+Na] + .

[0176] (vi) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-oxoundecyl cyclopentadecane-carboxylate (54). [ka]

[0177] To a reaction vial containing 3-cyclohexylpropanoic acid (386 mg, 2.47 mmol) was added DCM (3 mL), EDCI-HCl (592 mg, 3.1 mmol), and DMAP (377 mg, 3.1 mmol) under an inert atmosphere. Alcohol 65 (1.2 g, 2.06 mmol) was then added to the mixture. The mixture was stirred for 18 h and then washed with NaHCO (saturated, 2 × 10 mL). The aqueous phase was back-extracted with DCM (2 × 10 mL), collected, dried over NaSO, filtered, and evaporated. The residue was purified by silica chromatography (10% EtOAc / hexanes) to give the desired product (1.0 g, 67%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 5.02-4.82 (m, 1H), 4.04 (t, J = 6.61 Hz, 2H), 2.68-2.57 (m, 2H), 2.52 (m, 2H), 2.43-2.35 (m, 5H), 2.35-2.25 (m, 2H), 1.75-1.46 (m, 21H), 1.44-1.01 (m, 40H), 0.92-0.79 (m, 5H). LRMS m / z 743 [M+Na] + .

[0178] (vii) 11-((2-hydroxyoctyl)thio)-6-oxoundecyl 2-hexyldecanoate (66). [ka]

[0179] To a solution of 64 (500 mg, 1.45 mmol), EDCI-HCl (307 mg, 1.60 mmol), and DMAP (195 mg, 1.60 mmol) in DCM (6.00 mL) at room temperature under a nitrogen atmosphere was added 2-hexyldecanoic acid (372 mg, 1.45 mmol). The reaction was stirred at room temperature for 18 h, concentrated, and the residue was purified by silica chromatography (0–35% EtOAc in hexanes) to give 66 (692 mg, 82%). 1 H NMR (400 MHz, CDCl3) δ 4.06 (t, J = 6.6 Hz, 2H), 3.68-3.57 (m, 1H), 2.73 (dd, J = 13.6, 3.3 Hz, 1H), 2.52 (t, J = 7.3 Hz, 2H), 2.47-2.36 (m, 5H), 2.35- 2.24 (m, 1H), 1.73-1.18 (m, 46H), 0.94-0.82 (m, 9H).

[0180] (viii) 11-((2-(octanoyloxy)octyl)thio)-6-oxoundecyl 2-hexyldecanoate (55). [ka]

[0181] Prepared from 66 and octanoic acid according to the procedure in part (vii) above. 1H NMR (400 MHz, CDCl3) δ 5.02-4.89 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.65-2.61 (m, 2H), 2.53 (td, J = 7.3, 2.1 Hz, 2H), 2.39 (td, J = 7.4, 2.2 Hz, 4H), 2.34-2.26 (m, 3H), 1.71-1.13 (m, 55H), 0.94-0.80 (br t, 12H).

[0182] (ix) 11-((2-((6-methylheptanoyl)oxy)octyl)thio)-6-oxoundecyl 2-hexyldecanoate (56). [ka]

[0183] Prepared from 66 and 6-methylheptanoic acid according to the procedure in part (vii) above. 1 H NMR (400 MHz, CDCl3) δ 5.02-4.89 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.65-2.61 (m, 2H), 2.53 (td, J = 7.3, 2.1 Hz, 2H), 2.39 (td, J = 7.4, 2.2 Hz, 4H), 2.34-2.26 (m, 3H), 1.71-1.13 (m, 53H), 0.94-0.80 (m, 15H).

[0184] (x) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-oxoundecyl 2-hexyldecanoate (57). [ka]

[0185] Prepared from 66 and 3-cyclohexylpropanoic acid according to the procedure in part (vii) above. 1H NMR (400 MHz, CDCl3) δ 5.00-4.84 (m, 1H), 4.05 (t, J = 6.6 Hz, 2H), 2.68-2.57 (m, 1H), 2.57-2.48 (m, 2H), 2.39 (td, J = 7.4, 2.1 Hz, 4H), 2.34-2.25 (m, 4H), 1.78-1.05 (m, 57H), 0.95-0.81 (m, 11H).

[0186] (xi) 1-((11-((2-hexyldecanoyl)oxy)-6-oxoundecyl)thio)octan-2-ylcycloheptane-carboxylate (58). [ka]

[0187] Prepared from 66 and cycloheptanecarboxylic acid according to the procedure in part (vii) above. 1 H NMR (400 MHz, CDCl3) δ 4.93 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.70-2.59 (m, 2H), 2.58-2.53 (m, 4H), 2.52-2.44 (m, 1H), 2.41 (br t, 2H), 2.35-2.24 (m, 1H), 2.01-1.90 (m, 2H), 1.80-1.23 (m, 56H), 0.94-0.82 (m, 9H).

[0188] (xii) 11-((2-(2-cycloheptylacetoxy)octyl)thio)-6-oxoundecyl 2-hexyldecanoate (59). [ka]

[0189] Prepared from 66 and 2-cycloheptylacetic acid according to the procedure in part (vii) above. 1H NMR (400 MHz, CDCl3) δ 5.01-4.90 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.70-2.60 (m, 2H), 2.59-2.50 (m, 4H), 2.49-2.40 (m, 1H), 2.36-2.26 (m, 1H), 2.22 (d, J = 7.3 Hz, 2H), 2.06-1.95 (m, 1H), 1.78-1.15 (m, 59H), 0.91-0.84 (br t, 9H).

[0190] (xiii) 5-pentyloxepan-2-one (71). [ka]

[0191] To a solution of metachloroperoxybenzoic acid (16.24 gm, 94.08 mmol, 2 equiv.) in CHCl (30 mL) at 0° C. was added 4-pentylcyclohexanone (7.9 g, 47.04 mmol, 1.0 equiv.). After stirring at 40° C. for 5 days, the reaction mixture was filtered, washed with saturated aqueous NaSO, saturated aqueous NaHCO, and water, dried over NaSO, and concentrated in vacuo to give 71 (8.6 g, approximately quantitative) as a colorless oil, which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl3) 4.34-4.29 (m, 1H), 4.21-4.15 (m, 1H), 3.70-3.65 (m, 1H), 2.73-2.57 (m, 2H), 2.02-1.90 (m, 2H), 1.63-1.29 (s, 8H), 0.92-0.88 (t, 3H, J = 6.82 Hz).

[0192] (xiv) 3-(4-(2-hydroxyethyl)nonanoyl)-5-pentyloxepan-2-one (72). [ka]

[0193] Prepared from 71 according to the procedure in part (i) above. 1 H NMR (400 MHz, CDCl3) δ 7.81-7.79 (d, 2H, J = 6.96 Hz), 7.38-7.36 (d, 2H, J = 6.96 Hz), 4.41-4.25 (m, 2H), 4.10-4.01 (m, 2H), 3.69-3.66 (m, 1H), 2.65-2.57 (m, 1H), 2.49-2.35 (m, 4H), 2.20-2.16 (m, 1H), 2.02-1.99 (m, 1H),1.61-1.14 (s, 24H), 0.91-0.84 (m, 6H).

[0194] (xv) 3-(3-oxo-3-(2-oxo-5-pentyloxepan-3-yl)propyl)octyl 4-methylbenzenesulfonate (73). [ka]

[0195] Prepared from 72 according to the procedure in part (ii) above. 1 H NMR (400 MHz, CDCl3) δ 7.81-7.79 (d, 2H, J = 6.96 Hz), 7.38-7.36 (d, 2H, J = 6.96 Hz), 4.41-4.25 (m, 2H), 4.10-4.01 (m, 2H), 3.69-3.66 (m, 1H), 2.65-2.57 (m, 1H), 2.49-2.35 (m, 4H), 2.20-2.16 (m, 1H), 2.02-1.99 (m, 1H),1.61-1.14 (s, 24H), 0.91-0.84 (m, 6H).

[0196] (xvi) S-(3-(3-oxo-3-(2-oxo-5-pentyloxepan-3-yl)propyl)octyl)ethanethioate (74).

[0197] [ka]

[0198] Prepared from 73 according to the procedure in part (iii) above. 1 H NMR (400 MHz, CDCl3) δ 4.40-4.23 (m, 2H), 3.69-3.66 (m, 1H), 2.84 (br, J = 7.3 Hz, 2H), 2.65-2.57 (m, 1H), 2.49-2.35 (m, 4H), 2.31 (s, 3H), 2.20-2.16 (m, 1H), 2.02-1.99 (m, 1H),1.61-1.14 (s, 24H), 0.91-0.84 (m, 6H).

[0199] (xvii) 6-(2-hydroxyethyl)-12-(2-((2-hydroxyhexyl)thio)ethyl)heptadecan-9-one (75). [ka] Prepared from 74 and 1-hexene oxide according to the procedure in part (iv) above. 1 H NMR (400 MHz, CDCl3) δ 3.76-3.66 (m, 3H), 2.56-2.52 (m, 2H), 2.49-2.39 (m, 4H), 2.99 (brs, 2H), 1.67-1.27 (s, 34H), 0.95-0.88 (m, 9H).

[0200] (xviii) 6-(2-(((1R,2R)-2-hydroxycyclohexyl)thio)ethyl)-12-(2-hydroxyethyl)heptadecan-9-one (76). [ka]

[0201] Prepared from 74 and cyclohexene oxide according to the procedure in part (iv) above. 1H NMR (400 MHz, CDCl3) δ 3.76-3.65 (m, 2H), 3.35-3.29 (m, 1H), 2.61-2.53 (m, 2H), 2.47-2.34 (m, 4H), 2.19-2.08 (m, 3H), 1.79-1.73 (m, 3H), 1.67-1.43 (s, 11H), 1.32-1.27 (m, 20H), 0.92-0.88 (m, 6H).

[0202] (xix) 1-((9-(2-(octanoyloxy)ethyl)-6-oxo-3-pentyltetradecyl)thio)hexan-2-yl octanoate (67). [ka] A solution of 75 (1.03 g, 2.24 mmol), octanoic acid (806 mg, 5.60 mmol), EDCI-HCl (1.12 g, 5.83 mmol), and DMAP (712 mg, 5.83 mmol) in CHCl (20.0 mL) was stirred at room temperature under nitrogen for 18 h and then concentrated. The residue was purified by silica chromatography (0–10% EtOAc in hexanes) to give 67 (1.4 g, 1.95 mmol, 87%). 1 H NMR (400 MHz, CDCl3) δ 5.03-4.94 (m, 1H), 4.12-4.08 (t, 2H, J = 7.01 Hz), 2.67-2.65 (m, 2H), 2.57-2.51 (m, 2H), 2.43-2.39 (m, 4H), 2.34-2.28 (m, 4H), 1.68-1.27 (m, 55H), 0.93-0.88 (m, 12H).

[0203] (xx) 1-((9-(2-((3-cyclohexylpropanoyl)oxy)ethyl)-6-oxo-3-pentyltetradecyl)thio)-hexan-2-yl 3-cyclohexylpropanoate (68). [ka] Prepared from 75 and 3-cyclohexylpropanoic acid according to the procedure in part (xix) above. 1 H NMR (400 MHz, CDCl3) δ 4.99-4.93 (m, 1H), 4.10-4.08 (t, 2H, J = 7.06 Hz), 2.67-2.65 (m, 2H), 2.56-2.53 (m, 2H), 2.43-2.39 (m, 4H), 2.35-2.29 (m, 4H), 1.73-1.09 (m, 55H), 0.92-0.88 (m, 12H).

[0204] (xxi) 9-(2-(((1R*,2R*)-2-(octanoyloxy)cyclohexyl)thio)ethyl)-6-oxo-3-pentyltetradecyl octanoate (69). [ka]

[0205] Prepared from 76 and octanoic acid according to the procedure in part (xix) above. 1 H NMR (400 MHz, CDCl3) δ 4.80-4.74 (m, 1H), 4.12-4.08 (t, 2H, J = 7.06 Hz), 2.70-2.55 (m, 3H), 2.43-2.29 (m, 8H), 2.11-2.03 (m, 2H), 1.71-1.27 (m, 50H), 0.92-0.88 (m, 12H).

[0206] (xxii) 9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)cyclohexyl)thio)ethyl)-6-oxo-3-pentyltetradecyl 3-cyclohexylpropanoate (70). [ka]

[0207] Prepared from 76 and 3-cyclohexylpropanoic acid according to the procedure in part (xix) above.1 H NMR (400 MHz, CDCl3) δ 4.79-4.73 (m, 1H), 4.11-4.08 (m, 2H), 2.70-2.55 (m, 3H), 2.43-2.29 (m, 7H), 2.11-2.03 (m, 2H), 1.73-1.13 (m, 56H), 0.95-0.87 (m, 9H).

[0208] (xxiii) Methyl 6-(acetylthio)hexanoate (89). [ka]

[0209] To an RBF under an inert atmosphere was added DMF (225 mL) and ethyl 6-bromohexanoate (50.0 g, 40 mL, 224 mmol). The solution was degassed by sparging with N for 10 minutes, and then triethylamine (34.0 g, 44 mL, 336 mmol) and thioacetic acid (20.0 g, 19 mL, 336 mmol) were added sequentially at room temperature (an initial exothermic reaction (approximately 50 °C) was observed upon addition of the thioacetic acid). The mixture was stirred at 60 °C for 1.5 hours, then diluted with water (250 mL) and extracted with hexane (250 mL). The aqueous phase was back-extracted with hexane (2 × 150 mL). The combined organic phase was washed with water (300 mL), dried (NaSO), decolorized with carbon, filtered, and evaporated to give the crude product 89 (51.5 g, 236 mmol, >95% yield), which was carried forward without purification. 1 H NMR (400MHz, CDCl3) d = 4.11 (q, J = 7.2 Hz, 2 H), 2.85 (t, J = 7.3 Hz, 2 H), 2.31 (s, 3 H), 2.28 (t, J = 7.5 Hz, 2 H), 1.67 - 1.53 (m, 4 H), 1.43 - 1.33 (m, 2 H), 1.24 (t, J = 7.2 Hz, 3 H). 13C NMR (101MHz, CDCl3) d = 195.8, 173.5, 60.2, 34.1, 30.6, 29.1, 28.8, 28.2, 24.4, 14.2.

[0210] (xxiv) Methyl 6-((2-hydroxyhexyl)thio)hexanoate (90). [ka]

[0211] To a sealed RBF containing NaOMe (11.7 g, 217 mmol) under an inert atmosphere was added methanol (110 mL). The solution was degassed with N (needle sparged for 20 min), and then crude thioester 89 (25.5 g, 118 mmol) was added via syringe. The mixture was stirred at room temperature for 20 min, then cooled in a water bath while pure 1-hexene oxide (11 g, 110 mmol) was added (the addition of the epoxide was exothermic). The reaction was stirred for 15 min, then quenched with saturated NH Cl (200 mL) and extracted with hexane (300 mL). The layers were separated, and the organic phase was collected. The aqueous phase was back-extracted with hexane (2 × 100 mL). The combined organic phases were dried (Na SO ), filtered, and evaporated to give crude product 90 (27 g, 103 mmol, 87%) as a yellow oil. This product was carried on to the next step without purification. 1 H NMR (400MHz, CDCl3) d = 3.67 (s, 3 H), 3.66 - 3.59 (m, J = 3.5, 8.7 Hz, 1 H), 2.73 (dd, J = 3.3, 13.6 Hz, 1 H), 2.60 (br. s., 1 H), 2.53 (t, J = 7.3 Hz, 2 H), 2.43 (dd, J = 9.0, 13.6 Hz, 1 H), 2.32 (t, J = 7.4 Hz, 2 H), 1.69 - 1.56 (m, 4 H), 1.54 - 1.24 (m, 8 H), 0.88 (t, J = 6.5 Hz, 3 H).

[0212] (xxv) Methyl 6-((2-hydroxyoctyl)thio)hexanoate (91). [ka]

[0213] To a sealed RBF containing NaOMe (23.5 g, 435 mmol) under an inert atmosphere was added methanol (220 mL). The solution was degassed with N (needle sparged for 20 min), and then crude thioester 89 (51.5 g, 236 mmol) was added via syringe. The mixture was stirred at room temperature for 20 min, then cooled in a water bath while neat 1-octane oxide (28 g, 33 mL, 218 mmol) was added (the addition of the epoxide was exothermic). The reaction was stirred for 15 min, then quenched with saturated NH Cl (200 mL) and extracted with hexane (300 mL). The layers were separated, and the organic phase was collected. The aqueous phase was back-extracted with hexane (2 × 200 mL). The combined organic phases were dried (Na SO ), filtered, and evaporated to give crude product 91 (55.5 g, 191 mmol, 81%) as a yellow oil. This product was carried on to the next step without purification. 1 H NMR (400MHz, CDCl3) d = 3.67 (s, 3 H), 3.66 - 3.59 (m, J = 3.5, 8.7 Hz, 1 H), 2.73 (dd, J = 3.3, 13.6 Hz, 1 H), 2.60 (br. s., 1 H), 2.53 (t, J = 7.3 Hz, 2 H), 2.43 (dd, J = 9.0, 13.6 Hz, 1 H), 2.32 (t, J = 7.4 Hz, 2 H), 1.69 - 1.56 (m, 4 H), 1.54 - 1.24 (m, 12 H), 0.88 (t, J = 6.5 Hz, 3 H). 13 C NMR (101MHz, CDCl3) d = 174.0, 69.1, 51.5, 40.2, 36.2, 33.8, 31.9, 31.7, 29.3, 29.3, 28.2, 25.7, 24.4, 22.5, 14.0.

[0214] (xxvi) Methyl 6-((2-((tert-butyldimethylsilyl)oxy)hexyl)thio)hexanoate (92). [ka]

[0215] A solution of 90 (5 g, 19 mmol), TBS-Cl (3.3 g, 22.8 mmol, 1.2 equiv.), and imidazole (1.9 g, 28.5 mmol, 1.5 equiv.) in CHCl (15 mL) was stirred under nitrogen at room temperature for 18 h, at which point TLC and NMR indicated the reaction was complete. The mixture was diluted with NHCl (15 mL), and the CHCl phase was separated and retained. The aqueous phase was extracted with CHCl (2 × 10 mL). The combined organic phases were dried (NaSO) and concentrated to give crude product 92 (6.9 g, 18.3 mmol, >95% yield). This product was carried on to the next step without purification. 1 H NMR (400MHz, CDCl3) d 3.75 (s, 1 H), 3.67 (s, 3 H), 2.58 - 2.50 (m, 4 H), 2.32 (t, J = 7.5 Hz, 2 H), 1.69 - 1.55 (m, 6 H), 1.50 - 1.23 (m, 10 H), 0.93 - 0.85 (m, 8 H), 0.09 - 0.05 (m, 6 H).

[0216] (xxvii) Methyl 6-((2-((tert-butyldimethylsilyl)oxy)octyl)thio)hexanoate (93). [ka]

[0217] A solution of 91 (51.6 g, 178 mmol), TBS-Cl (31.2 g, 213 mmol, 1.2 equiv.), and imidazole (18.1 g, 267 mmol, 1.5 equiv.) in CHCl (180 mL) was stirred under nitrogen at room temperature for 18 h, at which point TLC and NMR indicated the reaction was complete. The mixture was diluted with NHCl (150 mL), and the CHCl phase was separated and retained. The aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic phases were dried (NaSO) and concentrated to give crude product 93 (71.9 g, 177 mmol, >95% yield). This product was carried on to the next step without purification. 1 H NMR (400MHz, CDCl3) d = 3.75 (s, 1 H), 3.67 (s, 3 H), 2.58 - 2.50 (m, 4 H), 2.32 (t, J = 7.5 Hz, 2 H), 1.69 - 1.55 (m, 6 H), 1.50 - 1.23 (m, 10 H), 0.93 - 0.85 (m, 12 H), 0.09 - 0.05 (m, 6 H).

[0218] (xxviii) 1,11-bis((2-hydroxyoctyl)thio)undecan-6-one (97). [ka]

[0219] Under a nitrogen atmosphere, a 9.1 M solution of TiCl4 (50.6 g, 267 mmol) in toluene (29 mL) was added (syringe pump) over 90 min to a chilled (-20 °C), well-stirred solution of crude 93 (72 g, 178 mmol) and Bu3N (59 g, 76 mL, 320 mmol) in toluene (285 mL). After the 90 min addition, TLC and NMR indicated completion. The mixture was removed from the cooling bath and first diluted with hexane (150 mL), followed by quenching by the slow addition of water (150 mL) with rapid stirring (insufficient stirring can cause precipitation of the Ti salt). The layers were separated, and the organic phase was collected. The aqueous phase was extracted with hexane (2 × 150 mL). The combined extracts were washed with brine (1 × 100 mL), dried (Na2SO4), filtered, and concentrated to give crude product 94 as a mixture of keto and enol tautomers. A solution of this crude material (approximately 70 g) in 1,4-dioxane (130 mL), kept under an inert atmosphere, was treated with 7.5 N NaOH (80 mL, 600 mmol) at room temperature. The mixture was stirred at room temperature for 18 h, then acidified to pH 5 with aqueous HCl and heated at 65 °C for 1.5 h, which resulted in decarboxylation of the intermediate beta-keto acid. The layers were separated, and the organic phase was collected. The aqueous phase was extracted with hexane (2 × 150 mL). The combined extracts were washed with brine (1 × 100 mL), dried (NaSO), filtered, and evaporated to give a mixture of 97 and its silylated derivative. Complete desilylation was achieved by redissolving this mixture in DCM (92 mL) and treating the solution with HF-pyridine (18.1 g, 16.5 mL, 183 mmol) at 0 °C. The mixture was stirred at 0 °C for 20 min, then it was quenched with saturated NaHCO3 and extracted with DCM (50 mL). The organic phase was collected. The aqueous phase was extracted with DCM (2 × 50 mL). The combined organic phase was washed with water (1 × 100 mL), 1 N HCl (1 × 100 mL), dried (Na2SO4), filtered, and evaporated to give the crude solid diol. The crude solid was suspended in hot hexane (100 mL), and then EtOAc was added slowly with stirring while heating with a heat gun to give a clear amber solution (final amount of EtOAc added was 50 mL).The solution was cooled in an ice bath to precipitate the diol, which was collected by filtration to give 97 (25.4 g, 52 mmol, 57% yield) as a beige solid. 1 H NMR (400MHz, CDCl3) d 3.67 - 3.59 (m, 2H), 2.73 (dd, J = 3.3, 13.6 Hz, 2H), 2.57 (br. s., 2H), 2.53 (t, J = 7.4 Hz, 4H), 2.47 - 2.37 (m, 6H), 1.66 - 1.22 (m, 32H), 0.89 (t, J = 6.7 Hz, 6H).

[0220] (xxix) 1,11-bis((2-hydroxyhexyl)thio)undecan-6-one (96). [ka]

[0221] Prepared from 92 by procedure xxviii above. 1 H NMR (400MHz, CDCl3) d 3.67-3.59 (m, 2 H), 2.73 (dd, J = 3.3, 13.6 Hz, 2 H), 2.57 (br. s., 2 H), 2.53 (t, J = 7.4 Hz, 4 H), 2.47-2.37 (m, 6 H), 1.66-1.22 (m, 24 H), 0.89 (t, J = 6.7 Hz, 6 H).

[0222] (xxx)1,11-dihydroxyundecan-6-one (100). [ka]

[0223] Titanium tetrachloride (TiCl4) (28.8 mL, 262.8 mmol, 1.3 equiv.) was added dropwise (syringe pump) over 30 min to a cold (-78 °C), well-stirred solution of caprolactone (19.4 mL, 175.2 mmol, 1.0 equiv.) and triethylamine (44 mL, 315.4 mmol, 1.5 equiv.) in dichloromethane (100 mL) under argon. The resulting mixture was allowed to warm to room temperature and stirring was continued for 5 h. The solution was cooled to 0 °C, and water (100 mL) was carefully added. The organic layer was removed, and the aqueous layer was further extracted with dichloromethane:methanol (95:5) (5 × 100 mL). The combined organic layers were evaporated in vacuo. The residue was diluted with 1 M HCl (50 mL), heated at 60 °C for 5 h, and then extracted with dichloromethane:methanol (95:5) (5 × 100 mL). The combined organic layers were washed with brine, dried (NaSO), filtered, and concentrated in vacuo. The solid residue was purified by crystallization from ethyl ether:n-hexane (2:1) to give 100 (16.1 g, 79.6 mmol, 91%) as an off-white solid, mp 57 °C (lit. mp 58.5 °C). 1 H NMR (400 MHz, CDCl3) δ 3.63 (t, J= 6.5 Hz, 4H), 2.41 (t, J= 7.4 Hz, 4H), 1.63-1.52 (m, 8H), 1.38-1.30 (m, 4H). 13 C NMR (100 MHz, CDCl3) δ 211.7, 62.5, 42.6, 32.3, 25.3, 23.4.

[0224] (xxxi) 6-oxoundecane-1,11-diyldimethanesulfonate (101). [ka]

[0225] To a solution of 100 (2.50 g, 12.4 mmol) and TEA (4.31 mL, 30.9 mmol) in DCM (30.0 mL) at 0 °C under nitrogen was added MsCl (2.10 mL, 27.2 mmol). The reaction was warmed to room temperature, stirred for 2 h, diluted with water, and extracted with DCM (3 × 20.0 mL). The combined extracts were dried (NaSO) and concentrated to give 101 (4.5 g, crude), which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl3) δ 4.21 (t, J = 6.4 Hz, 4H), 3.00 (s, 6H), 2.41 (t, J = 7.2 Hz, 4H), 1.81-1.68 (m, 4H), 1.66-1.54 (m, 4H), 1.46-1.34 (m, 4H).

[0226] (xxxii) S,S'-(6-oxoundecane-1,11-diyl)diethanethioate (102). [ka]

[0227] To a solution of 101 (4.5 g, crude) and TEA (2.30 mL, 32.6 mmol) in DMF (20.0 mL) was added thioacetic acid (2.30 mL, 32.6 mmol). The mixture was stirred at 60 °C for 16 h, diluted with water (50.0 mL), and extracted with hexane (3 × 40.0 mL). The combined extracts were washed (brine), dried (NaSO), and concentrated to give 102 (2.46 g, 62% over two steps). 1 H NMR (400 MHz, CDCl3) δ 2.84 (t, J = 7.3 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.31 (s, 6H), 1.56 (m, 8H), 1.39-1.27 (m, 4H).

[0228] (xxxiii) 1,11-bis((2-hydroxyoctyl)thio)undecan-6-one (97) from 102. [ka]

[0229] To a solution of 102 (2.46 g, 7.71 mmol), 2-hexyloxirane (2.94 mL, 19.3 mmol) in EtOH (20.0 mL) at room temperature under nitrogen was added NaOH (1.23 g, 30.8 mmol). The reaction was stirred at reflux for 4 h, cooled to room temperature, diluted with water (40.0 mL), and extracted with DCM (3 × 40.0 mL). The combined organics were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–50% EtOAc in hexanes) to give 97 (3.42 g, 90%). 1 H NMR (400 MHz, CDCl3) δ 3.65 - 3.54 (m, 2H), 2.71 (dd, J = 13.6, 3.3 Hz, 2H), 2.51 (t, J = 7.3 Hz, 4H), 2.47 - 2.32 (m, 6H), 1.70 - 1.18 (m, 32H), 0.92 - 0.82 (m, 6H).

[0230] (xxxiv) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dinonanoate (78). [ka]

[0231] A solution of 97 (1.10 g, 2.24 mmol), nonanoic acid (887 mg, 5.60 mmol), EDCI-HCl (1.12 g, 5.83 mmol), and DMAP (712 mg, 5.83 mmol) in DCM (20 mL) was stirred under nitrogen at room temperature for 18 h and then concentrated. The residue was purified by silica chromatography (0–10% EtOAc in hexanes) to give 78 (1.56 g, 90%). 1H NMR (400 MHz, CDCl3) δ 5.00 - 4.89 (m, 2H), 2.68 - 2.57 (m, 4H), 2.57 - 2.48 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.58 (m, 16H), 1.46 - 1.17 (m, 40H), 0.95 - 0.80 (m, 12H).

[0232] (xxxv) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(hexane-1,2-diyl)bis(3-cyclohexyl-propanoate) (79). [ka]

[0233] Prepared from 96 and 3-cyclohexylpropanoic acid by procedure (xxxiv) above. 1 H NMR (400 MHz, CDCl3) δ 5.16-4.78 (m, 2H), 2.68-2.58 (m, 4H), 2.57 - 2.48 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.2 Hz, 4H), 1.78-1.05 (m, 46H), 0.97-0.78 (m, 10H).

[0234] (xxxvi) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dioctanoate (77). [ka]

[0235] Prepared from 97 and octanoic acid by procedure (xxxiv) above. 1H NMR (400 MHz, CDCl3) δ 4.98 - 4.89 (m, 2H), 2.68 - 2.58 (m, 4H), 2.56 - 2.49 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.76 - 1.02 (m, 52H), 0.87 (t, J = 6.5 Hz, 12H).

[0236] (xxxvii) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (80). [ka]

[0237] Prepared from 97 and 3-cyclohexylpropanoic acid by the procedure in part (xxxvi) above. 1 H NMR (400 MHz, CDCl3) δ 5.16-4.78 (m, 2H), 2.68-2.58 (m, 4H), 2.57-2.48 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.2 Hz, 4H), 1.78-1.05 (m, 54H), 0.97-0.78 (m, 10H).

[0238] (xxxviii) 1-((11-((2-hydroxyoctyl)thio)-6-oxoundecyl)thio)octan-2-ylheptanoate (120). [ka]

[0239] A solution of 97 (1.10 g, 2.24 mmol), heptanoic acid (290 mg, 2.2 mmol), EDCI-HCl (500 g, 2.9 mmol), and DMAP (350 mg, 2.9 mmol) in CHCl (10.0 mL) was stirred at room temperature under nitrogen for 18 h and then concentrated. The residue was purified by silica chromatography (0–20% EtOAc in hexanes) to give 120 (810 mg, 60%). 1 H NMR (400 MHz, CDCl3) δ 4.98-4.89 (m, 1H), 3.65-3.54 (m, 1H), 2.72 (m, 2H), 2.68-2.58 (m, 2H), 2.56-2.49 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 2H), 1.76-1.02 (m, 40H), 0.87 (br t, 9H).

[0240] (xxxix) 1-((11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-oxoundecyl)thio)octan-2-ylheptanoate (81). [ka]

[0241] Prepared from 120 and 3-cyclohexylpropanoic acid according to procedure (xxxviii) above. 1 H NMR (400 MHz, CDCl3) δ 5.16-4.78 (m, 1H), 4.98-4.89 (m, 1H), 2.68-2.58 (m, 4H), 2.57-2.48 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.2 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.78-1.05 (m, 48H), 0.97-0.78 (m, 13H).

[0242] (xl) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dicycloheptane-carboxylate (82). [ka]

[0243] Prepared from 97 and cycloheptanecarboxylic acid by procedure (xxxiv) above. 1 H NMR (400 MHz, CDCl3) δ = 4.93 (dtd, J = 8.2, 6.1, 4.4 Hz, 2H), 2.70-2.59 (m, 4H), 2.58-2.53 (m, 4H), 2.52-2.44 (m, 2H), 2.41 (t, J = 7.4 Hz, 4H), 2.01-1.90 (m, 4H), 1.80-1.23 (m, 52H), 0.89 (td, J = 6.4, 3.0 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ = 210.9, 176.7, 72.5, 45.3, 42.6, 36.0, 33.2, 32.5, 31.7, 31.0, 30.9, 29.4, 29.1, 28.4, 28.3, 28.3, 26.4, 25.3, 23.4, 22.6, 14.1.

[0244] (xli) ((6-oxoundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)-bis(adamantane-1-carboxylate) (83). [ka]

[0245] Prepared from 97 and adamantanecarboxylic acid by procedure (xxxiv) above. 1H NMR (400 MHz, CDCl3) δ = 4.95-4.85 (m, 2H), 2.67-2.56 (m, 4H), 2.53 (t, J = 7.1 Hz, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.01 (s, 6H), 1.89 (d, J = 2.9 Hz, 12H), 1.77-1.65 (m, 14H), 1.63-1.52 (m, 10H), 1.41-1.20 (m, 20H), 0.91-0.85 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ = 211.1, 177.4, 72.4, 42.8, 40.9, 39.0, 36.7, 36.2, 33.2, 32.6, 31.8, 29.5, 29.2, 28.5, 28.1, 25.4, 23.5, 14.2.

[0246] (xlii) 4-((tert-butyldiphenylsilyl)oxy)butan-1-amine (122). [ka]

[0247] A solution of tert-butyl(chloro)diphenylsilane (TBDPSCl; 6.8 g, 24.7 mmol, 1.1 equiv.) in CHCl (4 mL) was added dropwise over 15 min to a well-stirred solution of 4-amino-1-butanol (2.0 g, 22.4 mmol, 1.0 equiv.) and imidazole (3.4 g, 49.3 mmol, 2.2 equiv.) in CHCl (5 mL). The mixture was stirred overnight at room temperature. The reaction mixture was washed sequentially with saturated aqueous NaHCO (2 × 5 mL), water (2 × 5 mL), and saturated aqueous NaCl (2 × 5 mL), then dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give 122 (6.72 g, 92%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.71-7.68 (m, 4H), 7.40-7.36 (m, 6H), 3.70 (t, J=6.0 Hz, 2H), 2.67 (t, J=6.6 Hz, 2H), 1.86 (s, 2H), 1.65-1.48 (m, 4H), 1.09 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ 135.4, 133.8, 129.4, 127.5, 63.6, 41.8, 29.9, 29.8, 26.7, 19.0.

[0248] (xliii) 2-(2-((tert-butyldiphenylsilyl)oxy)ethoxy)ethan-1-amine (131) [ka]

[0249] Prepared from 2-(2-aminoethoxy)ethan-1-ol by procedure (xlii) above. 1 H NMR (400 MHz, CDCl3) δ 7.75-7.64 (m, 4H), 7.45-7.34 (m, 6H), 3.81 (t, J = 5.2 Hz, 2H), 3.57 (m, 2H), 3.48 (t, J = 5.2 Hz, 2H), 2.82 (t, J = 5.2 Hz, 2H), 1.05 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 135.7, 133.9, 129.8, 127.8, 73.6, 72.4, 63.6, 42.1, 26.9, 19.3.

[0250] (xliv) 2-((2-((tert-butyldiphenylsilyl)oxy)ethyl)thio)ethan-1-amine (132). [ka]

[0251] Prepared from 2-((2-aminoethyl)thio)ethan-1-ol by procedure (xlii) above. 1 H NMR (400 MHz, CDCl3) δ 7.74-7.63 (m, 4H), 7.46-7.33 (m, 6H), 3.79 (t, J = 7.1 Hz, 2H), 2.74 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 7.1 Hz, 2H), 2.50 (t, J = 6.3 Hz, 2H), 1.06 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 135.7, 135.0, 129.9, 127.8, 64.0, 41.3, 36.7, 33.8, 26.9, 26.8, 19.3. (xlv) cis-3-((tert-butyldiphenylsilyl)oxy)cyclobutan-1-amine (133). [ka] Prepared from cis-3-aminocyclobutan-1-ol by procedure (xlii) above. 1 H NMR (400 MHz, CDCl3) δ 7.68-7.61 (m, 4H), 7.45-7.34 (m, 6H), 3.86 (tt, J = 7.7, 6.6 Hz, 1H), 2.77 (tt, J = 8.7, 6.8 Hz, 1H), 2.57-2.47 (m, 2H), 1.80-1.70 (m, 2H), 1.03 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 135.6, 134.3, 129.7, 127.7, 61.2, 45.2, 39.7, 26.9, 19.1. (B) Preparation of lipids 5–35. (a) General procedure for reducing ketones to alcohols

[0252] (i) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-hydroxyundecyl cyclopentadecanecarboxylate (121). [ka]

[0253] Solid NaBH4 (13.7 mg, 3.63 mmol) was added to a solution of 54 (200 mg, 0.277 mmol) in EtOH (2 mL) at room temperature under a nitrogen atmosphere. The mixture was stirred for 30 min, after which TLC showed complete conversion. The reaction was quenched with saturated aqueous NH4Cl (2 mL), diluted with water (5 mL), and extracted with CHCl2 (3 × 5 mL). The combined extracts were dried (Na2SO4) and concentrated to give 121 (198 mg, quantitative), which was used in the next step without purification. 1 H NMR (400 MHz, CDCl3) δ 4.99-4.87 (m, 1H), 4.06 (t, J = 6.64 Hz, 2H), 3.69-3.47 (m, 1H), 2.71-2.47 (m, 4H), 2.39 (t, J = 6.68 Hz, 1H), 2.34-2.26 (m, 2H), 1.78-1.09 (m, 66H), 0.98-0.75 (m, 5H). LRMS m / z 745 [M+Na] + . The following compounds were prepared in the same manner:

[0254] (ii) ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dioctanoate. [ka]

[0255] From ketone 77. 1 H NMR (400 MHz, CDCl3) δ 4.99-4.90 (m, 2H), 3.63-3.54 (m, 1H), 2.74-2.47 (m, 8H), 2.30 (t, J = 7.5 Hz, 4H), 1.76-1.17 (m, 56H), 0.94-0.80 (m, 12H).

[0256] (iii) ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dinonanoate. [ka]

[0257] From ketone 78. 1 H NMR (400 MHz, CDCl3) δ 5.02-4.87 (m, 2H), 3.64-3.53 (m, 1H), 2.74-2.45 (m, 8H), 2.30 (t, J = 7.5 Hz, 4H), 1.80-1.17 (m, 60H), 0.92-0.81 (m, 12H).

[0258] (iv) ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(hexane-1,2-diyl)bis(3-cyclohexyl-propanoate). [ka]

[0259] From ketone 79. 1 H NMR (400 MHz, CDCl3) δ 5.02-4.89 (m, 2H), 3.62-3.49 (m, 1H), 2.68-2.57 (m, 4H), 2.57-2.48 (m, 4H), 2.35-2.29 (m, 2H), 1.76-1.04 (m, 52H), 0.95-0.82 (m, 10H).

[0260] (v) ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0261] From ketone 80. 1H NMR (400 MHz, CDCl3) δ 5.02-4.89 (m, 2H), 3.62-3.49 (m, 1H), 2.68-2.57 (m, 4H), 2.57-2.48 (m, 4H), 2.35-2.29 (m, 2H), 1.76-1.04 (m, 60H), 0.95-0.82 (m, 10H). (b) General procedure for converting alcohols to type 1 ionizable head groups.

[0262] (i) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-((4-(dimethylamino)butanoyl)-oxy)-undecyl cyclopentadecanecarboxylate (5). [ka]

[0263] A solution of 121 (198 mg, 0.274 mmol), 4-(dimethylamino)butanoic acid hydrochloride (65.0 mg, 0.388 mmol), EDCI-HCl (99.2 mg, 0.517 mmol), and DMAP (63.2 mg, 0.517 mmol) in CHCl (2.00 mL) was stirred at room temperature under nitrogen for 18 h. The mixture was concentrated, and the residue was purified by silica chromatography (0–5% MeOH in CHCl) to give 5 (130 mg, 57%). 1 H NMR (400 MHz, CDCl3) δ 4.98-4.89 (m, 1H), 4.89-4.79 (m, 1H), 4.03 (t, J = 6.63 Hz, 2H), 2.69-2.54 (m, 2H), 2.54-2.45 (m, 2H), 2.43-2.23 (m, 7H), 2.21 (s, 6H), 1.81-1.44 (m, 16H), 1.44-1.03 (m, 51H), 0.96-0.80 (m, 5H). LRMS m / z 836 [M+H]+.

[0264] The following compounds were prepared in the same manner:

[0265] (ii) ((6-((4-(dimethylamino)butanoyl)oxy)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dioctanoate (16). [ka]

[0266] From ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dioctanoate. 1 H NMR (400 MHz, C6D6) δ 5.28 - 5.19 (m, 2H), 5.12 - 5.03 (m, 1H), 2.76 - 2.43 (m, 8H), 2.36 (t, J = 7.3 Hz, 2H), 2.31 - 2.21 (m, 4H), 2.16 (t, J = 6.9 Hz, 2H), 2.05 (s, 6H), 1.80 (p, J = 7.1 Hz, 2H), 1.74 - 1.11 (m, 56H), 0.97 - 0.84 (m, 12H).

[0267] (iii) ((6-((4-(dimethylamino)butanoyl)oxy)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dinonanoate (17). [ka]

[0268] From ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl) dinonanoate. 1H NMR (400 MHz, C6D6) δ 5.41-5.29 (m, 2H), 5.23-5.13 (m, 1H), 2.88-2.54 (m, 8H), 2.46 (t, J = 7.3 Hz, 2H), 2.43-2.30 (m, 4H), 2.26 (t, J = 6.9 Hz, 2H), 2.15 (s, 6H), 1.90 (p, J = 7.1 Hz, 2H), 1.85-1.25 (m, 60H), 1.08-0.92 (m, 12H).

[0269] (iv) ((6-((4-(dimethylamino)butanoyl)oxy)undecane-1,11-diyl)bis(sulfanediyl))bis(hexane-1,2-diyl)bis(3-cyclohexylpropanoate) (18). [ka]

[0270] From ((6-hydroxyundecane-1,11-diyl)bis-(sulfanediyl))bis(hexane-1,2-diyl) bis(3-cyclohexyl-propanoate). 1 H NMR (400 MHz, C6D6) δ 5.29-5.18 (m, 2H), 5.12-5.03 (m, 1H), 2.74-2.44 (m, 8H), 2.37 (t, J = 7.3 Hz, 2H), 2.33-2.26 (m, 4H), 2.16 (t, J = 6.9 Hz, 2H), 2.05 (s, 6H), 1.80 (p, J = 7.1 Hz, 3H), 1.76-1.00 (m, 49H), 0.89 (t, J = 6.8 Hz, 6H), 0.84-0.72 (m, 4H).

[0271] (v) ((6-((4-(dimethylamino)butanoyl)oxy)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (19). [ka]

[0272] From ((6-hydroxyundecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexyl-propanoate). 1 H NMR (400 MHz, C6D6) δ 5.29-5.18 (m, 2H), 5.12-5.03 (m, 1H), 2.74-2.44 (m, 8H), 2.37 (t, J = 7.3 Hz, 2H), 2.33-2.26 (m, 4H), 2.16 (t, J = 6.9 Hz, 2H), 2.05 (s, 6H), 1.80 (p, J = 7.1 Hz, 3H), 1.76-1.00 (m, 57H), 0.89 (t, J = 6.8 Hz, 6H), 0.84-0.72 (m, 4H).

[0273] (vi) ((6-((1-methylazetidine-3-carbonyl)oxy)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (20). [ka]

[0274] From ((6-hydroxyundecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexyl-propanoate) and 1-methylazetidine-3-carboxylic acid. 1H NMR (400 MHz, CDCl3) δ 4.97-4.90 (m, 2H), 4.87 (p, J = 6.4 Hz, 1H), 3.59-3.48 (m, 2H), 3.30-3.20 (m, 3H), 2.68-2.57 (m, 4H), 2.57-2.46 (m, 4H), 2.31 (t, J = 7.6 Hz, 4H), 2.30 (s, 3H), 1.76-1.46 (m, 25H), 1.42-1.06 (m, 32H), 0.94-0.82 (m, 10H). 13 C NMR (101 MHz, CDCl3) δ 173.8, 172.9, 74.5, 72.8, 58.8, 45.9, 37.2, 36.0, 34.0, 33.2, 33.0, 32.6, 32.4, 32.2, 31.7, 29.5, 29.1, 28.7, 26.6, 26.3, 25.3, 25.0, 22.6, 14.1. (b) General procedure for the reductive amination of ketones with primary amines.

[0275] (i) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-11-((2-(octanoyloxy)octyl)thio)-undecyl 2-hexyldecanoate (123). [ka]

[0276] A solution of 55 (460 mg, 0.648 mmol), amine 122 (318 mg, 0.972 mmol), sodium triacetoxyborohydride (247 mg, 1.17 mmol), and HOAc (1 drop) in 1,2-dichloroethane (4 mL) was stirred at room temperature under nitrogen for 18 h. The reaction was quenched with saturated aqueous NaHCO (2.00 mL), diluted with water (4.00 mL), and extracted with CHCl (3 × 5 mL). The combined extracts were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in CHCl) to give 123 (536 mg, 81%). 1H NMR (400 MHz, CDCl3) δ 7.69-7.62 (m, 4H), 7.46-7.33 (m, 6H), 5.03-4.88 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.66 (t, J = 5.9 Hz, 2H), 2.73-2.49 (m, 6H), 2.49-2.41 (m, 1H), 2.35-2.24 (m, 3H), 1.71-1.16 (m, 64H), 1.04 (s, 9H), 0.95-0.77 (m, 12H).

[0277] The following compounds were prepared in the same manner:

[0278] (ii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-11-((2-((6-methylheptanoyl)oxy)-octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0279] From 56 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.63 (m, 4H), 7.49-7.33 (m, 6H), 4.99-4.91 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.66 (t, J = 6.0 Hz, 2H), 2.68-2.60 (m, 2H), 2.58-2.49 (m, 4H), 2.47-2.39 (m, 1H), 2.37-2.27 (m, 3H), 1.80-1.13 (m, 61H), 1.04 (s, 9H), 0.92-0.84 (m, 15H).

[0280] (iii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-11-((2-((3-cyclohexylpropanoyl)oxy)-octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0281] From 57 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.72-7.63 (m, 4H), 7.47-7.33 (m, 6H), 4.99-4.89 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.74-3.61 (m, 2H), 2.74-2.40 (m, 7H), 2.40-2.23 (m, 3H), 1.75-1.09 (m, 65H), 1.04 (s, 9H), 0.94-0.82 (m, 11H).

[0282] (iv) 10-(5-((2-hexyldecanoyl)oxy)pentyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-yl cycloheptanecarboxylate. [ka]

[0283] From 58 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.61 (m, 4H), 7.49-7.34 (m, 6H), 4.99-4.87 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 5.9 Hz, 2H), 2.69-2.40 (m, 8H), 2.37-2.26 (m, 1H), 1.99-1.18 (m, 66H), 1.04 (s, 9H), 0.91-0.84 (m, 9H).

[0284] (v) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-11-((2-(2-cycloheptylacetoxy)-octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0285] From 59 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.70-7.64 (m, 4H), 7.44-7.32 (m, 6H), 5.02-4.90 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 5.9 Hz, 2H), 2.70-2.60 (m, 2H), 2.59-2.50 (m, 4H), 2.49-2.40 (m, 1H), 2.36-2.26 (m, 1H), 2.22 (d, J = 7.3 Hz, 2H), 2.06-1.95 (m, 1H), 1.78-1.15 (m, 66H), 1.04 (s, 9H), 0.91-0.84 (m, 9H).

[0286] (vi) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-11-((2-((3-cyclohexylpropanoyl)-oxy)octyl)thio)undecyl cyclopentadecanecarboxylate. [ka]

[0287] From 54 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.58 (m, 4H), 7.55-7.31 (m, 6H), 5.13-4.79 (m, 1H), 4.05 (t, J = 6.64 Hz, 2H), 3.83-3.48 (m, 2H), 2.63 (dd, J = 6.12, 2.16 Hz, 2H), 2.60-2.35 (m, 5H), 2.31 (dd, J = 8.41, 7.19 Hz, 2H), 1.76-1.48 (m, 21H), 1.45-1.16 (m, 50H), 1.04 (s, 9H), 0.94-0.79 (m, 5H). LRMS m / z 1032 [M+H]+.

[0288] (vii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-((2-(octanoyloxy)hexyl)-thio)ethyl)-3-pentyltetradecyl octanoate. [ka]

[0289] From 67 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.68-7.66 (m, 4H), 7.44-7.38 (m, 6H,), 4.97 (brs, 1H), 4.11-4.07 (m, 2H), 3.69-3.68 (m, 2H), 2.66-2.54 (m, 5H), 2.34-2.27 (m, 4H), 1.64-1.26 (m, 62H), 1.06 (m, 9H),0.91-0.88 (m, 15H).

[0290] (viii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-((2-((3-cyclohexylpropan-oyl)oxy)hexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. [ka]

[0291] From 68 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.68-7.66 (m, 4H), 7.44-7.37 (m, 6H,), 5.00 (m, 1H), 4.11-4.07 (m, 2H), 3.69-3.68 (m, 2H), 2.66-2.54 (m, 6H), 2.35-2.29 (m, 4H), 1.72-1.13 (m, 64H), 1.06 (m, 9H), 0.91-0.88 (m, 12H).

[0292] (ix) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyl-oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl octanoate. [ka]

[0293] From 69 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.68-7.66 (m, 4H), 7.44-7.37 (m, 6H,), 4.85-4.77 (m, 1H), 4.11-4.07 (m, 2H), 3.69 (br t, 2H), 2.68-2.52 (m, 5H), 2.35-2.29 (m, 4H), 1.72-1.13 (m, 63H), 1.06 (br s, 9H), 0.91-0.88 (m, 12H).

[0294] (x) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-(((1R*,2R*)-2-((3-cyclohexyl-propanoyl)oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. [ka]

[0295] From 70 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.68-7.66 (m, 4H), 7.44-7.37 (m, 6H,), 4.85-4.77 (m, 1H), 4.11-4.07 (m, 2H), 3.69-3.68 (m, 2H), 2.68-2.52 (m, 5H), 2.35-2.29 (m, 4H), 1.72-1.13 (m, 65H), 1.06 (br s, 9H), 0.91-0.88 (m, 10H).

[0296] (xi) ((6-((4-(tert-butyldiphenylsilyloxy)butyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dinonanoate. [ka]

[0297] From 78 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.64 (m, 4H), 7.48-7.32 (m, 6H), 4.96 (q, J = 6.5 Hz, 2H), 3.76-3.55 (m, 2H), 2.69-2.41 (m, 11H), 2.30 (t, J = 7.5 Hz, 4H), 1.42 (d, J = 115.3 Hz, 64H), 1.04 (s, 9H), 0.91-0.84 (m, 12H).

[0298] (xii) ((6-((4-(tert-butyldiphenylsilyloxy)butyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dioctanoate. [ka]

[0299] From 77 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.69-7.63 (m, 4H), 7.46-7.33 (m, 6H), 5.03-4.87 (m, 2H), 3.77-3.61 (m, 2H), 2.72-2.42 (m, 11H), 2.30 (t, J = 7.5 Hz, 4H), 1.84-1.14 (m, 60H), 1.04 (s, 9H), 0.92-0.81 (m, 12H).

[0300] (xiii) ((6-((4-(tert-butyldiphenylsilyloxy)butyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0301] From 80 and 122. 1 H NMR (400 MHz, C6D6) δ 7.88-7.79 (m, 4H), 7.33-7.24 (m, 6H), 5.30-5.19 (m, 2H), 3.73 (t, J = 6.3 Hz, 2H), 2.76-2.48 (m, 10H), 2.46-2.39 (m, 1H), 2.35-2.22 (m, 4H), 1.80-1.01 (m, 71H), 0.94-0.85 (m, 6H), 0.84-0.70 (m, 4H).

[0302] (xiv) ((6-((2-(2-tert-butyldiphenylsilyloxy)ethyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0303] From 80 and 131. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.64 (m, 4H), 7.45-7.33 (m, 6H), 4.98-4.89 (m, 2H), 3.81-3.77 (m, 2H), 3.59-3.55 (m, 4H), 2.76 (t, J = 5.2 Hz, 2H), 2.67-2.57 (m, 4H), 2.56-2.48 (m, 5H), 2.34-2.27 (m, 4H), 1.75-1.09 (m, 58H), 1.04 (s, 9H), 0.93-0.82 (m, 10H).

[0304] (xv) ((6-((2-((2-tert-butyldiphenylsilyloxy)thio)ethyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0305] From 80 and 132. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.64 (m, 4H), 7.45-7.33 (m, 6H), 4.98-4.89 (m, 2H), 3.81-3.77 (m, 2H), 2.76 (br t, J = 5.2, 2H), 2.67-2.57 (m, 8H), 2.56-2.48 (m, 5H), 2.34-2.27 (m, 4H), 1.75-1.09 (m, 58H), 1.04 (s, 9H), 0.93-0.82 (m, 10H).

[0306] (xvi) 10-(5-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)pentyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-ylheptanoate. [ka]

[0307] From 81 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.69-7.64 (m, 4H), 7.45-7.34 (m, 4H), 5.02-4.87 (m, 2H), 3.73-3.63 (m, 2H), 2.74-2.44 (m, 11H), 2.37-2.25 (m, 4H), 1.85-1.10 (m, 61H), 1.04 (s, 9H), 0.93-0.83 (m, 11H).

[0308] (xvii) ((6-((4-tert-butyldiphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dicycloheptanecarboxylate. [ka]

[0309] From 82 and 122. 1 H NMR (400 MHz, CDCl3) δ 7.85-7.77 (m, 4H), 7.30-7.23 (m, 6H), 5.27-5.17 (m, 2H), 3.73 (t, J = 6.2 Hz, 2H), 2.75-2.46 (m, 12H), 2.43 (s, 1H), 2.09-1.94 (m, 4H), 1.91-1.16 (m, 69H), 0.89 (t, J = 6.8 Hz, 6H).

[0310] (xviii) ((6-((4-tert-butyldiphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)-bis(adamantane-1-carboxylate). [ka]

[0311] From 83 and 122. 1H NMR (400 MHz, CDCl3) δ 7.84-7.79 (m, 4H), 7.30-7.24 (m, 6H), 5.22 (dt, J = 10.6, 5.4 Hz, 2H), 3.73 (t, J = 6.2 Hz, 2H), 2.74-2.49 (m, 10H), 2.46-2.38 (m, 1H), 2.10-2.06 (m, 12H), 1.91-1.86 (m, 6H), 1.77-1.18 (m, 52H), 1.21 (s, 9H), 0.92-0.86 (m, 6H).

[0312] (xix) ((6-cis-((3-((tert-butyldiphenylsilyl)oxy)cyclobutyl)amino)undecane-1,11-diyl)-bis(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0313] From 89 and 133. 1 H NMR (400 MHz, CDCl3) δ 7.67-7.62 (m, 4H), 7.44-7.33 (m, 6H), 5.00-4.89 (m, 2H), 3.96-3.88 (m, 1H), 2.72-2.57 (m, 4H), 2.57-2.41 (m, 6H), 2.35-2.27 (m, 4H), 1.87-1.06 (m, 62H), 1.02 (s, 9H), 0.93-0.80 (m, 10H). (c) General procedure for reductive methylation of secondary amines.

[0314] (i) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-(octanoyloxy)-octyl)thio)undecyl 2-hexyldecanoate (124). [ka]

[0315] A solution of 123 (274 mg, 0.268 mmol), sodium triacetoxyborohydride (284 mg, 1.34 mmol), and aqueous formaldehyde (37%, 0.75 mL) in THF (2 mL) was stirred under nitrogen for 2 days. The reaction was quenched with saturated aqueous NaHCO (2.00 mL), diluted with water (5 mL), and extracted with CHCl (3 × 5.00 mL). The combined organics were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in CHCl) to give 124 (217 mg, 78%). 1 H NMR (400 MHz, CDCl3) δ 7.69-7.62 (m, 4H), 7.46-7.33 (m, 6H), 5.03-4.88 (m, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.66 (t, J = 5.9 Hz, 2H), 2.73-2.49 (m, 6H), 2.49-2.41 (m, 1H), 2.35-2.24 (m, 3H), 2.12 (s. 3H). 1.71-1.16 (m, 64H), 1.04 (s, 9H), 0.95-0.77 (m, 12H).

[0316] The following compounds were prepared in the same manner:

[0317] (ii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-((6-methylheptan-oyl)oxy)octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0318] From 6-((4-((tert-butyldiphenylsilyl)oxy)-butyl)-amino)-11-((2-((6-methylheptanoyl)oxy)octyl)-thio)undecyl 2-hexyldecanoate. 1H NMR (400 MHz, CDCl3) δ 7.71-7.60 (m, 4H), 7.46-7.32 (m, 6H), 5.00-4.91 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.69-2.58 (m, 2H), 2.53 (td, J = 7.2, 1.3 Hz, 2H), 2.38-2.26 (m, 6H), 2.12 (s, 3H), 1.74-1.11 (m, 61H), 1.04 (s, 9H), 0.92-0.83 (m, 15H).

[0319] (iii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-((3-cyclohexyl-propanoyl)oxy)octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0320] From 6-((4-((tert-butyldiphenylsilyl)-oxy)butyl)amino)-11-((2-((3-cyclohexylpropanoyl)oxy)-octyl)thio)-undecyl 2-hexyldecanoate. 1 H NMR (400 MHz, CDCl3) δ 7.73-7.62 (m, 4H), 7.43-7.32 (m, 6H), 5.01-4.90 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.68-2.59 (m, 2H), 2.59-2.49 (m, 2H), 2.38-2.23 (m, 6H), 2.13 (s, 3H), 1.84-1.11 (m, 65H), 1.04 (s, 9H), 0.92-0.78 (m, 12H).

[0321] (iii) 10-(5-((2-hexyldecanoyl)oxy)pentyl)-2,2,9-trimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-yl cycloheptanecarboxylate. [ka]

[0322] From 10-(5-((2-hexyldecanoyl)oxy)-pentyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetra-cosan-18-yl cycloheptanecarboxylate. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.61 (m, 4H), 7.49-7.34 (m, 6H), 4.99-4.87 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 5.9 Hz, 2H), 2.69-2.40 (m, 8H), 2.37-2.26 (m, 1H), 2.13 (s, 3H), 1.99-1.18 (m, 66H), 1.04 (s, 9H), 0.91-0.84 (m, 9H).

[0323] (iv) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-(2-cycloheptyl-acetoxy)octyl)thio)undecyl 2-hexyldecanoate. [ka]

[0324] From 6-((4-((tert-butyldiphenylsilyl)oxy)-butyl)amino)-11-((2-(2-cycloheptylacetoxy)octyl)-thio)undecyl 2-hexyldecanoate. 1H NMR (400 MHz, CDCl3) δ 7.73-7.61 (m, 4H), 7.46-7.34 (m, 6H), 5.00-4.91 (m, 1H), 4.05 (t, J = 6.7 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.72-2.59 (m, 2H), 2.57-2.48 (m, 2H), 2.40-2.26 (m, 4H), 2.22 (d, J = 7.3 Hz, 2H), 2.12 (s, 3H), 2.06-1.95 (m, 1H), 1.78-1.12 (m, 66H), 1.04 (s, 9H), 0.93-0.83 (m, 9H).

[0325] (v) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-((3-cyclohexyl-propanoyl)oxy)octyl)thio)undecyl cyclopentadecanecarboxylate. [ka]

[0326] From 6-((4-((tert-butyldiphenyl-silyl)oxy)butyl)amino)-11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-undecyl cyclopenta-decanecarboxylate. 1 H NMR (400 MHz, CDCl3) δ 7.78-7.54 (m, 4H), 7.48-7.28 (m, 6H), 5.03-4.81 (m, 1H), 4.04 (t, J = 6.66 Hz, 2H), 3.81-3.45 (m, 2H), 2.63 (dd, J = 6.14, 2.09 Hz, 2H), 2.53 (t, J = 7.19 Hz, 2H), 2.44-2.27 (m, 5H), 2.12 (s, 3H), 1.76-1.44 (m, 20H), 1.44-1.09 (m, 50H), 1.04 (s, 9H), 0.95-0.80 (m, 5H). LRMS m / z 1048 [M+H]+ .

[0327] (vi) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-((2-(octanoyloxy)-hexyl)thio)ethyl)-3-pentyltetradecyl octanoate. [ka]

[0328] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)-amino)-9-(2-((2-(octanoyloxy)hexyl)thio)-ethyl)-3-pentyltetradecyl octanoate. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.25-5.24 (m, 1H), 4.30-4.17 (m, 2H), 3.80-3.45 (m, 2H), 2.91 (brs, 1H), 2.80-2.59 (m, 6H), 2.42 (br s, 3H), 2.34-2.23 (m, 4H), 1.77-1.21 (m, 60H), 1.04 (br s, 9H), 0.99-0.95 (br t, 6H), 0.90-0.86 (br t, 9H).

[0329] (vii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-((2-((3-cyclohexyl-propanoyl)oxy)hexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. [ka]

[0330] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-((2-((3-cyclohexylpropanoyl)oxy)-hexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.28-5.22 (m, 1H), 4.30-4.20 (m, 2H), 3.80-3.45 (m, 2H), 2.93 (brs, 1H), 2.78-2.64 (m, 5H), 2.46 (brs, 3H), 2.34-2.27 (m, 3H), 1.78-0.75 (m, 77H); 1.04 (br s, 9H)

[0331] (viii) 1-((9-(2-((3-cyclohexylpropanoyl)oxy)ethyl)-6-((4-hydroxybutyl)(methyl)-amino)-3-pentyltetradecyl)thio)hexan-2-yl 3-cyclohexylpropanoate (13). [ka]

[0332] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-((2-((3-cyclohexyl-propanoyl)-oxy)hexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. 1 H NMR (400 MHz, C6D6) δ 5.28-5.22 (m, 1H), 4.30-4.20 (m, 2H), 3.66-3.63 (m, 2H), 2.93 (brs, 1H), 2.78-2.64 (m, 5H), 2.46 (brs, 3H), 2.34-2.27 (m, 3H), 1.78-0.75 (m, 77H).

[0333] (viii) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyl-oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl octanoate. [ka]

[0334] From 6-((4-((tert-butyldiphenylsilyl)-oxy)butyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyloxy)cyclohexyl)thio)-ethyl)-3-pentyltetradecyl octanoate.

[0335] 1 H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.13-5.09 (m, 1H), 4.30-4.14 (m, 2H), 3.78-3.44 (m, 2H), 2.90 (brs, 1H), 2.77-2.58 (m, 4H), 2.41-2.23 (m, 6H), 2.13-2.09 (m, 2H),1.70-1.21 (m, 62H), 1.04 (br s, 9H), 0.98-0.95 (m, 6H), 0.91-0.86 (m, 6H).

[0336] (ix) 6-((4-hydroxybutyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyloxy)cyclohexyl)-thio)ethyl)-3-pentyltetradecyl octanoate (14). [ka]

[0337] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)-(methyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyloxy)cyclo-hexyl)thio)ethyl)-3-pentyltetradecyl octanoate. 1 H NMR (400 MHz, C6D6) δ 5.13-5.09 (m, 1H), 4.30-4.14 (m, 2H), 3.63-3.60 (m, 2H), 2.90 (brs, 1H), 2.77-2.58 (m, 4H), 2.41-2.23 (m, 6H), 2.13-2.09 (m, 2H),1.70-1.21 (m, 62H), 0.98-0.95 (m, 6H), 0.91-0.86(m, 6H).

[0338] (ix) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. [ka]

[0339] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-9-(2-(((1R*,2R*)-2-((3-cyclohexyl-propanoyl)oxy)-cyclohexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.12-5.05 (m, 1H), 4.29-4.17 (m, 2H), 3.79-3.45 (m, 2H), 2.89-2.24 (m, 11H), 2.14-2.08 (m, 2H), 1.67-1.05 (m, 64H), 1.04 (br s, 9H), 0.98-0.95 (m, 6H), 0.91-0.76 (m, 4H).

[0340] (x) 9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)cyclohexyl)thio)ethyl)-6-((4-hydroxy-butyl)(methyl)amino)-3-pentyltetradecyl 3-cyclohexylpropanoate (15). [ka]

[0341] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)-cyclohexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropan-oate. 1 H NMR (400 MHz, C6D6) δ 5.12-5.05 (m, 1H), 4.29-4.17 (m, 2H), 3.64-3.60 (m, 2H), 2.89-2.24 (m, 11H), 2.14-2.08 (m, 2H), 1.67-1.05 (m, 62H), 0.98-0.95 (m, 6H), 0.91-0.76 (m, 4H).

[0342] (x) ((6-((4-(tert-butyldiphenylsilyloxybutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)dinonanoate. [ka]

[0343] From ((6-((4-(tert-butyldiphenylsilyloxy)butyl)-amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dinonanoate. 1H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.00-4.91 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.72-2.57 (m, 4H), 2.57-2.48 (m, 4H), 2.39-2.26 (m, 7H), 2.12 (s, 3H), 1.84-1.11 (m, 64H), 1.04 (s, 9H), 0.97-0.83 (m, 12H).

[0344] (xi) ((6-((4-(tert-butyldiphenylsilyloxybutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)dioctanoate. [ka]

[0345] From ((6-((4-(tert-butyldiphenylsilyloxy)butyl)-amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dioctanoate. 1 H NMR (400 MHz, CDCl3) δ 7.73-7.59 (m, 4H), 7.47-7.32 (m, 6H), 5.06-4.88 (m, 2H), 3.66 (t, J = 6.2 Hz, 2H), 2.72-2.57 (m, 4H), 2.53 (dd, J = 7.9, 6.3 Hz, 4H), 2.38-2.25 (m, 7H), 2.12 (s, 3H), 1.76-1.10 (m, 60H), 1.04 (s, 9H), 0.94-0.83 (m, 12H).

[0346] (xii) ((6-((4-(tert-butyldiphenylsilyloxybutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0347] From ((6-((4-(tert-butyldiphenylsilyl-oxy)butyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexyl-propanoate). 1 H NMR (400 MHz, C6D6) δ 7.90-7.75 (m, 4H), 7.31-7.25 (m, 6H), 5.32-5.19 (m, 2H), 3.74 (t, J = 6.2 Hz, 2H), 2.76-2.48 (m, 8H), 2.43-2.34 (m, 3H), 2.33-2.24 (m, 4H), 2.13 (s, 3H), 1.82-0.98 (m, 71H), 0.96-0.86 (m, 6H), 0.83-0.71 (m, 4H).

[0348] (xiii) ((6-((2-(2-tert-butyldiphenylsilyloxy)ethoxy)ethyl)(methyl)amino)undecane-1,11-diyl)-bis(sulfanediyl))bis(octane-1,2-diyl) bis(3-cyclohexylpropanoate). [ka]

[0349] From ((6-((2-(2-tert-butyldiphenylsilyloxy)ethyl)amino)undecane-1,11-diyl)bis(sulfanediyl))-bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1H NMR (400 MHz, CDCl3) δ 7.70-7.62 (m, 4H), 7.46-7.32 (m, 6H), 4.95 (dtd, J = 8.1, 6.1, 4.4 Hz, 2H), 3.83-3.76 (m, 2H), 3.60-3.54 (m, 2H), 3.50 (t, J = 6.6 Hz, 2H), 2.69-2.58 (m, 4H), 2.58-2.46 (m, 7H), 2.31 (dd, J = 8.4, 7.1 Hz, 4H), 2.21 (s, 3H), 1.74-1.09 (m, 58H), 1.05 (s, 9H), 0.94-0.82 (m, 10H).

[0350] (xiv) ((6-((2-((2-tert-butyldiphenylsilyloxy)thio)ethyl)(methyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0351] From ((6-((2-((2-tert-butyldiphenylsilyloxy)thio)ethyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, CDCl3) δ 7.70-7.65 (m, 4H), 7.44-7.34 (m, 6H), 4.99-4.90 (m, 2H), 3.79 (t, J = 7.1 Hz, 2H), 2.70-2.60 (m, 6H), 2.54 (dd, J = 7.3, 1.6 Hz, 4H), 2.51 (s, 3H), 2.34-2.27 (m, 4H), 1.77-1.08 (m, 63H), 1.05 (s, 9H), 0.93-0.82 (m, 10H).

[0352] (xv) ((6-((4-tert-butyldiphenylsilyloxybutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)dicycloheptanecarboxylate. [ka]

[0353] From ((6-((4-tert-butyldiphenyl-silyloxybutyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dicycloheptane-carboxylate. 1 H NMR (400 MHz, CDCl3) δ 7.85-7.79 (m, 4H), 7.30-7.24 (m, 6H), 5.27-5.18 (m, 2H), 3.75 (t, J = 6.2 Hz, 2H), 2.74-2.48 (m, 10H), 2.42-2.33 (m, 3H), 2.13 (s, 3H), 2.07-1.95 (m, 4H), 1.90-1.52 (m, 20H), 1.46-1.12 (m, 50H), 0.89 (t, J = 6.8 Hz, 6H).

[0354] (xvi) ((6-((4-tert-butyldiphenylsilyloxybutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)-bis(adamantane-1-carboxylate). [ka]

[0355] From ((6-((4-tert-butyl-diphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)-bis(adamantane-1-carboxylate). 1H NMR (400 MHz, CDCl3) δ 7.84-7.79 (m, 4H), 7.30-7.24 (m, 6H), 5.26-5.18 (m, 2H), 3.75 (t, J = 6.2 Hz, 2H), 2.74-2.49 (m, 8H), 2.37 (q, J = 6.5 Hz, 3H), 2.13 (s, 3H), 2.10-2.06 (m, 12H), 1.89 (s, 6H), 1.82-0.99 (m, 52H), 1.22 (s, 9H), 0.89 (t, J = 6.9 Hz, 6H).

[0356] (xvii) ((6-cis-((3-((tert-butyldiphenylsilyl)oxy)cyclobutyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0357] From ((6-cis-((3-((tert-butyldiphenylsilyl)oxy)cyclobutyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, CDCl3) δ 7.68-7.63 (m, 4H), 7.45-7.34 (m, 6H), 4.99-4.90 (m, 2H), 3.96-3.85 (m, 1H), 3.78-3.71 (m, 1H), 2.69-2.58 (m, 4H), 2.57-2.49 (m, 4H), 2.48-2.40 (m, 1H), 2.37-2.21 (m, 6H), 1.96 (s, 3H), 2.01-1.05 (m, 60H), 1.02 (s, 9H), 0.93-0.82 (m, 10H). (d) General procedure for reductive alkylation of secondary amines. Same as procedure (i) in part (c) except that a formaldehyde analog was used, and the following compounds were prepared:

[0358] (i) ((6-(ethyl(4-tert-butyldiphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dinonanoate. [ka]

[0359] From ((6-(4-tert-butyldiphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)dinonanoate and acetaldehyde. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.60 (m, 4H), 7.47-7.34 (m, 6H), 5.00-4.91 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.72-2.57 (m, 4H), 2.57-2.48 (m, 4H), 2.39-2.26 (m, 7H), 2.12 (s, 3H), 1.84-1.11 (m, 64H), 1.04 (s, 9H), 0.97-0.83 (m, 12H).

[0360] (ii) 10-(5-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)pentyl)-9-ethyl-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-ylheptanoate. [ka]

[0361] From 10-(5-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)pentyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-ylheptanoate and acetaldehyde. 1 H NMR (400 MHz, CDCl3) δ 7.70-7.61 (m, 4H), 7.45-7.34 (m, 6H), 5.01-4.89 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 2.68-2.59 (m, 4H), 2.58-2.48 (m, 4H), 2.41-2.24 (m, 9H), 1.79-1.08 (m, 59H), 1.04 (s, 9H), 0.94 (t, J = 7.0 Hz, 3H), 0.91-0.85 (m, 11H).

[0362] (iii) ((6-(ethyl(4-tert-butyldiphenylsilyloxybutyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0363] From ((6-((4-(tert-butyldiphenylsilyloxy)-butyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropane-oate) and acetaldehyde. 1H NMR (400 MHz, CDCl3) δ 7.70-7.61 (m, 4H), 7.48-7.33 (m, 6H), 5.01-4.89 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 2.69-2.58 (m, 4H), 2.52 (t, J = 7.0 Hz, 4H), 2.43-2.27 (m, 9H), 1.80-1.08 (m, 62H), 1.04 (s, 9H), 0.94 (t, J = 7.0 Hz, 3H), 0.92-0.84 (m, 10H). (e) General procedure for perduteroalkylation of secondary amines.

[0364] (i) ((6-((4-tert-butyldiphenylsilyloxybutyl)(methyl-d3)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0365] To a reaction vial under an inert atmosphere was added ((6-((4-(tert-butyldiphenylsilyloxy)butyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (1.0 mmol, 1.07 g), DMF (1.0 mL), and iodomethane-d3 (1.5 mmol, 0.09 mL). The vial was covered with Al foil and stirred at room temperature for 18 h. The solution was then diluted with water (2 mL) and hexane (2 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexane (2 × 2 mL). The combined organics were washed with 0.1 M NaOH (1 × 5 mL), dried (Na2SO4), filtered, and evaporated to give a crude mixture of starting material, the desired tertiary amine, and the quaternary ammonium product. The crude mixture was diluted with CHCl (1.0 mL) under an inert atmosphere, followed by the addition of triethylamine (1.5 mmol, 0.2 mL) and Boc anhydride (1.0 mmol, 0.216 g). The mixture was stirred for 18 h and then diluted with water (2 mL) and hexanes (2 mL). The layers were separated and the organic phase was recovered. The aqueous phase was back-extracted with hexanes (2 × 2 mL). The combined organics were washed with 0.1 M NaOH (1 × 5 mL), dried (NaSO), filtered, and evaporated to give a mixture of the Boc-protected starting material and crude product. This was purified by automated chromatography (0–7% (9:1 MeOH:NHOH) in DCM over 12 CV) to give the desired tertiary amine (0.214 mmol, 0.234 g, 22% yield). 1 H NMR (400 MHz, CDCl3) δ 7.69-7.63 (m, 4H), 7.45-7.33 (m, 6H), 4.99-4.90 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.68-2.58 (m, 4H), 2.55-2.49 (m, 4H), 2.36-2.27 (m, 7H), 1.77-1.09 (m, 62H), 1.04 (s, 9H), 0.94-0.81 (m, 10H). 13C NMR (101 MHz, CDCl3) δ 173.8, 135.6, 134.1, 129.5, 127.6, 72.8, 63.9, 62.8, 53.2, 37.2, 36.0, 33.2, 33.0, 32.8, 32.4, 32.2, 31.7, 30.5, 29.8, 29.7, 29.3, 29.1, 27.1, 26.9, 26.6, 26.3, 25.3, 24.7, 22.6, 19.2, 14.1.

[0366] (ii) ((6-((4-tert-butyldiphenylsilyloxybutyl)(ethyl-d5)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). [ka]

[0367] To a reaction vial under an inert atmosphere was added ((6-((4-(tert-butyldiphenylsilyloxy)butyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (0.37 mmol, 0.403 g), DMF (0.37 mL), and iodoethane-d5 (0.45 mmol, 0.04 mL). The vial was covered with Al foil, stirred at room temperature for 18 h, and then diluted with water (2 mL) and hexane (2 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexane (2 × 2 mL). The combined organics were washed with 0.1 M NaOH (1 × 5 mL), dried (NaSO), filtered, and evaporated to give a crude mixture of starting material, the desired tertiary amine, and the quaternary ammonium product. The crude mixture was diluted with CHCl (0.37 mL) under an inert atmosphere, followed by the addition of triethylamine (0.56 mmol, 0.08 mL) and Boc anhydride (0.37 mmol, 0.082 g). The mixture was stirred for 18 h and then diluted with water (2 mL) and hexanes (2 mL). The layers were separated and the organic phase was recovered. The aqueous phase was back-extracted with hexanes (2 × 2 mL). The combined organics were washed with 0.1 M NaOH (1 × 5 mL), dried (NaSO), filtered, and evaporated to give a mixture of the Boc-protected starting material and crude product. This was purified by automated chromatography (0–7% (9:1 MeOH:NHOH) in DCM over 12 CV) to give the desired tertiary amine (0.127 mmol, 0.142 g, 34% yield). 1 H NMR (400 MHz, C6D6) δ 7.85-7.79 (m, 4H), 7.30-7.24 (m, 6H), 5.28-5.20 (m, 2H), 3.75 (t, J = 6.2 Hz, 2H), 2.70 (dd, J = 13.6, 6.3 Hz, 2H), 2.66-2.50 (m, 6H), 2.48-2.41 (m, 1H), 2.38 (t, J = 7.0 Hz, 2H), 2.33-2.26 (m, 4H), 1.76-1.03 (m, 71H), 0.89 (t, J = 6.9 Hz, 6H), 0.84-0.71 (m, 4H).13 C NMR (101 MHz, C6D6) δ = 173.2, 136.1, 134.6, 130.0, 128.1, 72.8, 64.4, 60.1, 49.9, 37.4, 36.5, 33.7, 33.2, 33.1, 32.9, 32.3, 32.1, 31.0, 30.8, 30.2, 29.6, 29.5, 27.6, 27.2, 26.9, 26.6, 26.3, 25.8, 23.0, 14.3. (e) General procedure for silyl group release.

[0368] (i) 6-((4-hydroxybutyl)(methyl)amino)-11-((2-(octanoyloxy)octyl)thio)undecyl 2-hexyldecanoate (6). [ka]

[0369] To a solution of 124 (188 mg, 0.182 mmol) in THF (2 mL) was added HF-pyridine (0.2 mL) under an inert atmosphere at 0 °C. The reaction was allowed to warm to room temperature and stirred for 18 h. Water (5 mL) was added and the mixture was extracted with CHCl (3 × 5 mL). The combined extracts were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in CHCl) to give 6 (103 mg, 71%). 1 H NMR (400 MHz, C6D6) δ 5.33-5.20 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.73-3.58 (m, 2H), 2.89-2.46 (m, 8H), 2.37 (s, 3H), 2.33-2.24 (m, 2H), 1.97-0.99 (m, 64H), 0.98-0.86 (m, 12H).

[0370] The following compounds were prepared in the same manner:

[0371] (ii) 6-((4-hydroxybutyl)(methyl)amino)-11-((2-((6-methylheptanoyl)oxy)octyl)thio)-undecyl 2-hexyldecanoate (7). [ka]

[0372] From 6-((4-((tert-butyldiphenylsilyl)oxy)-butyl)(methyl)amino)-11-((2-((6-methylheptanoyl)-oxy)octyl)thio)undecyl 2-hexyldecanoate. 1 H NMR (400 MHz, C6D6) δ 5.36-5.20 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.74-3.54 (m, 2H), 2.86-2.46 (m, 8H), 2.42-2.36 (m, 3H), 2.29 (td, J = 7.4, 3.0 Hz, 2H), 1.91-1.01 (m, 61H), 0.99-0.89 (m, 9H), 0.87 (d, J = 6.6 Hz, 6H).

[0373] (iii) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-((4-hydroxybutyl)(methyl)-amino)undecyl 2-hexyldecanoate (8). [ka]

[0374] From 6-((4-((tert-butyldiphenylsilyl)oxy)-butyl)(methyl)amino)-11-((2-((3-cyclohexyl-propanoyl)oxy)octyl)thio)undecyl 2-hexyl-decanoate. 1H NMR (400 MHz, C6D6) δ 5.34-5.19 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.75-3.58 (m, 2H), 2.89-2.44 (m, 8H), 2.43-2.27 (m, 5H), 1.97-1.01 (m, 65H), 1.00-0.85 (m, 9H), 0.87-0.73 (m, 2H).

[0375] (iv) 1-((11-((2-hexyldecanoyl)oxy)-6-((4-hydroxybutyl)(methyl)amino)undecyl)thio)-octan-2-ylcycloheptanecarboxylate (9). [ka]

[0376] From 10-(5-((2-hexyldecanoyl)oxy)pentyl)-2,2,9-trimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-yl cycloheptane-carboxylate. 1 H NMR (400 MHz, C6D6) δ 5.30-5.20 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.62 (t, J = 5.2 Hz, 2H), 2.92-2.45 (m, 9H), 2.35 (s, 3H), 2.13-1.96 (m, 2H), 1.90-1.08 (m, 64H), 0.97-0.85 (m, 9H).

[0377] (v) 11-((2-(2-cycloheptylacetoxy)octyl)thio)-6-((4-hydroxybutyl)(methyl)amino)-undecyl 2-hexyldecanoate (10). [ka]

[0378] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-11-((2-(2-cycloheptylacetoxy)octyl)thio)-undecyl 2-hexyldecanoate. 1 H NMR (400 MHz, C6D6) δ 5.30-5.23 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.70-3.60 (m, 2H), 2.85-2.47 (m, 8H), 2.34 (s, 3H), 2.25 (d, J = 6.5 Hz, 2H), 2.13 (s, 1H), 1.91-1.06 (m, 66H), 0.97-0.87 (m, 9H).

[0379] (vi) 11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-((4-hydroxybutyl)(methyl)-amino)undecyl cyclopentadecanecarboxylate (11). [ka]

[0380] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)-(methyl)amino)-11-((2-((3-cyclohexylpropanoyl)oxy)octyl)-thio)undecyl cyclopentadecane-carboxylate. 1 H NMR (400 MHz, CDCl3) δ 4.98-4.84 (m, 1H), 4.06 (t, J = 6.65 Hz, 2H), 3.73 (t, J = 5.30 Hz, 2H), 3.32-3.00 (m, 3H), 2.78 (s, 3H), 2.71-2.45 (m, 4H), 2.39 (p, J = 6.68 Hz, 1H), 2.34-2.24 (m, 2H), 2.02-1.85 (m, 2H), 1.82-1.47 (m, 16H), 1.47-0.98 (m, 51H), 0.94-0.75 (m, 5H). LRMS m / z 808 [M+H] + .

[0381] (vii) 6-((4-hydroxybutyl)(methyl)amino)-9-(2-((2-(octanoyloxy)hexyl)thio)ethyl)-3-pentyltetradecyl octanoate (12). [ka]

[0382] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)-amino)-9-(2-((2-(octanoyloxy)hexyl)-thio)ethyl)-3-pentyltetradecyl octanoate. 1 H NMR (400 MHz, C6D6) δ 5.25-5.24 (m, 1H), 4.30-4.17 (m, 2H), 3.64-3.61 (m, 2H), 2.91 (brs, 1H), 2.80-2.59 (m, 6H), 2.42 (brs, 3H), 2.42 (brs, 3H), 2.34-2.23 (m, 4H), 1.77-1.21 (m, 60H), 0.99-0.95 (m, 6H), 0.90-0.86 (m, 9H).

[0383] (viii) 1-((9-(2-((3-cyclohexylpropanoyl)oxy)ethyl)-6-((4-hydroxybutyl)(methyl)-amino)-3-pentyltetradecyl)thio)hexan-2-yl 3-cyclohexylpropanoate (13). [ka]

[0384] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-((2-((3-cyclohexylpropanoyl)-oxy)hexyl)-thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. 1H NMR (400 MHz, C6D6) δ 5.28-5.22 (m, 1H), 4.30-4.20 (m, 2H), 3.66-3.63 (m, 2H), 2.93 (brs, 1H), 2.78-2.64 (m, 5H), 2.46 (brs, 3H), 2.34-2.27 (m, 3H), 1.78-0.75 (m, 77H).

[0385] (ix) 6-((4-hydroxybutyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyloxy)cyclohexyl)-thio)ethyl)-3-pentyltetradecyl octanoate (14). [ka]

[0386] From 6-((4-((tert-butyldiphenylsilyl)-oxy)butyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-(octanoyl-oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl octanoate. 1 H NMR (400 MHz, C6D6) δ 5.13-5.09 (m, 1H), 4.30-4.14 (m, 2H), 3.63-3.60 (m, 2H), 2.90 (brs, 1H), 2.77-2.58 (m, 4H), 2.41-2.23 (m, 6H), 2.13-2.09 (m, 2H),1.70-1.21 (m, 62H), 0.98-0.95 (m, 6H), 0.91-0.86(m, 6H).

[0387] (x) 9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)cyclohexyl)thio)ethyl)-6-((4-hydroxy-butyl)(methyl)amino)-3-pentyltetradecyl 3-cyclohexylpropanoate (15). [ka]

[0388] From 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-9-(2-(((1R*,2R*)-2-((3-cyclohexylpropanoyl)oxy)cyclohexyl)thio)ethyl)-3-pentyltetradecyl 3-cyclohexylpropanoate. 1 H NMR (400 MHz, C6D6) δ 5.12-5.05 (m, 1H), 4.29-4.17 (m, 2H), 3.64-3.60 (m, 2H), 2.89-2.24 (m, 11H), 2.14-2.08 (m, 2H), 1.67-1.05 (m, 62H), 0.98-0.95 (m, 6H), 0.91-0.76 (m, 4H).

[0389] (xi) ((6-((4-hydroxybutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dinonanoate (23). [ka]

[0390] From ((6-((4-(tert-butyldiphenylsilyloxy-butyl)(methyl)amino)-undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dinonane-oate. 1 H NMR (400 MHz, C6D6) δ 5.32-5.23 (m, 2H), 3.68-3.61 (m, 2H), 2.88-2.80 (m, 1H), 2.80-2.47 (m, 10H), 2.37 (s, 3H), 2.35-2.24 (m, 4H), 1.88-1.02 (m, 64H), 1.00-0.86 (m, 12H).

[0391] (xii) ((6-((H-hydroxybutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dioctanoate (24). [ka]

[0392] From ((6-((4-(tert-butyldiphenylsilyloxy)(methyl)-amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)dioctanoate. 1 H NMR (400 MHz, C6D6) δ 5.37-5.16 (m, 2H), 3.67 (t, J = 5.4 Hz, 2H), 2.91-2.83 (m, 1H), 2.81-2.49 (m, 10H), 2.42 (s, 3H), 2.37-2.18 (m, 4H), 1.89-0.99 (m, 60H), 0.99-0.83 (m, 12H).

[0393] (xiii) ((6-(ethyl(4-hydroxybutyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dinonanoate (25). [ka]

[0394] From ((6-(ethyl(4-tert-butyldiphenylsilyloxybutyl)-amino)undecane-1,11-diyl)bis(sulfanediyl))-bis(octane-1,2-diyl)dinonanoate. 1 H NMR (400 MHz, C6D6) δ 5.31-5.19 (m, 2H), 3.66 (t, J = 5.5 Hz, 2H), 2.95-2.85 (m, 1H), 2.81-2.53 (m, 12H), 2.36-2.27 (m, 4H), 1.94-1.02 (m, 67H), 0.98-0.86 (m, 12H).

[0395] (xiv) 1-((11-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-(ethyl(4-hydroxybutyl)-amino)undecyl)thio)octan-2-ylheptanoate (26). [ka]

[0396] From 10-(5-((2-((3-cyclohexyl-propanoyl)oxy)-octyl)thio)pentyl)-9-ethyl-2,2-dimethyl-3,3-diphenyl-4-oxa-16-thia-9-aza-3-silatetracosan-18-ylheptanoate. 1 H NMR (400 MHz, C6D6) δ 5.33-5.20 (m, 2H), 3.73-3.63 (m, 2H), 2.96-2.86 (m, 1H), 2.83-2.51 (m, 12H), 2.38-2.24 (m, 4H), 1.87-1.03 (m, 59H), 0.97-0.74 (m, 14H).

[0397] (xv) ((6-((4-hydroxybutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (27). [ka]

[0398] From ((6-((4-(tert-butyldiphenylsilyloxybutyl)-(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexyl-propanoate). 1H NMR (400 MHz, C6D6) δ 5.38-5.20 (m, 2H), 3.75-3.58 (m, 2H), 2.87-2.79 (m, 1H), 2.80-2.45 (m, 10H), 2.41-2.26 (m, 7H), 1.93-0.98 (m, 62H), 0.94-0.88 (m, 6H), 0.88-0.73 (m, 4H).

[0399] (xvi) ((6-((4-hydroxybutyl)(methyl-d3)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (28). [ka]

[0400] From ((6-((4-tert-butyldiphenylsilyloxy-butyl)(methyl-d3)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, CDCl3) δ 5.00-4.89 (m, 2H), 3.55 (t, J = 4.9 Hz, 2H), 2.68-2.58 (m, 4H), 2.57-2.50 (m, 4H), 2.48-2.35 (m, 3H), 2.35-2.28 (m, 4H), 1.77-1.43 (m, 32H), 1.42-0.99 (m, 50H), 0.94-0.82 (m, 22H). 13 C NMR (101 MHz, CDCl3) δ 174.0, 73.0, 63.6, 62.9, 54.5, 37.3, 36.1, 34.8, 33.3, 33.1, 32.9, 32.6, 32.4, 32.3, 31.9, 31.7, 30.1, 29.7, 29.4, 29.2, 29.2, 27.4, 26.7, 26.4, 26.3, 25.4, 22.8, 22.7, 14.3, 14.2, 11.6.

[0401] (xvii) ((6-(ethyl(4-hydroxybutyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (29). [ka]

[0402] From ((6-(ethyl(4-tert-butyldiphenylsilyloxybutyl)-amino)undecane-1,11-diyl)bis(sulfanediyl))-bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, C6D6) δ 5.34-5.21 (m, 2H), 3.70 (t, J = 5.5 Hz, 2H), 2.98-2.88 (m, 1H), 2.82-2.51 (m, 12H), 2.33 (td, J = 7.5, 2.0 Hz, 4H), 1.90-1.05 (m, 64H), 0.95-0.74 (m, 13H).

[0403] (xviii) ((6-((ethyl-d5)(4-hydroxybutyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (30). [ka]

[0404] From ((6-((4-tert-butyldiphenylsilyloxy-butyl)(ethyl-d5)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1H NMR (400 MHz, C6D6) δ 5.31-5.19 (m, 2H), 3.57 (t, J = 5.9 Hz, 2H), 2.70 (dd, J = 13.7, 6.3 Hz, 2H), 2.66-2.52 (m, 6H), 2.50-2.44 (m, 1H), 2.37-2.24 (m, 6H), 1.80-1.02 (m, 62H), 0.89 (t, J = 6.5 Hz, 6H), 0.84-0.70 (m, 4H). 13 C NMR (101 MHz, C6D6) δ 173.3, 72.8, 62.9, 59.7, 50.0, 37.4, 36.5, 33.7, 33.2, 33.0, 32.9, 32.9, 32.4, 32.1, 32.0, 30.6, 30.1, 29.6, 29.5, 27.6, 26.9, 26.6, 25.8, 23.0, 14.3.

[0405] (xix) ((6-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (31). [ka]

[0406] From ((6-((2-(2-tert-butyldiphenylsilyloxy)ethoxy)ethyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))-bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1H NMR (400 MHz, CDCl3) δ 4.99-4.90 (m, 2H), 3.72-3.66 (m, 2H), 3.62-3.55 (m, 4H), 2.68-2.60 (m, 4H), 2.62-2.49 (m, 6H), 2.43-2.36 (m, 1H), 2.36-2.29 (m, 4H), 2.22 (s, 3H), 1.77-1.12 (m, 58H), 0.95-0.80 (m, 10H).

[0407] (xx) ((6-((2-((2-hydroxyethyl)thio)ethyl)(methyl)amino)undecane-1,11-diyl)bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (32). [ka]

[0408] From ((6-((2-((2-tert-butyldiphenylsilyloxy)thio)ethyl)(methyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, CDCl3) δ = 4.99-4.90 (m, 2H), 3.73 (t, J = 5.7 Hz, 2H), 2.74 (t, J = 5.7 Hz, 2H), 2.65-2.59 (m, 8H), 2.57-2.51 (m, 4H), 2.40-2.28 (m, 5H), 2.19 (s, 3H), 1.76-1.08 (m, 58H), 0.94-0.82 (m, 10H). 13C NMR (101 MHz, CDCl3) δ = 174.0, 73.0, 63.5, 61.4, 54.0, 37.3, 37.1, 36.3, 36.2, 33.3, 33.1, 32.9, 32.6, 32.3, 31.9, 31.2, 30.2, 29.8, 29.3, 29.2, 27.2, 26.7, 26.4, 25.4, 22.7, 14.2.

[0409] (xxi) ((6-((4-hydroxybutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dicycloheptanecarboxylate (33). [ka]

[0410] From ((6-((4-tert-butyldiphenylsilyloxy-butyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)dicycloheptane-carboxylate. 1 H NMR (400 MHz, C6D6) δ 5.26-5.18 (m, 2H), 3.61 (t, J = 5.6 Hz, 2H), 2.70 (dd, J = 13.6, 6.3 Hz, 2H), 2.66-2.49 (m, 8H), 2.40-2.32 (m, 1H), 2.29 (t, J = 6.1 Hz, 2H), 2.02 (s, 3H), 2.07-1.95 (m, 4H), 1.90-1.53 ​​(m, 18H), 1.52-1.05 (m, 43H), 0.89 (t, J = 6.8 Hz, 6H). 13C NMR (101 MHz, C6D6) δ 176.2, 72.5, 63.6, 62.8, 54.3, 45.6, 36.6, 35.6, 33.7, 32.9, 32.2, 32.1, 31.4, 31.3, 30.1, 30.0, 29.5, 29.5, 28.7, 28.6, 27.4, 26.6, 26.6, 25.8, 23.0, 14.3.

[0411] (xxii) ((6-((4-hydroxybutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)-bis(adamantane-1-carboxylate) (34). [ka]

[0412] From ((6-((4-tert-butyldiphenylsilyloxy-butyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)-bis(adamantane-1-carboxylate). 1 H NMR (400 MHz, C6D6) δ 5.23 (dtd, J = 8.4, 6.1, 4.2 Hz, 2H), 3.61 (t, J = 5.6 Hz, 2H), 2.75-2.50 (m, 8H), 2.42-2.33 (m, 1H), 2.30 (t, J = 6.1 Hz, 2H), 2.10-2.05 (m, 11H), 2.02 (s, 3H), 1.92-1.86 (m, 6H), 1.79-1.52 (m, 21H), 1.51-1.07 (m, 32H), 0.89 (t, J = 6.8 Hz, 6H). 13 C NMR (101 MHz, C6D6) δ 176.7, 72.3, 63.6, 62.8, 54.3, 41.2, 39.5, 36.8, 36.6, 35.6, 33.7, 32.9, 32.1, 30.1, 30.0, 29.5, 29.5, 28.5, 27.4, 25.8, 23.0, 14.3.

[0413] (xxiii) ((6-((cis-3-hydroxycyclobutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (35). [ka]

[0414] From ((6-cis-((3-((tert-butyldiphenylsilyl)oxy)cyclobutyl)(methyl)amino)undecane-1,11-diyl)bis(sulfanediyl))-bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate). 1 H NMR (400 MHz, CDCl3) δ (400 MHz, CDCl3) δ 5.01-4.89 (m, 2H), 3.94 (p, J = 7.4 Hz, 1H), 2.69-2.58 (m, 5H), 2.57-2.42 (m, 6H), 2.38-2.29 (m, 5H), 1.98 (s, 3H), 1.77-1.48 (m, 23H), 1.42-1.06 (m, 37H), 0.94-0.81 (m, 10H). 13 C NMR (101 MHz, CDCl3) δ 174.1, 72.9, 61.4, 47.8, 39.8, 37.3, 36.1, 36.1, 33.3, 33.1, 32.8, 32.8, 32.6, 32.3, 31.9, 31.0, 29.8, 29.7, 29.3, 29.2, 27.1, 26.7, 26.4, 25.4, 22.7, 14.2. (f) General procedure for converting ketones to type 2 ionizable head groups.

[0415] (i) (((4-(2-hydroxyethyl)-1,3-dioxolane-2,2-diyl)bis(pentane-5,1-diyl))bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (127). [ka]

[0416] A solution of 80 (655 mg, 0.868 mmol), 1,2,4-butanetriol (184 mg, 1.74 mmol), and pyridinium p-toluenesulfonate (44 mg, 0.174 mmol) in toluene (15 mL) was refluxed overnight under nitrogen with continuous removal of water (Dean-Stark trap). The mixture was cooled to room temperature and washed with water (2 × 10 mL), brine (10 mL), then dried (NaSO) and concentrated. The residue was purified by silica gel column chromatography (0–3% MeOH in DCM) to afford ketal 127 (474 ​​mg, 64%) as an oil. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.89 (m, 2H), 4.32-4.17 (m, 1H), 4.08 (dd, J = 7.9, 6.0 Hz, 1H), 3.80 (td, J = 5.9, 2.8 Hz, 2H), 3.52 (t, J = 8.0 Hz, 1H), 2.71-2.58 (m, 4H), 2.58-2.48 (m, 4H), 2.35-2.28 (m, 4H), 2.01=1.03 (m, 60H), 0.96-0.79 (m, 10H).

[0417] (ii) (((4-(2-(tosyloxy)ethyl)-1,3-dioxolane-2,2-diyl)bis(pentane-5,1-diyl))bis(sulfane-diyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (128). [ka]

[0418] To a solution of 127 (189 mg, 0.221 mmol), TEA (0.0462 mL, 0.332 mmol), and DMAP (2.70 mg, 0.0221 mmol) in CHCl (2.00 mL) under an inert atmosphere at 0 °C was added TsCl (50.6 mg, 0.265 mmol). The reaction was allowed to warm to room temperature and stirred for 18 h. The reaction was quenched with water (3.00 mL) and extracted with CHCl (3 × 4.00 mL). The combined organics were dried (NaSO) and concentrated to give 128 (221 mg, crude, quantitative), which was used in the next step without purification. 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 5.01-4.86 (m, 2H), 4.20-4.04 (m, 3H), 4.03-3.97 (m, 1H), 3.48-3.41 (m, 1H), 2.66-2.60 (m, 4H), 2.56-2.49 (m, 4H), 2.45 (s, 3H), 2.34-2.28 (m, 4H), 1.89 (q, J = 6.2 Hz, 2H), 1.78-1.04 (m, 58H), 0.95-0.81 (m, 10H).

[0419] (iii) (((4-(2-(dimethylamino)ethyl)-1,3-dioxolane-2,2-diyl)bis(pentane-5,1-diyl))bis-(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (22). [ka]

[0420] A solution of 128 (221 mg, crude), dimethylamine (3.00 mL, 2 M in THF), and MeOH (3.00 mL) was heated in a microwave reactor (110 °C, normal absorbance) for 15 min. The mixture was then concentrated, and the residue was purified by silica chromatography (0–5% MeOH in DCM) to afford lipid 22 (165 mg, 85% over two steps) as an oil.1 H NMR (400 MHz, C6D6) δ 5.29-5.17 (m, 2H), 4.14-4.00 (m, 1H), 3.97-3.87 (m, 1H), 3.40 (t, J = 7.6 Hz, 1H), 2.75-2.44 (m, 8H), 2.37-2.19 (m, 6H), 2.03 (s, 6H), 1.86-0.95 (m, 60H), 0.94-0.86 (m, 6H), 0.84-0.71 (m, 4H). (g) General procedure for converting alcohols to ionizable head groups of type 12.

[0421] (i) ((6-(2-bromo-1-ethoxyethoxy)undecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (126). [ka]

[0422] To a sealed reaction vial under an inert atmosphere was added ((6-hydroxyundecane-1,11-diyl)bis(sulfanediyl))bis(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (0.15 mmol, 116 mg), DCM (0.3 mL), (Z)-1-bromo-2-ethoxyethylene (0.75 mmol, 0.08 mL), and PPTS (0.015 mmol, 4 mg). The mixture was stirred at room temperature for 18 h and then diluted with hexane (3 mL) and saturated NH4Cl (3 mL). The layers were separated and the organic phase was collected. The aqueous phase was back-extracted with hexane (2 × 2 mL). The combined organics were dried (Na2SO4), filtered, and evaporated to give the crude product. This was purified by silica chromatography (0-10% EtOAc in hexanes over 12 CV) to give 126 (0.13 mmol, 122 mg, 88% yield). 1H NMR (400 MHz, CDCl3) δ 4.92 (dtd, J = 8.3, 6.2, 4.4 Hz, 2H), 4.69-4.61 (m, 1H), 3.74-3.44 (m, 3H), 3.38-3.26 (m, 2H), 2.67-2.57 (m, 4H), 2.56-2.45 (m, 4H), 2.33-2.27 (m, 4H), 1.76-1.00 (m, 61H), 0.93-0.79 (m, 10H). 13 C NMR (101 MHz, CDCl3) δ 173.9, 100.8, 77.7, 72.9, 61.7, 37.3, 36.1, 34.5, 33.8, 33.2, 33.1, 32.7, 32.7, 32.5, 32.5, 32.2, 31.8, 29.7, 29.6, 29.2, 29.1, 26.6, 26.3, 25.4, 25.1, 24.6, 22.7, 15.4, 14.2.

[0423] (ii) ((6-(2-(dimethylamino)-1-ethoxyethoxy)undecane-1,11-diyl)bis(sulfanediyl))bis-(octane-1,2-diyl)bis(3-cyclohexylpropanoate) (21). [ka]

[0424] To a solution of 126 (0.13 mmol, 122 mg) in a microwave vial under an inert atmosphere was added 2 M dimethylamine in THF (2.7 mmol, 1.3 mL). The mixture was heated at 110 °C for 45 min by microwave irradiation. The mixture was then concentrated, diluted with hexane (5 mL), and washed with 0.1 M NaOH (5 mL). The collected organic phase was dried (NaSO), filtered, and evaporated. The residue was purified by silica chromatography (0–7% MeOH in CHCl) to give 21 (0.07 mmol, 62 mg, 53% yield). 1H NMR (400 MHz, CDCl3) δ 4.96 (dtd, J = 8.0, 6.1, 4.4 Hz, 2H), 4.64 (dd, J = 6.3, 4.3 Hz, 1H), 3.73-3.63 (m, 1H), 3.61-3.50 (m, 2H), 2.71-2.59 (m, 4H), 2.60-2.50 (m, 5H), 2.40-2.26 (m, 4H), 2.30 (s, 6H), 1.82-1.08 (m, 61H), 0.97-0.83 (m, 10H). 13 C NMR (101 MHz, CDCl3) δ 173.9, 100.3, 72.9, 62.6, 60.9, 46.5, 37.3, 36.1, 34.6, 33.9, 33.3, 33.1, 32.9, 32.8, 32.6, 32.3, 31.8, 29.8, 29.8, 29.3, 29.3, 29.2, 26.7, 26.4, 25.4, 25.2, 24.9, 22.7, 15.5, 14.2. Example 2: mRNA-containing LNPs comprising ionizable cationic amino lipids of the present disclosure are shown to outperform the nor-MC3 benchmark in in vivo delivery of mRNA to the liver and spleen.

[0425] Lipid nanoparticle (LNP) formulations containing ionizable lipids 1, 5-13, and 16-35 / DSPC / chol / PEG-DMG at 50 / 10 / 38.5 / 1.5 mol% with a nitrogen-to-phosphorus ratio (N / P) of 6 and mRNA encoding luciferase were prepared as described in "Materials and Methods." Ionizable lipids 5-13 and 16-35 are described in Example 1 and Table 1. nor-MC3 benchmark lipid 1 is disclosed in co-owned and co-pending WO 2022 / 246571, which is incorporated herein by reference in its entirety. The polydispersity index (PDI), encapsulation efficiency, and size of the LNP formulations are shown in Figure 1.

[0426] The LNP formulation was then injected into CD-1 mice, and the in vivo transfection efficiency in the liver and spleen was tested. The mRNA dose was 1 mg / kg. The luminescence intensity in the liver and spleen was measured 4 hours after injection.

[0427] When the sulfur-containing ionized cationic amino lipids of the present disclosure were incorporated into lipid nanoparticles, a surprising increase in luminescence intensity per mg of liver and spleen was observed. Figure 2A shows the luminescence intensity per mg of liver for the ionized lipids of the present disclosure and the norMC3 benchmark. Figure 2B shows the luminescence intensity per mg of spleen compared to the norMC3 benchmark.

[0428] These examples are intended to illustrate the preparation and properties of specific ionized cationic amino lipid and lipid nanoparticle nucleic acid preparations and are not intended to limit the scope of the invention.

[0429] As used herein, the articles "a" or "an" are intended to include both the singular and the plural, unless otherwise specified.

Claims

1. An ionized cationic amino having the structure of Formula A: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof [In the formula, m, n, p, and q of Formula A are independently 2 to 8; R １ is a linear, branched, monocyclic or polycyclic, optionally substituted C ３ ~C ２０ is an alkyl group; E is an ester group that is —(C═O)O— or —O(C═O)—; R ２ is a linear or branched, optionally substituted C １ ~C １０ is an alkyl group, or R ２ is R ３ to form a ring structure shown by the dashed curved line; R ３ is H or a straight or branched, optionally substituted C １ ~C １０ alkyl group or R ３ is R ２ to form a ring structure shown by the dashed curved line; R ４ and R ５ are independently H or a straight-chain or branched, optionally substituted C １ ~C １０ alkyl group or R ４ and R ５ are bonded to each other to form a ring structure; R ６ and R ７ are independently H or a straight-chain or branched, optionally substituted C １ ~C １０ alkyl group or R ６ and R ７ are bonded to each other to form a ring structure; A is O, S, or carbonyl (C=O); When A is O, R ８ is an acyl group 【Chemistry 2】 where the wavy line represents the bond to A and R′ is R １ is as defined above; When A is carbonyl (C=O), R ８ teeth 【Transformation 3】 where the wavy line represents the bond to A and R′ is R １ is as defined above; If A is S, then R ８ is a radical of the structure: 【Chemistry 4】 Here, E is an ester group that is —(C═O)O— or —O(C═O)—, the wavy line represents a bond to A, and R ９ is R １ and R １０ is R ２ and R １１ is R ３ as defined for W １ and Y are bonded to each other or are not bonded to each other, W １ When and Y are bonded to each other, W １ is O or S; W ２ is O or S; X is CH; Y is (CH ２ ) ｔ where t is 1 or 2; Z is selected from any of the following structures a to c, where the wavy line represents the bond to X: a. 【Transformation 5】 Type 2 ionizable head groups, wherein n in said Type 2 ionizable head groups is 1 to 5; b. 【Transformation 6】 Type 3 ionizable head groups, wherein m and n of said Type 3 ionizable head groups are independently 1 to 5; c. 【Transformation 7】 Type 4 ionizable head groups, wherein m and n in said Type 4 ionizable head groups are independently 2 to 5, and R is C １ -C ６ alkyl or cycloalkyl; W １ and Y are not bonded to each other, W １ is H; W ２ is O, S, NH or NR １２ and R １２ is a C optionally substituted with an OH group １ ~C ４ is alkyl; Part of formula A 【Transformation 8】 is a group selected from any one of the following structures d to l, and the wavy line is W ２ represents a bond to: d. 【Chemistry 9】 Type 1 ionizable head groups, wherein n in said Type 1 ionizable head groups is 1 to 5; e. 【Chemistry 10】 Type 5 ionizable head groups, wherein m and n of said Type 5 ionizable head groups are independently 1 to 5; f. 【Chemistry 11】 Type 6 ionizable head groups, where m is 1 to 5, n is independently 2 to 5, and R is C １ -C ６ alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl; g. 【Chemistry 12】 Type 7 ionizable head groups, where W ２ is NH or NR １２ and n of the type 7 ionizable head group is 1 to 5, and (CH ２ ) ｎ Methylene (CH ２ ) is optionally substituted with a sulfur atom or an oxygen atom; h. 【Chemistry 13】 Ionizable head groups of type 8, wherein n in said type 8 ionizable head group is 1 to 5; i. 【Chemistry 14】 Type 9 ionizable head groups, wherein m and n in said Type 9 ionizable head groups are independently 1 to 5; j. 【Chemistry 15】 Type 10 ionizable head groups, wherein the curved lines represent atoms of a ring structure containing the N atom, said ring structure having 2 to 8 carbon atoms, and j in said Type 10 ionizable head groups is 0 to 5; k. 【Chemistry 16】 Type 11 ionizable head groups, where W ２ is NH or NR １２ wherein the circle represents a homocyclic or heterocyclic ring containing 3 to 8 atoms, and j in said Type 11 ionizable head group is 0 to 5; l. 【Chemistry 17】 Type 12 ionizable head group, where R=C １ -C ６ alkyl, cycloalkyl, deuterated alkyl or deuterated cycloalkyl, and j ranges from 1 to 5.

2. 10. The ionizable cationic amino lipid of claim 1, having the structure of any one of compounds 5-35 defined below, or a pharmaceutically acceptable salt thereof: [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 。

3. The ionized cationic amino lipid according to claim 2, having the structure of compounds 5 to 13, 16 to 19, 22 to 31 or 33 to 35, or a pharmaceutically acceptable salt thereof.

4. An ionized cationic amino lipid or a pharmaceutically acceptable salt thereof, a protonated amino head group; two lipophilic chains, the protonated amino head group having a central carbon atom to which each of the two lipophilic chains is directly attached; At least one of the two lipophilic chains has the structure of formula A.1; 【Chemistry 23】 where the wavy line represents the bond to the central carbon atom; m and n are independently 2 to 8; E is an ester group that is —(C═O)O— or —O(C═O)—; R １ is a linear, branched, monocyclic or polycyclic, optionally substituted, ring-shaped ring containing 0 to 2 carbon-carbon double bonds, C ３ ~C ２０ is an alkyl group; R ２ is a linear or branched, optionally substituted C １ ~C １０ is an alkyl group, or R ２ is R ３ to form a ring structure shown by the dashed curved line; R ３ is H or a straight or branched, optionally substituted C １ ~C １０ alkyl group or R ３ is R ２ to form a ring structure shown by the dashed curved line; R ４ and R ５ are independently H or a straight-chain or branched, optionally substituted C １ ~C １０ alkyl group or R ４ and R ５ are bonded to each other to form a ring structure; each lipophilic chain having a total of 15 to 40 carbon atoms; The lipid has (i) a pK between 6 and 7.5 ａ (ii) a log P of at least 11, or a pharmaceutically acceptable salt thereof.

5. The second of the two lipophilic chains attached to the central carbon atom of the head group has a structure defined by Formula D: 【Chemistry 24】 Formula D wherein the wavy line represents the bond of the head group to the central carbon atom; R ６ and R ７ are independently H or a straight-chain or branched, optionally substituted C １ ~C １０ alkyl group or R ６ and R ７ are bonded to each other to form a ring structure; A is O, S, or carbonyl (C=O); When A is O, R ８ is an acyl group 【Chemistry 23】 where the wavy line represents the bond to A and R′ is R １ is as defined above; When A is carbonyl (C=O), R ８ teeth 【Chemistry 24】 where the wavy line represents the bond to A and R′ is R １ is as defined above; If A is S, then R ８ is a group of formula E: 【Chemistry 25】 where E' is an ester group that is -(C=O)O- or -O(C=O)-; The wavy line represents a bond with A, and R ９ is R １ is as defined above, and R １０ is R ２ is as defined above, and R １１ is R ３ 5. The ionizable cationic amino lipid of claim 4, wherein:

6. 6. The ionized cationic amino lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the lipid, when formulated into lipid nanoparticles containing mRNA, results in at least about a 10-fold increase in biodistribution of the lipid nanoparticles in the spleen compared to otherwise identical lipid nanoparticles containing norDLin-MC3-DMA (nor-MC3), as measured by in vivo luminescence of the mRNA in the spleen.

7. 6. The ionized cationic amino lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the lipid, when formulated into lipid nanoparticles comprising mRNA, results in at least about a two-fold increase in biodistribution of the lipid nanoparticles in the liver compared to lipid nanoparticles comprising norDLin-MC3-DMA (nor-MC3), as measured by in vivo luminescence of the mRNA in the liver.

8. A lipid nanoparticle comprising the ionized cationic amino lipid according to any one of claims 1 to 7 and a nucleic acid.

9. The lipid nanoparticle of claim 8, comprising a helper lipid.

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

11. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing lipid nanoparticles described in claim 8, 9, or 10 containing the nucleic acid, and administering the lipid nanoparticles to the subject.

12. A method for delivering a nucleic acid molecule to a cell, the method comprising contacting the lipid nanoparticles of claim 8, 9, or 10 with the cell in vivo or in vitro.

13. Use of the ionized cationic amino lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or the lipid nanoparticle according to claim 8, 9, or 10, in the manufacture of a medicament for treating or preventing a disease, disorder, or condition treatable and / or preventable by a nucleic acid.

14. Use of the ionized cationic amino lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or the lipid nanoparticle according to claim 8, 9, or 10, for delivering a nucleic acid to a subject to treat or prevent a disease, disorder, or condition treatable or preventable by the nucleic acid.

15. 15. The use defined in claim 13 or 14, wherein said nucleic acid is mRNA.

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