Sulfur-containing ionizable lipids for the delivery of therapeutic agents

Abstract

Provided are novel sulfur-containing lipids, nanoparticles comprising such lipids and cargo molecules such as nucleic acids, methods for combining the lipids with nucleic acids to produce lipid nanoparticles, and chemical routes for producing the lipids. The lipids may have the structure of Formula A, as defined herein. JPEG2025532475000185.jpg5579 Formula A

Description

[Technical Field]

[0001] Provided herein are sulfur-containing lipids that can be incorporated into delivery vehicles to facilitate encapsulation of cargo, such as, but not limited to, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceuticals, and salts thereof. [Background technology]

[0002] (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.

[0003] The primary component of lipid nanoparticles (LNPs) is ionized lipids. Ionized lipids are typically 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 via 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.

[0004] An early example of an LNP product approved for clinical use that relies on ionizable lipids is Onpattro®, 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" (Figure 1). MC3 is an evolved form of a structurally related ionizable lipid, designated "KC2" (Figure 1). MC3 is considered the most advanced ionizable lipid for siRNA delivery, requiring approximately threefold less siRNA than KC2, but KC2 is superior in other applications, making it a valuable research tool.

[0005] The aforementioned ionizable lipids are particularly effective in delivering siRNA-containing LNPs to hepatocytes but are less effective in delivering mRNA-containing LNPs to the liver. For example, mRNA vaccines, including the COVID-19 vaccines from Pfizer / BioNTech and Moderna, rely on lipid nanoparticles to deliver mRNA to the cytoplasm of hepatocytes. After entering the host cell, the mRNA is transcribed to produce antigenic proteins. In the case of the COVID-19 vaccine, the mRNA encodes the highly immunogenic Sars-CoV-2 spike protein. However, such vaccines also incorporate other types of ionizable lipids besides MC3 or KC2. Notably, 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]

[0006] Furthermore, the lipids described above have been optimized for delivery of therapeutic nucleic acids to the liver. However, there remains a need to develop new 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 locations other than the liver would expand the clinical utility of LNPs to target disease states affecting tissues and organs other than the liver. There is also a continuing need to develop LNPs with improved capabilities for delivering nucleic acids or other charged cargoes to the liver.

[0007] 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

[0008] As used herein, a "Type 1 ionizing head" or "MC-type ionizing 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]

[0009] As used herein, the terms "type 2 ionization head" and "KC type ionization head" refer 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]

[0010] 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]

[0011] 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 is =C1-C6 alkyl or cycloalkyl, and m and n independently range from 2 to 5. [ka]

[0012] 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]

[0013] 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 is =C1-C6 alkyl or cycloalkyl, m ranges from 1 to 5, and n independently ranges from 2 to 5. [ka]

[0014] 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 an equivalent thereof, where R is =C1-C6 alkyl or cycloalkyl, and n ranges from 1 to 5. [ka]

[0015] 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 is =C1-C6 alkyl or cycloalkyl, and n ranges from 1 to 5. [ka]

[0016] As used herein, a "Type 9 ionizing 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]

[0017] 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.

[0018] The term "lipophilic chain" as used herein refers to an alkyl group attached to a nitrogen or carbon atom of a lipid, said alkyl group containing at least 6 C atoms, and optionally containing a C=C double bond, and / or a ring structure, and / or a carbonyl group, and / or heteroatoms such as N, O, S, such that the CLogP of the parent compound of said alkyl group is at least 6.

[0019] For example, lipid MC3,1 and lipid KC2,2 have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene with a CLogP of 9.25: [ka]

[0020] The lipid ALC-0315, 3, has a pair of lipophilic chains derived from hexyl 2-hexyldecanoate with a CLogP of 10.01: [ka]

[0021] 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-yl octanoate with a CLogP of 11.6: [ka]

[0022] As used herein, the term "alkyl" or "alkyl group" refers to a carbon-containing chain that is straight or branched, optionally contains a C=C double bond and / or a ring structure, and is optionally substituted.

[0023] As used herein, the term "C m ~C n Alkyl" or "C m ~C n An "alkyl group" refers to an optionally unsaturated, optionally substituted, straight 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.

[0024] The term "optionally substituted" with respect to alkyl 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 by one or more substituents containing a heteroatom selected from O, S, and NR', where R' is as defined below. Non-limiting examples of groups that can replace a hydrogen atom include halogen; alkyl groups; cycloalkyl groups; oxo groups (=O); hydroxyl groups (-OH); -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) xNR'R';-NR'S(O) x R'; and -S(O) x NR'R', where R', at each occurrence, is H, C1-C 15 alkyl or cycloalkyl; and x is 0, 1, or 2.

[0025] 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.

[0026] 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 that include helper lipids.

[0027] As used herein, the term "nanoparticle" refers to any suitable particle that can incorporate lipids and can contain one or more helper lipid components.The one or more lipid components can include ionized lipids prepared by the methods described herein, and / or can include additional lipid components, such as one or more helper lipid components.The term includes, but is not limited to, vesicles with one or more bilayers, including multilamellar vesicles, unilamellar vesicles, and vesicles with electron-dense cores.The term also includes polymer-lipid hybrids, which include particles in which lipids are attached to polymers.

[0028] As used herein, the term "encapsulation," with respect to incorporating a cargo molecule (e.g., mRNA) within a delivery vehicle, refers to any association of the cargo with any component or compartment of the delivery vehicle, such as a nanoparticle.

[0029] 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.

[0030] As used herein, the articles "a" or "an" are intended to include both the singular and the plural, unless otherwise specified. (overview)

[0031] The present disclosure is based, at least in part, on the surprising discovery that LNP formulations of nucleic acids containing ionizable lipids incorporating at least one lipophilic chain substituted with a sulfur atom and an ester moiety are more potent than the then-benchmark MC3 for hepatic delivery of therapeutic RNA. As further described herein, such lipids may exhibit organ selectivity distinct from known lipids. In particular, the non-limiting examples described herein demonstrate that such lipids promote selective mRNA delivery to the spleen 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.

[0032] 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.

[0033] According to one embodiment of the present disclosure, a lipid having the structure of Formula A: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, m is 4 to 8; n is 4 to 8; R 1 , R 2 , R 3 , and R 4 is a linear or branched, optionally substituted C3-C 20 alkyl, optionally containing 0 to 2 carbon-carbon double bonds; A is C or N, If A is C, then 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)q, where q is 1 or 2; Z is selected from one of the following structures a-c, where the wavy line represents the bond to X: a. [ka] Type 2 ionizable head group; b. [ka] Type 3 ionizable head group; c. [ka] Type 4 ionizable head group; W 1 If and Y are not bonded to each other, W 1 is H; W 2 is O, S, NH or NR 2a and R 2a is a C1-C4 alkyl optionally substituted with an OH group; Part of formula A [ka] is a group selected from the following structures d to h, and the wavy line represents W 2 represents a bond to: d. [ka] Type 1 ionizable head group; e. [ka] Type 5 ionizable head group; f. [ka] Type 6 ionizable head group; g. [ka] Type 7 ionizable head group; h. [ka] Type 8 ionizable head group; i. [ka] Type 9 ionizable head group; If A is N, W 1 and Y is absent; W 2 and X together form the structure (CR a R b ) p and R a and R b is independently H or C1-C5 alkyl or cycloalkyl, and p is 2 to 6; Z is OH or NR'R'', where R' and R'' are independently optionally substituted C1-C5 alkyl or cycloalkyl, or R' and R'' together with the N atom of NR'R'' form an optionally substituted heterocycle incorporating the N atom to which R' and R'' are each attached.

[0034] According to an embodiment of the aforementioned aspect, R 1 and R 4 At least one of may independently be a moiety of formula B: [ka] R' and R'' are independently straight-chain or branched optionally substituted C3-C 12 an alkyl group, optionally containing 0 to 2 carbon-carbon double bonds; R''' is H or a linear, branched or cyclic optionally substituted C1-C6 alkyl group; G 1 and G 2 However, independently, (CR a R b ) p and R a and R b are each independently selected from H or optionally substituted C1-C5 alkyl or cycloalkyl, and p is 0-6.

[0035] According to the aforementioned aspect or an embodiment thereof, A 3 can be N, and W 1 and Y does not exist and W 2 and X together form the structure (CR a R b ) r where Z is NR'R'' and the heterocyclic group incorporating the N atom to which R' and R'' are attached is pyrrolidine, piperidine or morpholine.

[0036] According to the aforementioned aspect or an embodiment thereof, A 3 can be a carbon atom.

[0037] According to the aforementioned aspect or an embodiment thereof, W 1 and Y are not bonded to each other.

[0038] According to the aforementioned aspect or an embodiment thereof, W 2 can be O.

[0039] In one embodiment, the moiety of formula A [ka] is structure d.

[0040] According to the foregoing aspect or an embodiment thereof, the lipid has the structure of any one of compounds 5-21 set forth in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0041] According to a further aspect of the present disclosure, with a protonatable amino head group; two lipophilic chains, wherein an amino head group has a central nitrogen or carbon atom to which each of the two lipophilic chains is directly attached; At least one of the lipophilic chains has the formula: [ka] In the formula, R 1 and R 2 are independently linear or branched optionally substituted C3-C 20 alkyl, optionally with varying degrees of unsaturation; n is 4 to 8; each lipophilic chain having a total of 15 to 40 carbon atoms; Lipids with (i) pK between 6 and 8 a and (ii) a logP of at least 11.

[0042] According to further embodiments of any of the foregoing aspects of the present disclosure, the lipid, when incorporated into lipid nanoparticles containing mRNA, results in at least about a 10% increase in biodistribution of the lipid nanoparticles in the liver and / or one or more extrahepatic tissues, such as the liver and / or spleen, as measured by in vivo luminescence of the mRNA in one or more extrahepatic tissues. The assay and formulation used to determine biodistribution are as described in Example 2.

[0043] In another aspect, there is provided a lipid nanoparticle comprising a lipid of any one of the preceding aspects or embodiments thereof and a nucleic acid.

[0044] The lipid particle may comprise a helper lipid and a hydrophilic polymer-lipid conjugate. The helper lipid may be selected from cholesterol, diacylglycerol, and sphingolipids.

[0045] According to another aspect: an ionizable lipid having two lipophilic chains directly attached to a central nitrogen or carbon atom, wherein at least one of the lipophilic chains has the formula: [ka] n is 4 to 8; * denotes a carbon branch point; R 5 and R 6 are each independently a linear or branched substituted C3-C 30 is an alkyl group; R 5 and R 6 One of the groups is substituted with an ester group, and R 5 and R 6 the other of which is substituted with a sulfur atom at the α, β, or γ position relative to the carbon branch point; with one or more helper lipids; with hydrophilic polymer-lipid conjugates; and a nucleic acid.

[0046] In a further aspect, there is provided a method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing a lipid nanoparticle as described in any of the preceding aspects or embodiments comprising a nucleic acid, and administering the lipid nanoparticle to the subject.

[0047] According to another aspect of the present disclosure, there is provided a method for delivering a cargo molecule to a cell, the method comprising contacting the cell in vivo or in vitro with a lipid nanoparticle described in any of the above aspects or embodiments, hi one embodiment, the cargo molecule is a nucleic acid.

[0048] According to a further aspect of the present disclosure, there is provided use of a lipid as defined in any of the above aspects or embodiments or a pharmaceutically acceptable salt thereof, or a lipid nanoparticle as defined in any of the above 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.

[0049] According to a further aspect of the present disclosure, there is provided the use of the lipid or its pharmaceutically acceptable salt as defined above, or the lipid nanoparticle of any one of the above aspects or embodiments, for treating or preventing diseases, disorders or conditions that can be treated or prevented by nucleic acid.In one embodiment, the nucleic acid is mRNA. [Brief explanation of the drawings]

[0050] [Figure 1] Bar graph showing the entrapment rate (%), particle size, and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) containing ionizable lipids 1 and 5–32 (Table 1). The LNPs were composed of 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG, with an amine-to-phosphate ratio (N / P) of 6.

[0051] [Figure 2A] Figure shows the luminescence intensity / mg in the liver of mRNA-containing LNPs containing ionized lipids 29, 28, 18, 19, 15, 17, 14, 20, 13, 21, 1, 7, 9, 8, 11, 10, 6, 25, 23, 30, 12, 24, 16, 32, 26, 22, 31, 27, and 5. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% ionized lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0052] [Figure 2B] Figure shows the luminescence intensity / mg in the spleen of mRNA-containing LNPs containing ionized lipids 19, 29, 28, 18, 10, 11, 1, 20, 21, 7, 17, 23, 12, 26, 27, 15, 8, 14, 13, 31, 24, 32, 25, 5, 16, 22, 6, 30, and 9, 4 h after intravenous administration to CD-1 mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% ionized lipid / DSPC / chol / PEG-DMG (N / P = 6). DETAILED DESCRIPTION OF THE INVENTION

[0053] (Detailed explanation) Various aspects and embodiments of the present disclosure relate to ionizable lipids having the structure of Formula A, and pharmaceutically acceptable salts thereof. [ka]

[0054] The formulations comprising such lipids are used for the delivery of nucleic acid to any target site.In some embodiments, such lipids are found to be particularly effective for the delivery of mRNA when formulated in suitable delivery vehicle.In further embodiments, such lipids can be easily synthesized and prepared by a process that is more economical than the known method for producing ionized lipids. Method for producing lipids of formula A

[0055] The lipid of formula A or its pharmaceutically acceptable salt can be prepared using any suitable method known to those skilled in the art. Particularly suitable methods are described below, but are not intended to be limiting, and are illustrated by the synthesis of compounds 5-32 in Table 1 below. Those skilled in the art will understand that alternative starting materials can be used in the same order to obtain analogs of compounds 5-32 defined by formula A. Therefore, the synthetic scheme shown below is merely illustrative of selected embodiments.

[0056] Specific steps in the synthesis of compounds such as 5 are described in detail in co-pending and commonly owned WO 2023 / 173203, which is incorporated herein by reference. As noted in the foregoing disclosure, one such step involves reacting a suitable amino alcohol, or an O-protected variant thereof, represented by general formula 33 in Scheme 2, with a suitable alkyl halide or sulfonate, represented by general formula 34 in Scheme 2: JPEG2025532475000031.jpg229170JPEG2025532475000032.jpg229170JPEG2025532475000033.jpg227170JPEG2025532475000034.jpg219170JPEG2025532475000035.jpg96170As a result, different products are formed depending on the conditions. Specifically, primary amines can be converted to the corresponding secondary amines by reaction in DMF at room temperature in the presence of K2CO3, which results in selective mono-N-alkylation of the starting amine, resulting in the formation of the product represented by general formula 35 in Scheme 2. Secondary amines such as 35 can then be N-alkylated a second time by reacting them with another alkyl halide or sulfonate of general formula 36 in Scheme 2 by heating in acetonitrile in the presence of KCO or NaCO. Thus, secondary amine 35 is converted to a tertiary amine of general structure 37. [ka]

[0057] Alternatively, primary amine 33 can be doubly alkylated in one step by heating with alkyl halide or sulfonate 34 in the presence of K2CO3 or Na2CO3 in acetonitrile, which results in double N-alkylation of the starting amine and converts 33 to a tertiary amine of general structure 38 (Scheme 3). If Z in 33 or 38 is a protecting group, a deprotection step can be used to convert compound 38 to 39. [ka]

[0058] The technique outlined in Scheme 3 can be used to synthesize lipid 5, where the primary amine is an O-protected derivative of 4-amino-1-butanol (such as O-TBDPS derivative 40), the alkyl halide is 41, and the product is 42 (Scheme 4), which can be converted to lipid 5 as described below. [ka]

[0059] The synthesis of representative lipid 5 continues with bis-epoxidation of the double bond of 42 with any suitable epoxidizing reagent. For example, reaction of 42 with peroxycarboxylic acids such as magnesium monoperoxyphthalate, peracetic acid, or meta-chloroperoxybenzoic acid (MCPBA) produces 43 as a mixture of unresolved epoxide diastereomers. Reaction of 43 with a thiol such as 1-pentanethiol under basic conditions results in selective nucleophilic cleavage of the epoxide at the CH2 position, generating bis-β-hydroxysulfide 44, which retains the N-oxide functionality. The N-oxide is then reduced to the corresponding tertiary amine 45 by reaction with triphenylphosphine (Scheme 5). [ka]

[0060] Compounds such as 45 can be converted to lipids such as 5 by esterifying the OH group with a carboxylic acid in the presence of a condensing agent, e.g., a carbodiimide such as EDCI, optionally in the presence of a catalyst such as 4-dimethylaminopyridine (DMAP), followed by release of the TBDPS group with a fluoride ion source, e.g., pyridine-HF complex. In the case of 5, the carboxylic acid is decanoic acid (Scheme 6). [ka]

[0061] Those skilled in the art will appreciate that the ionizable head groups present in lipids 6-32 in Table 1 can be introduced by starting with precursors of those lipids in which a ketone functional group is present in place of the ionizable head group. The ketone can then be converted to the appropriate ionizable head group by appropriate organic synthesis steps. Thus, the synthesis of 6-32 begins with the preparation of the appropriate ketone.

[0062] Representative lipids 6-32 can be prepared from, but are not limited to, a ketone having the general structure shown in Scheme 7 as 50. Specific steps in the synthesis of ketones such as 50 are described in detail in co-pending and co-owned WO 2022 / 246555, which is incorporated herein by reference. As described therein, one such step involves the Claisen condensation of an appropriate ester under Mukaiyama conditions, followed by hydrolysis of the resulting beta-ketoester and decarboxylation of the intermediate beta-ketoacid to form the ketone. In certain embodiments, these steps are most advantageously performed as a "one-pot operation," meaning that various synthetic intermediates can, but do not need to, be isolated. For example, ketone 50 can be prepared starting from the Claisen-Mukaiyama condensation of ester 47, with synthetic intermediates 48 and 49 being optional intermediates that do not need to be isolated. [ka]

[0063] Alternatively, ketones such as 50 and its congeners can be prepared by specific synthetic steps detailed in commonly owned and co-pending U.S. Provisional Patent Application No. 63 / 445,854, incorporated herein by reference. One such step involves double alkylation of a reagent such as tosylmethylisocyanide (TosMIC) by reaction with approximately two equivalents of an alkyl halide (chloride, bromide, or iodide) or sulfonate (tosylate, mesylate, triflate, etc.) under basic conditions, followed by acidic hydrolysis of the product. This is exemplified in Scheme 8 for the synthesis of 50 via the TosMIC method. [ka]

[0064] Ketones such as 50 are symmetrical, meaning that the alkyl groups attached to the carbonyl are identical. The provisional application teaches that unsymmetrical congeners of 50, in which two different alkyl groups are attached to the carbonyl group, can be prepared by sequential alkylation of TosMIC with two different alkyl halides or sulfonates, followed by hydrolysis of the product under acidic conditions (Scheme 9). [ka]

[0065] Conversion of ketones such as 50 to lipids of Formula A begins with epoxidation of the double bond using an appropriate epoxidation reagent. Suitable reagents include, but are not limited to, percarboxylic acids such as magnesium monoperoxyphthalate, performic acid, peracetic acid, and meta-chloroperoxybenzoic acid (MCPBA), which convert the double bond directly to an epoxide, or electrophilic halogen sources in aqueous media, such as chlorine, bromine, iodine, N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), and N-iodosuccinimide (NIS), which convert the double bond to a halohydrin that can be converted to an epoxide by treatment with base. In either case, the result is the formation of the bis-epoxide xx as a mixture of unresolved oxirane diastereomers. This is shown in Scheme 10, where 50 is treated with MCPBA to form 56. Subsequent regioselective ring-opening of the epoxide with a thiol, R-SH, under basic conditions generates the dihydroxyketone 57. [ka]

[0066] The dihydroxyketone is then esterified with an appropriate carboxylic acid. Depending on the conditions, the esterification reaction can be carried out to form symmetrical diester 58 as the major product, for example, by using at least two molar equivalents of the acid R'-COOH in the presence of a condensing agent, such as a carbodiimide, e.g., EDCI, and DMAP, or to form monoester 59 as the major product, for example, by using approximately one molar equivalent of the acid R'-COOH in the presence of a condensing agent, such as a carbodiimide, e.g., EDCI, and DMAP (Scheme 10). Furthermore, monoester 59 can be converted to asymmetrical diester 60 by subsequent reaction with a second carboxylic acid, R"-COOH, in the presence of a condensing agent, e.g., a carbodiimide, e.g., EDCI, and DMAP (Scheme 10). The ketone group of 58 or 60 can then be converted to any ionizable head group of types 1-9 (see definitions above) by chemical methods well known to those skilled in the art. Representative examples are provided below, but are not limited to these.

[0067] The ketone of type 50 required for the synthesis of lipid 6 is 61 (a variant of 50 with n = 3), the diepoxyketone obtained from 61 is 62 (a variant of 56 with n = 3), the thiol used in the epoxide cleavage reaction is cyclohexanethiol, and the acid used in the esterification of the resulting dihydroxyketone 63 is decanoic acid (Scheme 11). The ketone in product 64 is then converted to an ionizable head group of type 1 by selective reduction with a hydride reagent, such as sodium borohydride, in a suitable solvent, such as an alcohol, such as ethanol or isopropanol. The resulting alcohol 65 is subsequently esterified with 4-(dimethylamino)butanoic acid or its salt, such as the corresponding hydrochloride, in the presence of a condensing agent, such as a carbodiimide, e.g., EDCI, and DMAP, to form lipid 6. [ka]

[0068] The ketone of type 50 required for the synthesis of lipid 7 is 66 (a variant of 50 with n = 4), the diepoxyketone obtained from 66 is 67 (a variant of 56 with n = 4), the thiol utilized in the epoxide cleavage reaction is 1-hexanethiol, and the acid utilized in the esterification of the resulting dihydroxyketone 68 is decanoic acid (Scheme 12). The ketone product 69 is then converted to the ionizable head group of type 1 as shown above in Scheme 11.

[0069] The synthesis of lipid 8 (Scheme 13) involves esterifying compound 68 from Scheme 12 with 3-cyclohexylpropanoic acid, followed by conversion of the resulting ketone 71 to an ionizable head group of type 1 as shown in Scheme 11 above.

[0070] The synthesis of lipid 9 requires acid 74, which can be prepared, for example, by the method shown in Scheme 14: [ka] [ka] Details are described in commonly owned and co-pending U.S. Provisional Patent Application No. 63 / 410,273. Mono-esterification of dihydroxyketone 68 with decanoic acid and further esterification of the resulting 75 with acid 74 affords 76. Conversion of 76 to a ketone ionizable head group of type 1 is then achieved (Scheme 15), as shown above in Scheme 11. [ka]

[0071] The diepoxyketone required for the synthesis of lipid 10 is 67, the thiol utilized in the epoxide cleavage reaction is cyclohexanethiol, and the acid utilized in the esterification of the resulting dihydroxyketone 78 is decanoic acid (Scheme 16). The ketone of product 79 is then converted to an ionizable head group of type 1 as shown in Scheme 11 above. [ka]

[0072] The synthesis of lipid 11 (Scheme 17) involves esterifying dihydroxyketone 78 with nonanoic acid, followed by conversion of the ketone of the resulting diester 81 to an ionizable head group of type 1 as shown in Scheme 11 above.

[0073] The synthesis of lipid 12 (Scheme 18) begins with diepoxyketone 62. The thiol utilized in the epoxide cleavage reaction is 1-pentanethiol, and the acid utilized in the esterification of the resulting dihydroxyketone 83 is decanoic acid. The ketone in product 84 is then [ka] The process begins with the formation of ketal 85 by reaction with 1,2,4-butanetriol in the presence of an acid catalyst in a suitable solvent at moderately elevated temperatures. The ketal is then converted to a type 2 ionizable head group, preferably with continuous azeotropic removal of the water produced during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium para-toluenesulfonate (PPTS), using a Dean-Stark trap for water removal. The OH group in the product of the ketalization step, 85, is then converted to a leaving group such as a halide (chloride, bromide, or iodide) or a sulfonate ester (tosylate, mesylate, triflate, etc.) in preparation for the introduction of the dimethylamino moiety. For example, 85 can be converted to tosylate 86 by reaction with para-toluenesulfonyl chloride (TsCl) in the presence of a base such as triethylamine in a basic solvent such as pyridine, or in a non-basic solvent such as CHCl, optionally in the presence of a catalyst such as 4-dimethylaminopyridine. Reaction of tosylate 86 with dimethylamine in a suitable solvent or mixture of solvents, such as tetrahydrofuran (THF) and methanol, at a suitable elevated temperature, optionally in the presence of a base such as NaCO or KCO, and optionally with microwave activation, produces lipid 12. [ka]

[0074] The synthesis of lipid 13 (Scheme 19) demonstrates how ketones can be converted to ionizable head groups of type 4. Thus, ketone 84 can be converted to bromoketal 87 by reaction with 3-bromo-1,2-propanediol in a suitable solvent at moderately elevated temperatures in the presence of an acid catalyst, preferably with continuous azeotropic removal of the water produced during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium para-toluenesulfonate (PPTS), and a Dean-Stark trap can be used for water removal. Reaction of 87 with 4-methylamino-1-butanol in a suitable solvent or mixture of solvents, such as acetonitrile, at moderately elevated temperatures, optionally in the presence of a base such as NaCO or KCO, and optionally with microwave activation, produces 13. [ka]

[0075] Lipids 14 and 15 can be prepared in a similar manner from tosylate 86 by reaction with 4-ethylamino-1-butanol (Scheme 20) and piperidin-4-ol (Scheme 21), respectively. [ka] [ka]

[0076] Lipid 16 can be prepared from ketone 64 of Scheme 11 by converting the carbonyl group to an ionizable head group of type 2 by the method outlined in Scheme 18 (Scheme 22). [ka]

[0077] The head groups present in lipids 17 and 18 are variants of the Type 3 ionizable group, which can be generated by starting with the conversion of ketone 64 (Scheme 11) to ketal 90 by reaction with 2,2-bis(hydroxymethyl)propane-1,3-diol (pentaerythritol) in a suitable solvent at a moderately elevated temperature in the presence of an acid catalyst, preferably with continuous azeotropic removal of water generated during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium para-toluenesulfonate (PPTS), and a Dean-Stark trap can be used for water removal. Compound 90 is then monoesterified with 3-(dimethylamino)propanoic acid or its hydrochloride in the presence of a carbodiimide such as EDCI and, optionally, DMAP, to give 17. A similar esterification reaction with 4-(dimethylamino)butanoic acid or its HCl salt produces 18 (Scheme 23). [ka]

[0078] The synthesis of lipid 19 (Scheme 24) involves converting ketone compound 69 from Scheme 12 to an ionizable head group of type 2 in a manner similar to that shown in Scheme 19, except that dimethylamine is used in the final reaction. [ka]

[0079] Lipid 20 (Scheme 25) can be prepared by reacting bromoketal 91 with 4-(methylamino)-1-butanol according to the method previously shown in Scheme 19. [ka]

[0080] Ionizable groups such as type 4 of lipid 21 can be introduced by converting ketone 69 to ketal 92, followed by tosylation and displacement with 4-methylamino-1-butanol (Scheme 26). [ka]

[0081] The synthesis of lipids 22-32 demonstrates how to convert a ketone group into an ionizable head of type 7. Thus, lipid 22 can be prepared from ketone 84 (Scheme 18) by reductive amination with an O-protected form of 4-amino-1-butanol, such as a silyl ether (e.g., tert-butyldiphenylsilyl (TBDPS) ether), in a suitable solvent, e.g., 1,2-dichloroethane, in the presence of a reducing agent, e.g., a boronate reagent such as sodium triacetoxyborohydride or sodium cyanohydride, and optionally in the presence of a catalyst such as acetic acid (Scheme 27). The secondary amine thus formed, such as 94, is then N-alkylated to produce a tertiary amine. In the case of lipid 22, this is followed by N-methylation to give 95. This can be achieved by treating 94 with a reducing agent, such as, but not limited to, aqueous formaldehyde and sodium triacetoxyborohydride, in a suitable solvent such as THF, or with a methylating agent, such as a methyl halide (chloride, bromide, iodide), sulfate, sulfonate, sulfonium, or sulfoxonium reagent, also in a suitable solvent and under suitable conditions. Lipid 22 can be obtained by releasing the TBDPS group of 95 using a fluoride ion source, for example, HF-pyridine complex. [ka]

[0082] The synthesis of lipid 23 (Scheme 28) begins with the reaction of bis-epoxyketone 62 from Scheme 11 with 1-heptanethiol, followed by esterification of the resulting 96 with decanoic acid to produce 97. The latter compound is then converted to lipid 23 by the same method as shown in Scheme 27 above. [ka]

[0083] The synthesis of lipid 24 (Scheme 29) can be achieved in a similar manner, except that compound 96 is esterified with nonanoic acid. [ka]

[0084] Lipid 25 can be prepared by starting with the reaction of bis-epoxy ketone 67 of Scheme 12 with 1-pentanethiol and esterifying the resulting 103 with nonanoic acid (Scheme 30). The ketone 104 thus obtained can then be converted to lipid 25 by the method shown in Scheme 27 above. [ka]

[0085] Lipid 26 can be prepared in a similar manner starting from the esterification of compound 103 with decanoic acid (Scheme 31). [ka]

[0086] Lipid 27 can be obtained from ketone 69 of Scheme 12 by the method shown in Scheme 27 above (Scheme 32). [ka]

[0087] Lipid 28 can be prepared starting from N-propylation of secondary amine 110. This can be done, for example, by reacting 110 with propanal (propionaldehyde) in a suitable solvent such as 1,2-dichloroethane in the presence of a reducing agent such as a borohydride reagent, e.g., sodium triacetoxyborohydride, sodium cyanoborohydride, and the like, optionally in the presence of a catalyst such as acetic acid (Scheme 33). The resulting 112 is converted to lipid 28 by the same method as shown in Scheme 27 above.

[0088] Lipid 29 can be prepared starting from the N-isobutylation of secondary amine 110, for example, with a borohydride reagent in a suitable solvent such as 1,2-dichloroethane. [ka] For example, this can be achieved by reacting 110 with 2-methylpropanal (isobutyraldehyde) in the presence of a reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride, optionally in the presence of a catalyst such as acetic acid (Scheme 34). The resulting 113 is converted to lipid 29 by the same method as shown in Scheme 27 above. [ka]

[0089] Lipid 30 can be prepared from ketone 76 of Scheme 15 by the methods outlined in Scheme 27 above (Scheme 35). [ka]

[0090] Lipid 31 can be prepared by starting with the reaction of bis-epoxy ketone 67 of Scheme 12 with 1-heptanethiol and esterifying the resulting 116 with nonanoic acid (Scheme 36). The ketone 117 thus obtained can then be converted to lipid 31 by the method shown in Scheme 27 above. [ka]

[0091] Lipid 32 can be prepared from ketone 81 of Scheme 17 by the methods outlined in Scheme 27 above (Scheme 37). [ka] Formulating the lipids into a delivery vehicle

[0092] 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.

[0093] In one embodiment, lipids having the structure of Formula A of the present disclosure are formulated into a delivery vehicle by mixing them 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).

[0094] As mentioned above, helper lipids include sterols, diacylglycerols, ceramides, or derivatives thereof.

[0095] 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.

[0096] 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.

[0097] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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).

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0127] 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.

[0128] 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.

[0129] For example, the lipids described herein may also facilitate the incorporation of proteins and peptides into delivery vehicles that include ribonucleoproteins, including both linear and non-linear peptides, proteins, or ribonucleoproteins.

[0130] Although pharmaceutical compositions are described above, the lipids described herein can be ingredients in any nutritional, cosmetic, cleaning, or food product. Pharmaceutical preparations

[0131] 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.

[0132] 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.

[0133] 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.

[0134] The pharmaceutical composition comprises a pharmaceutically acceptable salt and / or excipient.

[0135] The compositions described herein can be administered to a patient. As used herein, the term "patient" includes human or non-human subjects.

[0136] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0137] material 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.

[0138] Luciferase activity was assayed using mRNA encoding firefly luciferase purchased from APExBIO Technology LLC (Houston, TX). method Preparation of mRNA-containing lipid nanoparticles (LNPs)

[0139] Lipid 1 or 2, 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 acid (siRNA or mRNA) was 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 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), at a flow ratio of 3:1 (v / v; respectively) and a total flow rate of 20 mL / min. 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

[0140] 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

[0141] 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 5-32.

[0142] Unless otherwise specified, reagents and solvents were commercially available and used without 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 performed under an inert atmosphere (nitrogen or argon). Reaction mixtures from aqueous workups were dried by passing 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 silica gel plates (Merck 60 F254 plates). Visualization of the developed chromatograms was achieved by staining with I or potassium permanganate solution. Chromatographic purifications were performed on a Biotage ISCO system. 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.

[0143] Synthesis of lipid 5 from 4-amino-1-butanol was carried out as follows. As discussed, synthesis of type 5 lipids involves mono- or di-N-alkylation of 4-amino-1-butanol with specific alkyl halides or sulfonates under appropriate conditions. This technique is as described in co-owned and co-pending WO2023 / 173203 (incorporated herein by reference).

[0144] Lipids 5-32 were synthesized 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 2022 / 246555 (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

[0145] (i) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-N-(hept-6-en-1-yl)hept-6-en-1-amine (42). [ka] A mixture of protected O-TBDPS 4-amino-1-butanol 40 (841 mg, 2.57 mmol), 7-bromohept-1-ene (1.00 g, 5.65 mmol), and K2CO3 (859 mg, 6.22 mmol) in MeCN (15.0 mL) was stirred at 80 °C for 18 h in a sealed reaction vessel. The mixture was cooled, diluted with water (15 mL), and extracted with C2Cl2 (3 × 15 mL). The combined organics were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give amine 42 (802 mg, 60%) as an oil. 1 H NMR (400 MHz, CDCl3) δ 7.72-7.57 (m, 4H), 7.48-7.31 (m, 6H), 5.89-5.69 (m, 2H), 5.06-4.86 (m, 4H), 3.78-3.63 (m, 2H), 3.01-2.28 (m, 6H), 2.04 (q, J = 7.1 Hz, 0H), 1.60-1.21 (m, 20H), 1.04 (s, 9H).

[0146] (ii) Trideca-1,12-dien-7-one ( 61 ). [ka] To a solution of methyl hept-6-enoate (5.95 g, 41.9 mmol) and NBu3 (18.0 mL, 75.4 mmol) in toluene (80.0 mL) was added dropwise a solution of TiCl4 (6.89 mL, 62.9 mmol) in toluene (40.0 mL) at 0 °C under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and stirred for 2 h. Water (40 mL) was added at 0 °C. The biphasic mixture was extracted with toluene (2 × 40 mL). The combined organics were concentrated, the residue was dissolved in EtOH (70 mL), and 25% NaOH (25 mL) was added. The mixture was stirred for 2 h, concentrated to 25% volume, acidified to pH 2 with concentrated HCl, and extracted with 50:50 hexane / Et2O (3 × 40 mL). The combined organics were washed with brine, dried (Na2SO4), and concentrated. The residue was purified by silica chromatography (0-8% EtOAc in hexanes) to give ketone 61 as an oil (3.7 g, 91%). 1 H NMR (400 MHz, CDCl3) δ 5.79 (ddt, J = 16.9, 10.2, 6.7 Hz, 2H), 5.07-4.86 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.20-1.98 (m, 4H), 1.71-1.49 (m, 4H), 1.49-1.33 (m, 4H).

[0147] (iii) Pentadeca-1,14-dien-8-one (66). [ka] Prepared from methyl oct-7-enoate by the procedure in part (ii) above. 1H NMR (400 MHz, CDCl3) δ 5.79 (ddt, J = 16.9, 10.2, 6.6 Hz, 2H), 5.07-4.84 (m, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.10-1.99 (m, 4H), 1.62-1.52 (m, 4H), 1.45-1.34 (m, 4H), 1.34-1.23 (m, 4H).

[0148] (iv) 1,9-di(oxiran-2-yl)nonan-5-one (62). [ka] Solid mCPBA (13.9 g, approximately 50% purity) was added to a solution of ketone 61 (3.9 g, 20.0 mmol) in DCM (70.0 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 2 h. The mixture was cooled to 0 °C, quenched with sat. aq. sodium sulfite, and diluted with water (20 mL). The layers were separated, and the organics were washed with 1 N NaOH (3 × 30 mL), dried (NaSO), and concentrated to give bis-epoxide 62 (4.1 g, 90%). 1 H NMR (400 MHz, CDCl3) δ 2.94-2.87 (m, 2H), 2.74 (dd, J = 5.0, 3.9 Hz, 2H), 2.46 (dd, J = 5.0, 2.7 Hz, 2H), 2.41 (t, J = 7.3 Hz, 4H), 1.73-1.34 (m, 12H).

[0149] (v).1,11-Di(oxiran-2-yl)undecan-6-one (67). [ka] Prepared from ketone 66 by procedure (iv) above. 1H NMR (400 MHz, CDCl3) δ 2.94-2.83 (m, 2H), 2.76-2.69 (m, 2H), 2.44 (dd, J = 5.0, 2.7 Hz, 2H), 2.38 (td, J = 7.5, 1.6 Hz, 4H), 1.65-1.20 (m, 16H).

[0150] (vi) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-5-(oxiran-2-yl)-N-(5-(oxiran-2-yl)pentyl)-pentan-1-amine oxide (43). [ka] Solid mCPBA (1.59 g, approximately 50% purity) was added to a solution of 42 (800 mg, 1.54 mmol) in DCM (10.0 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 3 h. The mixture was cooled to 0 °C, quenched with sat. aq. sodium sulfite, and diluted with water (10.0 mL). The layers were separated, and the organics were washed with 1 N NaOH (3 × 30.0 mL), dried (NaSO), and concentrated to give bis-epoxide 43 as a waxy white solid (629 mg, 72%), which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.60 (m, 4H), 7.51-7.34 (m, 6H), 3.71 (t, J = 5.9 Hz, 2H), 3.32-3.12 (m, 6H), 2.92-2.84 (m, 2H), 2.78-2.70 (m, 2H), 2.49-2.41 (m, 2H), 2.01-1.34 (m, 20H), 1.04 (s, 9H).

[0151] (vii) General procedure for epoxide ring-opening with thiols. To a well-stirred solution of bis-epoxide (1 mmol) and thiol (2.2 mmol, 2.2 equiv.) in EtOH (10 mL) kept under an inert atmosphere was added solid NaOH (4 equiv.). The mixture was heated to reflux for 2 h, cooled, diluted with water (20 mL), and extracted with DCM (3 × 15.0 mL). The combined organics were dried (NaSO4) and concentrated. The residue was purified by silica chromatography (0–50% EtOAc in hexanes) to give the desired product (75–80%). The following compounds were then prepared:

[0152] (viii) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-6-hydroxy-N-(6-hydroxy-7-(pentylthio)hept-yl)-7-(pentylthio)heptan-1-amine oxide (44) and 9-(6-hydroxy-7-(pentylthio)heptyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-17-thia-9-aza-3-siladocosan-15-ol (45). [ka] Compound 44 was obtained from 43 and 1-pentanethiol by the general procedure in part (vii). 1 H NMR (400 MHz, CDCl) δ 3.69-3.55 (m, 2H), 2.73 (dd, J = 13.6, 3.3 Hz, 2H), 2.51 (t, J = 7.4 Hz, 4H), 2.46-2.34 (m, 6H), 1.67-1.23 (m, 28H), 0.90 (t, J = 7.0 Hz, 6H). A solution of amine oxide 44 (510 mg, 0.657 mmol) and triphenylphosphine (517 mg, 1.97 mmol) in glacial HOAc (8.00 mL) was heated to reflux for 4 h. The mixture was then diluted with DCM (10.0 mL), washed with water (3 × 15.0 mL), brine (15.0 mL), dried (NaSO), and concentrated. The residue was purified by silica chromatography (0-40% MeOH in DCM) to give amine 45 (255 mg, 51%).1 H NMR (400 MHz, CDCl3) δ 7.69 - 7.62 (m, 4H), 7.47 - 7.33 (m, 6H), 3.67 (t, J = 5.9 Hz, 2H), 3.65 - 3.58 (m, 2H), 2.71 (dd, J = 13.6, 3.5 Hz, 2H), 2.66 - 2.56 (m, 6H), 2.51 (t, J = 7.5 Hz, 4H), 2.43 (dd, J = 13.6, 8.9 Hz, 2H), 1.66 - 1.26 (m, 32H), 1.04 (s, 9H), 0.89 (t, J = 7.1 Hz, 6H).

[0153] (ix) 1,13-bis(cyclohexylthio)-2,12-dihydroxytridecan-7-one (63). [ka] Prepared from 62 and cyclohexanethiol according to the procedure in part (vii). 1 H NMR (400 MHz, CDCl3) δ 3.66-3.56 (m, 2H), 2.78 (dd, J = 13.5, 3.4 Hz, 2H), 2.69-2.57 (m, 2H), 2.49-2.37 (m, 6H), 2.03-1.89 (m, 6H), 1.81-1.71 (m, 4H), 1.67-1.20 (m, 22H).

[0154] (x) 1,15-bis(hexylthio)-2,14-dihydroxypentadecan-8-one (68). [ka] Prepared from 67 and 1-hexanethiol according to the procedure in part (vii). 1H NMR (400 MHz, CDCl3) δ 3.69-3.55 (m, 2H), 2.73 (dd, J = 13.6, 3.3 Hz, 2H), 2.51 (t, J = 7.4 Hz, 4H), 2.46-2.34 (m, 6H), 1.67-1.23 (m, 32H), 0.90 (t, J = 7.0 Hz, 6H).

[0155] (xi) 1,15-bis(cyclohexylthio)-2,14-dihydroxypentadecan-8-one (78). [ka] Prepared from 67 and cyclohexanethiol according to the procedure in part (vii). 1 H NMR (400 MHz, CDCl3) δ 3.65-3.53 (m, 2H), 2.83-2.73 (m, 2H), 2.70-2.57 (m, 2H), 2.55-2.30 (m, 6H), 2.07-1.85 (m, 4H), 1.82-1.70 (m, 4H), 1.58 (tdt, J = 14.8, 7.3, 3.9 Hz, 6H), 1.51-1.39 (m, 6H), 1.39-1.17 (m, 16H).

[0156] (xii) 2,12-dihydroxy-1,13-bis(pentylthio)tridecan-7-one (83). [ka] Prepared from 62 and 1-pentanethiol according to the procedure in part (vii). 1 H NMR (400 MHz, CDCl3) δ 3.68 - 3.55 (m, 2H), 2.79 - 2.68 (m, 2H), 2.58 - 2.36 (m, 10H), 1.66 - 1.52 (m, 8H), 1.53 - 1.43 (m, 6H), 1.41 - 1.25 (m, 10H), 0.95 - 0.85 (m, 6H).

[0157] (xiii) 1,13-bis(heptylthio)-2,12-dihydroxytridecan-7-one (96). [ka] Prepared from 62 and 1-heptanethiol according to the procedure in part (vii). 1 H NMR (400 MHz, CDCl3) δ 3.70-3.56 (m, 2H), 2.72 (dd, J = 13.6, 3.3 Hz, 2H), 2.58-2.34 (m, 10H), 1.70-1.53 ​​(m, 8H), 1.53-1.42 (m, 6H), 1.42-1.22 (m, 18H), 0.92-0.83 (m, 6H).

[0158] (xiv) 2,14-dihydroxy-1,15-bis(pentylthio)pentadecan-8-one (103). [ka] Prepared from 67 and 1-pentanethiol according to the procedure in part (vii). 1 H NMR (400 MHz, CDCl3) δ 3.69-3.55 (m, 2H), 2.73 (dd, J = 13.6, 3.3 Hz, 2H), 2.51 (t, J = 7.4 Hz, 4H), 2.46-2.34 (m, 6H), 1.67-1.23 (m, 28H), 0.90 (t, J = 7.0 Hz, 6H).

[0159] (xv) 1,15-bis(heptylthio)-2,14-dihydroxypentadecan-8-one (116). [ka] Prepared from 67 and 1-heptanethiol according to the procedure in part (vii). 1H NMR (400 MHz, CDCl3) δ 3.69-3.55 (m, 2H), 2.73 (dd, J = 13.6, 3.3 Hz, 2H), 2.51 (t, J = 7.4 Hz, 4H), 2.46-2.34 (m, 6H), 1.67-1.23 (m, 36H), 0.90 (t, J = 7.0 Hz, 6H). (B) Preparation of symmetric diester derivatives of the dihydroxy compounds of part (A).

[0160] (i) General procedure. A solution of dihydroxy compound (1 mmol, 1 equiv.), carboxylic acid (2.4 mmol, 1.2 equiv.), EDCI*HCl (2.5 mmol, 1.25 equiv.), and DMAP (2.4 mmol) in DCM (4 mL) was stirred at room temperature under a nitrogen atmosphere for 18 hours. The reaction was concentrated, and the residue was purified by silica chromatography (0-10% EtOAc in hexanes) to give the diester (80-90%) as an oil, and the following compounds were obtained:

[0161] (ii) 9-(6-(decanoyloxy)-7-(pentylthio)heptyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-17-thia-9-aza-3-siladocosan-15-yl decanoate (46). [ka] Obtained from 45 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 7.74-7.62 (m, 4H), 7.48-7.33 (m, 6H), 4.99-4.89 (m, 2H), 3.72-3.62 (m, 2H), 2.69-2.57 (m, 4H), 2.53 (td, J = 7.3, 1.8 Hz, 4H), 2.45-2.24 (m, 10H), 1.77-1.15 (m, 60H), 1.04 (s, 9H), 0.94-0.82 (m, 12H).

[0162] (iii) 1,13-bis(cyclohexylthio)-7-oxotridecane-2,12-diyl bis(decanoate) ( 64 ). [ka] 63 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 4.98-4.81 (m, 2H), 2.77-2.57 (m, 6H), 2.38 (t, J = 7.4 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 2.00-1.91 (m, 4H), 1.81-1.18 (m, 56H), 0.95-0.81 (m, 6H).

[0163] (iv) 1,15-bis(hexylthio)-8-oxopentadecane-2,14-diyl bis(decanoate) (69). [ka] It was obtained from 68 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 4.94 (dtd, J = 8.1, 6.1, 4.4 Hz, 2H), 2.69-2.58 (m, 4H), 2.53 (td, J = 7.3, 1.3 Hz, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 1.78-1.47 (m, 16H), 1.46-1.20 (m, 44H), 0.93-0.81 (m, 12H).

[0164] (v) 1,15-bis(hexylthio)-8-oxopentadecane-2,14-diyl bis(3-cyclohexylpropanoate) (71). [ka] It was obtained from 68 and 3-cyclohexylpropanoic acid. 1H NMR (400 MHz, CDCl3) δ 5.00-4.88 (m, 2H), 2.69-2.58 (m, 4H), 2.53 (td, J = 7.2, 1.4 Hz, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.33-2.28 (m, 4H), 1.79-1.06 (m, 54H), 0.95-0.83 (m, 10H).

[0165] (vi) 1,15-bis(cyclohexylthio)-8-oxopentadecane-2,14-diyl bis(decanoate) (79). [ka] Obtained from 78 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 4.91 (dtd, J = 8.1, 6.2, 4.3 Hz, 2H), 2.73-2.58 (m, 6H), 2.36 (t, J = 7.4 Hz, 6H), 2.28 (t, J = 7.5 Hz, 4H), 1.99-1.89 (m, 4H), 1.81-1.48 (m, 16H), 1.37-1.19 (m, 42H), 0.92-0.82 (m, 6H).

[0166] (vii) 1,15-bis(cyclohexylthio)-8-oxopentadecane-2,14-diyl dinonanoate (81). [ka] Obtained from 78 and nonanoic acid. 1 H NMR (400 MHz, CDCl3) δ 4.91 (dtd, J = 8.2, 6.2, 4.3 Hz, 2H), 2.76-2.58 (m, 6H), 2.37 (t, J = 7.4 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 1.96 (dd, J = 8.9, 5.2 Hz, 4H), 1.80-1.48 (m, 18H), 1.37-1.20 (m, 38H), 0.91-0.83 (m, 6H).

[0167] (viii) 7-oxo-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (84). [ka] 83 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 5.00-4.87 (m, 2H), 2.70-2.57 (m, 4H), 2.53 (td, J = 7.3, 1.5 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.34-2.26 (m, 4H), 1.78-1.47 (m, 18H), 1.38-1.22 (m, 34H), 0.95-0.84 (m, 12H).

[0168] (ix) 1,13-bis(heptylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (97). [ka] 96 and decanoic acid. 1 H NMR (400 MHz, CDCl3) δ 5.08-4.85 (m, 2H), 2.69-2.57 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.79-1.47 (m, 18H), 1.42-1.20 (m, 38H), 0.88 (t, J = 6.8 Hz, 12H).

[0169] (x) 1,13-bis(heptylthio)-7-oxotridecane-2,12-diyl dinonanoate (100). [ka] It was obtained from 96 and nonanoic acid. 1H NMR (400 MHz, CDCl3) δ 5.08-4.85 (m, 2H), 2.69-2.57 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.79-1.47 (m, 18H), 1.42-1.20 (m, 38H), 0.88 (t, J = 6.8 Hz, 12H).

[0170] (xi) 8-oxo-1,15-bis(pentylthio)pentadecane-2,14-diyl dinonanoate (104). [ka] Obtained from 103 and nonanoic acid. 1 H NMR (400 MHz, CDCl3) δ 4.94 (dtd, J = 8.1, 6.1, 4.3 Hz, 2H), 2.70-2.58 (m, 4H), 2.59-2.47 (m, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.80-1.48 (m, 16H), 1.46-1.20 (m, 36H), 0.98-0.78 (m, 12H).

[0171] (xii) 8-oxo-1,15-bis(pentylthio)pentadecane-2,14-diyl bis(decanoate) (107). [ka] Obtained from 103 and decanoic acid. 1H NMR (400 MHz, CDCl3) δ 5.05-4.85 (m, 2H), 2.71-2.58 (m, 4H), 2.53 (td, J = 7.3, 1.5 Hz, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.78-1.49 (m, 16H), 1.39-1.21 (m, 40H), 0.96-0.83 (m, 12H).

[0172] (xiii) 1,15-bis(heptylthio)-8-oxopentadecane-2,14-diyl dinonanoate (117). [ka] Obtained from 116 and nonanoic acid 1 H NMR (400 MHz, CDCl3) δ 5.01-4.87 (m, 2H), 2.70-2.57 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.76-1.49 (m, 16H), 1.42-1.18 (m, 44H), 0.92-0.84 (m, 12H). (C) Preparation of unsymmetrical diester derivatives of the dihydroxy compounds of part (A).

[0173] (i) Exemplary procedure for the monoesterification of a dihydroxy compound: 1,15-bis(hexylthio)-14-hydroxy-8-oxopentadecane-2-yl decanoate (75). [ka] A solution of 68 (0.15 g, 0.306 mmol), decanoic acid (0.0526 g, 0.306 mmol), EDCI-HCl (0.0879 g, 0.458 mmol), and DMAP (0.0261 g, 0.214 mmol) in CHCl (15 mL) was stirred under inert atmosphere for 16 h, then quenched with water and extracted with CHCl (3 × 30 mL). The combined extracts were dried (NaSO) and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography using a gradient of EtOAc in hexanes (0 to 50% EtOAc in hexanes) to give pure 75 in 51% yield. 1 H NMR (400 MHz, CDCl3) δ 4.93 (m, 1H), 3.61 (m, 1H), 2.73 (m, 1H), 2.63 (m, 2H), 2.52 (m, 4H), 2.45-2.34 (m, 5H), 2.29 (tr, J = 7.5 Hz, 2H), 2.07 (br, 1H), 1.73-1.44 (m, 14), 1.41-1.19 (m, 32H), 0.91-0.84 (tr, 9H).

[0174] (ii) Methyl N-hexanoyl-N-methylglycinate (73). [ka] To a solution of hexanoic acid (1.3 g, 11.1 mmol) in CHCl (50 mL) was added EDCI·HCl (3.19 g, 16.6 mmol), followed by HOBt (2.55 g, 16.6 mmol), sarcosine methyl ester hydrochloride (1.55 g, 11.1 mmol), and diisopropylethylamine (3.9 mL, 22.2 mmol). The mixture was stirred under an inert atmosphere for 16 h, then quenched with water and extracted with CHCl (3 × 30 mL). The combined extracts were dried (NaSO) and concentrated under reduced pressure. The orange residue was purified by column chromatography (0–25% ethyl acetate in hexanes) to give pure 73 in 60% yield. 1H NMR (400 MHz, CDCl3, rotamers) δ 4.14-4.00 (s, 2H), 3.79-3.66 (m, 3H), 3.09-2.91 (s, 3H), 2.39-2.16 (tr, 2H), 1.69-1.63 (m, 2H), 1.35-1.25 (m, 4H), 0.93-0.85 (m, 3H).

[0175] (iii) N-Hexanoyl-N-methylglycine (74). A solution of LiOH·HO (1.56 g, 37 mmol) in water (10 mL) was added to a solution of 73 (3 g, 15 mmol) in THF (30 mL). The mixture was stirred at reflux under an inert atmosphere. The reaction was complete in 4 h, at which point it was cooled, acidified to pH ∼1 with 1 M HCl, and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate and concentrated under reduced pressure to give the product in 90% yield. 1 H NMR (400 MHz, CDCl3, rotamers) δ 4.20-4.01 (m, 2H), 3.14-2.92 (m, 3H), 2.42-2.19 (m, 2H), 1.70-1.54 (m, 2H), 1.38-1.19 (m, 4H), 0.94-0.83 (m, 3H).

[0176] (iv) Exemplary procedure for esterification of hydroxy monoester: 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-oxopentadecane-2-yl decanoate (76). [ka] A solution of alcohol 75 (0.532 g, 0.825 mmol), acid 74 (0.178 g, 0.948 mmol), EDCI·HCl (0.237 g, 1.24 mmol), and DMAP (0.0705 g, 0.577 mmol) in CHCl (20 mL) was stirred at room temperature for 16 h, then quenched with water and extracted with CHCl (3 × 30 mL). The combined organics were dried (NaSO) and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography using a gradient of EtOAc in hexanes to give pure 76 in 75% yield. 1 H NMR (400 MHz, CDCl3 rotamer) δ 5.10-4.87 (m, 2H), 4.23-4.00 (m, 2H), 3.07 and 2.98 (s, 3H), 2.67- 2.59 (m, 4H), 2.56- 2.48 (m, 4H), 2.41- 2.21 (m, 8H), 1.76-1.49 (m, 16H), 1.40-1.19 (m, 36H), 0.93- 0.82 (tr, 12H) (D) General procedure for release of the silyl group: ((4-hydroxybutyl)azanediyl)bis(1-(pentylthio)heptane-7,2-diyl) bis(decanoate) (5). [ka]

[0177] To a cold (0 °C) solution of protected TBDPS 46 (1.1 g, 1 mmol) in THF (5 mL) kept under an inert atmosphere was added HF-pyridine (1 mL). The reaction was allowed to warm to room temperature and stirred for 18 h. Water (10 mL) was added, and the mixture was extracted with CHCl (3 × 15 mL). The combined extracts were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give lipid 5 (630 mg, 76%) as an oil. (E) Preparation of lipids with type 1 ionizable head groups: synthesis of lipids 6–11.

[0178] (i) General procedure for ketone reduction. To a solution (0 °C) of ketone (0.5 mmol) in EtOH (3 mL) was added solid NaBH (0.5 mmol). The mixture was warmed to room temperature and stirred for 30 min. The reaction was quenched with sat. aq. NH Cl (2 mL), diluted with water (3 mL), and extracted with DCM (3 × 5 mL). The combined extracts were dried (Na SO ) and concentrated to give the corresponding alcohol (quantitative) as an oil, which was used in the next step without further purification. The following compounds were obtained:

[0179] (ii) 1,13-bis(cyclohexylthio)-7-hydroxytridecane-2,12-diyl bis(decanoate) ( 65 ). [ka] Obtained from ketone 64. 1 H NMR (400 MHz, CDCl3) δ 5.00-4.85 (m, 2H), 3.65-3.49 (m, 1H), 2.80-2.60 (m, 6H), 2.30 (t, J = 7.5 Hz, 4H), 2.11-1.90 (m, 4H), 1.83-1.14 (m, 60H), 0.98-0.77 (m, 6H).

[0180] (iii) 1,15-bis(hexylthio)-8-hydroxypentadecane-2,14-diyl bis(decanoate) ( 70 ). [ka] Obtained from ketone 69. 1 H NMR (400 MHz, CDCl3) δ 5.03-4.88 (m, 2H), 3.60 - 3.52 (m, 1H), 2.70 - 2.61 (m, 4H), 2.59 - 2.49 (m, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.85 - 1.19 (m, 65H), 0.88 (td, J = 6.9, 2.7 Hz, 12H).

[0181] (iv) 1,15-bis(hexylthio)-8-hydroxypentadecane-2,14-diyl bis(3-cyclohexylpropanoate) (72). [ka] Obtained from ketone 71. 1 H NMR (400 MHz, CDCl3) δ 5.00-4.89 (m, 2H), 3.66-3.51 (m, 1H), 2.72-2.59 (m, 4H), 2.53 (td, J = 7.2, 1.4 Hz, 4H), 2.39-2.26 (m, 4H), 1.86-1.02 (m, 58H), 0.97-0.81 (m, 10H).

[0182] (v) 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-hydroxypentadecan-2-yl decanoate (77). [ka] Obtained from ketone 76. 1 H NMR (400 MHz, CDCl3, rotamers) δ 5.11-4.89 (m, 2H), 4.29-3.95 (m, 2H), 3.56 (m, 1H), 3.08 and 2.98 (s, 3H), 2.68-2.60 (m, 4H), 2.57-2.48 (m, 4H), 2.36 (tr, J=7.6, 2H), 2.30 (tr, J=7.5, 7.5, 2H), 1.76-1.50 (m, 13H), 1.45- 1.21 (m, 44H), 0.92-0.83 (t, 12H).

[0183] (vi) 1,15-bis(cyclohexylthio)-8-hydroxypentadecane-2,14-diyl bis(decanoate) (80). [ka] Obtained from ketone 79. 1H NMR (400 MHz, CDCl3) δ 4.98-4.86 (m, 2H), 3.63-3.53 (m, 1H), 2.77-2.55 (m, 6H), 2.30 (t, J = 7.5 Hz, 4H), 2.04-1.90 (m, 4H), 1.82-1.18 (m, 64H), 0.94-0.81 (m, 6H).

[0184] (vii) 1,15-bis(cyclohexylthio)-8-hydroxypentadecane-2,14-diyl dinonanoate (82). [ka] Obtained from ketone 81. 1 H NMR (400 MHz, CDCl3) δ 4.98-4.88 (m, 2H), 3.63-3.51 (m, 1H), 2.80-2.57 (m, 6H), 2.29 (t, J = 7.5 Hz, 4H), 1.96 (dd, J = 8.8, 5.0 Hz, 4H), 1.84-1.16 (m, 60H), 0.93-0.82 (m, 6H).

[0185] (viii) General procedure for alcohol esterification. A solution of alcohol (0.5 mmol, 1 equiv.), 4-(dimethylamino)butyric acid hydrochloride (0.65 mmol, 1.3 equiv.), EDCI-HCl (0.7 mmol, 1.4 equiv.), and DMAP (23.1 mg, 0.7 mmol, 1.4 equiv.) in DCM (5 mL) was stirred at room temperature under an inert atmosphere for 18 hours and then concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give the lipid (75–85%) as an oil. The following lipid was obtained:

[0186] (ix) 1,13-bis(cyclohexylthio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis-(decanoate) (6). [ka] Obtained from Alcohol 65. 1 H NMR (400 MHz, C6D6) δ 5.29-5.17 (m, 2H), 5.14-5.04 (m, 1H), 2.81-2.58 (m, 6H), 2.43-2.35 (m, 2H), 2.29 (td, J = 7.4, 1.9 Hz, 4H), 2.17 (t, J = 6.8 Hz, 2H), 2.07 (s, 6H), 2.05-1.94 (m, 4H), 1.90-1.02 (m, 62H), 0.92 (t, J = 6.9 Hz, 6H).

[0187] (x) 8-((4-(dimethylamino)butanoyl)oxy)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(decanoate) (7). [ka] Obtained from Alcohol 70. 1 H NMR (400 MHz, CDCl3) δ 4.99-4.90 (m, 2H), 4.84 (p, J = 6.4 Hz, 1H), 2.70-2.59 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.42-2.22 (m, 14H), 1.92-1.19 (m, 66H), 0.88 (td, J = 6.9, 2.6 Hz, 12H).

[0188] (xi) 8-((4-(dimethylamino)butanoyl)oxy)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(3-cyclohexylpropanoate) (8). [ka] Obtained from Alcohol 72. 1H NMR (400 MHz, C6D6) δ 5.33-5.18 (m, 2H), 5.16-5.05 (m, 1H), 2.77-2.43 (m, 8H), 2.37 (t, J = 7.3 Hz, 2H), 2.33-2.27 (m, 4H), 2.16 (t, J = 6.9 Hz, 2H), 2.05 (s, 6H), 1.89-0.98 (m, 60H), 0.87 (t, J = 7.0 Hz, 6H), 0.83-0.72 (m, 4H).

[0189] (xii) 8-((4-(dimethylamino)butanoyl)oxy)-14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis-(hexylthio)pentadecan-2-yl decanoate (9). [ka] Obtained from Alcohol 77. 1 H NMR (400 MHz, CDCl3, rotamers) δ 5.09-4.89 (m, 2H), 4.84 (m, 1H), 4.23-4.02 (m, 2H), 3.07 and 2.98 (s, 3H), 2.68-2.60 (m, 4H), 2.56-2.46 (m, 4H), 2.40- 2.21 (m, 12H), 1.81 (m, 2H), 1.76-1.42 (m, 18H), 1.42- 1.17 (m, 40H), 0.93-0.83 (tr, J = 7.1 Hz, 12H).

[0190] (xiii) 1,15-bis(cyclohexylthio)-8-((4-(dimethylamino)butanoyl)oxy)pentadecane-2,14-diyl bis(decanoate) (10). [ka] Obtained from Alcohol 80. 1H NMR (400 MHz, CDCl3) δ 4.98-4.87 (m, 2H), 4.87-4.79 (m, 1H), 2.74-2.59 (m, 6H), 2.53-2.22 (m, 14H), 2.06-1.42 (m, 28H), 1.28 (d, J = 9.3 Hz, 42H), 0.92-0.83 (m, 6H).

[0191] (xiv) 1,15-bis(cyclohexylthio)-8-((4-(dimethylamino)butanoyl)oxy)pentadecane-2,14-diyl dinonanoate (11). [ka] Obtained from Alcohol 82. 1 H NMR (400 MHz, CDCl3) δ 4.95-4.87 (m, 2H), 4.87-4.79 (m, 1H), 2.73-2.57 (m, 6H), 2.35-2.25 (m, 8H), 2.22 (s, 6H), 2.04-1.90 (m, 4H), 1.88-1.41 (m, 20H), 1.36-1.20 (m, 42H), 0.92-0.83 (m, 6H). (F) Preparation of lipids with ketal-type ionizable head groups: synthesis of lipids 12–21.

[0192] (i) General procedure for ketone ketalization. A solution of ketone (1 mmol, 1 equiv.), diol (2 mmol, 2 equiv.), and pyridinium p-toluenesulfonate (PPTS, 0.2 mmol, 0.2 equiv.) in toluene (10.0 mL) was refluxed under nitrogen with continuous removal of water (Dean-Stark trap) until TLC and NMR indicated complete conversion to the product (12 h–4 d, depending on the diol). The mixture was cooled to room temperature, washed with water (2 × 10 mL) and brine (10 mL), dried (NaSO), and concentrated. The residue was purified by silica gel column chromatography (0–2% MeOH in CHCl) to afford the ketal (65–85%) as an oil. The following compounds were obtained:

[0193] (ii) (4-(2-hydroxyethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (85). [ka] Obtained from ketone 84 and 1,2,4-butanetriol. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.89 (m, 2H), 4.28-4.18 (m, 1H), 4.07 (t, J = 7.2 Hz, 1H), 3.83-3.77 (m, 2H), 3.51 (td, J = 8.1, 2.8 Hz, 1H), 2.71-2.58 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 2.02-1.16 (m, 58H), 0.88 (q, J = 7.0 Hz, 12H).

[0194] (iii) (4-(bromomethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl) bis(decanoate) (87). [ka] Obtained from ketone 84 and 3-bromo-1,2-propanediol. 1H NMR (400 MHz, CDCl3) δ 5.02-4.86 (m, 2H), 4.37-4.26 (m, 1H), 4.13 (dd, J = 8.5, 6.2 Hz, 1H), 3.76 (dd, J = 8.6, 6.1 Hz, 1H), 3.44 (dd, J = 10.0, 4.6 Hz, 1H), 3.29 (dd, J = 10.0, 8.1 Hz, 1H), 2.71-2.57 (m, 4H), 2.57-2.51 (m, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.84-1.16 (m, 56H), 1.03-0.80 (m, 12H).

[0195] (iv) (4-(2-hydroxyethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis-(decanoate) (88). [ka] Obtained from ketone 64 and 1,2,4-butanetriol. 1 H NMR (400 MHz, CDCl3) δ 4.99-4.84 (m, 2H), 4.32-4.15 (m, 1H), 4.11-4.03 (m, 1H), 3.79 (t, J = 5.6 Hz, 2H), 3.50 (td, J = 8.0, 2.6 Hz, 1H), 2.78-2.58 (m, 6H), 2.29 (t, J = 7.5 Hz, 4H), 2.03-1.11 (m, 66H), 0.96-0.81 (m, 6H).

[0196] (v) (5,5-bis(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis-(decanoate) (90). [ka] It was obtained from ketone 64 and pentaerythritol. 1H NMR (400 MHz, CDCl3) δ 5.01-4.87 (m, 2H), 3.75 (s, 4H), 3.70 (s, 4H), 2.79-2.59 (m, 6H), 2.30 (t, J = 7.6 Hz, 4H), 2.01-1.88 (m, 4H), 1.85-1.15 (m, 60H), 0.88 (t, J = 6.7 Hz, 6H).

[0197] (vi) (4-(bromomethyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl) bis(decanoate) (91). [ka] Obtained from ketone 69 and 3-bromo-1,2-propanediol. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.88 (m, 2H), 4.37-4.28 (m, 1H), 4.13 (dd, J = 8.5, 6.2 Hz, 1H), 3.77 (dd, J = 8.6, 6.1 Hz, 1H), 3.45 (dd, J = 10.0, 4.6 Hz, 1H), 3.29 (dd, J = 10.0, 8.1 Hz, 1H), 2.73-2.59 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.75-1.49 (m, 18H), 1.45-1.20 (m, 46H), 0.88 (m, 12H).

[0198] (vii) (5-(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl) bis(decanoate) (92). [ka] It was obtained from ketone 69 and 2-hydroxymethyl-1,3-propanediol. 1H NMR (400 MHz, CDCl3) δ 5.00-4.91 (m, 2H), 3.98 (dd, J = 11.8, 4.0 Hz, 2H), 3.80-3.71 (m, 4H), 2.70-2.60 (m, 4H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.61 (m, 19H), 1.44-1.19 (m, 46H), 0.99-0.83 (m, 12H).

[0199] (viii) General procedure for hydroxyketal tosylation. Solid TsCl (1.3 mmol, 1.3 equiv.) was added to a cold (0 °C) solution of hydroxyketal (1 mmol, 1 equiv.), EtN (1.5 mmol, 1.5 equiv.), and DMAP (0.1 mmol, 0.1 equiv.) in CHCl (3 mL) under a nitrogen atmosphere. The reaction was allowed to warm to room temperature and analyzed by TLC and HPLC. 1 The mixture was stirred until complete conversion to the product was confirmed by H NMR. The reaction was quenched with water (10 mL) and extracted with CHCl (3×10 mL). The combined extracts were dried (NaSO) and concentrated to give the crude tosylate (approximately quantitative), which was used in the next step without purification, and the following compound was obtained:

[0200] (ix) (4-(2-(tosyloxy)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl) bis(decanoate) (86). [ka] Obtained from hydroxyketal 85. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 5.01-4.88 (m, 2H), 4.19-4.04 (m, 3H), 4.03-3.98 (m, 1H), 3.43 (t, J = 7.8 Hz, 1H), 2.71-2.58 (m, 4H), 2.57-2.51 (m, 4H), 2.46 (s, 3H), 2.29 (td, J = 7.6, 2.2 Hz, 4H), 1.96-1.18 (m, 58H), 0.94-0.83 (m, 12H).

[0201] (x) (4-(2-(tosyloxy)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis-(decanoate) (89). [ka] Obtained from hydroxyketal 88. 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 4.98-4.86 (m, 2H), 4.26-4.05 (m, 3H), 4.04-3.97 (m, 1H), 3.43 (t, J = 7.7 Hz, 1H), 2.75-2.56 (m, 6H), 2.45 (s, 3H), 2.34-2.22 (m, 4H), 2.06-1.04 (m, 66H), 0.95-0.81 (m, 6H).

[0202] (xi) (5-((tosyloxy)methyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl) bis-(decanoate) (93). [ka] 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 5.03-4.87 (m, 2H), 4.17 (d, J = 7.2 Hz, 2H), 3.95 (dd, J = 11.9, 3.6 Hz, 2H), 3.64 (dd, J = 12.2, 4.0 Hz, 2H), 2.69-2.59 (m, 4H), 2.54 (ddt, J = 8.2, 6.1, 2.5 Hz, 4H), 2.45 (s, 3H), 2.33-2.27 (m, 4H), 1.94-1.15 (m, 65H), 0.93-0.83 (m, 12H).

[0203] (xii) Exemplary procedure for the displacement of bromides / tosylates with low-boiling amines: (4-(2-(dimethylamino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl) bis-(decanoate) (12). [ka] A solution of tosylate 86 (49.0 mg, 0.0481 mmol), dimethylamine (2 M in THF, 1 mL), and MeOH (1 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 give lipid 12 (31 mg, 74%) as an oil. 1 H NMR (400 MHz, C6D6) δ 5.31-5.20 (m, 2H), 4.13-4.01 (m, 1H), 3.93 (dd, J = 7.7, 5.9 Hz, 1H), 3.41 (t, J = 7.9 Hz, 1H), 2.74-2.41 (m, 8H), 2.33-2.17 (m, 6H), 2.05 (s, 6H), 1.84-1.12 (m, 58H), 1.01-0.81 (m, 12H).

[0204] (xiii) Exemplary procedure for the displacement of bromides / tosylates with high-boiling amines: (4-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl) bis(decanoate) (13). [ka] A mixture of bromoketal 87 (75 mg, 0.0854 mmol), 4-(methylamino)-1-butanol (11 mg, 0.111 mmol), and K2CO3 (15.3 mg, 0.111 mmol) in MeCN (1 mL) was stirred at 80 °C for 18 h in a sealed reaction vessel. The mixture was cooled, diluted with water (2 mL), and extracted with C2Cl2 (3 × 3 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in C2Cl2) to give lipid 13 (49 mg, 64%) as an oil. 1 H NMR (400 MHz, C6D6) δ 5.31-5.19 (m, 2H), 4.27-4.18 (m, 1H), 3.96 (dd, J = 8.0, 6.2 Hz, 1H), 3.66-3.52 (m, 2H), 3.47 (t, J = 7.9 Hz, 1H), 2.72-2.41 (m, 9H), 2.34-2.23 (m, 6H), 2.18-2.10 (m, 1H), 2.09 (s, 3H), 1.82-1.17 (m, 60H), 0.92 (t, J = 6.9 Hz, 6H), 0.85 (t, J = 7.0 Hz, 6H).

[0205] (xiv) (4-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)-hexane-6,2-diyl) bis(decanoate) (14). [ka] Prepared from bromoketal 87 and 4-(ethylamino)-1-butanol by procedure (xiii) above.1 H NMR (400 MHz, C6D6) δ 5.30-5.19 (m, 2H), 4.06-3.94 (m, 1H), 3.88 (t, J = 6.8 Hz, 1H), 3.62 (t, J = 5.3 Hz, 2H), 3.37 (t, J = 7.8 Hz, 1H), 2.80-2.12 (m, 18H), 1.82-1.17 (m, 62H), 0.96-0.89 (m, 9H), 0.88=0.82 (m, 6H).

[0206] (xv) (4-(2-(4-hydroxypiperidin-1-yl)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl) bis(decanoate) (15). [ka] Prepared from bromoketal 87 and 4-piperidinol by procedure (xiii) above. 1 H NMR (400 MHz, C6D6) δ 5.32-5.17 (m, 2H), 4.12-4.02 (m, 1H), 3.99-3.92 (m, 1H), 3.53-3.29 (m, 2H), 2.73-2.41 (m, 10H), 2.38-2.17 (m, 8H), 1.96-1.15 (m, 62H), 0.92 (t, J = 6.9 Hz, 6H), 0.85 (t, J = 6.9 Hz, 6H).

[0207] (xvi) (4-(2-(dimethylamino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis(decanoate) (16). [ka] Prepared from tosylate 89 and dimethylamine by procedure (xii) above. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 4.98-4.86 (m, 2H), 4.26-4.05 (m, 3H), 4.04-3.97 (m, 1H), 3.43 (t, J = 7.7 Hz, 1H), 2.75-2.56 (m, 6H), 2.45 (s, 3H), 2.34-2.22 (m, 4H), 2.06-1.04 (m, 66H), 0.95-0.81 (m, 6H).

[0208] (xvii) (5-(((3-(dimethylamino)propanoyl)oxy)methyl)-5-(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis(decanoate) (17). [ka] Prepared from dihydroxyketal 90 and 3-(dimethylamino)propanoic acid hydrochloride according to procedure (viii) in Part E above, except that 0.8 equivalents of acid and 1 equivalent of EDCI-HCl / DMAP were used. 1 H NMR (400 MHz, C6D6) δ 5.37-5.15 (m, 2H), 4.39 (s, 2H), 3.79-3.67 (m, 4H), 3.55 (s, 2H), 2.83-2.61 (m, 6H), 2.32-2.26 (m, 4H), 2.23 (t, J = 6.4 Hz, 2H), 2.11 (t, J = 6.4 Hz, 2H), 2.06-1.94 (m, 4H), 1.91 (s, 6H), 1.83-1.04 (m, 60H), 0.92 (t, J = 6.9 Hz, 6H).

[0209] (xviii) (5-(((4-(dimethylamino)butanoyl)oxy)methyl)-5-(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl) bis(decanoate) (18). [ka] Prepared from dihydroxyketal 90 and 4-(dimethylamino)butanoic acid hydrochloride according to procedure (viii) in Part E above, except that 0.8 equivalents of acid and 1 equivalent of EDCI-HCl / DMAP were used. 1 H NMR (400 MHz, C6D6) δ 5.39-5.21 (m, 2H), 4.33 (s, 2H), 3.81-3.68 (m, 4H), 3.47 (s, 2H), 2.81-2.61 (m, 6H), 2.28 (t, J = 7.3 Hz, 4H), 2.16 (t, J = 7.1 Hz, 2H), 2.10-1.95 (m, 12H), 1.84-1.05 (m, 62H), 0.92 (t, J = 6.9 Hz, 6H).

[0210] (xix) (4-((dimethylamino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl) bis(decanoate) (19). [ka] Prepared from bromoketal 91 by procedure (xii) above). 1 H NMR (400 MHz, CDCl3) δ 5.05-4.87 (m, 2H), 4.22 (p, J = 6.5 Hz, 1H), 4.07 (dd, J = 7.9, 6.2 Hz, 1H), 3.50 (t, J = 7.8 Hz, 1H), 2.71-2.59 (m, 4H), 2.59-2.45 (m, 5H), 2.43-2.23 (m, 11H), 1.79-1.46 (m, 18H), 1.47-1.15 (m, 46H), 0.88 (m, 12H).

[0211] (xx) (4-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)-heptane-7,2-diyl) bis(decanoate) (20). [ka] Prepared from bromoketal 91 and 4-(methylamino)-1-butanol by procedure (xiii) above. 1 H NMR (400 MHz, CDCl3) δ 5.04-4.89 (m, 2H), 4.35-4.19 (m, 1H), 4.10 (dd, J = 8.0, 6.2 Hz, 1H), 3.67-3.55 (m, 2H), 3.49 (t, J = 7.9 Hz, 1H), 2.70-2.41 (m, 12H), 2.38-2.24 (m, 7H), 1.77-1.47 (m, 20H), 1.44-1.18 (m, 48H), 0.95-0.82 (m, 12H).

[0212] (xxi) (5-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)-heptane-7,2-diyl) bis(decanoate) (21). [ka] Prepared from tosylate 93 and 4-(methylamino)-1-butanol by procedure (xiii) above. 1H NMR (400 MHz, C6D6) δ 5.36-5.16 (m, 2H), 3.91 (dd, J = 11.7, 4.0 Hz, 2H), 3.65 (dd, J = 11.7, 6.4 Hz, 2H), 3.55 (t, J = 5.4 Hz, 2H), 2.75-2.42 (m, 8H), 2.37-2.20 (m, 4H), 2.16 (d, J = 7.3 Hz, 2H), 2.09 (t, J = 6.0 Hz, 2H), 1.90 (s, 3H), 1.88-1.12 (m, 69H), 0.99-0.83 (m, 12H). (G) Synthesis of lipid precursors (lipids 22–35) with type 7 ionizable head groups.

[0213] (i) General procedure for the reductive amination of ketones with primary amines to form secondary amines. To a solution of ketone (1 mmol) and primary amine (2 mmol, 2 equiv.) in 1,2-dichloroethane (10 mL) was added NaBH(OAc) (1.8 mmol, 1.8 equiv.) and HOAc (0.1 mL). The resulting mixture was stirred under nitrogen at room temperature for 18 h, then quenched with saturated aqueous NaHCO (3 mL), diluted with water (5.00 mL), and extracted with DCM (3 × 10 mL). The combined extracts were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to afford the secondary amine (70–75%) as an oil. The following compounds were obtained:

[0214] (ii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (94). [ka] It was obtained from ketone 84 and OTBDPS-protected 4-amino-1-butanol 40. 1H NMR (400 MHz, CDCl3) δ 7.77-7.52 (m, 4H), 7.45-7.34 (m, 6H), 5.03-4.85 (m, 2H), 3.70-3.63 (m, 2H), 2.71-2.39 (m, 11H), 2.30 (t, J = 7.5 Hz, 4H), 1.88-1.18 (m, 60H), 1.04 (s, 9H), 0.97-0.79 (m, 12H).

[0215] (iii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl bis(decanoate) (98). [ka] It was obtained from ketone 97 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl3) δ 7.72-7.59 (m, 4H), 7.46-7.34 (m, 6H), 5.02-4.82 (m, 2H), 3.77-3.56 (m, 2H), 2.81-2.44 (m, 11H), 2.30 (t, J = 7.5 Hz, 4H), 1.85-1.18 (m, 68H), 1.04 (s, 9H), 0.93-0.77 (m, 12H).

[0216] (iv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl dinonanoate (101). [ka] It was obtained from ketone 100 and OTBDPS-protected 4-amino-1-butanol 40. 1H NMR (400 MHz, CDCl3) δ 7.68-7.62 (m, 4H), 7.45-7.34 (m, 6H), 5.00-4.88 (m, 2H), 3.69-3.62 (m, 2H), 2.73-2.38 (m, 11H), 2.30 (t, J = 7.5 Hz, 4H), 1.86-1.17 (m, 64H), 1.04 (s, 9H), 0.91-0.81 (m, 12H).

[0217] (v) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl dinonanoate (105). [ka] It was obtained from ketone 104 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl3) δ 7.75-7.60 (m, 4H), 7.49-7.34 (m, 6H), 5.01-4.91 (m, 2H), 3.72-3.66 (m, 2H), 2.79-2.46 (m, 11H), 2.32 (t, J = 7.5 Hz, 4H), 1.79-1.18 (m, 60H), 1.06 (s, 9H), 0.95-0.80 (m, 12H).

[0218] (vi) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl bis(decanoate) (108). [ka] It was obtained from ketone 107 and OTBDPS-protected 4-amino-1-butanol 40. 1H NMR (400 MHz, CDCl3) δ 7.72-7.61 (m, 4H), 7.46-7.35 (m, 6H), 5.02-4.86 (m, 2H), 3.72-3.59 (m, 2H), 2.89-2.39 (m, 11H), 2.29 (t, J = 7.5 Hz, 4H), 1.93-1.18 (m, 64H), 1.04 (s, 9H), 0.97-0.76 (m, 12H).

[0219] (vii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(decanoate) (110). [ka] It was obtained from ketone 69 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl3) δ 7.73-7.60 (m, 4H), 7.45-7.33 (m, 6H), 5.01-4.84 (m, 2H), 3.67 (t, J = 5.6 Hz, 2H), 2.74-2.44 (m, 11H), 2.29 (t, J = 7.5 Hz, 4H), 1.84-1.18 (m, 68H), 1.04 (s, 9H), 0.95-0.76 (m, 12H).

[0220] (viii) 10-(6-((N-hexanoyl-N-methylglycyl)oxy)-7-(hexylthio)heptyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-18-thia-9-aza-3-silatetracosan-16-yl decanoate (114). [ka] It was obtained from ketone 76 and OTBDPS-protected 4-amino-1-butanol 40. 1H NMR (400 MHz, CDCl3, rotamers) δ 7.66 (m, 4H), 7.47-7.33 (m, 6H), 5.10-4.88 (m, 2H), 4.26-4.01 (m, 2H), 3.66 (tr, J=5.8, 2H), 3.06 and 2.98 (s, 3H), 2.68-2.60 (m, 4H), 2.58-2.50 (m, 6H), 2.43 (br,1H), 2.36 (tr, J= 7.7, 2H), 2.33-2.22 (m, 3H), 1.77-1.48 (m, 16H), 1.41-1.21 (m, 44H), 1.04 (s, 9H), 0.91-0.85 (tr, 12H).

[0221] (ix) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(heptylthio)pentadecane-2,14-diyl dinonanoate (118). [ka] It was obtained from ketone 117 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl3) δ 7.76-7.62 (m, 4H), 7.46-7.34 (m, 6H), 5.01-4.87 (m, 2H), 3.70-3.62 (m, 2H), 2.75-2.43 (m, 11H), 2.29 (t, J = 7.5 Hz, 4H), 1.79-1.17 (m, 68H), 1.04 (s, 9H), 0.91-0.83 (m, 12H).

[0222] (x) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(cyclohexylthio)pentadecane-2,14-diyl dinonanoate (120). [ka] It was obtained from ketone 81 and OTBDPS-protected 4-amino-1-butanol 40.1 H NMR (400 MHz, CDCl3) δ 7.76-7.65 (m, 4H), 7.59-7.36 (m, 6H), 4.99-4.86 (m, 2H), 3.69 (t, J = 6.0 Hz, 2H), 2.97-2.47 (m, 9H), 2.32 (t, J = 7.5 Hz, 4H), 2.01-1.88 (m, 4H), 1.83-1.16 (m, 58H), 1.06 (s, 9H), 0.96-0.79 (m, 12H).

[0223] (xi) General procedure for reductive methylation of secondary amines. A solution of secondary amine (1 mmol), aq. formaldehyde (37%, 6 mL), and NaBH(OAc)3 (5 mmol) in THF (10 mL) was stirred at room temperature under an inert atmosphere for 3 days. The reaction was then quenched with sat. aq. NaHCO3 (10 mL), diluted with water (10 mL), and extracted with CHCl2 (3 × 15 mL). The combined extracts were dried (NaSO4) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give the tertiary methylamine (75–85%) as an oil.

[0224] (xii) General procedure for reductive alkylation of secondary amines. To a solution of a secondary amine (1 mmol) and an aldehyde (5 mmol) in DCE (15 mL) was added NaBH(OAc) (5 mmol) and HOAc (0.2 mL). The resulting mixture was stirred at room temperature under an inert atmosphere for 18 h, then quenched with saturated aqueous NaHCO (5 mL), diluted with water (15 mL), and extracted with DCM (3 × 15 mL). The combined extracts were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give the tertiary amine (70–80%) as an oil.

[0225] (xiii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (95). [ka] Obtained from secondary amine 94 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.74-7.56 (m, 4H), 7.49-7.31 (m, 6H), 5.05-4.91 (m, 2H), 3.66 (t, J = 6.2 Hz, 2H), 2.66-2.60 (m, 4H), 2.53 (td, J = 7.3, 1.7 Hz, 4H), 2.39-2.23 (m, 7H), 2.11 (s, 3H), 1.74-1.12 (m, 60H), 1.04 (s, 9H), 0.93-0.83 (m, 12H).

[0226] (xiv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl bis(decanoate) (99). [ka] Obtained from secondary amine 98 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.73-7.62 (m, 4H), 7.47-7.36 (m, 6H), 5.03-4.93 (m, 2H), 3.68 (t, J = 6.1 Hz, 2H), 2.75-2.62 (m, 4H), 2.55 (td, J = 7.3, 1.6 Hz, 4H), 2.42-2.26 (m, 7H), 2.14 (s, 3H), 1.78-1.14 (m, 68H), 1.07 (s, 9H), 0.99-0.83 (m, 12H).

[0227] (xv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl dinonanoate (102). [ka] Obtained from secondary amine 101 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.80-7.59 (m, 4H), 7.52-7.37 (m, 6H), 5.06-4.90 (m, 2H), 3.68 (t, J = 6.2 Hz, 2H), 2.72-2.62 (m, 4H), 2.55 (td, J = 7.3, 1.6 Hz, 4H), 2.40-2.27 (m, 7H), 2.14 (s, 3H), 1.80-1.15 (m, 64H), 1.07 (s, 9H), 0.95-0.87 (m, 12H).

[0228] (xvi) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl dinonanoate (106). [ka] Obtained from secondary amine 105 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.79-7.60 (m, 4H), 7.52-7.32 (m, 6H), 5.01-4.91 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.75-2.60 (m, 4H), 2.53 (td, J = 7.2, 1.7 Hz, 4H), 2.40 - 2.23 (m, 7H), 2.12 (s, 3H), 1.77 - 1.13 (m, 60H), 1.04 (s, 9H), 0.99 - 0.82 (m, 12H).

[0229] (xvii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl bis(decanoate) (109). [ka] Obtained from secondary amine 108 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.77-7.60 (m, 4H), 7.47-7.32 (m, 6H), 5.03-4.90 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.71-2.60 (m, 4H), 2.53 (td, J = 7.3, 1.7 Hz, 4H), 2.44-2.22 (m, 7H), 2.12 (s, 9H), 1.83-1.16 (m, 64H), 1.04 (s, 3H), 0.98-0.77 (m, 12H).

[0230] (xviii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(decanoate) (111). [ka] Obtained from secondary amine 110 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.73-7.60 (m, 4H), 7.45-7.33 (m, 6H), 5.01-4.84 (m, 2H), 3.67 (t, J = 5.6 Hz, 2H), 2.74-2.44 (m, 11H), 2.29 (t, J = 7.5 Hz, 4H), 1.84-1.18 (m, 68H), 1.04 (s, 9H), 0.95-0.76 (m, 12H).

[0231] (xix) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(propyl)amino)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(decanoate) (112). [ka] Obtained from secondary amine 110 and ethanal (acetaldehyde) by procedure (xii). 1H NMR (400 MHz, CDCl3) δ 7.74-7.61 (m, 4H), 7.48-7.32 (m, 6H), 5.02-4.87 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 2.67-2.60 (m, 4H), 2.53 (td, J = 7.3, 1.6 Hz, 4H), 2.38-2.21 (m, 9H), 1.80-1.10 (m, 70H), 1.04 (s, 9H), 0.88 (td, J = 6.9, 2.6 Hz, 12H), 0.83 (t, J = 7.4 Hz, 3H).

[0232] (xx) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(isobutyl)amino)-1,15-bis(hexylthio)pentadecane-2,14-diyl bis(decanoate) (113). [ka] Obtained from secondary amine 110 and 2-methyl-propanal (isobutyraldehyde) by procedure (xii). 1 H NMR (400 MHz, CDCl3) δ 7.71-7.61 (m, 4H), 7.48-7.33 (m, 6H), 5.01-4.90 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 2.70 - 2.60 (m, 4H), 2.53 (td, J = 7.2, 1.6 Hz, 4H), 2.28 (q, J = 7.2 Hz, 7H), 2.06 (d, J = 7.1 Hz, 2H), 1.86-1.09 (m, 69H), 1.04 (s, 9H), 0.91-0.85 (m, 12H), 0.82 (d, J = 6.5 Hz, 6H).

[0233] (xxi) 10-(6-((N-hexanoyl-N-methylglycyl)oxy)-7-(hexylthio)heptyl)-2,2,9-trimethyl-3,3-diphenyl-4-oxa-18-thia-9-aza-3-silatetracosan-16-yl decanoate (115). [ka] Obtained from secondary amine 114 by procedure (xi). 1 H NMR (400 MHz, CDCl3, rotamers) δ 7.66 (m, 4H), 7.44-7.34 (m, 6H), 5.11-4.87 (m, 2H), 4.25-4.00 (m, 2H), 3.66 (tr, J=5.8, 2H), 3.06 and 2.98 (s, 3H), 2.68-2.59 (m, 4H), 2.56-2.49 (m, 4H), 2.41-2.21 (m, 7H), 2.15 (s, 3H), 1.71-1.46 (m, 17H), 1.40-1.20 (m, 43H), 1.04 (s, 9H), 0.92-0.83 (tr, 12H).

[0234] (xxii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(heptylthio)pentadecane-2,14-diyl dinonanoate (119). [ka] Obtained from secondary amine 118 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.72-7.58 (m, 4H), 7.48-7.33 (m, 6H), 5.03-4.90 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.63 (d, J = 6.0 Hz, 4H), 2.53 (td, J = 7.3, 1.7 Hz, 4H), 2.31 (dt, J = 15.0, 7.6 Hz, 7H), 2.12 (s, 3H), 1.80-1.10 (m, 68H), 1.04 (s, 9H), 0.98-0.82 (m, 12H).

[0235] (xxiii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(cyclohexylthio)-pentadecane-2,14-diyl dinonanoate (121). [ka] Obtained from secondary amine 120 by procedure (xi). 1 H NMR (400 MHz, CDCl3) δ 7.73-7.61 (m, 4H), 7.50-7.35 (m, 6H), 5.06-4.86 (m, 2H), 3.66 (t, J = 6.1 Hz, 2H), 2.79-2.59 (m, 6H), 2.47-2.23 (m, 7H), 2.15 (s, 3H), 2.03-1.93 (m, 4H), 1.82-1.13 (m, 64H), 1.04 (s, 9H), 0.96-0.81 (m, 6H).

[0236] (H) General Procedure for Release of Silyl Protecting Group: Synthesis of Lipids 5 and 22–35. To a solution of TBDPS-protected compound (1 mmol) in THF (5 mL) was added HF-pyridine (1 mL) at 0°C under an inert atmosphere. The reaction was allowed to warm to room temperature and stirred for 18 hours. Water (10 mL) was added, and the mixture was extracted with DCM (3 × 15 mL). The combined organics were dried (NaSO) and concentrated. The residue was purified by silica chromatography (0–5% MeOH in DCM) to give the corresponding lipids (55–70%) as oils. The following lipids were obtained:

[0237] (i) ((4-hydroxybutyl)azanediyl)bis(1-(pentylthio)heptane-7,2-diyl) bis(decanoate) (5). [ka] Obtained from compound 46. 1H NMR (400 MHz, CDCl3) δ 5.00-4.89 (m, 2H), 3.62 (t, J = 4.9 Hz, 2H), 2.78-2.57 (m, 10H), 2.53 (td, J = 7.3, 1.5 Hz, 4H), 2.30 (t, J = 7.6 Hz, 4H), 1.83-1.49 (m, 20H), 1.43-1.17 (m, 40H), 0.96-0.82 (m, 12H).

[0238] (ii) 7-((4-hydroxybutyl)(methyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (22). [ka] Obtained from compound 95. 1 H NMR (400 MHz, CDCl3) δ 5.06-4.88 (m, 2H), 3.73-3.58 (m, 2H), 2.76-2.61 (m, 7H), 2.53 (td, J = 7.3, 1.4 Hz, 4H), 2.39-2.26 (m, 7H), 1.81-1.20 (m, 60H), 0.94-0.81 (m, 12H).

[0239] (iii) 1,13-bis(heptylthio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (23). [ka] Obtained from compound 99. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.89 (m, 2H), 3.61 (t, J = 4.9 Hz, 2H), 2.69-2.57 (m, 7H), 2.53 (td, J = 7.2, 1.4 Hz, 4H), 2.36-2.25 (m, 7H), 1.79-1.18 (m, 68H), 0.92-0.85 (m, 12H).

[0240] (iv) 1,13-bis(heptylthio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl dinonano-ate (24). [ka] Obtained from compound 102. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.88 (m, 2H), 3.65-3.52 (m, 2H), 2.75-2.60 (m, 4H), 2.59-2.38 (m, 7H), 2.30 (t, J = 7.5 Hz, 4H), 2.16 (s, 3H), 1.82-1.12 (m, 64H), 0.96-0.83 (m, 12H).

[0241] (v) 8-((4-hydroxybutyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl dinonano-ate (25). [ka] Obtained from compound 106. 1 H NMR (400 MHz, CDCl3) δ 5.03-4.88 (m, 2H), 3.67-3.55 (m, 2H), 2.69-2.62 (m, 4H), 2.60-2.49 (m, 7H), 2.30 (t, J = 7.5 Hz, 4H), 2.24 (s, 3H), 1.78-1.20 (m, 60H), 0.93-0.84 (m, 12H).

[0242] (vi) 8-((4-hydroxybutyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl bis-(decanoate) (26). [ka] Obtained from compound 109. 1H NMR (400 MHz, CDCl3) δ 5.03-4.85 (m, 2H), 3.65-3.52 (m, 2H), 2.72-2.59 (m, 4H), 2.53 (td, J = 7.3, 1.6 Hz, 7H), 2.30 (t, J = 7.5 Hz, 4H), 2.18 (s, 3H), 1.79-1.15 (m, 64H), 0.95 - 0.84 (m, 12H).

[0243] (vii) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecane-2,14-diyl bis-(decanoate) (27). [ka] Obtained from compound 111. 1 H NMR (400 MHz, CDCl3) δ 5.01-4.88 (m, 2H), 3.65-3.55 (m, 2H), 2.70-2.48 (m, 11H), 2.36-2.20 (m, 7H), 1.81-1.22 (m, 68H), 0.96-0.84 (m, 12H).

[0244] (viii) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(propyl)amino)pentadecane-2,14-diyl bis-(decanoate) (28). [ka] Obtained from compound 112. 1 H NMR (400 MHz, CDCl3) δ 5.44-5.33 (m, 2H), 3.73-3.64 (m, 2H), 2.88-2.72 (m, 5H), 2.71-2.52 (m, 6H), 2.51-2.44 (m, 2H), 2.40 (t, J = 7.4 Hz, 4H), 1.99-1.20 (m, 70H), 1.07-0.94 (m, 15H).

[0245] (ix) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(isobutyl)amino)pentadecane-2,14-diyl bis-(decanoate) (29). [ka] Obtained from compound 113. 1 H NMR (400 MHz, C6D6) δ 5.33-5.21 (m, 2H), 3.54-3.48 (m, 2H), 2.67 (qd, J = 13.6, 6.2 Hz, 4H), 2.59-2.44 (m, 5H), 2.42-2.33 (m, 2H), 2.33-2.24 (m, 4H), 2.16 (d, J = 7.1 Hz, 2H), 1.87-1.11 (m, 69H), 1.02-0.81 (m, 18H).

[0246] (x) 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-((4-hydroxybutyl)(methyl)-amino)pentadecan-2-yl decanoate (30). [ka] Obtained from compound 115. 1 H NMR (400 MHz, CDCl3, rotamers) δ 5.02 (m, 1H), 4.94 (m, 1H), 4 .32-3.87 (m, 2H), 3.77 (m, 1H), 3.67 (m, 1H), 3.25-2.94 (6H), 2.84-2.72 (m, 3H), 2.64 (d, J=6 Hz, 4H), 2.58-2.50 (m, 4H), 2.37 (m, 2H), 2.31 (m, 2H), 2.00 (1H), 1.87 (2H), 1.79-1.50 (m, 16H), 1.47-1.21 (m, 40H), 0.94-0.81 (tr, 12H).

[0247] (xi) 1,15-bis(heptylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecane-2,14-diyl dinonanoate (31). [ka] Obtained from compound 119. 1 H NMR (400 MHz, CDCl3) δ 5.02-4.89 (m, 2H), 3.68-3.54 (m, 2H), 2.69-2.60 (m, 4H), 2.58-2.46 (m, 7H), 2.30 (t, J = 7.5 Hz, 4H), 2.21 (s, 3H), 1.78-1.20 (m, 68H), 0.96-0.82 (m, 12H).

[0248] (xii) 1,15-bis(cyclohexylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecane-2,14-diyl dinonanoate (32). [ka] Obtained from compound 121 and characterized as the HCl salt. 1 H NMR (400 MHz, CDCl3) δ 4.97-4.86 (m, 2H), 3.73 (t, J = 5.3 Hz, 2H), 3.16-3.11 (m, 1H), 3.07 (t, J = 6.6 Hz, 2H), 2.73 (s, 3H), 2.70-2.56 (m, 6H), 2.30 (t, J = 7.6 Hz, 4H), 2.07-1.13 (m, 68H), 0.93-0.73 (m, 6H). Example 2: mRNA-containing LNPs containing ionizable lipids 1 or 2 demonstrate superior in vivo delivery of mRNA to the liver and spleen compared to the MC3 benchmark

[0249] After injecting CD-1 mice with an LNP formulation containing ionizable lipids / 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, the in vivo transfection efficiency in the liver and spleen was tested. The mRNA dose was 1 mg / kg. The luminescence intensity of the liver and spleen was measured 4 h after injection. The ionizable lipids were 1 and 5–32 (Table 1).

[0250] The results in Figure 2A show that the luminescence intensity per mg of liver for lipids 7, 9, 8, 11, 10, 6, 25, 23, 30, 12, 24, 30, 12, 24, 16, 32, 26, 22, 31, 27, and 5 was higher than the MC3 benchmark. The luminescence intensity per mg of spleen for lipids 20, 21, 7, 17, 23, 12, 26, 27, 15, 8, 14, 13, 31, 24, 32, 25, 5, 16, 22, 6, 30, and 9 was higher than the MC3 benchmark (Figure 2B). ……………

Claims

1. A lipid having the structure of Formula A: 【Chemical 1】 Formula A or a pharmaceutically acceptable salt thereof. [In the formula, m is 4 to 8; n is 4 to 8; R １ , R ２ , R ３ , and R ４ is a linear or branched optionally substituted C ３ ~C ２０ alkyl, optionally containing 0 to 2 carbon-carbon double bonds; A is C or N; If A is C, then 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 ) q, where q is 1 or 2; Z is selected from one of the following structures a-c, where the wavy line represents the bond to X: a. 【Chemistry 2】 Type 2 ionizable head groups; b. 【Chemistry 3】 Type 3 ionizable head groups; c. 【Chemistry 4】 Type 4 ionizable head groups; W １ and Y are not bonded to each other, W １ is H; W ２ is O, S, NH or NR ２ａ and R ２ａ is C optionally substituted with an OH group １ ~C ４ is alkyl; Part of formula A 【Chemistry 5】 is a group selected from the following structures d to h, and the wavy line represents W ２ represents a bond to: d. 【Chemistry 6】 Type 1 ionizable head group; e. 【Chemistry 7】 Type 5 ionizable head groups; f. 【Chemistry 8】 Type 6 ionizable head groups; g. 【Chemistry 9】 Type 7 ionizable head group; h. 【Chemistry 10】 Type 8 ionizable head group; i. 【Chemistry 11】 Type 9 ionizable head group; If A is N, W １ and Y is absent; W ２ and X together form the structure (CR ａ R ｂ ) ｐ and R ａ and R ｂ are independently H or C １ ~C ５ alkyl or cycloalkyl, and p is 2 to 6; Z is OH or NR'R'', and R' and R'' are independently optionally substituted C １ ~C ５ alkyl or cycloalkyl, or R′ and R″ together with the N atom of NR′R″ form an optionally substituted heterocycle incorporating the N atom to which said R′ and R″ are each attached.

2. R １ and R ４ is independently a moiety of formula B; 【Chemistry 12】 Formula B R′ and R″ are independently linear or branched optionally substituted C ３ ~C １２ an alkyl group, optionally containing 0 to 2 carbon-carbon double bonds; R''' is H or a linear, branched or cyclic optionally substituted C １ ~C ６ is an alkyl group; G １ and G ２ However, independently, (CR ａ R ｂ ) ｐ and R ａ and R ｂ are each independently H or optionally substituted C １ ~C ５ 2. The lipid of claim 1, or a pharmaceutically acceptable salt thereof, wherein p is selected from alkyl or cycloalkyl and p is 0 to 6.

3. A ３ is N and W １ and Y is absent and W ２ and X together form the structure (CR ａ R ｂ ) ｒ wherein Z is NR'R'' and the heterocyclic group incorporating the N atom to which R' and R'' are attached is pyrrolidine, piperidine or morpholine, or a pharmaceutically acceptable salt thereof.

4. A ３ The lipid or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein is a carbon atom.

5. W １ 5. The lipid or pharmaceutically acceptable salt of claim 4, wherein Y and Y are not bonded to each other.

6. W ２ The lipid or pharmaceutically acceptable salt of claim 5, wherein is O.

7. Part of formula A 【Chemistry 13】 The lipid or pharmaceutically acceptable salt of claim 6, wherein: is structure d.

8. 2. The lipid of claim 1, having the structure of any one of the following compounds 5-32: 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】

9. A lipid or a pharmaceutically acceptable salt thereof, a protonatable amino head group; two lipophilic chains, the amino head group having a central nitrogen or carbon atom to which each of the two lipophilic chains is directly attached; At least one of the lipophilic chains has the formula: 【Chemistry 20】 In the formula, R １ and R ２ are independently linear or branched optionally substituted C ３ ~C ２０ alkyl, optionally with varying degrees of unsaturation; n is 4 to 8; Each lipophilic chain has a total of 15 to 40 carbon atoms; The lipid has (i) a pK between 6 and 8 ａ (ii) a log P of at least 11, or a pharmaceutically acceptable salt thereof.

10. 10. The lipid or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the lipid, when formulated into lipid nanoparticles comprising mRNA, results in at least about a 10% increase in biodistribution of the lipid nanoparticles in the liver and / or one or more extrahepatic tissues compared to lipid nanoparticles comprising DLin-MC3-DMA, as measured by in vivo luminescence of the mRNA in the liver and / or one or more extrahepatic tissues.

11. A lipid nanoparticle comprising the lipid according to any one of claims 1 to 10 and a nucleic acid.

12. The lipid nanoparticle of claim 11, comprising a helper lipid and optionally a hydrophilic polymer-lipid conjugate.

13. The lipid nanoparticle of claim 12, wherein the helper lipid is selected from cholesterol, diacylglycerol, glycero-phospholipid-cholesterol conjugates and sphingolipids.

14. An ionizable lipid having two lipophilic chains directly attached to a central nitrogen or carbon atom, wherein at least one of the lipophilic chains has the formula 【Chemical 21】 and an ionizable lipid having: n is 4 to 8; * represents a carbon branch point; R ５ and R ６ are each independently a linear or branched substituted C ３ ~C ３０ is an alkyl group; R ５ and R ６ one of which is substituted with an ester group, and R ５ and R ６ the other of which is substituted with a sulfur atom at the α, β, or γ position relative to said carbon branch point; one or more helper lipids; optionally a hydrophilic polymer-lipid conjugate; A lipid nanoparticle comprising: a nucleic acid;

15. 15. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing lipid nanoparticles according to any one of claims 10 to 14 comprising the nucleic acid, and administering the lipid nanoparticles to the subject.

16. A method for delivering a cargo molecule to a cell, said method comprising contacting the lipid nanoparticles of any one of claims 11 to 14 with said cell in vivo or in vitro.

17. 17. The method of claim 16, wherein the cargo molecule is a nucleic acid.

18. Use of the lipid according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or the lipid nanoparticle according to any one of claims 11 to 14, in the manufacture of a medicament for treating or preventing a disease, disorder or condition treatable and / or preventable by a nucleic acid.

19. Use of the lipid according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, or the lipid nanoparticle according to any one of claims 11 to 14, for delivering a nucleic acid to a subject for treating or preventing a disease, disorder or condition treatable or preventable by the nucleic acid.

20. 20. The use defined in claim 18 or 19, wherein said nucleic acid is mRNA.

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