Sulfur-containing ionizable lipids for delivery of therapeutic agents

Abstract

Novel sulfur-containing lipids and nanoparticles containing the lipids and cargo molecules, such as nucleic acids, methods of formulating the lipids with nucleic acids to produce lipid nanoparticles, and chemical pathways for preparing the lipids, are provided. The lipid may have a structure of Formula A as defined herein. # imgabs0 #

Description

Technical Field

[0001] Provided herein are sulfur-containing lipids that can be formulated in delivery vehicles to facilitate encapsulation of cargo such as, but not limited to, nucleic acids (eg, RNA or DNA), proteins, peptides, drugs, and salts thereof.

[0002] background

[0003] Nucleic acid-based therapeutics have enormous potential in medicine. However, to realize this potential, nucleic acids must be delivered to the target site in the patient. This presents a challenge because nucleic acids are rapidly degraded by enzymes in the plasma upon administration. Even if nucleic acids are delivered to the disease site, there remains the challenge of intracellular delivery. To address these issues, lipid nanoparticles have been developed that protect nucleic acids from this degradation and facilitate delivery across the cell membrane to access the intracellular compartment where the relevant translation machinery resides.

[0004] The key component of lipid nanoparticle (LNP) is ionizable lipid.Ionizable lipid is usually positively charged under low pH, which helps to combine with negatively charged nucleic acid.However, ionizable lipid is neutral under physiological pH, making it more biocompatible in biological systems.In addition, it has been proposed that after LNP is absorbed by endocytosis by cells, the ionizability of these lipids under low pH allows endosome escape.This makes nucleic acid can be released into the intracellular compartment again.

[0005] Earlier examples of LNP products approved for clinical use and relying on ionizable lipids are It is a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of polyneuropathy induced by hereditary transthyretin amyloidosis. Relying on an ionizable lipid that the researchers call "DLin-MC3-DMA" or more commonly "MC3," 1( Figure 1 ). In addition, MC3 represents an evolution of a structurally related ionizable lipid, referred to by the researchers as “KC2”, 2( Figure 1 ). MC3 is considered a state-of-the-art ionizable lipid for delivery of siRNA, requiring approximately 3-fold less siRNA than KC2, although KC2 is superior in other applications and it remains a valuable research tool.

[0006] While the aforementioned ionizable lipids are particularly effective for delivering LNPs containing siRNA to hepatocytes, they are much less effective for delivering LNPs containing mRNA to the liver. For example, mRNA vaccines, including the COVID-19 Pfizer / BioNTech and Moderna vaccines, rely on lipid nanoparticles to deliver mRNA to the cytoplasm of hepatocytes. Once inside 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, this vaccine combines other types of ionizable lipids in addition to MC3 or KC2. Specifically, 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.

[0007]

[0008] Solution 1

[0009] In addition, the above lipids are optimized to deliver therapeutic nucleic acids to the liver. However, it is still necessary to develop new lipids for delivering charged goods such as nucleic acids to other organs such as the spleen, lungs, bone marrow, skin, etc. The delivery of therapeutic agents outside the liver will expand the clinical utility of LNPs to target disease states of tissues and organs outside the liver. It is also necessary to develop LNPs with improved nucleic acids or other charged goods to the delivery of the liver.

[0010] The present disclosure seeks to address one or more of the above-identified problems and / or to provide useful alternatives to known products and / or compositions for delivering nucleic acids or other charged cargoes.

[0011] definition

[0012] As used herein, "Type 1 ionizable head" or "MC-type ionizable head" refers to a moiety of a lipid head group having the following Formula I or its equivalent, with n ranging from 1 to 5:

[0013]

[0014] As used herein, "Type 2 ionizable head" or "Type KC ionizable head" refers to a moiety having a head group of a lipid of Formula II below or its equivalent, with n ranging from 1 to 5:

[0015]

[0016] As used herein, "Type 3 ionizable head" refers to a moiety of a head group of the structure defined by Formula III below, or its equivalent, with m and n independently ranging from 1 to 5:

[0017]

[0018] As used herein, "Type 4 ionizable head" refers to a moiety of a head group of the structure defined by Formula IV below, or its equivalent, wherein R = C 1 -C 6 alkyl or cycloalkyl, and m and n are independently in the range of 2 to 5:

[0019]

[0020] As used herein, "5-type ionizable head" refers to a moiety of a head group of the structure defined by Formula V below, or its equivalent, with m and n independently ranging from 1 to 5:

[0021]

[0022] As used herein, "6-type ionizable head" refers to a moiety or equivalent of a head group having a structure as defined by Formula VI below, wherein R = C 1 -C 6 alkyl or cycloalkyl and m is in the range of 1 to 5, and n is independently in the range of 2 to 5:

[0023]

[0024] As used herein, "7-type ionizable head" refers to a moiety or equivalent of a head group having a structure as defined by Formula VII below, wherein R = C 1 -C 6 Alkyl or cycloalkyl, and n is in the range of 1 to 5:

[0025]

[0026] As used herein, "8-type ionizable head" refers to a moiety of a head group of the structure defined by Formula VIII, or its equivalent, wherein R = C 1 -C 6 Alkyl or cycloalkyl, and n is in the range of 1 to 5:

[0027]

[0028] As used herein, "9-type ionizable head" refers to a moiety of a head group of the structure defined by Formula IX below, or its equivalent, wherein m and n are independently in the range of 1 to 5:

[0029]

[0030] As used herein, the term "ionizable lipid" refers to a lipid that is in an electrostatically neutral form at a given pH and can accept or donate protons to become electrostatically charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) greater than 8.

[0031] As used herein, the term "lipophilic chain" refers to an alkyl group bound to a nitrogen or carbon atom of a lipid, the alkyl group comprising at least 6 C atoms and optionally comprising a C=C double bond, and / or a ring structure, and / or a carbonyl group, and / or a heteroatom such as N, O, S, and such that the parent compound of the alkyl group has a cLogP of at least 6.

[0032] For example, lipid MC3,1 and lipid KC2,2 have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene, which have a cLogP of 9.25:

[0033]

[0034] Lipid ALC-0315,3 has a pair of lipophilic chains derived from 2-hexyldecanoate hexyl ester with a cLogP of 10.01:

[0035]

[0036] Lipid SM-102,4 has one lipophilic chain derived from undecyl hexanoate, which has a cLogP of 7.59, and one lipophilic chain derived from heptadecan-9-yl octanoate, which has a cLogP of 11.6:

[0037]

[0038] As used herein, the term "alkyl" or "alkyl group" is a carbon-containing chain that is linear or branched and optionally contains a C=C double bond and / or a ring structure and is optionally substituted.

[0039] As used herein, the term “C m To C n Alkyl" or "C m To C n "Alkyl group" refers to a straight or branched carbon chain having a total of at least m carbon atoms and at most n carbon atoms, and which is optionally unsaturated and optionally substituted. For example, "C 1 To C 3 Alkyl" or "C 1 To C 3 An "alkyl group" is an alkyl group having 1 to 3 carbon atoms.

[0040] The term "optionally substituted" in reference to an alkyl group means that at least one hydrogen atom of the alkyl group may be replaced by a non-hydrogen atom or group of atoms (i.e., a "substituent"), and / or the alkyl group is interrupted by one or more substituents comprising heteroatoms selected from O, S, and NR', wherein R' is as defined below. Non-limiting examples of groups that may replace a hydrogen atom include halogen; an alkyl group; a cycloalkyl group; an oxo group (=O); a hydroxyl group (-OH); -(C≡O)OR'; -O(C≡O)R'; -C(O)R'; -OR'; -S(O)xR'; -S-SR'; -C(O)SR'; -SC(O)R'; -NR'R'; -NR'C(O)R'; -C(O)NR'R'; -NR'C(O)NR'R'; -OC(O)NR'R'; -NR'C(O)OR'; -NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', wherein R' at each occurrence is independently selected from H, C 1 -C 15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0041] As used herein, the term "helper lipid" refers to a compound selected from the following: sterols, such as cholesterol or its derivatives; diacylglycerols or their derivatives, such as glycerophospholipids, including phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), etc.; and sphingolipids, such as ceramides, sphingomyelins, cerebrosides, gangliosides or reduced analogs thereof, 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 by a cholesterol-containing moiety. The term includes naturally occurring or synthetic lipids.

[0042] As used herein, the term "delivery vehicle" includes any formulation in which the lipids described herein can be formulated, and includes, but is not limited to, delivery vehicles comprising a helper lipid.

[0043] As used herein, the term "nanoparticle" is any suitable particle in which a lipid can be formulated and which can include one or more auxiliary lipid components. The one or more lipid components can include ionizable lipids prepared by the methods described herein and / or can include additional lipid components such as one or more auxiliary lipid components. The term includes, but is not limited to, vesicles having one or more bilayers, including multilamellar vesicles, unilamellar vesicles, and vesicles with electron-dense cores. The term also includes polymer-lipid hybrids, including particles of lipids bound to a polymer.

[0044] As used herein, the term "encapsulated" in reference to the incorporation of a cargo molecule (eg, mRNA) into a delivery vehicle refers to any association of the cargo with any component or compartment of the delivery vehicle, such as a nanoparticle.

[0045] The term "pharmaceutically acceptable salt" in reference to the lipid forms of the present disclosure in protonated form (ie, charged) and / or as part of a pharmaceutical formulation for formulating LNPs refers to salts prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.

[0046] Unless otherwise stated, the articles "a" or "an" as used herein are intended to include both the singular and the plural.

[0047] Overview

[0048] The disclosure is based at least in part on surprising discovery, i.e., the LNP preparation of nucleic acid comprising ionizable lipid is more effective than the benchmark MC3 for the liver delivery of therapeutic RNA, and the ionizable lipid is mixed with at least one lipophilic chain substituted by sulphur atom and ester moiety. As further described herein, this lipid can show different organ selectivity relative to known lipids. Especially, non-limiting examples as described herein prove that this lipid promotes mRNA selective delivery to spleen more effectively than other known lipids. In addition, the chemical synthesis of the lipid of some embodiments herein is more direct and / or economical than the chemical synthesis of known lipids.

[0049] Other objects, features and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description and accompanying drawings.

[0050] In one aspect of the present disclosure, a lipid having a structure of Formula A is provided:

[0051]

[0052] or a pharmaceutically acceptable salt thereof;

[0053] in

[0054] m is 4 to 8; and

[0055] n is 4 to 8;

[0056] R 1 , R 2 , R 3 and R 4 is a linear or branched optionally substituted C 3 To C 20 alkyl, and optionally containing 0 to 2 carbon-carbon double bonds;

[0057] A is C or N, and

[0058] If A is C, then W1 and Y are bound to each other or not bound to each other, and

[0059] If W 1 and Y combine with each other, then

[0060] W 1 is O or S;

[0061] W 2 is O or S;

[0062] X is CH;

[0063] Y is (CH 2 ) q , where q is 1 or 2;

[0064] Z is selected from one of the following structures a to c, where the wavy line represents the bond to X:

[0065] a. Type 2 ionizable head group;

[0066] b. Type 3 ionizable head group;

[0067] c. Type 4 ionizable head group;

[0068] If W 1 and Y do not bind to each other, then

[0069] W 1 is H;

[0070] W 2 O, S, NH or NR 2a , where R 2a is C optionally substituted by an OH group 1 To C 4 Alkyl; and

[0071] Formula A The moiety is a group selected from the following structures d to h, wherein the wavy line represents the 2 Keys:

[0072] d. Type 1 ionizable head group;

[0073] e. Type 5 ionizable head group;

[0074] f. Type 6 ionizable head group;

[0075] g. (CH 2 ) n OH 7 type ionizable head group;

[0076] h. Type 8 ionizable head group;

[0077] i. Type 9 ionizable head group;

[0078] If A is N, then

[0079] W 1 and Y does not exist;

[0080] W 2 Together with X, it forms the structure (CR a R b ) p A group in which R a and R b independently H or C 1 -C 5 alkyl or cycloalkyl, and wherein p is 2 to 6; and

[0081] Z is OH or NR'R", wherein R' and R" are independently optionally substituted C 1 -C 5 alkyl or cycloalkyl, or wherein R' and R" together with the N atom of NR'R" form an optionally substituted heterocyclic ring incorporated into the N atom to which R' and R" are each bound.

[0082] According to one embodiment of the aforementioned aspect, R 1 and R 4 At least one of them may independently be a moiety of formula B, wherein:

[0083]

[0084] R' and R" are independently linear or branched optionally substituted C 3 To C 12 alkyl, and optionally containing 0 to 2 carbon-carbon double bonds;

[0085] R'' is H or a linear, branched or cyclic optionally substituted C 1 To C 6 Alkyl; and

[0086] G 1 and G 2 Independently (CR a R b ) p , where R a and R b are each independently selected from H or optionally substituted C 1 -C 5 Alkyl or cycloalkyl, wherein p is 0 to 6.

[0087] According to the aforementioned aspect or embodiments thereof, A 3 Can be N, W 1 and Y does not exist, W 2 Together with X, it forms the structure (CR a R b ) r and Z is NR'R", and the heterocyclic group in which the N atom to which R' and R" are bonded is pyrrolidine, piperidine or morpholine.

[0088] According to the aforementioned aspects or embodiments thereof, A 3 It may be a carbon atom.

[0089] According to the aforementioned aspect or embodiments thereof, W 1 and Y do not bind to each other.

[0090] According to the aforementioned aspect or embodiments thereof, W 2 It can be O.

[0091] In one embodiment, wherein the Part is structure d.

[0092] According to the aforementioned aspects or embodiments thereof, the lipid has the structure of any one of Compounds 5 to 21 listed in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0093] Another aspect of the present disclosure provides a lipid or a pharmaceutically acceptable salt thereof, comprising:

[0094] a protonatable amino head group;

[0095] two lipophilic chains, wherein the amino head group has a central nitrogen or carbon atom, each of the two lipophilic chains being directly bonded to the central nitrogen or carbon atom;

[0096] At least one of the lipophilic chains has the formula:

[0097]

[0098] Where R 1 and R 2 is independently a linear or branched optionally substituted C 3 -C 20 alkyl, and optionally with varying degrees of unsaturation;

[0099] n is 4 to 8;

[0100] Each lipophilic chain has a total of 15 to 40 carbon atoms; and

[0101] wherein the lipid has (i) a pK of 6 to 8 a ; and (ii) a logP of at least 11.

[0102] According to other embodiments of any of the foregoing aspects of the present disclosure, when formulated in a lipid nanoparticle comprising mRNA, the lipid causes the biodistribution of the lipid nanoparticle in the liver and / or one or more extrahepatic tissues such as the spleen relative to the mRNA luminescence of the lipid nanoparticle comprising DLin-MC3-DMA in the liver and / or one or more extrahepatic tissues to increase by at least about 10%. The assay and formulation for determining biodistribution are as described in Example 2.

[0103] The lipid nanoparticles may comprise a helper lipid and a hydrophilic polymer-lipid conjugate. The helper lipid may be selected from cholesterol, diacylglycerol and sphingolipid.

[0104] In another aspect, there is provided a lipid nanoparticle comprising:

[0105] An ionizable lipid having two lipophilic chains directly bonded to a central nitrogen or carbon atom, wherein at least one of the lipophilic chains has the formula:

[0106]

[0107] n is 4 to 8;

[0108] Where * indicates carbon branch point;

[0109] Where R 5 and R 6 Each independently is a linear or branched substituted C 3 -C 30 alkyl;

[0110] Where R 5 and R 6 One of them is substituted by an ester group, and R 5 and R 6 the other of which is substituted by a sulfur atom in the α, β, or γ position relative to the carbon branch point;

[0111] one or more helper lipids;

[0112] optionally a hydrophilic polymer-lipid conjugate; and

[0113] Nucleic acid.

[0114] In another aspect, a method for administering a nucleic acid to an individual is provided, the method comprising preparing or providing a lipid nanoparticle comprising a nucleic acid as described in any of the preceding aspects or embodiments, and administering the lipid nanoparticle to the individual, the individual being in need of the method.

[0115] Another aspect of the present disclosure provides a method of delivering a cargo molecule to a cell, the method comprising contacting the lipid nanoparticle described in any of the above aspects or embodiments with the cell in vivo or in vitro. In one embodiment, the cargo molecule is a nucleic acid.

[0116] In another aspect of the present disclosure, there is provided a use of a lipid as defined above or a pharmaceutically acceptable salt thereof, or a lipid nanoparticle as defined in any aspect or embodiment above, in the preparation of a medicament for treating or preventing a disease, disorder or condition that can be treated and / or prevented by nucleic acid.

[0117] Another aspect of the present disclosure provides the use of a lipid as defined above or a pharmaceutically acceptable salt thereof or a lipid nanoparticle in any aspect or embodiment described above in delivering a nucleic acid to a patient to treat or prevent a disease, disorder or condition that can be treated or prevented by a nucleic acid. In one embodiment, the nucleic acid is mRNA.

[0118] BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 Is a bar graph showing entrapment (%), particle size and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising ionizable lipids 1 and 5 to 32 (Table 1). The LNPs consist of 50 / 10 / 38.5 / 1.5 mol% ionizable lipids / DSPC / chol / PEG-DMG and an amine to phosphate ratio (N / P) of 6.

[0120] Figure 2A The luminescence intensity / mg in the liver of mRNA containing LNPs containing ionizable 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 is shown after 4 hours of intravenous administration to CD-1 mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipids / DSPC / chol / PEG-DMG (N / P=6).

[0121] Figure 2BThe luminescence intensity / mg in spleen of mRNA containing LNPs containing ionizable 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 is shown after 4 hours of intravenous administration to CD-1 mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipids / DSPC / chol / PEG-DMG (N / P=6).

[0122] Details

[0123] Various aspects and embodiments of the present disclosure relate to ionizable lipids having the structure of Formula A and pharmaceutically acceptable salts thereof.

[0124]

[0125] The preparation comprising this lipid can be used for nucleic acid delivery to any interested target site. In certain embodiments, it has been found that when prepared in a suitable delivery vehicle, this lipid is particularly effective for the delivery of mRNA. In other embodiments, this lipid can be easily synthesized and prepared by a method with improved economy relative to the known methods for the preparation of ionizable lipids.

[0126] Method for producing lipid of formula A

[0127] The lipid of formula A or its pharmaceutically acceptable salt can be prepared using any suitable method known to those skilled in the art. A particularly suitable method is described below, which is exemplified by the synthesis of compounds 5 to 32 of Table 1 below, but is not intended to be limiting. It will be appreciated by those skilled in the art that alternative starting materials can be used in the same order to obtain the congeners of compounds 5 to 32 as defined by formula A. Therefore, the synthetic schemes set forth below are merely examples of selected embodiments.

[0128] Certain steps of the synthesis of compounds such as 5 are described in detail in co-pending and co-owned WO2023 / 173203, which is incorporated herein by reference. As described in the aforementioned disclosure, one such step entails reacting a suitable amino alcohol represented by general formula 33 in Scheme 2 or an O-protected variant thereof with a suitable alkyl halide or sulfonate represented by general formula 34 in Scheme 2, resulting in the formation of different products depending on the conditions. Specifically, by reacting at room temperature in DMF at K 2 CO 3 In the presence of K, the selective mono-N-alkylation of the starting amine occurs, resulting in the formation of a product represented by the general formula 35 in Scheme 2, which can convert the primary amine into the corresponding secondary amine. 2 CO 3Or Na 2 CO 3 A secondary amine such as 35 can be N-alkylated a second time by reacting it with another alkyl halide or sulfonate represented by the general formula 36 in Scheme 2 in the presence of 35 by heating in acetonitrile. Thus, the secondary amine 35 is converted to a tertiary amine of the general formula 37.

[0129]

[0130] Table 1

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Solution 2

[0137] Alternatively, primary amine 33 can be oxidized by 2 CO 3 Or Na 2 CO 3 The bis-alkylation of the starting amine occurs in a single step by heating with an acetonitrile solution of an alkyl halide or sulfonate 34 in the presence of , whereby bis-N-alkylation of the starting amine occurs, leading to the conversion of 33 to a tertiary amine of general formula 38 (Scheme 3). In the case where Z in 33 or 38 is a protecting group, a deprotection step can be used to convert compound 38 to 39.

[0138]

[0139] Solution 3

[0140] The technique outlined in Scheme 3 can be used to synthesize lipid 5, in this case 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.

[0141]

[0142] Solution 4

[0143] The synthesis of representative lipid 5 proceeds with diepoxidation of the double bond in 42 with any suitable epoxidation reagent. For example, reaction of 42 with a peroxycarboxylic acid such as, but not limited to, magnesium monoperoxyphthalate, peracetic acid, meta-chloroperbenzoic acid (MCPBA), etc., produces an inseparable mixture of epoxide diastereomers 43. Reaction of compound 43 with a thiol such as 1-pentanethiol under basic conditions results in the epoxide being fused to the CH 2 Position-selective nucleophilic ring opening leads to the formation of the 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).

[0144]

[0145] Solution 5

[0146] Compounds such as 45 can be converted to lipids such as 5 by esterification of the OH group with a carboxylic acid in the presence of a condensing agent such as a carbodiimide (e.g. 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 such as a pyridine-HF complex. In the case of 5, the carboxylic acid is decanoic acid (Scheme 6).

[0147]

[0148] Solution 6

[0149] Those skilled in the art will appreciate that the ionizable head groups present in lipids 6 to 32 of Table 1 can be introduced starting from a precursor of the lipid in which a ketone functional group is present instead of an ionizable head group. The ketone can then be converted to a suitable ionizable head group by appropriate organic synthesis steps. Thus, the synthesis of 6 to 32 starts with the preparation of an appropriate ketone.

[0150] Representative but non-limiting lipids 6 to 32 can be prepared from ketones having the general structure shown in Scheme 7 as 50. Certain steps of 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 in the disclosure, one such step requires Claisen condensation of an appropriate ester under Mukaiyama conditions, followed by hydrolysis of the resulting β-ketoester and decarboxylation of the intermediate β-ketoacid, resulting in the formation of a ketone. In certain embodiments, these steps are most advantageously performed in a "one-pot operation", meaning that the various synthetic intermediates, while separable, do not need to be separated. For example, ketone 50 can be prepared starting from the Claisen-Mukaiyama condensation of ester 47, and the synthetic intermediates that optionally do not need to be separated are 48 and 49.

[0151]

[0152] Solution 7

[0153] Alternatively, ketones such as 50 and their analogs can be prepared by certain synthetic procedures that are described in detail in co-owned and co-pending U.S. Provisional Patent Application No. 63 / 445,854, which is incorporated herein by reference. One such procedure requires dialkylation of a reagent such as tosylmethyl isonitrile (TosMIC) by reaction with about 2 equivalents of an alkyl halide (chloride, bromide, or iodide) or sulfonate (toluenesulfonate, mesylate, triflate, etc.) under basic conditions followed by acidic hydrolysis of the product. This is illustrated in Scheme 8 for the synthesis of 50 by the TosMIC method.

[0154]

[0155] Solution 8

[0156] Ketones such as 50 are symmetrical, meaning that the alkyl groups attached to the carbonyl are the same. The aforementioned provisional application teaches that asymmetric analogs of 50 with two different alkyl groups attached to the carbonyl can be prepared by sequentially alkylating TosMIC with two different alkyl halides or sulfonates, followed by hydrolysis of the products under acidic conditions (Scheme 9).

[0157]

[0158] Solution 9

[0159] The conversion of a ketone such as 50 to a lipid of formula A begins with epoxidation of the double bond with a suitable epoxidation reagent. Without wishing to be limited, suitable reagents include peroxycarboxylic acids such as magnesium monoperoxyphthalate, performic acid, peracetic acid, metachloroperoxybenzoic acid (MCPBA), etc., which convert the double bond directly to an epoxide, or an electrophilic halogen source such as chlorine, bromine, iodine, N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), N-iodosuccinimide (NIS), etc., which in aqueous media convert the double bond to a halohydrin that can be converted to an epoxide by treatment with a base. In either case, the result is the formation of a diepoxide xx as a mixture of oxirane diastereoisomers that are not separated. This is illustrated in Scheme 10, where 56 is formed by treating 50 with MCPBA. Subsequent regioselective ring opening of the epoxide with a thiol R-SH under alkaline conditions produces the dihydroxy ketone 57.

[0160]

[0161] Solution 10

[0162] The dihydroxy ketone is then esterified with an appropriate carboxylic acid. Depending on the conditions, the esterification reaction can be carried out so that a symmetrical diester 58 is formed as the main product, for example, by using at least 2 molar equivalents of acid R'-COOH in the presence of a condensing agent, for example, such as EDCI and DMAP, or so that a monoester 59 is formed as the main product, for example, by using about 1 molar equivalent of acid R'-COOH in the presence of a condensing agent, for example, such as EDCI and DMAP. In addition, the monoester 59 can be converted to an asymmetrical diester 60 (Scheme 10) by subsequent reaction with a second carboxylic acid R"-COOH in the presence of a condensing agent, for example, such as EDCI and DMAP. The keto group in 58 or 60 can then be converted to any ionizable head group of type 1 to 9 (see above definition) by chemical methods well known to those skilled in the art. Representative but by no means limiting examples are set forth below.

[0163] The ketone of type 50 required for the synthesis of lipid 6 is 61 (a variant of 50 wherein n=3), the diepoxy ketone obtained from 61 is 62 (a variant of 56 wherein n=3), the thiol used in the epoxide ring-opening reaction is cyclohexanethiol, and the acid used in the esterification of the resulting dihydroxy ketone 63 is decanoic acid (Scheme 11). The ketone in product 64 is then converted to a type 1 ionizable head group by selective reduction using a hydride reagent such as sodium borohydride in an appropriate solvent such as an alcohol (e.g., ethanol or isopropanol), followed by esterification of the resulting alcohol 65 using 4-(dimethylamino)butyric acid or a salt thereof (e.g., the corresponding hydrochloride) in the presence of a condensing agent such as a carbodiimide (e.g., EDCI) and DMAP, resulting in the formation of lipid 6.

[0164]

[0165] Solution 11

[0166] The ketone of type 50 required for the synthesis of lipid 7 is 66 (a variant of 50 wherein n=4), the diepoxy ketone obtained from 66 is 67 (a variant of 56 wherein n=4), the thiol used in the epoxide ring-opening reaction is 1-hexanethiol, and the acid used in the esterification of the resulting dihydroxy ketone 68 is decanoic acid (Scheme 12). The ketone in product 69 is then converted to a type 1 ionizable head group as shown in Scheme 11 above.

[0167] The synthesis of lipid 8 (Scheme 13) involved esterification of compound 68 of Scheme 12 with 3-cyclohexylpropionic acid followed by conversion of the resulting ketone in 71 to an ionizable head group of type 1 as shown in Scheme 11 above.

[0168] The synthesis of lipid 9 requires acid 74, which can be prepared, for example, by the method shown.

[0169]

[0170] Solution 12

[0171]

[0172] Solution 13

[0173]

[0174] Solution 14

[0175] In Scheme 14, details of which are in co-owned and co-pending U.S. Provisional Patent Application No. 63 / 410,273, monoesterification of dihydroxy ketone 68 with decanoic acid and further esterification of the resulting 75 with acid 74 affords 76. Conversion of the ketone in 76 to a Type 1 ionizable head group is then accomplished as shown in Scheme 11 above (Scheme 15).

[0176]

[0177] Solution 15

[0178] The diepoxy ketone required for the synthesis of lipid 10 was 67, the thiol used in the epoxide ring-opening reaction was cyclohexanethiol, and the acid used in the esterification of the resulting dihydroxy ketone 78 was decanoic acid (Scheme 16). The ketone in product 79 was then converted to a type 1 ionizable head group as shown in Scheme 11 above.

[0179]

[0180] Scheme 16

[0181] The synthesis of lipid 11 (Scheme 17) involves the esterification of dihydroxyketone 78 with nonanoic acid, followed by conversion of the ketone in the resulting diester 81 to an ionizable head group of type 1 as shown in Scheme 11 above.

[0182] The synthesis of lipid 12 (Scheme 18) started with the diepoxy ketone 62. The thiol used in the epoxide ring-opening reaction was 1-pentanethiol, and the acid used in the esterification of the resulting dihydroxy ketone 83 was decanoic acid. The ketone in the product 84 was then converted to a type 2 ionizable head group, starting with the formation of the ketal 85 by reaction with a 1,2,4-

[0183]

[0184] Solution 17

[0185] Butanetriol in a suitable solvent, at a suitably elevated temperature, and in the presence of an acid catalyst, preferably with continuous azeotropic removal of water formed during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium p-toluenesulfonate (PPTS), and a Dean-Stark water trap can be used to remove water. The OH group in the product 85 of the ketalization step is then converted to a leaving group such as a halide (chloride, bromide or iodide) or a sulfonate (toluenesulfonate, mesylate, triflate, etc.) to prepare for the introduction of the dimethylamino moiety. For example, 85 can be prepared by reacting with p-toluenesulfonyl chloride (TsCl) in an alkaline solvent such as pyridine, or in an aqueous solution such as CH 2 Cl 2 The tosylate 86 is converted to the tosylate 86 by reaction in a non-basic solvent in the presence of a base such as triethylamine, in either case optionally in the presence of a catalyst such as 4-dimethylaminopyridine. The tosylate 86 is reacted with dimethylamine in a suitable solvent or solvent mixture such as tetrahydrofuran (THF) and methanol at a suitably elevated temperature, optionally in the presence of a catalyst such as Na 2 CO 3 or K 2 CO 3 The reaction is carried out in the presence of a base, optionally under microwave activation, to produce lipid 12.

[0186]

[0187] Scheme 18

[0188] The synthesis of lipid 13 (Scheme 19) demonstrates a method for converting ketones to ionizable head groups of type 4. Thus, ketone 84 is converted to bromoketal 87 by reaction with 3-bromo-1,2-propanediol in a suitable solvent, at a suitably elevated temperature and in the presence of an acid catalyst, preferably with continuous azeotropic removal of the water formed during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium p-toluenesulfonate (PPTS), and a Dean-Stark trap can be used to remove the water. 87 is reacted with 4-methylamino-1-butanol in a suitable solvent or solvent mixture, such as acetonitrile, at a suitably elevated temperature, optionally in the presence of a solvent such as Na 2 CO 3 or K 2 CO 3 In the presence of a base, optionally under microwave activation, 13 is produced.

[0189]

[0190] Solution 19

[0191] 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.

[0192]

[0193] Solution 20

[0194]

[0195] Solution 21

[0196] Lipid 16 can be prepared from ketone 64 of Scheme 11 by converting the carbonyl group to a type 2 ionizable head group via the method outlined in Scheme 18 (Scheme 22).

[0197]

[0198] Solution 22

[0199] The head group present in lipids 17 and 18 is a variant of the ionizable ionizable group of type 3 and can be produced starting from the conversion of ketone 64 of Scheme 11 to ketal 90 by reaction with 2,2-bis(hydroxymethyl)propane-1,3-diol (pentaerythritol) in a suitable solvent at a suitably elevated temperature and in the presence of an acid catalyst, preferably with continuous azeotropic removal of the water formed during the reaction. For example, the reaction can be carried out in refluxing toluene in the presence of pyridinium p-toluenesulfonate (PPTS) and a Dean-Stark trap can be used to remove the water. Compound 90 is then monoesterified with 3-(dimethylamino)propionic acid or its hydrochloride salt, for example in the presence of a carbodiimide such as EDCI and optionally DMAP, to give 17. The same esterification reaction with 4-(dimethylamino)butyric acid or its HCl salt gives 18 (Scheme 23).

[0200]

[0201] Solution 23

[0202] The synthesis of lipid 19 (Scheme 24) involved the conversion of keto compound 69 of Scheme 12 to a Type 2 ionizable head group by a method similar to that shown in Scheme 19, except that dimethylamine was used in the final reaction.

[0203]

[0204] Solution 24

[0205] Lipid 20 (Scheme 25) can be prepared by reacting bromoketal 91 with 4-(methylamino)-1-butanol by the method shown in Scheme 19 above.

[0206]

[0207] Solution 25

[0208] The 4-type ionizable group 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).

[0209]

[0210] Scheme 26

[0211] The synthesis of lipids 22 to 32 demonstrates a method for converting a keto group into an ionizable head of type 7. Thus, lipid 22 can be prepared from ketone 84 of Scheme 18, starting from reductive amination with an O-protected form of 4-amino-1-butanol, for example a silyl ether such as tert-butyldiphenylsilyl (TBDPS) ether, in a suitable solvent such as 1,2-dichloroethane, in the presence of a reducing agent, for example a borohydride reagent such as sodium triacetoxyborohydride, sodium cyanoborohydride, and the like, 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 requires N-methylation to give 95. This can be achieved by treating 94 with aqueous formaldehyde and a reducing agent such as, but not limited to, 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 in a suitable solvent and under suitable conditions. By releasing the TBDPS group in 95 with a fluoride ion source such as HF-pyridine complex, lipid 22 is obtained.

[0212]

[0213] Scheme 27

[0214] The synthesis of lipid 23 (Scheme 28) started with the reaction of bis-epoxyketone 62 of Scheme 11 with 1-heptylthiol and esterification of the resulting 96 with decanoic acid to produce 97. The latter compound was then converted to lipid 23 by the same method as shown in Scheme 27 above.

[0215]

[0216] Scheme 28

[0217] The synthesis of lipid 24 (Scheme 29) can be achieved in a similar manner, except that compound 96 is esterified with nonanoic acid.

[0218]

[0219] Scheme 29

[0220] Lipid 25 can be prepared starting from the reaction of bis-epoxy ketone 67 of Scheme 12 with 1-pentanethiol, followed by esterification with nonanoic acid to obtain 103 (Scheme 30). The ketone 104 thus obtained can then be converted to lipid 25 by the method shown in Scheme 27 above.

[0221]

[0222] Scheme 30

[0223] Lipid 26 can be prepared in a similar manner starting from the esterification of compound 103 with decanoic acid (Scheme 31).

[0224]

[0225] Scheme 31

[0226] Lipid 27 can be obtained from ketone 69 of Scheme 12 by the method shown in Scheme 27 above (Scheme 32).

[0227]

[0228] Scheme 32

[0229] Lipid 28 can be prepared starting from N-propylation of secondary amine 110. This can be accomplished, for example, by reacting 110 with 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, etc., optionally in the presence of a catalyst such as acetic acid (Scheme 33). The resulting 112 is then converted to lipid 28 by the same method shown in Scheme 27 above.

[0230] Lipid 29 can be prepared starting from N-isobutylation of secondary amine 110. This can be accomplished, for example, by reacting 110 with 2-methylpropanal (isobutyraldehyde) in a suitable solvent such as 1,2-dichloroethane in the presence of a reducing agent such as borohydride.

[0231]

[0232] Scheme 33

[0233] Reagents such as sodium triacetoxyborohydride, sodium cyanoborohydride, etc., optionally in the presence of a catalyst such as acetic acid (Scheme 34). The resulting 113 is then converted to lipid 29 by the same method shown in Scheme 27 above.

[0234]

[0235] Scheme 34

[0236] Lipid 30 can be prepared from ketone 76 of Scheme 15 by the method outlined in Scheme 27 above (Scheme 35).

[0237]

[0238] Scheme 35

[0239] Lipid 31 can be prepared starting from the reaction of bis-epoxy ketone 67 of Scheme 12 with 1-heptylthiol, followed by esterification with nonanoic acid to give 116 (Scheme 36). The ketone 117 thus obtained can then be converted to lipid 31 by the method shown in Scheme 27 above.

[0240]

[0241] Scheme 36

[0242] Lipid 32 can be prepared from ketone 81 of Scheme 17 by the method outlined in Scheme 27 above (Scheme 37).

[0243]

[0244] Scheme 37

[0245] Formulation of the above lipids in a delivery vehicle

[0246] The lipids of the present disclosure can be formulated in various drug delivery vehicles known to those of ordinary skill in the art (also referred to herein as "delivery vehicles"). Examples of delivery vehicles are lipid nanoparticles, including liposomes, lipid complexes, polymer nanoparticles comprising lipids, polymer-based nanoparticles, emulsions, and micelles.

[0247] In one embodiment, a lipid having a structure of Formula A of the present disclosure is formulated in a delivery vehicle by mixing it with additional lipids including auxiliary lipids such as vesicle-forming lipids and, optionally, aggregation-inhibiting lipids such as hydrophilic polymer-lipid conjugates (e.g., PEG-lipids).

[0248] As mentioned above, the helper lipids include sterols, diacylglycerols, ceramides or their derivatives.

[0249] Examples of sterol include cholesterol or cholesterol derivatives such as cholestanol, cholestanone, cholestanone, coprostanol, cholestyl-2'-hydroxyethyl ether, cholestyl-4'-hydroxybutyl ether, β-sitosterol, fucoxantranol and the like.

[0250] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyl-phosphatidylcholine (POPC), palmitoyl-phosphatidylethanolamine (POPE), palmitoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dinonanoyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, DSPC-cholesterol conjugates, or mixtures thereof. These lipids can be synthesized or obtained from natural sources such as from eggs. The DSPC-cholesterol conjugate is a lipid in which one of the acyl chains is replaced with a cholesterol moiety which is linked to the head group via a succinate linker.

[0251] Suitable ceramide derivatives are egg sphingomyelin or dihydrosphingomyelin.

[0252] The delivery vehicle incorporated into the lipid of the present disclosure can be prepared using a variety of well-described formulation methodologies known to those skilled in the art, including but not limited to extrusion, ethanol injection and pipeline mixing. In one embodiment, the preparation method is a pipeline mixing technology, wherein an aqueous solution and an organic solution are mixed using a fast mixing device as described in Kulkarni et al., 2018, ACS Nano, 12: 4787 and Kulkarni et al., 2017, Nanoscale, 36: 133347, each of which is incorporated herein by reference in its entirety.

[0253] Delivery carrier can also be nanoparticle, it is the lipid complex that comprises the lipid core that is stabilized by surfactant.The lipid that forms vesicle can be used as stabilizing agent.In another embodiment, lipid nanoparticle is polymer-lipid mixed system, and it comprises the polymer nanoparticle core that is surrounded by stable lipid.Nanoparticle comprising lipid of the present disclosure can be prepared by lipid-free polymer alternatively.Such nanoparticle can comprise the condensed core of therapeutic agent, it is surrounded by polymer shell or can have solid or liquid dispersed in whole polymer matrix.

[0254] Lipids as described herein can also be incorporated into emulsions, which are drug delivery vehicles containing oil droplets or oil cores. Emulsions can be lipid-stabilized. For example, an emulsion can include a core of an oil-filled stabilized by an emulsifying component such as a monolayer or bilayer lipid.

[0255] The lipids described herein can be incorporated into micelles. Micelles are self-assembled particles composed of amphiphilic lipid or polymer components that are used to deliver agents present in a hydrophobic core.

[0256] Delivery of nucleic acids, genetic material, proteins, peptides or other charged agents

[0257] The lipids disclosed herein can facilitate the incorporation of compounds or molecules carrying a net negative or positive charge (also referred to herein as "cargo" or "cargo molecules") into delivery vehicles and subsequent delivery to target cells in vitro or in vivo.

[0258] In one embodiment, the cargo molecule is a 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 carrier DNA or linear DNA. The nucleic acid length can vary and can include nucleic acids with a length of 5 to 50,000 nucleotides. The nucleic acid can be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or a hybrid thereof. Single-stranded nucleic acids include antisense oligonucleotides.

[0259] In one embodiment, the cargo is mRNA, which includes a polynucleotide encoding 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 WO 2022 / 251953 A1, which is incorporated herein by reference.

[0260] mRNA used herein includes 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, produced and optionally purified using a recombinant expression system, or can be chemically synthesized.

[0261] In those embodiments where the mRNA is a chemically synthesized molecule, the mRNA can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In certain embodiments, the mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiopyrimidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2 -aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose / arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

[0262] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, in certain embodiments, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase and / or RNAse inhibitor.

[0263] In certain embodiments, in vitro synthesized mRNA can be purified prior to encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0264] The present disclosure can be used to encapsulate mRNAs of various lengths. In certain 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.

[0265] Typically, mRNA synthesis includes the addition of a "cap" at the 5' end and a "tail" at the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation.

[0266] In certain embodiments, mRNA includes 5' and / or 3' untranslated regions. In certain embodiments, 5' untranslated regions include one or more elements that affect mRNA stability or translation, such as iron response elements. In certain embodiments, the length of 5' untranslated regions can be between about 50 and 500 nucleotides.

[0267] In certain embodiments, the 3' untranslated region includes one or more polyadenylation signals, binding sites for proteins that affect the stability of mRNA localization in cells, or one or more binding sites for miRNA. In certain embodiments, the length of the 3' untranslated region can be between 50 and 500 nucleotides or longer.

[0268] In another embodiment, the mRNA is circular. Advantageously, this mRNA lacks 5' and 3' ends and is therefore more stable in vivo due to its resistance to exonuclease degradation. Circular mRNA can be prepared by any known method, including Deviatkin et al., 2023, "Cap-Independent Circular mRNA Translation Efficiency", Vaccines, 11 (2), 238, which is incorporated herein by reference. The translation of circular mRNA is carried out by a cap-independent initiation mechanism.

[0269] Although mRNA provided by an in vitro transcription reaction may be desirable in certain embodiments, other sources of mRNA are also contemplated, such as mRNA produced by bacteria, fungi, plants, and / or animals.

[0270] The mRNA sequence may contain a reporter gene sequence, although inclusion of a reporter gene sequence in a pharmaceutical formulation for administration is optional. Such a sequence may be incorporated into the mRNA for in vitro studies or for in vivo studies in animal models to assess biodistribution.

[0271] In another embodiment, the cargo is siRNA. siRNA is incorporated into endogenous cellular machinery to cause mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to the target site.

[0272] The siRNA encompassed by the embodiments of the present disclosure can be used to specifically inhibit the expression of a variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides can be easily prepared according to methods known in the art. The siRNA target site can be selected, and the corresponding siRNA can be chemically synthesized, produced by in vitro transcription, or expressed from a vector or PCR product. A variety of different siRNA molecules can be used to target specific genes or transcripts. The siRNA can be a double-stranded RNA, or a hybrid molecule comprising RNA and DNA, such as an RNA chain and a DNA chain. The siRNA can have a variety of lengths, such as a length of 15 to 30 nucleotides or a length of 20 to 25 nucleotides. In certain embodiments, the siRNA is double-stranded and has a 3' overhang or a 5' overhang. In certain embodiments, the overhang is UU or dTdt3'. In a specific embodiment, the siRNA comprises a stem-loop structure.

[0273] In other embodiments, the cargo molecule is a microRNA or a small nuclear RNA. MicroRNA (miRNA) is a short, non-coding RNA molecule that is transcribed from genomic DNA but is not translated into protein. It is believed that these RNA molecules play a role in the regulation of gene expression by binding to the region of the target mRNA. The binding of miRNA to the target mRNA can, for example, downregulate gene expression by inducing translational inhibition, deadenylation or degradation of the target mRNA. Small nuclear RNA (snRNA) is generally a longer non-coding RNA molecule that participates in gene splicing. snRNA molecules may have therapeutic importance in diseases that are the result of splicing defects.

[0274] In another embodiment, the cargo is a DNA vector as described in co-owned and co-pending WO 2022 / 251959, which is incorporated herein by reference. The DNA vector may be administered to an individual in order to repair, enhance, or block or reduce the expression of a cellular protein or peptide. Thus, the nucleotide polymer can be a nucleotide sequence comprising genomic DNA, cDNA, or RNA.

[0275] As will be appreciated by those skilled in the art, a vector may encode a promoter region, an operator region, or a structural region. A DNA vector may contain double-stranded DNA or may consist of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replication systems such as vector DNA.

[0276] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have extended activity, single-stranded nucleic acids will preferably have some or all of the nucleotide bonds substituted by stable non-phosphodiester bonds, including, for example, phosphorothioate, phosphorodithioate, phosphoselenate or O-alkyl phosphotriester bonds.

[0277] DNA vectors can include nucleic acids modified in one or more sugar moieties and / or one or more pyrimidine or purine bases. Such sugar modifications can include replacing one or more hydroxyls with halogen, alkyl, amine, azido or functionalized to ether or ester. In another embodiment, the entire sugar can be substituted by spatially and electronically similar structures, including aza-sugars and carbocyclic sugar analogs. The modification of purine or pyrimidine base moieties includes, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substituents well known to those skilled in the art.

[0278] In certain embodiments, a modified molecule such as a peptide, protein, steroid or sugar moiety can be used to modify the DNA vector. Modification of the DNA vector with such a molecule can facilitate delivery to a target site of interest. In certain embodiments, this modification causes the DNA vector to translocate across the nucleus of a target cell. As an example, a modifier can bind to a specific portion of the DNA vector (usually not encoding a target gene), but also has a peptide or other modifier with a nuclear homing effect such as a nuclear localization signal. A non-limiting example of a regulator is a steroid-peptide nucleic acid conjugate. As described in Rebuffat et al., 2002, Faseb J.16(11):1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. A promoter, an enhancer, a promoter regulated by stress or chemicals, an antibiotic sensitive region or a nutrient sensitive region, and a sequence encoding a therapeutic protein may be included as needed. Non-coding sequences may also be present in the DNA vector.

[0279] The nucleic acid that is used for the inventive method can be separated from natural sources, obtains from sources such as ATCC or GenBank library or prepares by synthetic methods.Synthetic nucleic acid can be prepared by various solutions or solid phase methods.Usually, solid phase synthesis is preferred.The detailed description of the method for solid phase synthesis of nucleic acid by phosphite-triester, phosphotriester and H-phosphonate chemical reaction is widely available.

[0280] In one embodiment, the DNA vector is double-stranded DNA and comprises more than 700 base pairs, more than 800 base pairs, or more than 900 base pairs, or more than 1000 base pairs.

[0281] In another embodiment, the DNA vector is a nanoparticle or a minicircle.

[0282] Gene editing systems can also be incorporated into delivery vectors comprising charged lipids. This includes Cas9-CRISPR, TALEN and zinc finger nuclease gene editing systems. In the case of Cas9-CRISPR, guide RNA (gRNA) can be incorporated into a delivery vector comprising lipids described herein together with a plasmid or mRNA encoding the Cas9 protein. Optionally, a ribonucleoprotein complex can be incorporated into a delivery vector comprising lipids described herein. Similarly, the present disclosure includes embodiments in which genetic materials encoding the DNA binding and cleavage domains of zinc finger nucleases or TALEN systems are incorporated into a delivery vector together with the lipids of the present disclosure.

[0283] Although a variety of nucleic acid cargo molecules are described above, it should be understood that the above examples are non-limiting and the present disclosure is not to be considered limited to the particular cargo molecules encapsulated in the delivery vector.

[0284] For example, the lipids described herein can also facilitate the incorporation of proteins and peptides into delivery vehicles, including ribonucleoproteins. This includes linear and non-linear peptides, proteins or ribonucleoproteins.

[0285] Although pharmaceutical compositions are described above, the lipids described herein may be a component of any nutritional, cosmetic, cleaning, or food product.

[0286] Pharmaceutical preparations

[0287] Ionizable lipids of the present disclosure may exist in salt form. Salts are typically pharmaceutically acceptable salts. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium and zinc. In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, etc.

[0288] In certain embodiments, the delivery vehicle comprising the cargo molecule is part of a pharmaceutical composition and is administered to treat and / or prevent a disease state. Treatment can provide prophylactic (preventive), ameliorative or therapeutic benefits. The pharmaceutical composition will be administered in any suitable dose.

[0289] In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intra-arterially, intravenously, subcutaneously, or intramuscularly. In another embodiment, the pharmaceutical composition is for intratumoral or intrauterine administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinally, intrathecally, or by other topical routes.

[0290] The pharmaceutical composition comprises a pharmaceutically acceptable salt and / or an excipient.

[0291] The compositions described herein can be administered to a patient. The term patient as used herein includes human or non-human individuals.

[0292] The following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Example

[0293] Material

[0294] Lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (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). Ionizable amino-lipids were synthesized as previously described in WO 2022 / 246555, which is incorporated herein by reference.

[0295] Luciferase activity was analyzed using mRNA encoding firefly luciferase purchased from APExBIO Technology LLC (Houston, TX).

[0296] method

[0297] Preparation of lipid nanoparticles (LNPs) containing mRNA

[0298] Lipid 1 or 2, DSPC, cholesterol and PEG-DMG described herein are dissolved in ethanol in appropriate proportions to a final concentration of 10 mM total lipid. The nucleic acid (siRNA or mRNA) is dissolved in an appropriate buffer, such as 25 mM sodium acetate pH 4 or sodium citrate pH 4, to a concentration required to obtain an appropriate amine to phosphate ratio. The aqueous solution and the organic solution are mixed using a rapid mixing device as described in Kulkarni et al., 2018, ACS Nano, 12: 4787 and Kulkarniet al., 2017, Nanoscale, 36: 133347 (each incorporated herein by reference) at a flow rate ratio of 3: 1 (v / v; respective). The resulting mixture is dialyzed directly with 1000 volumes of PBS pH 7.4. Use Amicon TM All preparations were concentrated by centrifugal filter units and analyzed using the methods described below.

[0299] LNP analysis

[0300] Using the Malvern Zetasizer TMParticle size analysis of LNPs in PBS was performed by backscattering measurements of dynamic light scattering at Wako Diagnostics (Worcestershire, UK). The reported particle sizes correspond to the number-weighted mean diameter (nm). Total lipid concentration was determined by extrapolation from cholesterol content, which was measured using cholesterol E-total cholesterol assay (Wako Diagnostics, Richmond, VA) according to the manufacturer's recommendations. Quant-iT RiboGreen TM The encapsulation efficiency of the formulations was determined using the ELISA kit (Invitrogen, Waltham, MA). Briefly, the total siRNA or mRNA content in the solution was measured by lysing the lipid nanoparticles in TE solution containing 2% Triton Tx-100, and the RiboGreen TM Fluorescence is used to measure the free DNA carrier in the solution (outside LNP). The total siRNA or mRNA content in the preparation is measured using a modified Bligh-Dyer extraction procedure. In brief, the LNP preparation containing siRNA or mRNA is dissolved in a mixture of chloroform, methanol and PBS to obtain a single phase and the absorbance at 260nm is measured using a spectrophotometer.

[0301] In vivo analysis in CD-1 mice

[0302] LNP-mRNA encoding firefly luciferase was injected intravenously (tail vein) into 6 to 8 week old CD-1 mice. 4 hours after injection, animals were euthanized and liver and spleen were separated. Tissue was homogenized in Glo lysis buffer and luciferase assay was performed using Steady Glo luciferase assay kit (according to the manufacturer's recommendation).

[0303] 5-32 Organic Synthesis of Lipids

[0304] Unless otherwise stated, reagents and solvents were commercial products and used without purification, except for THF (freshly distilled from Na / benzophenone under Ar), CH 2 Cl 2 (From CaH under Ar 2 The "dry methanol" was freshly distilled from the magnesium turnings. All reactions were performed under an inert atmosphere (nitrogen or argon). The reaction mixture from the aqueous workup was passed through anhydrous Na 2 SO 4 The precipitate was dried and concentrated on a rotary evaporator under reduced pressure. Thin layer chromatography was performed on silica gel plates coated with silica gel (Merck 60F254 plates). 2Chromatographic purification was performed on a Biotage ISCO system. 3 Recording in solution 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR spectra referenced to residual CHCl 3 (7.26ppm), 13 C NMR spectra referenced to CDCl 3 The center line of the triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the delta scale. Multiplicities are reported as "s" (singlet), "d" (doublet), "t" (triplet), "q" (quartet), "m" (multiplet), and further defined as "app" (apparent) and "br" (broad). Low-resolution and high-resolution mass spectra (m / z) were obtained in electrospray (ESI) and field desorption / field ionization (FD / FI) modes.

[0305] As described below, the synthesis of lipid 5 from 4-amino-1-butanol was performed. As discussed, the synthesis of type 5 lipids includes mono-N-alkylation or di-N-alkylation of 4-amino-1-butanol with certain alkyl halides or sulfonates under appropriate conditions. This technology is described in co-owned and co-pending WO 2023 / 173203 (incorporated herein by reference).

[0306] As described below, the synthesis of lipids 5 to 32 was performed. As discussed, the synthesis of the lipids includes subjecting certain esters or lactones to Claisen condensation under Mukaiyama conditions. This technology is described in co-owned and co-pending WO 2022 / 246555 (incorporated herein by reference). The product of this Claisen reaction is then converted to the final product as outlined in the above scheme and described below.

[0307] Example 1: Method for chemically synthesizing ionizable lipids

[0308] (A) Preparation of building blocks

[0309] (i) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-N-(hept-6-en-1-yl)hept-6-en-1-amine (42)

[0310]

[0311] O-TBDPS protected 4-amino-1-butanol 40 (841 mg, 2.57 mmol), 7-bromohept-1-ene (1.00 g, 5.65 mmol) and K2 CO 3 A mixture of (859 mg, 6.22 mmol) in MeCN (15.0 mL) was stirred in a sealed reaction vessel at 80 °C for 18 h. The mixture was cooled, diluted with water (15 mL) and washed with CH 2 Cl 2 (3×15 mL) extraction. The combined organic matter was dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to afford amine 42 (802 mg, 60%) as an oil. 1 H NMR (400 MHz, CDCl 3 )δ7.72-7.57(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=7.1Hz,0H),1.60-1.21(m,20H),1.04(s,9H).

[0312] (ii) Trideca-1,12-dien-7-one (61)

[0313]

[0314] At 0°C, under nitrogen atmosphere, methyl hept-6-enoate (5.95 g, 41.9 mmol) and NBu 3 TiCl (18.0 mL, 75.4 mmol) in toluene (80.0 mL) was added dropwise 4 (6.89 mL, 62.9 mmol) in toluene (40.0 mL). The reaction was warmed to room temperature and stirred for 2 hours. 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 hours, concentrated to 25% volume and acidified to pH 2 with concentrated hydrochloric acid. The reaction was washed with 50:50 hexane / Et 2 O (3 × 40 mL). The combined organics were washed with brine, dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 8% EtOAc in hexanes) to give ketone 61 (3.7 g, 91%) as an oil. 1 H NMR (400 MHz, CDCl 3)δ5.79(ddt,J=16.9,10.2,6.7Hz,2H),5.07-4.86(m,4H),2.39(t,J=7.4Hz,4H),2.20-1.98(m,4H),1.71-1.49(m,4H),1.49-1.33(m,4H).

[0315] (iii) Pentadecadien-1,14-dien-8-one (66)

[0316]

[0317] Prepared from methyl octanoate by the procedure of part (ii) above. 1 H NMR (400 MHz, CDCl 3 )δ5.79(ddt,J=16.9,10.2,6.6Hz,2H),5.07-4.84(m,4H),2.38(t,J=7.4Hz,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).

[0318] (iv) 1,9-Di(oxiran-2-yl)nonan-5-one (62)

[0319]

[0320] Solid mCPBA (13.9 g, about 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 hours. The mixture was cooled to 0°C and quenched with saturated aqueous sodium sulfite and diluted with water (20 mL). The layers were separated and the organics were washed with 1N NaOH (3×30 mL), dried (Na 2 SO 4 ) and concentrated to afford diepoxide 62 (4.1 g, 90%). 1 H NMR (400 MHz, CDCl 3 )δ2.94-2.87(m,2H),2.74(dd,J=5.0,3.9Hz,2H),2.46(dd,J=5.0,2.7Hz,2H),2.41(t,J=7.3Hz,4H),1.73-1.34(m,12H).

[0321] (v) 1,11-Di(oxiran-2-yl)undecane-6-one (67)

[0322]

[0323] Prepared from ketone 66 by procedure (iv) above. 1 H NMR (400 MHz, CDCl 3 )δ2.94-2.83(m,2H),2.76-2.69(m,2H),2.44(dd,J=5.0,2.7Hz,2H),2.38(td,J=7.5,1.6Hz,4H),1.65-1.20(m,16H).

[0324] (vi) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-5-(oxiran-2-yl)-N-(5-(oxiran-2-yl)pentyl)-pentan-1-amine oxide (43)

[0325]

[0326] Solid mCPBA (1.59 g, about 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 hours. The mixture was cooled to 0°C and quenched with saturated aqueous sodium sulfite and diluted with water (10.0 mL). The layers were separated and the organics were washed with 1N NaOH (3×30.0 mL), dried (Na 2 SO 4 ) and concentrated to afford diepoxide 43 (629 mg, 72%) as a waxy white solid which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 )δ7.71-7.60(m,4H),7.51-7.34(m,6H),3.71(t,J=5.9Hz,2H),3.32-3.12(m,6H),2.9 2-2.84(m,2H),2.78-2.70(m,2H),2.49-2.41(m,2H),2.01-1.34(m,20H),1.04(s,9H).

[0327] (vii) General procedure for epoxide ring opening using thiols. To a well stirred solution of diepoxide (1 mmol) and thiol (2.2 mmol, 2.2 eq) in EtOH (10 mL) maintained under an inert atmosphere was added solid NaOH (4 eq). The mixture was heated at reflux for 2 h, cooled, diluted with water (20 mL) and extracted with DCM (3 x 15.0 mL). The combined organics were dried (NaSO 4) and concentrated. The residue was purified by silica gel chromatography (0 to 50% EtOAc in hexanes) to give the desired product (75 to 80%). The following compounds were thus prepared:

[0328] (viii) N-(4-((tert-butyldiphenylsilyl)oxy)butyl)-6-hydroxy-N-(6-hydroxy-7-(pentylthio)heptyl)-7-(pentylthio)heptyl-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)

[0329]

[0330] Compound 44 was obtained from 43 and 1-pentanethiol by the general procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.69-3.55 (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 hours. The mixture was then diluted with DCM (10.0 mL) and washed with water (3×15.0 mL), brine (15.0 mL), and dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 40% MeOH in DCM) to afford amine 45 (255 mg, 51%). 1 H NMR (400 MHz, CDCl 3 )δ7.69-7.62(m,4H),7.47-7.33(m,6H),3.67(t,J=5.9Hz,2H),3.65-3.58(m,2H),2.71(dd,J=13.6,3.5Hz,2H),2.66-2 .56(m,6H),2.51(t,J=7.5Hz,4H),2.43(dd,J=13.6,8.9Hz,2H),1.66-1.26(m,32H),1.04(s,9H),0.89(t,J=7.1Hz,6H).

[0331] (ix) 1,13-Bis(cyclohexylthio)-2,12-dihydroxytridecane-7-one (63)

[0332]

[0333] Prepared from 62 and cyclohexanethiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.66-3.56(m,2H),2.78(dd,J=13.5,3.4Hz,2H),2.69-2.57(m,2H),2.4 9-2.37(m,6H),2.03-1.89(m,6H),1.81-1.71(m,4H),1.67-1.20(m,22H).

[0334] (x) 1,15-Bis(hexylthio)-2,14-dihydroxypentadecan-8-one (68)

[0335]

[0336] Prepared from 67 and 1-hexanethiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.69-3.55(m,2H),2.73(dd,J=13.6,3.3Hz,2H),2.51(t,J=7.4Hz,4H),2.46-2.34(m,6H),1.67-1.23(m,32H),0.90(t,J=7.0Hz,6H).

[0337] (xi) 1,15-Bis(cyclohexylthio)-2,14-dihydroxypentadecan-8-one (78)

[0338]

[0339] Prepared from 67 and cyclohexanethiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.65-3.53(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=14.8,7.3,3.9Hz,6H),1.51-1.39(m,6H),1.39-1.17(m,16H).

[0340] (xii) 2,12-Dihydroxy-1,13-bis(pentylthio)tridecyl-7-one (83)

[0341]

[0342] Prepared from 62 and 1-pentanethiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.68-3.55(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).

[0343] (xiii) 1,13-Bis(heptylthio)-2,12-dihydroxytridecane-7-one (96)

[0344]

[0345] Prepared from 62 and 1-heptylthiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.70-3.56(m,2H),2.72(dd,J=13.6,3.3Hz,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).

[0346] (xiv) 2,14-Dihydroxy-1,15-bis(pentylthio)pentadecan-8-one (103)

[0347]

[0348] Prepared from 67 and 1-pentanethiol by the procedure of part (vii). 1 H NMR (400 MHz, CDCl 3 )δ3.69-3.55(m,2H),2.73(dd,J=13.6,3.3Hz,2H),2.51(t,J=7.4Hz,4H),2.46-2.34(m,6H),1.67-1.23(m,28H),0.90(t,J=7.0Hz,6H).

[0349] (xv) 1,15-Bis(heptylthio)-2,14-dihydroxypentadecan-8-one (116)

[0350]

[0351] Prepared from 67 and 1-heptylthiol by the procedure of part (vii). 1H NMR (400 MHz, CDCl 3 )δ3.69-3.55(m,2H),2.73(dd,J=13.6,3.3Hz,2H),2.51(t,J=7.4Hz,4H),2.46-2.34(m,6H),1.67-1.23(m,36H),0.90(t,J=7.0Hz,6H).

[0352] (B) Preparation of symmetrical diester derivatives of dihydroxy compounds of part (A)

[0353] (i) General operation. Under nitrogen atmosphere, a solution of dihydroxy compound (1 mmol, 1 equivalent), carboxylic acid (2.4 mmol, 1.2 equivalent), EDCI·HCl (2.5 mmol, 1.25 equivalent) and DMAP (2.4 mmol) in DCM (4 mL) was stirred at room temperature for 18 hours. The reaction was concentrated and the residue was purified by silica gel chromatography (0 to 10% EtOAc in hexane) to give an oily diester (80 to 90%). The following compounds were obtained:

[0354] (ii) 9-(6-(Decanyloxy)-7-(pentylthio)heptyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-17-thia-9-aza-3-siladocosan-15-yldecanoate (46)

[0355]

[0356] Obtained from 45 and decanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ7.74-7.62(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=7.3,1.8Hz,4H),2.45-2.24(m,10H),1.77-1.15(m,60H),1.04(s,9H),0.94-0.82(m,12H).

[0357] (iii) 1,13-Bis(cyclohexylthio)-7-oxotridecan-2,12-diylbis(decanoate) (64)

[0358]

[0359] Obtained from 63 and decanoic acid. 1 H NMR (400 MHz, CDCl 3)δ4.98-4.81(m,2H),2.77-2.57(m,6H),2.38(t,J=7.4Hz,4H),2.29(t, J=7.5Hz,4H),2.00-1.91(m,4H),1.81-1.18(m,56H),0.95-0.81(m,6H).

[0360] (iv) 1,15-bis(hexylthio)-8-oxopentadecane-2,14-diyl bis(decanoate) (69)

[0361]

[0362] Obtained from 68 and decanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ4.94(dtd,J=8.1,6.1,4.4Hz,2H),2.69-2.58(m,4H),2.53(td,J=7.3,1.3Hz,4H),2.37(t,J= 7.4Hz, 4H), 2.29 (t, J = 7.5Hz, 4H), 1.78-1.47 (m, 16H), 1.46-1.20 (m, 44H), 0.93-0.81 (m, 12H).

[0363] (v) 1,15-bis(hexylthio)-8-oxopentadecane-2,14-diyl bis(3-cyclohexylpropionate) (71)

[0364]

[0365] Obtained from 68 and 3-cyclohexylpropionic acid. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.88(m,2H),2.69-2.58(m,4H),2.53(td,J=7.2,1.4Hz,4H),2.37( t,J=7.4Hz,4H),2.33-2.28(m,4H),1.79-1.06(m,54H),0.95-0.83(m,10H).

[0366] (vi) 1,15-bis(cyclohexylthio)-8-oxopentadecane-2,14-diyl bis(decanoate) (79)

[0367]

[0368] Obtained from 78 and decanoic acid. 1 H NMR (400 MHz, CDCl 3)δ4.91(dtd,J=8.1,6.2,4.3Hz,2H),2.73-2.58(m,6H),2.36(t,J=7.4Hz,6H),2.28(t,J= 7.5Hz,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).

[0369] (vii) 1,15-bis(cyclohexylthio)-8-oxopentadecane-2,14-diyl dinonanoate (81)

[0370]

[0371] Obtained from 78 and nonanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ4.91(dtd,J=8.2,6.2,4.3Hz,2H),2.76-2.58(m,6H),2.37(t,J=7.4Hz,4H),2.29(t,J=7.5H z, 4H), 1.96 (dd, J = 8.9, 5.2Hz, 4H), 1.80-1.48 (m, 18H), 1.37-1.20 (m, 38H), 0.91-0.83 (m, 6H).

[0372] (viii) 7-Oxo-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (84)

[0373]

[0374] Obtained from 83 and decanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.87(m,2H),2.70-2.57(m,4H),2.53(td,J=7.3,1.5Hz,4H),2.38(t,J=7.4Hz ,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).

[0375] (ix) 1,13-Bis(heptylthio)-7-oxotridecan-2,12-diylbis(decanoate) (97)

[0376]

[0377] Obtained from 96 and decanoic acid. 1 H NMR (400 MHz, CDCl3 )δ5.08-4.85(m,2H),2.69-2.57(m,4H),2.53(td,J=7.3,1.4Hz,4H),2.38(t,J=7.4Hz,4H ), 2.30 (t, J = 7.5Hz, 4H), 1.79-1.47 (m, 18H), 1.42-1.20 (m, 38H), 0.88 (t, J = 6.8Hz, 12H).

[0378] (x) 1,13-bis(heptylthio)-7-oxotridecan-2,12-diyl dinonanoate (100)

[0379]

[0380] Obtained from 96 and nonanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ5.08-4.85(m,2H),2.69-2.57(m,4H),2.53(td,J=7.3,1.4Hz,4H),2.38(t,J=7.4Hz,4H ), 2.30 (t, J = 7.5Hz, 4H), 1.79-1.47 (m, 18H), 1.42-1.20 (m, 38H), 0.88 (t, J = 6.8Hz, 12H).

[0381] (xi) 8-Oxo-1,15-bis(pentylthio)pentadecan-2,14-diylbis(nonanoic acid) ester (104)

[0382]

[0383] Obtained from 103 and nonanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ4.94(dtd,J=8.1,6.1,4.3Hz,2H),2.70-2.58(m,4H),2.59-2.47(m,4H),2.37(t,J=7.4H z, 4H), 2.30 (t, J = 7.5Hz, 4H), 1.80-1.48 (m, 16H), 1.46-1.20 (m, 36H), 0.98-0.78 (m, 12H).

[0384] (xii) 8-Oxo-1,15-bis(pentylthio)pentadecan-2,14-diyl bis(decanoate) (107)

[0385]

[0386] Obtained from 103 and decanoic acid. 1H NMR (400 MHz, CDCl 3 )δ5.05-4.85(m,2H),2.71-2.58(m,4H),2.53(td,J=7.3,1.5Hz,4H),2.37(t,J=7.4Hz, 4H), 2.30 (t, J=7.5Hz, 4H), 1.78-1.49 (m, 16H), 1.39-1.21 (m, 40H), 0.96-0.83 (m, 12H).

[0387] (xiii) 1,15-bis(heptylthio)-8-oxopentadecane-2,14-diyl dinonanoate (117)

[0388]

[0389] Obtained from 116 and nonanoic acid. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.87(m,2H),2.70-2.57(m,4H),2.53(td,J=7.3,1.4Hz,4H),2.37(t,J=7.4Hz, 4H), 2.30 (t, J = 7.5Hz, 4H), 1.76-1.49 (m, 16H), 1.42-1.18 (m, 44H), 0.92-0.84 (m, 12H).

[0390] (C) Preparation of the Asymmetric Diester Derivatives of the Dihydroxy Compounds of Part (A)

[0391] (i) Exemplary operation for monoesterification of dihydroxy compounds: 1,15-bis(hexylthio)-14-hydroxy-8-oxopentadec-2-yldecanoate (75)

[0392]

[0393] Under an inert atmosphere, 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) were dissolved in CH 2 Cl 2 A solution of 10% ethanol (15 mL) was stirred for 16 h, then quenched with water and quenched with CH 2 Cl 2 (3 × 30 mL) extraction. The combined extracts were dried (Na 2 SO 4) and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0 to 50% EtOAc in hexanes) to afford pure 75 in 51% yield. 1 HNMR (400MHz, CDCl 3 )δ4.93(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=7.5Hz,2H),2.07(br,1H),1.73-1.44(m,14),1.41-1.19(m,32H),0.91-0.84(tr,9H).

[0394] (ii) N-hexanoyl-N-methylglycine methyl ester (73)

[0395]

[0396] To hexanoic acid (1.3 g, 11.1 mmol) CH 2 Cl 2 EDCI·HCl (3.19 g, 16.6 mmol) was added to a solution of 1% ethyl acetate (50 mL), 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 heated with CH 2 Cl 2 (3 × 30 mL) extraction. The combined extracts were dried (Na 2 SO 4 ) and concentrated under reduced pressure. The orange residue was purified by column chromatography (0 to 25% ethyl acetate in hexanes) to give pure 73 in 60% yield. 1 H NMR (400 MHz, CDCl 3 , rotational isomers) 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).

[0397] (iii) N-hexanoyl-N-methylglycine (74)

[0398]

[0399] LiOH·H 2A solution of 0 (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 completed within 4 hours, at which time it was cooled, acidified to pH about 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, CDCl 3 , rotamer)δ4.20-4.01(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).

[0400] (iv) Exemplary procedure for esterification of hydroxy-monoester: 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-oxopentadec-2-yldecanoate (76)

[0401]

[0402] The alcohol 75 (0.532 g, 0.825 mmol), the acid 74 (0.178 g, 0.948 mmol), EDCI·HCl (0.237 g, 1.24 mmol) and DMAP (0.0705 g, 0.577 mmol) were dissolved in CH 2 Cl 2 The solution (20 mL) was stirred at room temperature for 16 h, then quenched with water and quenched with CH 2 Cl 2 (3×30 mL) extraction. The combined organic matter was dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography using a gradient of EtOAc in hexanes to afford pure 76 in 75% yield. 1 H NMR (400 MHz, CDCl 3 Rotational isomers) 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).

[0403] (D) General procedure for silyl group release: ((4-hydroxybutyl)azanediyl)bis(1-(pentylthio)heptane-7,2-diyl)bis(decanoate) (5)

[0404]

[0405] To a cold (0°C) solution of TBDPS protected 46 (1.1 g, 1 mmol) in THF (5 mL) maintained under an inert atmosphere was added HF-pyridine (1 mL). The reaction was warmed to room temperature and stirred for 18 h. Water (10 mL) was added and the mixture was washed with CH 2 Cl 2 (3 × 15 mL) extraction. The combined extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give lipid 5 (630 mg, 76%) as an oil.

[0406] (E) Preparation of lipids with type 1 ionizable head groups: Synthesis of lipids 6-11

[0407] (i) General procedure for ketone reduction. To a cold (0°C) solution of ketone (0.5 mmol) in EtOH (3 mL) was added solid NaBH 4 (0.5 mmol). The mixture was warmed to room temperature and stirred for 30 minutes. The reaction was heated with saturated NH 4 The Cl solution was quenched, diluted with water (3 mL), and extracted with DCM (3×5 mL). The combined extracts were dried (Na 2 SO 4 ) 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 thus obtained:

[0408] (ii) 1,13-Bis(cyclohexylthio)-7-hydroxytridecane-2,12-diyl bis(decanoate) (65)

[0409]

[0410] Obtained from ketone 64. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.85(m,2H),3.65-3.49(m,1H),2.80-2.60(m,6H),2.30(t,J=7.5Hz,4H),2.11-1.90(m,4H),1.83-1.14(m,60H),0.98-0.77(m,6H).

[0411] (iii) 1,15-bis(hexylthio)-8-hydroxypentadecan-2,14-diyl bis(decanoate) (70)

[0412]

[0413] Obtained from ketone 69. 1 H NMR (400 MHz, CDCl 3 )δ5.03-4.88(m,2H),3.60-3.52(m,1H),2.70-2.61(m,4H),2.59-2.49(m,4 H), 2.30 (t, J = 7.5Hz, 4H), 1.85-1.19 (m, 65H), 0.88 (td, J = 6.9, 2.7Hz, 12H).

[0414] (iv) 1,15-bis(hexylthio)-8-hydroxypentadecan-2,14-diyl bis(3-cyclohexylpropionate) (72)

[0415]

[0416] Obtained from ketone 71. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.89(m,2H),3.66-3.51(m,1H),2.72-2.59(m,4H),2.53(td,J=7. 2,1.4Hz,4H),2.39-2.26(m,4H),1.86-1.02(m,58H),0.97-0.81(m,10H).

[0417] (v) Decanoic acid 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-hydroxyhexadecane-2-yl ester (77)

[0418]

[0419] Obtained from ketone 76. 1 H NMR (400 MHz, CDCl 3 ,rotamers)δ5.11-4.89(m,2H),4.29-3.95(m,2H),3.56(m,1H),3.08and 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(t r,J=7.5,7.5,2H),1.76-1.50(m,13H),1.45-1.21(m,44H),0.92-0.83(t,12H).

[0420] (vi) 1,15-Bis(cyclohexylthio)-8-hydroxypentadecan-2,14-diyl bis(decanoate) (80)

[0421]

[0422] Obtained from ketone 79. 1 H NMR (400 MHz, CDCl 3 )δ4.98-4.86(m,2H),3.63-3.53(m,1H),2.77-2.55(m,6H),2.30(t,J=7.5Hz,4H),2.04-1.90(m,4H),1.82-1.18(m,64H),0.94-0.81(m,6H).

[0423] (vii) 1,15-bis(cyclohexylthio)-8-hydroxypentadecan-2,14-diyl dinonanoate (82)

[0424]

[0425] Obtained from ketone 81. 1 H NMR (400 MHz, CDCl 3 )δ4.98-4.88(m,2H),3.63-3.51(m,1H),2.80-2.57(m,6H),2.29(t,J=7.5H z, 4H), 1.96 (dd, J = 8.8, 5.0Hz, 4H), 1.84-1.16 (m, 60H), 0.93-0.82 (m, 6H).

[0426] (viii) General procedure for alcohol esterification. Under an inert atmosphere, a solution of alcohol (0.5 mmol, 1 eq.), 4-(dimethylamino)butyric acid hydrochloride (0.65 mmol, 1.3 eq.), EDCI-HCl (0.7 mmol, 1.4 eq.) and DMAP (23.1 mg, 0.7 mmol, 1.4 eq.) in DCM (5 mL) was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give an oily lipid (75 to 85%). The following lipids were thus obtained:

[0427] (ix) 1,13-bis(cyclohexylthio)-7-((4-(dimethylamino)butanoyl)oxy)tridecane-2,12-diyl bis(decanoate) (6)

[0428]

[0429] Obtained from alcohol 65. 1H NMR (400 MHz, C 6 D 6 )δ5.29-5.17(m,2H),5.14-5.04(m,1H),2.81-2.58(m,6H),2.43-2.35(m,2H),2.29(td,J=7.4,1.9Hz, 4H), 2.17(t,J=6.8Hz,2H),2.07(s,6H),2.05-1.94(m,4H),1.90-1.02(m,62H),0.92(t,J=6.9Hz,6H).

[0430] (x) 8-((4-(dimethylamino)butanoyl)oxy)-1,15-bis(hexylthio)pentadecan-2,14-diyl bis(decanoate) (7)

[0431]

[0432] Obtained from alcohol 70. 1 H NMR (400 MHz, CDCl 3 )δ4.99-4.90(m,2H),4.84(p,J=6.4Hz,1H),2.70-2.59(m,4H),2.53(td,J=7.3,1 .4Hz,4H),2.42-2.22(m,14H),1.92-1.19(m,66H),0.88(td,J=6.9,2.6Hz,12H).

[0433] (xi) 8-((4-(dimethylamino)butanoyl)oxy)-1,15-bis(hexylthio)pentadecan-2,14-diyl bis(3-cyclohexylpropionate) (8)

[0434]

[0435] Obtained from alcohol 72. 1 H NMR (400 MHz, C 6 D 6 )δ5.33-5.18(m,2H),5.16-5.05(m,1H),2.77-2.43(m,8H),2.37(t,J=7.3Hz,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).

[0436] (xii) 8-((4-(dimethylamino)butanoyl)oxy)-14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis-(hexylthio)pentadecan-2-yldecanoate (9)

[0437]

[0438] Obtained from alcohol 77. 1 H NMR (400 MHz, CDCl 3 ,rotamers)δ5.09-4.89(m,2H),4.84(m,1H),4.23-4.02(m,2H),3.07and 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=7.1Hz,12H).

[0439] (xiii) 1,15-bis(cyclohexylthio)-8-((4-(dimethylamino)butanoyl)oxy)pentadecan-2,14-diyl bis(decanoate) (10)

[0440]

[0441] Obtained from alcohol 80. 1 H NMR (400 MHz, CDCl 3 )δ4.98-4.87(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=9.3Hz,42H),0.92-0.83(m,6H).

[0442] (xiv) 1,15-bis(cyclohexylthio)-8-((4-(dimethylamino)butanoyl)oxy)pentadecan-2,14-diyldinonanoate (11)

[0443]

[0444] Obtained from alcohol 82. 1 H NMR (400 MHz, CDCl 3)δ4.95-4.87(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).

[0445] (F) Preparation of lipids with ketal-type ionizable head groups: Synthesis of lipids 12 to 21

[0446] (i) General procedure for ketalization. A solution of ketone (1 mmol, 1 eq.), diol (2 mmol, 2 eq.) and pyridinium p-toluenesulfonate (PPTS, 0.2 mmol, 0.2 eq.) 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 product (12 h to 4 days, depending on the diol). The mixture was cooled to room temperature, washed with water (2×10 mL) and brine (10 mL), and dried (Na 2 O 4 3 O 4 4 0 ... 2 SO 4 ) and concentrated. The product was purified by silica gel column chromatography (0 to 2% MeOH in CH 2 Cl 2 The residue was purified by purifying the ketal (65 to 85%) as an oil. The following compounds were thus obtained:

[0447] (ii) (4-(2-hydroxyethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (85)

[0448]

[0449] Obtained from ketone 84 and 1,2,4-butanetriol. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.89(m,2H),4.28-4.18(m,1H),4.07(t,J=7.2Hz,1H),3.83-3.77(m,2H),3.51(td,J=8.1,2.8Hz,1H),2 .71-2.58(m,4H),2.53(td,J=7.3,1.4Hz,4H),2.30(t,J=7.5Hz,4H),2.02-1.16(m,58H),0.88(q,J=7.0Hz,12H).

[0450] (iii) (4-(bromomethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (87)

[0451]

[0452] Obtained from ketone 84 and 3-bromo-1,2-propanediol. 1 H NMR (400 MHz, CDCl 3 )δ5.02-4.86(m,2H),4.37-4.26(m,1H),4.13(dd,J=8.5,6.2Hz,1H),3.76(dd,J=8.6,6.1Hz,1H),3.44(dd,J=10.0,4.6Hz,1H),3 .29(dd,J=10.0,8.1Hz,1H),2.71-2.57(m,4H),2.57-2.51(m,4H),2.30(t,J=7.5Hz,4H),1.84-1.16(m,56H),1.03-0.80(m,12H).

[0453] (iv) (4-(2-hydroxyethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl)bis(decanoate) (88)

[0454]

[0455] Obtained from ketone 64 and 1,2,4-butanetriol. 1 H NMR (400 MHz, CDCl 3 )δ4.99-4.84(m,2H),4.32-4.15(m,1H),4.11-4.03(m,1H),3.79(t,J=5.6Hz,2H),3.50(td,J=8 .0,2.6Hz,1H),2.78-2.58(m,6H),2.29(t,J=7.5Hz,4H),2.03-1.11(m,66H),0.96-0.81(m,6H).

[0456] (v) (5,5-bis(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl)bis(decanoate) (90)

[0457]

[0458] Obtained from ketone 64 and pentaerythritol. 1 H NMR (400 MHz, CDCl 3)δ5.01-4.87(m,2H),3.75(s,4H),3.70(s,4H),2.79-2.59(m,6H),2.30(t, J=7.6Hz,4H),2.01-1.88(m,4H),1.85-1.15(m,60H),0.88(t,J=6.7Hz,6H).

[0459] (vi) (4-(bromomethyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl)bis(decanoate) (91)

[0460]

[0461] Obtained from ketone 69 and 3-bromo-1,2-propanediol. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.88(m,2H),4.37-4.28(m,1H),4.13(dd,J=8.5,6.2Hz,1H),3.77(dd,J=8.6,6.1Hz,1H),3.45(dd,J=10.0,4.6Hz,1H),3.29(dd,J=1 0.0,8.1Hz,1H),2.73-2.59(m,4H),2.53(td,J=7.3,1.4Hz,4H),2.30( t,J=7.5Hz,4H),1.75-1.49(m,18H),1.45-1.20(m,46H),0.88(m,12H).

[0462] (vii) (5-(Hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl)bis(decanoate) (92)

[0463]

[0464] Obtained from ketone 69 and 2-hydroxymethyl-1,3-propanediol. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.91(m,2H),3.98(dd,J=11.8,4.0Hz,2H),3.80-3.71(m,4H),2.70-2.60(m,4H),2.53(td ,J=7.3,1.4Hz,4H),2.30(t,J=7.5Hz,4H),1.61(m,19H),1.44-1.19(m,46H),0.99-0.83(m,12H).

[0465] (viii) General procedure for tosylation of hydroxyketal. Under nitrogen atmosphere, solid TsCl (1.3 mmol, 1.3 eq.) was added to hydroxyketal (1 mmol, 1 eq.), Et 3 N (1.5 mmol, 1.5 eq.) and DMAP (0.1 mmol, 0.1 eq.) in a cold (0 °C) CH 2 Cl 2 The reaction was warmed to room temperature and stirred until TLC and 1H NMR showed complete conversion to product. The reaction was quenched with water (10 mL) and quenched with CH 2 Cl 2 (3×10 mL) extraction. The combined extracts were dried (Na 2 SO 4 ) and concentrated to give the crude tosylate (quantitative), which was used in the next step without purification. The following compounds were thus obtained:

[0466] (ix) (4-(2-(tosyloxy)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (86)

[0467]

[0468] Obtained from hydroxyketal 85. 1 H NMR (400 MHz, CDCl 3 )δ7.79(d,J=8.3Hz,2H),7.35(d,J=8.1Hz,2H),5.01-4.88(m,2H),4.19-4.04(m,3H),4.03-3.98(m,1H),3.43(t,J=7.8Hz,1 H),2.71-2.58(m,4H),2.57-2.51(m,4H),2.46(s,3H),2.29(td,J=7.6,2.2Hz,4H),1.96-1.18(m,58H),0.94-0.83(m,12H).

[0469] (x)(4-(2-(Tosyloxy)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl)bis(decanoate)(89)

[0470]

[0471] Obtained from hydroxyketal 88. 1 H NMR (400 MHz, CDCl 3)δ7.79(d,J=8.3Hz,2H),7.35(d,J=7.8Hz,2H),4.98-4.86(m,2H),4.26-4.05(m,3H),4.04-3.97(m,1H),3.4 3(t,J=7.7Hz,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).

[0472] (xi) (5-((Tosyloxy)methyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl)bis-(decanoate) (93)

[0473]

[0474] 1 H NMR (400 MHz, CDCl 3 )δ7.80(d,J=8.2Hz,2H),7.36(d,J=8.0Hz,2H),5.03-4.87(m,2H),4.17(d,J=7.2Hz,2H),3.95(dd,J=11.9,3.6Hz,2H),3.64(dd,J=12.2, 4.0Hz,2H),2.69-2.59(m,4H),2.54(ddt,J=8.2,6.1,2.5Hz,4H),2.45(s,3H),2.33-2.27(m,4H),1.94-1.15(m,65H),0.93-0.83(m,12H).

[0475] (xii) Exemplary procedure for bromide / toluenesulfonate substitution using low boiling point amines: (4-(2-(dimethylamino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (12)

[0476]

[0477] A solution of tosylate 86 (49.0 mg, 0.0481 mmol), dimethylamine (2M 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 gel chromatography (0 to 5% MeOH in DCM) to give lipid 12 (31 mg, 74%) as an oil. 1 H NMR (400 MHz, C 6 D 6)δ5.31-5.20(m,2H),4.13-4.01(m,1H),3.93(dd,J=7.7,5.9Hz,1H),3.41(t,J=7.9Hz,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).

[0478] (xiii) Exemplary procedure using bromide / toluenesulfonate displacement with high boiling point amines: (4-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (13)

[0479]

[0480] In a sealed reaction vessel, bromoketal 87 (75 mg, 0.0854 mmol), 4-(methylamino)-1-butanol (11 mg, 0.111 mmol) and K 2 CO 3 A solution of a mixture of (15.3 mg, 0.111 mmol) in MeCN (1 mL) was stirred at 80 °C for 18 h. The mixture was cooled, diluted with water (2 mL) and washed with CH 2 Cl 2 (3 × 3 mL) extraction. The combined extracts were dried (Na 2 SO 4 ) and concentrated. The product was purified by silica gel chromatography (0 to 5% MeOH in CH 2 Cl 2 The residue was purified by HPLC-MS / MS (HPLC-MS / MS / MS / 500 μL) to obtain lipid 13 (49 mg, 64%) as an oil. 1 H NMR (400 MHz, C 6 D 6 )δ5.31-5.19(m,2H),4.27-4.18(m,1H),3.96(dd,J=8.0,6.2Hz,1H),3.66-3.52(m,2H),3.47(t,J=7.9Hz,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=6.9Hz,6H),0.85(t,J=7.0Hz,6H).

[0481] (xiv) (4-(2-(ethyl(4-hydroxybutyl)amino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)-hexane-6,2-diyl)bis(decanoate) (14)

[0482]

[0483] Prepared from bromoketal 87 and 4-(ethylamino)-1-butanol by procedure (xiii) above. 1 H NMR (400 MHz, C 6 D 6 )δ5.30-5.19(m,2H),4.06-3.94(m,1H),3.88(t,J=6.8Hz,1H),3.62(t,J=5.3Hz,2H),3.37(t ,J=7.8Hz,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).

[0484] (xv) (4-(2-(4-hydroxypiperidin-1-yl)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(pentylthio)hexane-6,2-diyl)bis(decanoate) (15)

[0485]

[0486] Prepared from bromoketal 87 and 4-piperidinol by procedure (xiii) above. 1 H NMR (400 MHz, C 6 D 6 )δ5.32-5.17(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=6.9Hz,6H),0.85(t,J=6.9Hz,6H).

[0487] (xvi) (4-(2-(dimethylamino)ethyl)-1,3-dioxolane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl)bis(decanoate) (16)

[0488]

[0489] Prepared from tosylate 89 and dimethylamine by procedure (xii) above. 1 H NMR (400 MHz, CDCl3 )δ7.79(d,J=8.3Hz,2H),7.35(d,J=7.8Hz,2H),4.98-4.86(m,2H),4.26-4.05(m,3H),4.04-3.97(m,1H),3.4 3(t,J=7.7Hz,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).

[0490] (xvii) (5-(((3-(dimethylamino)propionyl)oxy)methyl)-5-(hydroxymethyl)-1,3-dioxane-2,2-diyl)bis(1-(cyclohexylthio)hexane-6,2-diyl)bis(decanoate) (17)

[0491]

[0492] Prepared from dihydroxy ketal 90 and 3-(dimethylamino)propionic acid hydrochloride by the procedure (viii) of Part E above, except using 0.8 equivalent of acid and 1 equivalent of EDCI-HCl / DMAP. 1 H NMR (400 MHz, C 6 D 6 )δ5.37-5.15(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=6.4Hz,2H),2.11(t,J=6.4Hz,2H),2.06-1.94(m,4H),1.91(s,6H),1.83-1.04(m,60H),0.92(t,J=6.9Hz,6H).

[0493] (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)

[0494]

[0495] Prepared from dihydroxy ketal 90 and 4-(dimethylamino)butyric acid hydrochloride by the procedure (viii) of Part E above, except using 0.8 equivalent of acid and 1 equivalent of EDCI-HCl / DMAP. 1 H NMR (400 MHz, C 6 D 6)δ5.39-5.21(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= 7.3Hz, 4H), 2.16 (t, J = 7.1Hz, 2H), 2.10-1.95 (m, 12H), 1.84-1.05 (m, 62H), 0.92 (t, J = 6.9Hz, 6H).

[0496] (xix) (4-((dimethylamino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)heptane-7,2-diyl)bis(decanoate) (19)

[0497]

[0498] Prepared from bromoketal 91 by procedure (xii) above. 1 H NMR (400 MHz, CDCl 3 )δ5.05-4.87(m,2H),4.22(p,J=6.5Hz,1H),4.07(dd,J=7.9,6.2Hz,1H),3.50(t,J=7.8Hz,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).

[0499] (xx)(4-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxolane-2,2-diyl)bis(1-(hexylthio)-heptane-7,2-diyl)bis(decanoate)(20)

[0500]

[0501] Prepared from bromoketal 91 and 4-(methylamino)-1-butanol by procedure (xiii) above. 1 H NMR (400 MHz, CDCl 3 )δ5.04-4.89(m,2H),4.35-4.19(m,1H),4.10(dd,J=8.0,6.2Hz,1H),3.67-3.55(m,2H),3.49(t,J=7.9Hz ,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).

[0502] (xxi) (5-(((4-hydroxybutyl)(methyl)amino)methyl)-1,3-dioxane-2,2-diyl)bis(1-(hexylthio)-heptane-7,2-diyl)bis(decanoate) (21)

[0503]

[0504] Prepared from tosylate 93 and 4-(methylamino)-1-butanol by procedure (xiii) above. 1 H NMR (400 MHz, C 6 D 6 )δ5.36-5.16(m,2H),3.91(dd,J=11.7,4.0Hz,2H),3.65(dd,J=11.7,6.4Hz,2H),3.55(t,J=5.4Hz,2H),2.75-2.42(m,8H ),2.37-2.20(m,4H),2.16(d,J=7.3Hz,2H),2.09(t,J=6.0Hz,2H),1.90(s,3H),1.88-1.12(m,69H),0.99-0.83(m,12H).

[0505] (G) Synthesis of lipid precursors with type 7 ionizable head groups (lipids 22 to 35).

[0506] (i) General procedure for the reductive amination of ketones with primary amines to give secondary amines. To a solution of ketone (1 mmol) and primary amine (2 mmol, 2 equivalents) in 1,2-dichloroethane (10 mL) was added NaBH(OAc) 3 (1.8 mmol, 1.8 equiv) and HOAc (0.1 mL). The resulting mixture was stirred at room temperature for 18 h under nitrogen and then washed with saturated NaHCO 3 The mixture was quenched with aqueous solution (3 mL). Diluted with water (5.00 mL) and extracted with DCM (3 x 10 mL). The combined extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the secondary amine as an oil (70 to 75%). The following compounds were thus obtained:

[0507] (ii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (94)

[0508]

[0509] Obtained from ketone 84 and OTBDPS-protected 4-amino-1-butanol 40.1 H NMR (400 MHz, CDCl 3 )δ7.77-7.52(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=7.5Hz,4H),1.88-1.18(m,60H),1.04(s,9H),0.97-0.79(m,12H).

[0510] (iii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl bis(decanoate) (98)

[0511]

[0512] Obtained from ketone 97 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 )δ7.72-7.59(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=7.5Hz,4H),1.85-1.18(m,68H),1.04(s,9H),0.93-0.77(m,12H).

[0513] (iv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diylbis(nonanoic acid)phosphate (101)

[0514]

[0515] Obtained from ketone 100 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 )δ7.68-7.62(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=7.5Hz,4H),1.86-1.17(m,64H),1.04(s,9H),0.91-0.81(m,12H).

[0516] (v) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(pentylthio)pentadecan-2,14-diylbis(nonanoic acid)phosphate (105)

[0517]

[0518] Obtained from ketone 104 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 )δ7.75-7.60(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=7.5Hz,4H),1.79-1.18(m,60H),1.06(s,9H),0.95-0.80(m,12H).

[0519] (vi) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(pentylthio)pentadecan-2,14-diyl bis(decanoate) (108)

[0520]

[0521] Obtained from ketone 107 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 )δ7.72-7.61(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=7.5Hz,4H),1.93-1.18(m,64H),1.04(s,9H),0.97-0.76(m,12H).

[0522] (vii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(hexylthio)pentadecan-2,14-diyl bis(decanoate) (110)

[0523]

[0524] Obtained from ketone 69 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3)δ7.73-7.60(m,4H),7.45-7.33(m,6H),5.01-4.84(m,2H),3.67(t,J=5.6Hz,2H),2.74-2 .44(m,11H),2.29(t,J=7.5Hz,4H),1.84-1.18(m,68H),1.04(s,9H),0.95-0.76(m,12H).

[0525] (viii) 10-(6-((N-hexanoyl-N-methylglycyl)oxy)-7-(hexylthio)heptyl)-2,2-dimethyl-3,3-diphenyl-4-oxa-18-thia-9-aza-3-silacosan-16-yldecanoate (114)

[0526]

[0527] Obtained from ketone 76 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 ,rotamers)δ7.66(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),3.06and 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),2.3 3-2.22(m,3H),1.77-1.48(m,16H),1.41-1.21(m,44H),1.04(s,9H),0.91-0.85(tr,12H).

[0528] (ix) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(heptylthio)pentadecan-2,14-diylbis(nonanoic acid) acid ester (118)

[0529]

[0530] Obtained from ketone 117 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3)δ7.76-7.62(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=7.5Hz,4H),1.79-1.17(m,68H),1.04(s,9H),0.91-0.83(m,12H).

[0531] (x) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-1,15-bis(cyclohexylthio)pentadecan-2,14-diylbis(nonanoic acid)ester (120)

[0532]

[0533] Obtained from ketone 81 and OTBDPS-protected 4-amino-1-butanol 40. 1 H NMR (400 MHz, CDCl 3 )δ7.76-7.65(m,4H),7.59-7.36(m,6H),4.99-4.86(m,2H),3.69(t,J=6.0Hz,2H),2.97-2.47(m,9 H), 2.32 (t, J = 7.5Hz, 4H), 2.01-1.88 (m, 4H), 1.83-1.16 (m, 58H), 1.06 (s, 9H), 0.96-0.79 (m, 12H).

[0534] (xi) General procedure for reductive methylation of secondary amines. Under an inert atmosphere, a secondary amine (1 mmol), aqueous formaldehyde (37%, 6 mL) and NaBH(OAc) were mixed. 3 A solution of (5 mmol) in THF (10 mL) was stirred at room temperature for 3 days. The reaction was then washed with saturated NaHCO 3 The mixture was quenched with aqueous solution (10 mL), diluted with water (10 mL) and washed with CH 2 Cl 2 (3 × 15 mL) extraction. The combined extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the tert-methylamine as an oil (75 to 85%).

[0535] (xii) General procedure for reductive alkylation of secondary amines. To a solution of secondary amine (1 mmol) and aldehyde (5 mmol) in DCE (15 mL) was added NaBH(OAc) 3(5 mmol) and HOAc (0.2 mL). The resulting mixture was stirred at room temperature for 18 h under an inert atmosphere and then washed with saturated NaHCO 3 The mixture was quenched with aqueous solution (5 mL), diluted with water (15 mL) and extracted with DCM (3 x 15 mL). The combined extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the tertiary amine as an oil (70 to 80%).

[0536] (xiii) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (95)

[0537]

[0538] Obtained from secondary amine 94 by procedure (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.74-7.56(m,4H),7.49-7.31(m,6H),5.05-4.91(m,2H),3.66(t,J=6.2Hz,2H),2.66-2.60(m,4H),2.53( td,J=7.3,1.7Hz,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).

[0539] (xiv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diyl bis(decanoate) (99)

[0540]

[0541] Obtained from secondary amine 98 by procedure (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.73-7.62(m,4H),7.47-7.36(m,6H),5.03-4.93(m,2H),3.68(t,J=6.1Hz,2H),2.75-2.62(m,4H),2.55( td,J=7.3,1.6Hz,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).

[0542] (xv) 7-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,13-bis(heptylthio)tridecane-2,12-diylbis(nonanoic acid)phosphate (102)

[0543]

[0544] Obtained from secondary amine 101 by operation (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.80-7.59(m,4H),7.52-7.37(m,6H),5.06-4.90(m,2H),3.68(t,J=6.2Hz,2H),2.72-2.62(m,4H),2.55( td,J=7.3,1.6Hz,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).

[0545] (xvi) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diylbis(nonanoic acid)ester (106)

[0546]

[0547] Obtained from secondary amine 105 by operation (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.79-7.60(m,4H),7.52-7.32(m,6H),5.01-4.91(m,2H),3.66(t,J=6.1Hz,2H),2.75-2.60(m,4H),2.53( td,J=7.2,1.7Hz,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).

[0548] (xvii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(pentylthio)pentadecane-2,14-diyl bis(decanoate) (109)

[0549]

[0550] Obtained from secondary amine 108 by operation (xi). 1H NMR (400 MHz, CDCl 3 )δ7.77-7.60(m,4H),7.47-7.32(m,6H),5.03-4.90(m,2H),3.66(t,J=6.1Hz,2H),2.71-2.60(m,4H),2.53( td,J=7.3,1.7Hz,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).

[0551] (xviii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(hexylthio)pentane-2,14-diyl bis(decanoate) (111)

[0552]

[0553] Obtained from secondary amine 110 by operation (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.73-7.60(m,4H),7.45-7.33(m,6H),5.01-4.84(m,2H),3.67(t,J=5.6Hz,2H),2.74-2 .44(m,11H),2.29(t,J=7.5Hz,4H),1.84-1.18(m,68H),1.04(s,9H),0.95-0.76(m,12H).

[0554] (xix) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(propyl)amino)-1,15-bis(hexylthio)pentadecan-2,14-diyl bis(decanoate) (112)

[0555]

[0556] Obtained by operation (xii) from secondary amine 110 and acetaldehyde (acetaldehyde). 1 H NMR (400 MHz, CDCl 3)δ7.74-7.61(m,4H),7.48-7.32(m,6H),5.02-4.87(m,2H),3.65(t,J=6.3Hz,2H),2.67-2.60(m,4H),2.53(td,J=7.3 ,1.6Hz,4H),2.38-2.21(m,9H),1.80-1.10(m,70H),1.04(s,9H),0.88(td,J=6.9,2.6Hz,12H),0.83(t,J=7.4Hz,3H).

[0557] (xx) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(isobutyl)amino)-1,15-bis(hexylthio)pentane-2,14-diylbis(decanoate) (113)

[0558]

[0559] Obtained by procedure (xii) from secondary amine 110 and 2-methyl-propionaldehyde (isobutyraldehyde). 1 H NMR (400 MHz, CDCl 3 )δ7.71-7.61(m,4H),7.48-7.33(m,6H),5.01-4.90(m,2H),3.65(t,J=6.3Hz,2H),2.70-2.60(m,4H),2.53(td,J=7.2,1.6Hz, 4H), 2.28 (q, J = 7.2Hz, 7H), 2.06 (d, J = 7.1Hz, 2H), 1.86-1.09 (m, 69H), 1.04 (s, 9H), 0.91-0.85 (m, 12H), 0.82 (d, J = 6.5Hz, 6H).

[0560] (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-silacosan-16-yldecanoate (115)

[0561]

[0562] Obtained from secondary amine 114 by operation (xi). 1 H NMR (400 MHz, CDCl 3, rotamer) δ7.66 (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), 3.06and2.98(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).

[0563] (xxii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(heptylthio)pentane-2,14-diylbis(nonanoic acid)phosphate (119)

[0564]

[0565] Obtained from secondary amine 118 by operation (xi). 1 H NMR (400 MHz, CDCl 3 )δ7.72-7.58(m,4H),7.48-7.33(m,6H),5.03-4.90(m,2H),3.66(t,J=6.1Hz,2H),2.63(d,J=6.0Hz,4H),2.53(td, J=7.3,1.7Hz,4H),2.31(dt,J=15.0,7.6Hz,7H),2.12(s,3H),1.80-1.10(m,68H),1.04(s,9H),0.98-0.82(m,12H).

[0566] (xxiii) 8-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-1,15-bis(cyclohexylthio)-pentadecan-2,14-diylbis(nonanoic acid) acid ester (121)

[0567]

[0568] Obtained from secondary amine 120 by operation (xi). 1 H NMR (400 MHz, CDCl 3)δ7.73-7.61(m,4H),7.50-7.35(m,6H),5.06-4.86(m,2H),3.66(t,J=6.1Hz,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).

[0569] (H) General procedure for the release of silyl protecting groups: Synthesis of lipids 5 and 22 to 35. To a solution of the 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 warmed 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 (Na 2 O 4 3 O 4 5 N 2 4 4 4 N 2 O 4 2 ... 2 SO 4 ) and concentrated. The residue was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the corresponding lipids as oils (55 to 70%). The following lipids were thus obtained:

[0570] (i) ((4-Hydroxybutyl)azanediyl)bis(1-(pentylthio)heptane-7,2-diyl)bis(decanoate) (5)

[0571]

[0572] Obtained from compound 46. 1 H NMR (400 MHz, CDCl 3 )δ5.00-4.89(m,2H),3.62(t,J=4.9Hz,2H),2.78-2.57(m,10H),2.53(td,J=7.3,1.5Hz, 4H), 2.30 (t, J=7.6Hz, 4H), 1.83-1.49 (m, 20H), 1.43-1.17 (m, 40H), 0.96-0.82 (m, 12H).

[0573] (ii) 7-((4-Hydroxybutyl)(methyl)amino)-1,13-bis(pentylthio)tridecane-2,12-diyl bis(decanoate) (22)

[0574]

[0575] Obtained from compound 95. 1 H NMR (400 MHz, CDCl 3)δ5.06-4.88(m,2H),3.73-3.58(m,2H),2.76-2.61(m,7H),2.53(td,J=7. 3,1.4Hz,4H),2.39-2.26(m,7H),1.81-1.20(m,60H),0.94-0.81(m,12H).

[0576] (iii) 1,13-bis(heptylthio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (23)

[0577]

[0578] Obtained from compound 99. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.89(m,2H),3.61(t,J=4.9Hz,2H),2.69-2.57(m,7H),2.53(td,J= 7.2,1.4Hz,4H),2.36-2.25(m,7H),1.79-1.18(m,68H),0.92-0.85(m,12H).

[0579] (iv) 1,13-bis(heptylthio)-7-((4-hydroxybutyl)(methyl)amino)tridecane-2,12-diylbis(nonanoic acid) (24)

[0580]

[0581] Obtained from compound 102. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.88(m,2H),3.65-3.52(m,2H),2.75-2.60(m,4H),2.59-2.38(m,7H) ,2.30(t,J=7.5Hz,4H),2.16(s,3H),1.82-1.12(m,64H),0.96-0.83(m,12H).

[0582] (v) 8-((4-Hydroxybutyl)(methyl)amino)-1,15-bis(pentylthio)pentadecan-2,14-diylbis(nonanoic acid)phosphate (25)

[0583]

[0584] Obtained from compound 106. 1 H NMR (400 MHz, CDCl 3)δ5.03-4.88(m,2H),3.67-3.55(m,2H),2.69-2.62(m,4H),2.60-2.49(m,7H) ,2.30(t,J=7.5Hz,4H),2.24(s,3H),1.78-1.20(m,60H),0.93-0.84(m,12H).

[0585] (vi) 8-((4-Hydroxybutyl)(methyl)amino)-1,15-bis(pentylthio)pentadecan-2,14-diyl bis(decanoate) (26)

[0586]

[0587] Obtained from compound 109. 1 H NMR (400 MHz, CDCl 3 )δ5.03-4.85(m,2H),3.65-3.52(m,2H),2.72-2.59(m,4H),2.53(td,J=7.3,1.6Hz ,7H),2.30(t,J=7.5Hz,4H),2.18(s,3H),1.79-1.15(m,64H),0.95-0.84(m,12H).

[0588] (vii) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecan-2,14-diyl bis(decanoate) (27)

[0589]

[0590] Obtained from compound 111. 1 H NMR (400 MHz, CDCl 3 )δ5.01-4.88(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).

[0591] (viii) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(propyl)amino)pentadecan-2,14-diyl bis(decanoate) (28)

[0592]

[0593] Obtained from compound 112. 1 H NMR (400 MHz, CDCl 3)δ5.44-5.33(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=7.4Hz,4H),1.99-1.20(m,70H),1.07-0.94(m,15H).

[0594] (ix) 1,15-bis(hexylthio)-8-((4-hydroxybutyl)(isobutyl)amino)pentadecan-2,14-diyl bis(decanoate) (29)

[0595]

[0596] Obtained from compound 113. 1 H NMR (400 MHz, C 6 D 6 )δ5.33-5.21(m,2H),3.54-3.48(m,2H),2.67(qd,J=13.6,6.2Hz,4H),2.59-2.44(m,5H),2.42- 2.33(m,2H),2.33-2.24(m,4H),2.16(d,J=7.1Hz,2H),1.87-1.11(m,69H),1.02-0.81(m,18H).

[0597] (x) Decanoic acid 14-((N-hexanoyl-N-methylglycyl)oxy)-1,15-bis(hexylthio)-8-((4-hydroxybutyl)(methyl)-amino)pentadecan-2-yl ester (30)

[0598]

[0599] Obtained from compound 115. 1 H NMR (400 MHz, CDCl 3 ,rotamers)δ5.02(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=6Hz ,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).

[0600] (xi) 1,15-bis(heptylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecan-2,14-diyl dinonanoate (31)

[0601]

[0602] Obtained from compound 119. 1 H NMR (400 MHz, CDCl 3 )δ5.02-4.89(m,2H),3.68-3.54(m,2H),2.69-2.60(m,4H),2.58-2.46(m,7H) ,2.30(t,J=7.5Hz,4H),2.21(s,3H),1.78-1.20(m,68H),0.96-0.82(m,12H).

[0603] (xii) 1,15-bis(cyclohexylthio)-8-((4-hydroxybutyl)(methyl)amino)pentadecan-2,14-diylbis(nonanoic acid) (32)

[0604]

[0605] Obtained from compound 121 and characterized as the HCl salt. 1 H NMR (400 MHz, CDCl 3 )δ4.97-4.86(m,2H),3.73(t,J=5.3Hz,2H),3.16-3.11(m,1H),3.07(t,J=6.6Hz,2H),2.73 (s,3H),2.70-2.56(m,6H),2.30(t,J=7.6Hz,4H),2.07-1.13(m,68H),0.93-0.73(m,6H).

[0606] Example 2: mRNA-containing LNPs comprising ionizable lipids 1 or 2 exhibit superior mRNA delivery to the liver and spleen in vivo over the MC3 benchmark

[0607] The in vivo transfection efficiency of LNP formulations containing 50 / 10 / 38.5 / 1.5 mol% of ionizable lipids / DSPC / chol / PEG-DMG (nitrogen to phosphorus ratio (N / P) of 6) and mRNA encoding luciferase was tested in the liver and spleen after injection into CD-1 mice. The mRNA dose was 1 mg / kg. The luminescence intensity in the liver and spleen was measured 4 hours after injection. The ionizable lipids were 1 and 5 to 32 (Table 1).

[0608] Figure 2AThe results in the study showed that the luminescence intensity per mg liver was higher for lipids 7, 9, 8, 11, 10, 6, 25, 23, 30, 12, 24, 30, 12, 24, 16, 32, 26, 22, 31, 27, and 5 compared to the MC3 benchmark. The luminescence intensity results per mg 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 were found to be higher than the MC3 benchmark ( Figure 2B ).

Claims

1. A lipid having the structure of formula A: or a pharmaceutically acceptable salt thereof; wherein m is from 4 to 8; and n is from 4 to 8; R 1 、R 2 、R 3 and R 4 are straight-chain or branched-chain optionally substituted C 3 to C 20 alkyl groups, and optionally contain 0 to 2 carbon-carbon double bonds; A is C or N, and If A is C, then W 1 and Y may or may not be combined with each other, and If W 1 and Y are combined with each other, then 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 to c, where the wavy line represents the bond to X: a. Type 2 ionizable head group; b. Type 3 ionizable head group; c. Type 4 ionizable head group; If W 1 and Y do not combine with each other, then W 1 is H; W 2 is O, S, NH or NR 2a , where R 2a is C 1 to C 4 alkyl optionally substituted by an OH group; and of formula A is a group selected from the following structures d to h, where the wavy line represents the bond to W 2 : d. Type 1 ionizable head group; e. Type 5 ionizable head group; f. Type 6 ionizable head group; g. Type 7 ionizable head group; h. Type 8 ionizable head group; i. Type 9 ionizable head group; If A is N, then W 1 and Y do not exist; W 2 Together with X forms a structure (CR a R b ) p group, wherein R a and R b are independently H or C 1 -C 5 alkyl or cycloalkyl, and wherein p is from 2 to 6; and Z is OH or NR’R”, where R’ and R” are independently optionally substituted C 1 -C 5 alkyl or cycloalkyl, or where R’ and R” together with the N atom of NR’R” form an optionally substituted heterocycle which is fused to the N atom to which R’ and R” are each attached.

2. The lipid or pharmaceutically acceptable salt according to claim 1, wherein R 1 and R 4 at least one of which is independently a moiety of formula B wherein: R’ and R” are independently straight-chain or branched, optionally substituted C 3 to C 12 alkyl groups, and optionally contain 0 to 2 carbon-carbon double bonds; R”’ is H or a linear, branched or cyclic optionally substituted C 1 to C 6 alkyl; and G 1 and G 2 are independently (CR a R b ) p , where R a and R b are each independently selected from H or optionally substituted C 1 -C 5 -alkyl or cycloalkyl, where p is from 0 to 6.

3. The lipid or pharmaceutically acceptable salt according to claim 1 or 2, wherein A 3 is N, W 1 and Y are absent, and W 2 and X together form a structure (CR a R b ) r group, and Z is NR’R”, and the heterocyclic group incorporating the N atom bonded to R’ and R” is pyrrolidine, piperidine or morpholine.

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

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

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

7. The lipid or pharmaceutically acceptable salt according to claim 6, wherein the moiety of formula A is structure d.

8. The lipid or pharmaceutically acceptable salt according to claim 1, having the structure of any one of the following compounds 5 to 32: or 9. A lipid or a pharmaceutically acceptable salt thereof, comprising: a protonatable amino head group; two lipophilic chains, wherein the amino head group has a central nitrogen atom or carbon atom, and each of the two lipophilic chains is directly bonded to the central nitrogen atom or carbon atom; at least one of the lipophilic chains has the following formula: wherein R 1 and R 2 are independently straight-chain or branched-chain optionally substituted C 3 -C 20 alkyl groups and optionally have different degrees of unsaturation; n is from 4 to 8; each lipophilic chain has a total of 15 to 40 carbon atoms; and wherein the lipid has (i) a pK of 6 to 8 a ; and (ii) a logP of at least 11.

10. The lipid or pharmaceutically acceptable salt according to any one of claims 1 to 9, wherein when formulated in a lipid nanoparticle containing mRNA, the lipid causes the lipid nanoparticle to have a biodistribution in the liver and / or one or more extrahepatic tissues that is at least about 10% increased as measured by mRNA luminescence of a lipid nanoparticle containing DLin-MC3-DMA 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 according to claim 11, comprising a helper lipid and an optional hydrophilic polymer-lipid conjugate.

13. The lipid nanoparticle according to claim 12, wherein the helper lipid is selected from cholesterol, diacylglycerol, glycerophospholipid-cholesterol conjugate, and sphingolipid.

14. A lipid nanoparticle comprising: an ionizable lipid having two lipophilic chains directly bonded to a central nitrogen or carbon atom, wherein at least one of the lipophilic chains has the following formula: n is from 4 to 8; where * represents a carbon branch point; wherein R 5 and R 6 each independently is a straight-chain or branched-chain substituted C 3 -C 30 alkyl; wherein one of R 5 and R 6 is substituted by an ester group, and the other of R 5 and R 6 is substituted by a sulfur atom at the α, β or γ position relative to the carbon branching point; one or more helper lipids; optionally a hydrophilic polymer-lipid conjugate; and a nucleic acid.

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

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

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

18. Use of the lipid or pharmaceutically acceptable salt according to any one of claims 1 to 10 or the lipid nanoparticle according to any one of claims 11 to 14 in the preparation of a medicament for the treatment or prevention of a disease, disorder, or medical condition treatable and / or preventable by nucleic acid therapy.

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

20. Use according to claim 18 or 19, wherein the nucleic acid is mRNA.

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