Ionizable lipids

Abstract

The present invention relates to ionizable lipids. Formulations of ionizable lipids can be used for the delivery of one or more nucleic acid therapeutic substances (e.g., mRNA, siRNA, or miRNA). The composition may further contain other lipids, such as noncationic lipids, PEG-modified lipids, and optionally cholesterol.

Description

Cross-reference of related applications

[0001] This application claims the interests of U.S. Provisional Application No. 63 / 504,257, filed on 25 May 2023, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] This invention relates to ionizable lipid compounds and their use in the formation of lipid nanoparticles. [Background technology]

[0003] (Background of the invention) Nucleic acids are useful for the treatment of various diseases and disorders. For example, RNA interference (RNAi) has become an important target for research and clinical development. Messenger RNA (mRNA) therapy is an important option for treating various diseases, particularly those associated with the deficiency of one or more proteins. Small non-coding RNAs that control gene expression can be useful for the treatment of various diseases and disorders. Based on their biological roles and structures, small non-coding RNAs can be classified into three major categories: miRNAs, siRNAs, and piRNAs. In addition to therapeutic use, gene silencing by siRNAs is an important tool for accurately identifying causative genes involved in specific pathological conditions. RNA interference tools such as siRNAs can be used in the study of mammalian cell signaling pathways. When siRNA binds sequence-specifically to mRNA and causes site-specific cleavage, downregulation or inhibition of genes involved in cancer or other pathological symptoms occurs. However, when using siRNA and mRNA in therapy, there are several hurdles (e.g., degradation by ribonuclease enzymes, stability of siRNA molecules under physiological conditions, inflammatory responses, site-specific and controlled release of siRNAs, and efficient delivery means). To succeed in using mRNA and siRNA as therapeutic agents, it is necessary to overcome these hurdles [Nitin Bharat Charbe, et. al., Small interfering RNA for cancer treatment: overcoming hurdles in delivery, Acta Pharmaceutica Sinica B, 10 (11) 2020, 2075-2109].

[0004] (Summary of the Invention) The present disclosure provides an ionizable lipid compound selected from Table 1: [Table 1]

[0005] This application further provides a pharmaceutical composition comprising one or more ionizable lipids of Table 1. The formulation can be used to deliver one or more nucleic acid therapeutics, such as, for example, mRNA, siRNA or miRNA. The composition can optionally comprise another lipid (e.g., a non-cationic lipid, a PEGylated lipid, and optionally cholesterol). Detailed Description of the Invention

[0006] This disclosure provides an ionizable lipid compound selected from Table 1: [Table 2] [Table 3]

[0007] This application further provides a pharmaceutical composition comprising one or more ionizable lipids of Table 1 or Table 2. The formulation can be used for the delivery of one or more nucleic acid therapeutics, such as, for example, mRNA, siRNA or miRNA. The composition can optionally further comprise another lipid (e.g., a non-cationic lipid, a PEGylated lipid, optionally cholesterol).

[0008] In one aspect, provided is a method for encapsulating mRNA in lipid nanoparticles, the method comprising the step of mixing one or more lipids of Table 1 or Table 2 and optionally one or more additional lipids in a lipid solution with one or more mRNAs in an mRNA solution to form mRNA encapsulated within the LNP.

[0009] The following is a definition of terms used in this specification and the appended claims. As used herein, the first definition of a group or term applies to that group or term, individually or as part of another group, throughout this specification and the claims unless otherwise indicated.

[0010] ​In some embodiments, lipid nanoparticle formulations are provided comprising ionizable lipids and one or more mRNA components as shown in Table 1 or 2. In some embodiments, the mRNA is modified mRNA. For example, modification can improve resistance to nuclease digestion in vivo. As used herein, the terms “modified” and “modified” in relation to nucleic acids provided herein preferably include at least one modification that enhances stability and makes the mRNA more stable (e.g., resistant to nuclease digestion) than wild-type or naturally occurring forms of mRNA. As used herein, the terms “stable” and “stability” in relation to nucleic acids of the present invention, and in particular with respect to mRNA, mean increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that can normally degrade such mRNA. Furthermore, the terms "modified" and "modified" in relation to mRNA in this invention are intended to refer to modifications that improve or enhance the translation of mRNA nucleic acids, and include, for example, sequences that function in the initiation of protein translation (e.g., Kozak consensus sequence) (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

[0011] In one embodiment, a lipid nanoparticle composition is provided that is manufactured to optimize the delivery of mRNA to target cells. For example, if the target cells are hepatocytes, the properties of the lipid nanoparticles can be optimized to effectively transport such delivery vehicles to the target cells to reduce immune clearance and / or promote retention in those target cells. In one embodiment, the composition of the present invention may be used in combination with an agent that promotes the delivery of exogenous mRNA (e.g., an agent that promotes the delivery of exogenous mRNA to target cells by disrupting or improving blood-brain barrier permeability).

[0012] The process of incorporating a target substance (e.g., nucleic acid) into lipid nanoparticles is often called "loading" (Lasic, et al., FEBS Lett., 312:255-258, 1992). Nucleic acids incorporated into LNPs can exist, all or part, within the internal space of the LNP, within the bilayer membrane of the LNP, or bound to the outer surface of the LNP membrane. The purpose of incorporating mRNA into lipid nanoparticles is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade nucleic acids, and / or systems or receptors that rapidly eliminate them. Therefore, in preferred embodiments of the present invention, a selected delivery vehicle can enhance the stability of the mRNA contained therein. Liposomes can enable the transport of encapsulated mRNA to target cells and / or selective delivery of encapsulated mRNA to target cells, or they can restrict the delivery of mRNA to other sites or cells where the presence of administered mRNA may be unhelpful or undesirable. Furthermore, by incorporating mRNA into a delivery vehicle (e.g., a cationic liposome), it becomes easier to deliver the mRNA to target cells.

[0013] Ideally, liposome delivery vehicles are manufactured to encapsulate one or more desired mRNAs so that the composition exhibits high transfection efficiency and enhanced stability. While liposomes can facilitate the delivery of nucleic acids to target cells, the addition of polycations (e.g., poly-L-lysine and protamine) as copolymers can enhance the transfection efficiency of several cationic liposomes in many cell lines, both in vitro and in vivo, sometimes by a significant 2- to 28-fold increase (NJ Caplen, et al., Gene Ther. 1995;2:603;S. Li, et al., Gene Ther. 1997;4, 891).

[0014] Lipid nanoparticles In preferred embodiments of the present invention, the delivery vehicle is formulated as lipid nanoparticles. As used herein, the term “lipid nanoparticles” refers to a delivery vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more mRNAs to one or more target cells. Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). It is also conceivable that polymers be used alone as a delivery vehicle or in combination with other delivery vehicles. Suitable polymers include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, dendrimers, and polyethyleneimines. In one embodiment, the delivery vehicle is selected based on its ability to facilitate the transfection of mRNA into target cells.

[0015] When used herein, the following words, phrases, and symbols are generally intended to have the meanings set forth below, unless the context in which they are used indicates a different meaning.

[0016] The compounds of the present invention may have one or more chiral centers. Unless otherwise stated, all chiral (enantiomers and diastereomers) and racemic forms of the compounds of the present invention are included in the compounds of the present invention. Many geometric isomers, such as olefins and C=N double bonds, can also exist in the compounds of the present invention, and all such stable isomers are within the scope of the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can also be isolated as isomer mixtures or as distinct isomeric forms. The compounds of the present invention can be isolated as optically active or racemic forms. Methods for producing optically active forms (e.g., resolution of racemic forms or synthesis from optically active starting materials) are well known in the art. Unless a specific stereochemistry or isomeric form is specifically indicated, all chiral forms (enantiomers and diastereomers) and racemic forms, as well as all structural geometric isomers, are within the scope of this specification.

[0017] The compounds in Table 1 or 2 may exist in free form (unionized) or as salts, and these are within the scope of the present invention. Unless otherwise stated, references to the compounds of the present invention are understood to include references to the free form and its salts. The term “salt” refers to acidic and / or basic salts formed using inorganic and / or organic acids and bases. Furthermore, the term “salt” may include zwitterions (internal salts) if, for example, the compounds in Table 1 contain both a basic moiety (e.g., an amine, pyridine, or imidazole ring) and an acidic moiety (e.g., a carboxylic acid). Medicinally acceptable (in other words, non-toxic and physiologically acceptable) salts are preferred, for example, acceptable metal salts and amine salts in which the cation does not significantly contribute to the toxicity or biological activity of the salt. However, other salts are considered to be within the scope of the present invention, for example, because they may be useful in isolation or purification steps that may be used during manufacturing. Salts of the compounds described herein can be formed, for example, by reacting the compound with a certain amount (e.g., an equal amount) of acid or base in a medium in which the salt precipitates or in an aqueous medium, and then freeze-drying it.

[0018] Exemplary acid addition salts include acetates [e.g., salts formed with acetic acid or trihaloacetic acid (e.g., trifluoroacetic acid)], adipines, alginates, ascorbic acid, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, borates, butyrates, citrates, camphor sulfates, camphor sulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanates, glycerophosphates, hemisulfonates, heptanates, hexanoates, hydrochlorides (formed with hydrochloric acid), and hydrobromides (formed with hydrogen bromide). Examples include hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate (formed with maleic acid), methanesulfonate (formed with methanesulfonic acid), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (formed with sulfuric acid), sulfonate (listed herein), tartrate, thiocyanate, toluenesulfonate (e.g., tosylate), and undecanoate.

[0019] Examples of basic salts include ammonium salts, alkali metal salts (e.g., sodium salts, lithium salts, and potassium salts); alkaline earth metal salts (e.g., calcium salts, and magnesium salts); barium salts, zinc salts, and aluminum salts; and salts with organic bases [e.g., trialkylamines such as organic amines (e.g., triethylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-efenamine, N,N'-dibenzylethylenediamine, dehydroabiethylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine, etc.) or similar pharmaceutically acceptable amines] and salts with amino acids (e.g., arginine, lysine, etc.). The basic nitrogen-containing group may be quaternized with substances such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long-chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), and aralkyl halides (e.g., benzyl and phenethyl bromide). Preferred salts include monohydrochloride, hydrogen sulfate, methanesulfonate, phosphate, or nitrate.

[0020] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is commensurate with a reasonable benefit-to-risk ratio, within the bounds of ordinary medical judgment.

[0021] As used herein, “pharmaceutically acceptable salt” refers to a derivative of the compound of the present invention modified by the parent compound forming an acidic or basic salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic salts or organic acid salts of basic groups such as amines, and alkali salts or organic acid salts of acidic groups such as carboxylic acids. Pharmacochemically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as those formed from non-toxic inorganic or organic acids. For example, conventional non-toxic salts include salts obtained from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid); and salts produced from organic acids (e.g., acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, maleic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isethionic acid, etc.).

[0022] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic group. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both; aqueous solvents such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are usually preferred. A list of suitable salts is provided in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, PA (1990), the disclosure of which is incorporated herein by reference.

[0023] In this specification, “to treat” or “to treat” encompasses the treatment of disease conditions in mammals, particularly humans, and includes: (a) preventing the development of a disease condition in a mammal, in particular preventing the development of a disease condition when the mammal is prone to the disease condition but has not yet been diagnosed with it; (b) inhibiting a disease condition, i.e., preventing its development; and / or (c) alleviating a disease condition, i.e., causing regression of the disease condition.

[0024] All stereoisomers of the compounds of the present invention are intended to be mixtures, or in pure or substantially pure forms. Stereoiomers include compounds that become optical isomers due to the presence of one or more chiral atoms, as well as compounds that become optical isomers due to the restriction of the rotation of one or more bonds (atropisomers). The definition of a compound according to the present invention encompasses all possible stereoisomers and mixtures thereof. In particular, it encompasses racemates and isolated optical isomers having specific activity. Racemates can be separated by physical methods such as fractional crystallization, separation or crystallization of diastereomer derivatives, or resolution by chiral column chromatography. Individual optical isomers can be obtained from racemates using conventional methods, such as crystallization after salt formation with an optically active acid.

[0025] This invention is intended to include isotopes of all atoms present in the compound of this application. Isotopes include atoms with the same atomic number but different mass numbers. Common examples, but not limited to them, include, hydrogen isotopes such as deuterium and tritium. Carbon isotopes include, 13 C and 14 C is present. The isotope-labeled compounds of the present invention can generally be prepared by means of the prior art known to those skilled in the art, or by methods similar to those described herein, using a suitable isotope-labeling reagent instead of an unlabeled reagent used in other methods.

[0026] Manufacturing method Examples The methods and conditions used in these examples, as well as the actual compounds produced in these examples, are not intended to limit the scope but to illustrate methods by which compounds can be produced. If the starting materials and reagents used in these examples are not produced by the methods described herein, they may be commercially available, reported in the chemical literature, or produced using procedures described in the chemical literature.

[0027] Example 1: Synthesis of Lipid-1 [ka] Step 1: Synthesis of ethyl(9Z,12Z)-octadeca-9,12-dienoate (intermediate-2) Concentrated H2SO4 (0.1 V) was added to ethanol (250 mL, 5 V) at 0-5°C. A solution of intermediate-1 (50 g, 0.178 mol) pre-dissolved in ethanol (250 mL, 5 V) was added dropwise to the above mixture at 0-5°C. The resulting reaction mixture was heated under reflux at 80°C for 18 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was cooled to 0°C and the pH was adjusted to ~8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol, the product was extracted with DCM (2 x 500 mL), the organic layer was washed with water, dried over Na2SO4, and concentrated under reduced pressure at 40°C to obtain crude ethyl (9Z,12Z)-octadeca-9,12-dienoate (intermediate-2) as a light brown liquid. Yield: 53 g (crude product); 1 Characterization by H-NMR

[0028] Step 2: Synthesis of (6Z,9Z,27Z,30Z)-19-hydroxyhexatriaconta-6,9,27,30-tetraen-18-one (intermediate-3) TMSCl (69.2 mL, 0.545 mol) was added dropwise to a round-bottom flask (RB) containing metallic sodium (14.92 g, 0.649 mol) and toluene (2.5 V) at 25-30°C. The resulting reaction mixture was heated to 40°C. A solution of intermediate-2 (40 g, 0.129 mol) pre-dissolved in toluene (6 V) was added dropwise at 40°C. After the addition was complete, the reaction mixture was refluxed at 115°C for 5 hours. Monitoring the reaction by TLC showed that intermediate-2 had been completely consumed. The reaction mixture was cooled to 0-5°C and quenched by slowly adding methanol at 0-5°C. After quenching, the reaction mixture was stirred at 25-30°C for 30 minutes. The reaction mixture was filtered through a Celite bed and washed with MTBE (1000 mL). The combined filtrate was collected and stirred with saturated ammonium chloride aqueous solution (500 mL) for 18 hours. The organic layer was separated, dried over NaSO4, and concentrated under reduced pressure at 40°C to obtain the crude product. The crude product was purified by passing through a combiflush, and the compound was eluted with 3-4% ethyl acetate:hexane. The pure fraction was evaporated under reduced pressure at 40°C to obtain (6Z,9Z,27Z,30Z)-19-hydroxyhexatriaconta-6,9,27,30-tetraen-18-one (intermediate-3) as a pale yellow liquid. Yield: 10.8 g (15.7%); 1 Characterization by H-NMR

[0029] Step 3: Synthesis of (6Z,9Z,27Z,30Z)-Hexatriaconta-6,9,27,30-tetraene-18,19-diol (intermediate-4) To a chilled solution of intermediate-3 (3.9 g, 0.0073 mol) / DCM (29.6 mL, 7.6 V) and methanol (29.6 mL, 7.6 V), NaHB4 (0.418 g, 0.011 mol) was added at 0–5°C. The resulting reaction mixture was stirred at 0–5°C for 30 minutes, and then stirred at ambient temperature for 16 hours. The reaction was monitored by TLC, which showed the formation of new polar spots along with intermediate-3. The reaction mixture was quenched with purified water (30 mL) at 0–5°C, the product was extracted with DCM (3 x 50 mL), the organic layer was dried over Na2SO4, and concentrated under reduced pressure at 40°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 6% ethyl acetate:hexane. The pure fraction was evaporated under reduced pressure at 40°C to obtain (6Z,9Z,27Z,30Z)-hexatriaconta-6,9,27,30-tetraene-18,19-diol (intermediate-4) as a white semi-solid. Yield: 1.79 g (45.7%) 1 Characterization by H-NMR

[0030] Step 4: Synthesis of (3-{4,5-bis[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-1,3-dioxolan-2-yl}propyl)dimethylamine (lipid-1) To a stirred solution of intermediate-4 (750 mg, 0.0014 mol) and intermediate-6 (357 mg, 0.0017 mol) in toluene (50 mL), PTSA monohydrate (338 mg, 0.0017 mol) was added at 25–30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The reaction was monitored by TLC. The reaction mixture was evaporated to remove toluene, and the resulting residue was diluted with ethyl acetate (60 mL) and washed with aqueous sodium bicarbonate (20 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to obtain the crude product. The product was purified by combiflash, and the compound was eluted with 3.5–4.5% methanol:DCM. The pure fraction was evaporated at 40°C to obtain (3-{4,5-bis[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-1,3-dioxolan-2-yl}propyl)dimethylamine (lipid-1) as a pale yellow liquid. Yield: 408 mg (45.0%) 1 Characterization by H-NMR and LCMS LCMS: (EI, m / z) C 43 H 77 Calculated as NO2[M+H]: 640.9; HPLC purity: 91.03% 1 H NMR (400 MHz, CDCl3): 5.40 - 5.30 (8H, m), 5.20 - 4.95 (1H, m), 4.09 - 3.98 (1H, m), 3.52 - 3.62 (1H, m), 2.79 - 2.76 (6H, t, J = 6.4 Hz), 2.08 - 2.03 (10H, m), 1.91 (4H, m), 1.51 - 1.34 (4H, m), 1.30 - 1.26 (36H, m), 0.89 - 0.88 (6H, t, J = 3.6 Hz)

[0031] Example 2: Synthesis of Lipid-2

Chemical formula

[0032] Step 2: Synthesis of (9E,12E)-octadeca-9,12-diene-1-ol (intermediate-3) DiBAL-H (12.28 mL, 0.025, 2M in THF) was slowly added over 15 minutes at -72 to -75°C to a solution of intermediate-2 (4 g, 0.012 mol) in THF (40 mL, 10 V). The resulting reaction mixture was stirred at -72 to -75°C for 2 hours. The progress of the reaction was monitored by TLC. The reaction mixture was quenched at 0 to 5°C with saturated ammonium chloride aqueous solution (30 mL), diluted with ethyl acetate (100 mL), and stirred at 25 to 30°C for 30 minutes. The reaction mixture was passed through a Celite bed and filtered. The filtrate was washed with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain crude (9E,12E)-octadeca-9,12-dien-1-ol (intermediate-3) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 3.2 g (crude product); 1 Characterization by H-NMR

[0033] Step 3: Synthesis of (9E)-octadeca-9-enal (intermediate-4) Intermediate-3 (3.2 g, 0.011 mol) was dissolved in DCM (64 mL, 20 V) to an ice-cold solution, to which Des-Martin periodinane (5.56 g, 0.0114 mol) was added at 0–5°C. The reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-3. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate aqueous solution (30 mL, 10 V) at 0–5°C, and the product was extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combifling with 3–4% siRNA / hexane to obtain (9E)-octadeca-9-enal (intermediate-4) as a colorless liquid. Yield: 2.05 g (70.6%) 1 Characterization by H-NMR

[0034] Step 4: Synthesis of (9E,27E)-hexatriaconta-9,27-diene-18,19-diol (intermediate-5) To a stirred solution of zinc powder (1.33 g, 0.02 mol) / 1,4-dioxane (15 mL, 7.5 V) and DCM (15 mL, 7.5 V), titanium chloride (1.66 mL, 0.014 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. A solution of intermediate-4 (2.0 g, 0.07 mol) pre-dissolved in 1,4-dioxane (20 mL, 10 V) was added to the reaction mixture at 5–10°C, and the reaction mixture was heated to 25–30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate aqueous solution at 0–5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by a combifling (registered trademark) using 6-8% ethyl acetate / hexane to obtain (9E,27E)-hexatriaconta-9,27-diene-18,19-diol (intermediate-5) as a white semi-solid. Yield: 1.16 g (37%); 1 Characterization by H-NMR

[0035] Step 5: Synthesis of (2-{4,5-bis[(8E)-heptadeca-8-en-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-2) To a toluene (25 mL, 35 V) solution of intermediate-5 (1.15 g, 0.0021 mol) and intermediate-7 (470 mg, 0.0026 mol), PTSA monohydrate (514 mg, 0.0026 mol) was added at 25-30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with aqueous sodium bicarbonate (20 mL). The product was extracted with ethyl acetate (3 x 30 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the product was eluted with 25-45% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4,5-bis[(8E)-heptadeca-8-en-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-2) as a light brown liquid. Yield: 660 mg (49.6%) 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 79 Calculated as NO2[M+H]: 618.8 HPLC purity: 99.21% 1 H NMR (400MHz, CDCl3):5.43-5.34 (4H, m), 5.03-5.00 (1H, t, J= 4.8Hz), 4.02-3.50 (2H, m), 2.43-2.40 (2H, q, J1= 8.8Hz, J2= 8.8Hz), 2.24 (6H, s), 1.97-1.94 (2H, t, J= 4.8Hz), 1.85-1.82 (2H, q, J1= 8.8Hz, J2= 8.8Hz), 1.54-1.53 ​​(6H, m), 1.34-1.18 (42H, m), 0.90-0.88 (6H, m);

[0036] Example 3: Synthesis of Lipid-3 [ka] Step 1: Synthesis of ethyl(9Z)-octadeca-9-enoate (intermediate-2) Concentrated H2SO4 (0.5 mL, 0.1 V) was added dropwise to cold ethanol (15 mL, 5 V) at 0–5°C. A solution of intermediate-1 (20 g, 0.0708 mol) pre-mixed in ethanol (100 mL, 5 V) was added dropwise at 0–5°C. The resulting reaction mixture was refluxed at 80°C for 18 hours. The reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was cooled to 0–5°C, and the pH was adjusted to 8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol, extracted with DCM (2 x 150 mL), the organic layer was washed with water, dried on anhydrous sodium sulfate, and evaporated at 40°C to obtain crude ethyl(9Z)-octadeca-9-enoate (intermediate-2) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 21 g (crude product) 1 Characterization by H-NMR

[0037] Step 2: Synthesis of (9Z,27Z)-19-hydroxyhexatriaconta-9,27-dien-18-one (intermediate-3) TMSCl (30.9 g, 0.143 mol) was added dropwise to a mixed solution of metallic sodium (7.75 g, 0.17 mol) and toluene (2.5 V at 25-30°C). The reaction mixture was heated to 40°C. A solution of intermediate-2 (21 g, 0.067 mol) pre-dissolved in toluene (6 V) was added dropwise at 40°C, and the reaction mixture was refluxed at 115°C for 18 hours. The reaction was monitored by TLC, which showed that intermediate-2 was completely consumed. The reaction mixture was cooled to 0-5°C and quenched by slowly adding methanol at 0-5°C. After quenching, the reaction mixture was stirred at 25-30°C for 30 minutes. The reaction mixture was filtered through a Celite bed and washed with MTBE (500 mL). The filtrate was collected and stirred with saturated ammonium chloride aqueous solution (280 mL) for 18 hours. The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated at 40°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the compound was eluted with 1-1.5% ethyl acetate:hexane. The pure fraction was evaporated at 45°C to obtain (9Z,27Z)-19-hydroxyhexatriaconta-9,27-dien-18-one (intermediate-3) as a pale yellow liquid. Yield: 3.8 g (10.55%); 1 Characterization by H-NMR

[0038] Step 3: Synthesis of (9Z,27Z)-hexatriaconta-9,27-diene-18,19-diol (intermediate-4) To a cold solution of intermediate-3 (3.8 g, 0.0071 mol) in dichloromethane (22.8 mL, 6 V) and methanol (22.8 mL, 6 V), NaBH4 (0.404 g, 0.0106 mol) was added at 0–5°C. The reaction mixture was stirred at 0–5°C for 30 minutes, and then stirred at 25–30°C for 18 hours. The reaction was monitored by TLC, which showed the formation of new polar spots along with unreacted intermediate-3. The reaction mixture was quenched with purified water at 0–5°C, the product was extracted with DCM (3 x 50 mL), the organic layer was dried over anhydrous sodium sulfate, and evaporated at 40°C to obtain the crude product. The crude product was purified by combiflash, and the compound was eluted with 6–8% ethyl acetate:hexane. The pure fraction was evaporated to obtain (9Z,27Z)-hexatriaconta-9,27-diene-18,19-diol (intermediate-4) as a white semi-solid. Yield: 1.52 g (39.8%) 1 Characterization by H-NMR

[0039] Step 4: Synthesis of (2-{4,5-bis[(8Z)-heptadeca-8-en-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-3) To a toluene (50 mL, 50 V) solution of intermediate-4 (1.2 g, 0.0024 mol) and intermediate-5 (488 mg, 0.0028 mol) was added, PTSA monohydrate (534 mg, 0.0028 mol) was added at 25-30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with an aqueous sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 50 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the product was eluted with 30-40% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4,5-bis[(8Z)-heptadeca-8-en-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-3) as a colorless liquid. Yield: 650 mg (47.1%)1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 79 Calculated as NO2[M+H]: 620.8 HPLC purity: 99.8% 1 H NMR (400MHz, CDCl3):5.40~5.30 (4H, m), 5.18-4.93 (1H, m), 4.02-3.55 (2H, m), 2.53 (2H, m), 2.32 (6H, s), 2.02-2.00 (12H, m), 1.30-1.27 (46H, m), 0.90-0.86 (6H, t, J= 8.8Hz)

[0040] Example 4: Synthesis of Lipid-4 [ka] Step 1: Synthesis of (9Z,12Z)-octadeca-9,12-dienal (intermediate-2) To an ice-cold solution of Intermediate-1 (10 g, 0.037 mol) / DCM (200 mL, 20 V), Des-Martin periodinane (17.50 g, 0.041 mol) was added at 0-5°C. The resulting reaction mixture was maintained at 25-30°C and stirred for 1.5 hours. The reaction was monitored by TLC, and completion of the reaction was indicated by TLC after 1.5 hours. The reaction mixture was quenched at 0-5°C with 10% NaHCO3 solution (100 mL, 10 V), and the product was extracted with DCM (3 x 100 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 3-4% ethyl acetate:hexane. The pure fraction was evaporated to obtain ethyl(9Z,12Z)-octadeca-9,12-dienal (intermediate-2) as a colorless liquid. Yield: 6.8 g (68.8%) 1 Characterization by H-NMR

[0041] Step 2: Synthesis of octadecanal (intermediate-4) Des-Martin periodinane (28.3 g, 0.066 mol) was added at 0-5°C to an ice-cold solution of Intermediate-3 (15 g, 0.0554 mol) / DCM (300 mL, 20 V). The reaction mixture was brought to 25-30°C and stirred at the same temperature for 1.5 hours. The progress of the reaction was monitored by TLC, and after 1.5 hours, TLC indicated completion of the reaction. The reaction mixture was quenched at 0-5°C with 10% NaHCO3 solution (150 mL, 10 V), and the product was extracted with DCM (3 x 150 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure at 45°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 3-4% ethyl acetate:hexane. The pure fraction was evaporated to obtain octadecanal (intermediate-4) as a white solid. Yield: 11.6 g (77.9%); 1 Characterization by H-NMR

[0042] Step 3: Synthesis of (6Z,9Z)-hexatriaconta-6,9-diene-18,19-diol (intermediate-5) To a stirred solution of zinc powder (10.8 g, 0.166 mol) / 1,4-dioxane (120 mL, 15 V) and DCM (120 mL, 15 V), titanium chloride (132.5 mL, 0.121 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. Solutions of intermediate-2 (8 g, 0.030 mol) and intermediate-4 (8.1 g, 0.030 mol) pre-dissolved in 1,4-dioxane (120 mL, 15 V) were added to the reaction mixture at 5–10°C. The reaction mixture was then heated to 25–30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched at 0-5°C with a 10% potassium carbonate aqueous solution, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. Purification by CombiFlash® with a 5-6% ethyl acetate hexane solution yielded (6Z,9Z)-hexatriaconta-6,9-diene-18,19-diol (intermediate-5) as a white semi-solid. Yield: 600 mg (46.5%); 1 Characterization by H-NMR

[0043] Step 4: Synthesis of (2-{4-[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-4) To a solution of intermediate-4 (4.5 g, 0.0084 mol) and intermediate-5 (488 mg, 0.00105 mol) in toluene (140 mL, 31 V), PTSA monohydrate (2 g, 0.0105 mol) was added at 25-30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 100 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 40-50% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4-[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-4) as a brown liquid. Yield: 2 g (38.4%) 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 79 Calculation for NO2[M+H]: 618.8; HPLC purity: 74.09% 1 H NMR (400MHz, CDCl3):5.40-5.32 (4H, m), 5.04-5.01 (1H, t, J= 4.8Hz), 3.56-3.55 (2H, m), 2.79-2.76 (2H, t, J= 6.4Hz), 2.58-2.40 (2H, m), 2.31 (6H, s), 2.08-2.03 (4H, q, J1= 13.6Hz, J2= 13.6Hz), 1.89-1.87 (2H, t, J= 3.2Hz), 1.54-1.53 ​​(4H, m), 1.44-1.26 (44H, m), 0.91-0.86 (6H, m).

[0044] Example 5: Synthesis of Lipid-5 [ka] Step 1: Synthesis of ethyl hexa-5-inoate (intermediate-2) To a stirred solution of Intermediate-1 (20 g, 0.178 mol) / ethanol (50 mL, 2.5 V), concentrated H2SO4 (0.1 V) was slowly added at 0-5°C. The resulting reaction mixture was heated under reflux at 80°C for 18 hours. The progress of the reaction was monitored by TLC. After 18 hours, TLC indicated completion of the reaction. The reaction mixture was cooled to 0-5°C and adjusted to pH 8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol and extracted with ethyl acetate (2 x 200 mL). The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain the crude product, ethyl hexa-5-isoate (Intermediate-2), as a light brown liquid. The crude product was used directly for the next reaction. Yield: 20 g (crude product); 1 Characterization by H-NMR

[0045] Step 2: Synthesis of toluene-4-sulfonic acid octa-2-inyl ester (intermediate-4) To a stirred solution of intermediate-3 (20 g, 0.15 mol) in acetone (100 mL, 5 V), p-toluenesulfonyl chloride (42.2 g, 0.22 mol) was added at 0–5°C. To the above reaction mixture, an aqueous solution of KOH (13.3 g, 0.23 mol) and K2CO3 (10.9 g, 0.079 mol) premixed in water (100 mL, 5 V) was slowly added at 0–5°C. The resulting suspension was stirred at 25–30°C for 18 hours. The progress of the reaction was monitored by TLC. After 18 hours, TLC showed the formation of new nonpolar spots. The reaction was quenched with 10% NaCl aqueous solution and extracted with DCM (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated at 45°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the compound was eluted with 6-8% ethyl acetate:hexane. The pure fraction was evaporated to obtain toluene-4-sulfonic acid octa-2-inyl ester (intermediate-4) as a colorless liquid. Yield: 15.8 g (36%); 1 Characterization by H-NMR

[0046] Step 3: Synthesis of tetradeca-5,8-diinone ethyl ester (intermediate-5) To a stirred suspension of sodium iodide (11.7 g, 0.078 mol) and copper iodide (14.96 g, 0.078 mol) / DMF (75 mL, 15 V) (pre-purged with argon), K2CO3 (9.87 g, 0.122 mol) was added at 25-30°C. Intermediate-2 (5 g, 0.035 mol) was added, followed by intermediate-4 (12 g, 0.042 mol), slowly at 25-30°C. The resulting pale yellow suspension was stirred at 25-30°C for 18 hours. The reaction was monitored by TLC. After 18 hours, TLC showed complete consumption of intermediate-2. The reaction mixture was quenched with saturated NH4Cl solution and filtered through a Celite bed. The filtrate was collected and extracted with MTBE (3 x 100 mL). The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain the crude product. The crude product was purified by passing it through a combifling fountain, and the compound was eluted with 6-8% ethyl acetate:hexane. The pure fraction was evaporated at 45°C to obtain tetradeca-5,8-diinone ethyl ester (intermediate-5) as a brown liquid. Yield: 6.36 g (70.8%) 1 Characterization by H-NMR

[0047] Step 4: Synthesis of (5Z,8Z)-tetradeca-5,8-dienoic acid ethyl ester (intermediate-6) Nickel acetate tetrahydrate (12.48 g, 0.05 mol) was added to ethanol (189 mL, 100 V) continuously purged with H2 gas, and the mixture was cooled to 10°C. Sodium borohydride (1.9 g, 0.05 mol) was added at 10-12°C. To the resulting black reaction mixture, ethylenediamine (6 mL, 0.093 mol) and then intermediate-5 (6.3 g, 0.022 mol) were added at 10°C. The reaction mixture was stirred at 5-10°C for 3 hours while continuously purging with H2 gas. The progress of the reaction was monitored by TLC (the reaction should be monitored every 30 minutes). After 3 hours, TLC indicated completion of the reaction. The reaction was quenched with 1.5 M hydrochloric acid and filtered through Celite. The filtrate was collected and extracted with MTBE. The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 0-2% ethyl acetate:hexane. The pure fraction was evaporated to obtain (5Z,8Z)-tetradeca-5,8-dienoic acid ethyl ester (intermediate-6) as a brown liquid. Yield: 4.3 g (77%); 1 Characterization by H-NMR

[0048] Step 5: Synthesis of (5Z,8Z)-tetradeca-5,8-diene-1-ol (intermediate-7) To a stirred solution of intermediate-6 (4.3 g, 0.017 mol) / THF (43 mL, 10 V), LiAlH4 (54.26 mL, 0.0085 mol, 2 M in THF) was added over 10 minutes at -25 to -30°C. The reaction mixture was stirred at the same temperature for 1 hour. The reaction was monitored by TLC. After 1 hour, TLC indicated completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride aqueous solution at 0 to -5°C, treated with ethyl acetate, and stirred at 25 to 30°C for 30 minutes. The reaction mixture was filtered through a Celite bed. The filtrate was washed with water and extracted with ethyl acetate. The combined organic layers were evaporated to obtain crude (5Z,8Z)-tetradeca-5,8-dien-1-ol (intermediate-7) as a pale yellow viscous oil. The crude product was used directly in the next step. Yield: 3.18 g (crude product);1 Characterization by H-NMR

[0049] Step 6: Synthesis of (5Z,8Z)-tetradeca-5,8-dienal (intermediate-8) Des-Martin periodinane (6.88 g, 0.016 mol) was added at 0–5°C to an ice-cold solution of intermediate-7 (3.1 g, 0.014 mol) / DCM (62 mL, 20 V). The reaction mixture was raised to 25–30°C and stirred at the same temperature for 1.5 hours. The reaction was monitored by TLC. After 1.5 hours, TLC indicated completion of the reaction. The reaction mixture was quenched at 0–5°C with 10% NaHCO3 solution (80 mL, 16 V), and the product was extracted with dichloromethane (3 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated at 45°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 3–4% ethyl acetate:hexane. The pure fraction was evaporated at 45°C to obtain (5Z,8Z)-tetradeca-5,8-dienal (intermediate-8) as a white solid. Yield: 2.0 g (65.1%) 1 Characterization by H-NMR

[0050] Step 7: (6Z,9Z,19Z,22Z)-octacosa-6,9,19,22-tetraene-14,15-diol (intermediate-9) synthesis To a stirred solution of zinc powder (1.7 g, 0.026 mol) / 1,4-dioxane (15 mL, 7.5 V) and DCM (15 mL, 7.5 V), titanium chloride (2.1 mL, 0.019 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. A solution of intermediate-8 (2.0 g, 0.0096 mol) pre-dissolved in 1,4-dioxane (20 mL, 10 V) was added at 5–10°C, and the reaction mixture was brought to 25–30°C and stirred at the same temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate solution at 0–5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. Purification by combifling with 6-8% ethyl acetate / hexane yielded (6Z,9Z,19Z,22Z)-octacosa-6,9,19,22-tetraen-14,15-diol (intermediate-9) as a white semi-solid. Yield: 0.83 g (41.5%)

[0051] Step 8: Synthesis of {2-[4,5-bis-((4Z,7Z)-trideca-4,7-dienyl)-[1,3]dioxolan-2-yl]-ethyl}dimethylamine (lipid-5) To a toluene (25 mL, 41 V) solution of intermediate-9 (600 mg, 0.0014 mol) and intermediate-10 (342 mg, 0.0017 mol), PTSA monohydrate (342 mg, 0.0017 mol) was added at 25-30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 50 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the product was eluted with 40-50% ethyl acetate:hexane. The pure fraction was evaporated to obtain {2-[4,5-bis-((4Z,7Z)-trideca-4,7-dienyl)-[1,3]dioxolan-2-yl]-ethyl}dimethylamine (lipid-5) as a brown liquid. Yield: 438 mg (60.9%); 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 33 H 59 Calculation for NO2[M+H]: 502.5 HPLC purity: 76.16% 1 H NMR (400MHz, CDCl3):5.41-5.34 (8H, m), 5.02 (1H, m), 3.57-3.56 (2H, m), 2.79-2.76 (2H, t, J= 6.0Hz), 2.41-2.39 (2H, t, J= 2.4Hz), 2.24 (6H, s), 2.12-2.02 (8H, m), 1.84-1.82 (2H, m), 1.57-1.54 (4H, m), 1.43-1.32 (2H, m), 1.30-1.26 (16H, m), 0.89-0.88 (6H, t, J= 4.0Hz).

[0052] Example 6: Synthesis of Lipid-6 [ka] Step 1: Synthesis of octa-7-ic acid (intermediate-5) To a stirred suspension of lithium acetylide, ethylenediamine complex (23 g, 0.23 mol) / DMSO (50 mL, 5 V), a solution of intermediate-4 (15 g, 0.076 mol) dissolved in DMSO (75 mL, 5 V) was added at 0-5°C. The resulting reaction mixture was stirred at 25-30°C for 2.5 hours. The progress of the reaction was monitored by TLC. After 2.5 hours, TLC indicated completion of the reaction. The reaction was quenched by pouring it into ice-cold saline solution, acidified to approximately pH 4-5 with 1.5 M hydrochloric acid, and the product was extracted with DCM (3 x 150 mL). The combined organic layers were dried on sodium sulfate and evaporated under reduced pressure at 45°C to obtain crude octa-7-ic acid (intermediate-5) as a brown liquid. The crude product was used directly in the next reaction. Yield: 18.1 g (crude product); 1 Characterization by H-NMR

[0053] Step 2: Synthesis of ethyl octa-7-inoate (intermediate-6) To a stirred solution of intermediate-5 (18 g, 0.12 mol) / ethanol (90 mL, 5 V), concentrated H2SO4 (0.1 V) was slowly added at 0-5°C. The resulting reaction mixture was heated under reflux at 80°C for 18 hours. The progress of the reaction was monitored by TLC. After 18 hours, TLC indicated completion of the reaction. The reaction mixture was cooled to 0-5°C, and the pH was adjusted to 8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol and extracted with ethyl acetate (2 x 75 mL). The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain crude ethyl octa-7-inoate (intermediate-6) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 10.38 g (crude product); 1 Characterization by H-NMR

[0054] Step 3: Synthesis of octa-2-in-1-yl 4-methylbenzene-1-sulfonate (intermediate-8) To a stirred solution of intermediate-7 (15 g, 0.118 mol) in acetone (75 mL, 5 V), p-toluenesulfonyl chloride (31.7 g, 0.116 mol) was added at 0–5°C. To the above reaction mixture, a pre-mixed solution of KOH (10 g, 0.178 mol) and K2CO3 (8.2 g, 0.059 mol) in water (75 mL, 5 V) was slowly added at 0–5°C. The resulting suspension was stirred at 25–30°C for 18 hours. The progress of the reaction was monitored by TLC. After 18 hours, TLC showed the formation of new nonpolar spots. The reaction was quenched with 10% NaCl aqueous solution and extracted with DCM (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated at 45°C to obtain the crude product. The crude product was purified by passing it through a combiflush, and the compound was eluted with 6-8% ethyl acetate:hexane. The pure fraction was evaporated to obtain octa-2-in-1-yl 4-methylbenzene-1-sulfonate (intermediate-8) as a colorless liquid. Yield: 15 g (60.9%) 1 Characterization by H-NMR

[0055] Step 4: Synthesis of ethyl tetradeca-5,8-diinoate (intermediate-9) To a stirred suspension of sodium iodide (20.6 g, 0.13 mol) and copper iodide (25.19 g, 0.13 mol) / DMF (155 mL, 15 V) (pre-purged with argon), K2CO3 (16.9 g, 0.122 mol) was added at 25-30°C. Intermediate-6 (10.3 g, 0.061 mol), followed by intermediate-8 (20.6 g, 0.073 mol), was slowly added at 25-30°C. The resulting pale yellow suspension was stirred at 25-30°C for 18 hours. The reaction was monitored by TLC. After 18 hours, TLC showed complete consumption of intermediate-6. The reaction mixture was quenched with saturated NH4Cl solution and filtered through a Celite bed. The filtrate was collected and extracted with MTBE (3 x 100 mL). The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain the crude product. The crude product was purified through a combiflush, and the compound was eluted with 6-8% ethyl acetate:hexane. The pure fraction was evaporated at 45°C to obtain ethyl tetradeca-5,8-diinoate (intermediate-9) as a brown liquid. Yield: 6.1 g (impurities); 1 Characterization by H-NMR

[0056] Step 5: Synthesis of ethyl(7Z,10Z)-hexadeca-7,10-dienoate (intermediate-1) Nickel acetate tetrahydrate (5.4 g, 0.021 mol) was added to ethanol (300 mL, 100 V) continuously purged with H2 gas, and the mixture was cooled to 10°C. Sodium borohydride (1.64 g, 0.043 mol) was added at 10-12°C. To the resulting black reaction mixture, ethylenediamine (6 g, 0.093 mol), followed by intermediate-9 (6 g, 0.217 mol), was added at 10°C. The reaction mixture was stirred at 5-10°C for 3 hours while continuously purging with H2 gas. The progress of the reaction was monitored by TLC (the reaction needed to be monitored every 30 minutes). After 3 hours, TLC indicated completion of the reaction. The reaction was quenched with 1.5 M hydrochloric acid, and the mixture was filtered through Celite. The filtrate was collected and extracted with MTBE. The combined organic layers were dried over sodium sulfate and concentrated at 45°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 0-2% ethyl acetate:hexane. The pure fraction was evaporated to obtain the brown liquid ethyl(7Z,10Z)-hexadeca-7,10-dienoate (intermediate-1). Yield: 1.4 g (46%); 1 Characterization by H-NMR

[0057] Step 6: Synthesis of (7Z,10Z)-hexadeca-7,10-diene-1-ol (intermediate-2) To a stirred solution of Intermediate-1 (3 g, 0.0104 mol) / THF (30 mL, 10 V), LiAlH4 (5.2 mL, 0.0104 mol, 2 M in THF) was added dropwise over 10 minutes at -25 to -30°C. The reaction mixture was stirred at the same temperature for 1 hour. The reaction was monitored by TLC. After 1 hour, TLC indicated completion of the reaction. The reaction mixture was quenched with saturated ammonium chloride aqueous solution at 0 to -5°C, treated with ethyl acetate, and stirred at 25 to 30°C for 30 minutes. The reaction mixture was filtered through a Celite bed. The filtrate was washed with water and extracted with ethyl acetate. The combined organic layers were dried on sodium sulfate and concentrated at 45°C to obtain crude (7Z,10Z)-hexadeca-7,10-dien-1-ol (Intermediate-2) as a pale yellow viscous oil. The crude product was used directly in the next step. Yield: 2.4 g (crude product); 1 Characterization by H-NMR

[0058] Step 7: Synthesis of Octadecanal (Intermediate-4) To a 48 mL, 20 V ice-cold solution of intermediate-3 (2.4 g, 0.010 mol) in DCM, des-martin periodinane (4.69 g, 0.011 mol) was added at 0–5°C. The reaction mixture was brought to 25–30°C and stirred at the same temperature for 1.5 hours. The reaction was monitored by TLC. After 1.5 hours, TLC indicated completion of the reaction. The reaction mixture was quenched at 0–5°C with 40 mL of 10% NaHCO3 solution (16 V), and the product was extracted with dichloromethane (3 x 40 mL). The combined organic layers were dried over Na2SO4 and concentrated at 45°C to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 3–4% ethyl acetate:hexane. The pure fraction was evaporated at 45°C to obtain octadecanal (intermediate-4) as a white solid. Yield: 1.9 g (80.16%); 1 Characterization by H-NMR

[0059] Step 8: Synthesis of (6Z,9Z,23Z,26Z)-dotriaconta-6,9,23,26-tetraene-16,17-diol (intermediate-10) To a stirred solution of zinc powder (1.43 g, 0.022 mol) / 1,4-dioxane (14.3 mL, 15 V) and DCM (14.3 mL, 15 V), titanium chloride (1.78 mL, 0.016 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. A solution of intermediate-3 (1.9 g, 0.008 mol) pre-dissolved in 1,4-dioxane (38 mL, 20 V) was added at 5–10°C. The reaction mixture was then heated to 25–30°C and stirred at this temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate solution at 0–5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. Purification by combifling with 6-8% ethyl acetate / hexane yielded (6Z,9Z,23Z,26Z)-dotriaconta-6,9,23,26-tetraen-16,17-diol (intermediate-10) as a white semi-solid. Yield: 1.1 g (55%)

[0060] Step 9: Synthesis of (2-{4,5-bis[(6Z,9Z)-pentadeca-6,9-dien-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-6) To a solution of intermediate-10 (600 mg, 0.0012 mol) and intermediate-11 (275 mg, 0.0015 mol) in toluene (25 mL, 41 V), PTSA monohydrate (286.5 mg, 0.0015 mol) was added at 25-30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combifling, and the product was eluted with 40-50% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4,5-bis[(6Z,9Z)-pentadeca-6,9-dien-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-6) as a light brown liquid. Yield: 245 mg (35%); 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 37 H 67 Calculation for NO2[M+H]: 558.7; HPLC purity: 63.42 1 H NMR (400MHz, CDCl3):5.39-5.38 (8H, m), 5.01 (1H, m), 3.56-3.55 (2H, m), 2.79-2.76 (2H, t, J= 6.8Hz), 2.42-2.40 (2H, t, J= 2.4Hz), 2.24 (6H, s), 2.07-2.03 (8H, m), 1.84-1.82 (2H, t, J= 9.2Hz), 1.48-1.40 (6H, m), 1.30-1.27 (24H, m), 0.89-0.88 (6H, t, J= 4.0Hz)

[0061] Example 7: Synthesis of Lipid-7 [ka] Step 1: Synthesis of ethyl (9E)-octadeca-9-enoate (intermediate-2) Concentrated H2SO4 (0.5 mL, 0.1 V) was added dropwise to cold ethanol (50 mL, 10 V) at 0–5°C. A solution of intermediate-1 (5.0 g, 0.017 mol) pre-mixed in ethanol (25 mL, 5 V) was added dropwise at 0–5°C. The resulting reaction mixture was refluxed at 80°C for 18 hours. The reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was cooled to 0–5°C, and the pH was adjusted to 8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol, extracted with dichloromethane (2 x 50 mL), the organic layer was washed with water, dried on anhydrous sodium sulfate, and evaporated at 40°C to obtain crude ethyl(9E)-octadeca-9-enoate (intermediate-2) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 5.0 g (crude product)

[0062] Step 2: Synthesis of (9E)-octadeca-9-en-1-ol (intermediate-5) LiAlH4 (8 mL, 0.032 mol, 2M in THF) was slowly added over 5 minutes at -25 to -30°C to a solution of intermediate-2 (5 g, 0.016 mol) in THF (30 mL, 10 V). The resulting reaction mixture was stirred at -25 to -30°C for 1 hour. The progress of the reaction was monitored by TLC. The reaction mixture was quenched with saturated ammonium chloride aqueous solution (60 mL) at 0 to 5°C, diluted with ethyl acetate (200 mL), and stirred at 25 to 30°C for 30 minutes. The reaction mixture was filtered through a Celite bed. The filtrate was washed with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain crude (9E)-octadeca-9-en-1-ol (intermediate-5) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 3.92 g (crude product)

[0063] Step 3: Synthesis of (9E)-octadeca-9-enal (intermediate-6) Des-Martin periodinane (6.78 g, 0.015 mol) was added at 0–5°C to an ice-cold solution of intermediate-5 (3.9 g, 0.014 mol) / DCM (78 mL, 20 V). The reaction mixture was stirred at 25–30°C. The progress of the reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-3. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate solution (25 mL, 10 V) at 0–5°C, and the product was extracted with DCM (3 x 60 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by a combifling (registered trademark) using 3-4% ethylacetate / hexane to obtain (9E)-octadeca-9-enal (intermediate-6) as a colorless liquid. Yield: 3.0 g (77%)

[0064] Step 4: Synthesis of octadecanal (intermediate-4) To an ice-cold solution of intermediate-3 (5 g, 0.018 mol) in DCM (100 mL, 20 V), des-martin periodinane (8.64 g, 0.020 mol) was added at 0–5°C. The reaction mixture was stirred at 25–30°C. The progress of the reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-3. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate solution (30 mL, 10 V) at 0–5°C, and the product was extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combiflase (registered trademark) with 3–4% Â / hexane to obtain octadecanal (intermediate-6) as a colorless liquid. Yield: 4.2 g (82%)

[0065] Step 5: Synthesis of (9E)-hexatriaconta-9-ene-18,19-diol (intermediate-7) To a stirred solution of zinc powder (3.34 g, 0.051 mol) / 1,4-dioxane (37.5 mL, 15 V) and DCM (37.5 mL, 15 V), titanium chloride (4.15 mL, 0.037 mol) was added over 10 minutes. The reaction mixture was stirred at 5-10°C for 30 minutes. Intermediate-4 (2.5 g, 0.093 mol) and intermediate-6 (2.48 g, 0.093 mol) were pre-dissolved in 1,4-dioxane (50 mL, 20 V) and added to the reaction mixture at 5-10°C. The reaction mixture was brought to 25-30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched at 0-5°C with a 10% aqueous potassium carbonate solution, filtered through a Celite plug, and the filtrate was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by a combifling (registered trademark) using 6-8% ethyl acetate / hexane to obtain (9E)-hexatriaconta-9-ene-18,19-diol (intermediate-7) as a white semi-solid. Yield: 1.3 g (26%)

[0066] Step 6: Synthesis of (2-{4-[(8E)-heptadeca-8-en-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-7) To a toluene (50 mL, 38 V) solution of intermediate-7 (1.3 g, 0.0024 mol) and intermediate-8 (530 mg, 0.003 mol) was added, PTSA monohydrate (693 mg, 0.0036 mol) was added at 25–30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution (20 mL). The product was extracted with ethyl acetate (3 x 40 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combiflush, and the product was eluted with 25–45% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4-[(8E)-heptadeca-8-en-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-7) as a light brown liquid. Yield: 800 mg (55%); 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 81 Calculated as NO2[M+H]: 620.8 HPLC purity: 55.29% 1 H NMR (400MHz, CDCl3):5.39-5.38 (2H, t, J= 3.6Hz), 5.03-5.00 (1H, t, J= 4.8Hz), 3.56-3.55 (2H, m), 2.44-2.39 (2H, m), 2.24 (6H, s), 1.97-1.94 (5H, t, J= 4.8Hz), 1.85-1.75 (3H, m), 1.62-1.60 (6H, m), 1.34-1.18 (48H, m), 0.90-0.88 (6H, m)

[0067] Example 8: Synthesis of Lipid-8 [ka] Step 1: Synthesis of (9Z)-octadeca-9-enal (intermediate-2) Des-Martin periodinane (8.68 g, 0.020 mol) was added at 0–5°C to an ice-cold solution of intermediate-1 (5 g, 0.018 mol) / DCM (100 mL, 20 V). The reaction mixture was stirred at 25–30°C for 1.5 hours. The reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-1. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate solution (50 mL, 10 V) at 0–5°C, and the product was extracted with DCM (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. Purification by combiflush® with 3–4% siRNA / hexane yielded (9Z)-octadeca-9-enal (intermediate-2) as a colorless liquid. Yield: 3.5 g (70.5%)

[0068] Step 2: Synthesis of octadecanal (intermediate-4) Des-Martin periodinane (28.3 g, 0.066 mol) was added at 0–5°C to an ice-cold solution of intermediate-3 (15 g, 0.0554 mol) / DCM (300 mL, 20 V). The reaction mixture was stirred at 25–30°C for 1.5 hours. The reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-3. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate solution (150 mL, 10 V) at 0–5°C, and the product was extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combifling with 3–4% Â / hexane to obtain octadecanal (intermediate-4) as a white solid. Yield: 11.6 g (77.9%)

[0069] Step 3: Synthesis of (9Z)-hexatriaconta-9-ene-18,19-diol (intermediate-5) Zinc powder (4.68 g, 0.071 mol) was stirred in 1,4-dioxane (52 mL, 15 V) and DCM (52 mL, 15 V) at 5-10°C, to which titanium chloride (5.8 mL, 0.052 mol) was added over 10 minutes. The reaction mixture was stirred at 5-10°C for 30 minutes. Intermediate-2 (3.5 g, 0.013 mol) and intermediate-4 (3.47 g, 0.013 mol) were added at 5-10°C, along with solutions pre-dissolved in 1,4-dioxane (70 mL, 20 V). The reaction mixture was heated to 25-30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate solution at 0-5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combifling with 5-6% ethyl acetate / hexane to obtain (9Z)-hexatriaconta-9-ene-18,19-diol (intermediate-5) as a white semi-solid. Yield: 3 g (42.8%) 1 Characterization by H-NMR

[0070] Step 4: Synthesis of (2-{4-[(8Z)-heptadeca-8-en-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-8) To a stirred solution of intermediate-5 (1.4 g, 0.0026 mol) and intermediate-6 (574 mg, 0.0032 mol) in toluene (50 mL, 35 V), PTSA monohydrate (623 mg, 0.0032 mol) was added at 25-30°C. The reaction mixture was refluxed at 130°C for 24 hours in a Dean-Stark apparatus. The reaction was monitored by TLC, and after completion, the reaction mixture was evaporated at 40°C to remove toluene. The resulting residue was treated with sodium bicarbonate solution, and the product was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combifling with 25-45% siRNA / hexane to obtain lipid-8 (600 mg, 37.5%) as a brown liquid. Yield: 600 mg (37.5%) 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 81 Calculation for NO2[M+H]: 620.8 HPLC purity: 61.38 1 H NMR (400MHz, CDCl3):5.36-5.34 (2H, m), 5.01 (1H, m), 3.56-3.55 (2H, m), 2.45-2.41 (2H, q, J1= 8.8Hz, J2= 8.8Hz), 2.25 (6H, s), 2.02-1.99 (4H, t, J= 6.0Hz), 1.85-1.81 (2H, m), 1.56-1.40 (6H, m), 1.44-1.26 (50H, m), 0.91-0.86 (6H, m)

[0071] Example 9: Synthesis of Lipid-9 [ka] Step 1: Synthesis of ethyl(9E,12E)-octadeca-9,12-dienoate (intermediate-2) Concentrated H2SO4 (0.5 mL, 0.1 V) was added dropwise to cold ethanol (15 mL, 5 V) at 0–5°C. A solution of intermediate-1 (3.0 g, 0.01 mol) pre-mixed in ethanol (15 mL, 5 V) was added dropwise at 0–5°C. The resulting reaction mixture was refluxed at 80°C for 18 hours. The reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was cooled to 0–5°C, and the pH was adjusted to 8 using a 10% NaHCO3 solution. The reaction mixture was evaporated to remove excess ethanol, extracted with dichloromethane (2 x 50 mL), the organic layer was washed with water, dried on anhydrous sodium sulfate, and evaporated at 40°C to obtain crude ethyl (9E,12E)-octadeca-9,12-dienoate (intermediate-2) (3.0 g) as a light brown liquid. The crude product was used directly in the next reaction. Yield: 3.0 g (crude product)

[0072] Step 2: Synthesis of (9E,12E)-octadeca-9,12-diene-1-ol (intermediate-3) LiAlH4 (4.8 mL, 0.0026, 2M in THF) was slowly added to a solution of intermediate-2 (3 g, 0.0026 mol) / THF (30 mL, 10 V) over 5 minutes at -25 to -30°C. The resulting reaction mixture was stirred at -25 to -30°C. The progress of the reaction was monitored by TLC. The reaction mixture was quenched with saturated ammonium chloride aqueous solution (100 mL) at 0 to 5°C, diluted with ethyl acetate (100 mL), and stirred at 25 to 30°C for 30 minutes. The reaction mixture was filtered through a Celite layer. The filtrate was washed with water and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain crude (9E,12E)-octadeca-9,12-dien-1-ol (intermediate-3) as a pale yellow viscous oil. The crude product was used directly in the next reaction. Yield: 2.32 g (crude product)

[0073] Step 3: Synthesis of (9E,12E)-octadeca-9,12-dienal (intermediate-4) To an ice-cold solution of intermediate-3 (2.25 g, 0.0084 mol) / DCM (45 mL, 20 V), des-martin periodinane (4.3 g, 0.010 mol) was added at 0–5°C. The reaction mixture was stirred at 25–30°C for 1.5 hours. The progress of the reaction was monitored by TLC. After 1.5 hours, TLC showed complete consumption of intermediate-3. The reaction mixture was cooled to 0–5°C, quenched with saturated sodium bicarbonate solution (25 mL, 11 V), and the product was extracted with DCM (3 x 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by a combifling (registered trademark) using 3-4% ethylacetate / hexane to obtain (9E,12E)-octadeca-9,12-dienal (intermediate-4) as a colorless liquid. Yield: 1.5 g (67%)

[0074] Step 4: Synthesis of (6E,9E,27E,30E)-Hexatriaconta-6,9,27,30-tetraene-18,19-diol (intermediate-5) To a stirred solution of zinc powder (1.01 g, 0.015 mol) / 1,4-dioxane (11.25 mL, 15 V) and DCM (11.25 mL, 15 V), titanium chloride (1.24 mL, 0.052 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. A solution of intermediate-4 (0.75 g, 0.013 mol) in 1,4-dioxane (70 mL, 20 V) was added to the reaction mixture at 5–10°C. The reaction mixture was then heated to 25–30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate solution at 0–5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. Purification by combifling with 5-6% ethyl acetate / hexane yielded (6E,9E,27E,30E)-hexatriaconta-6,9,27,30-tetraen-18,19-diol (intermediate-5) as a white semi-solid. Yield: 690 mg (46%)

[0075] Step 5: Synthesis of (2-{4,5-bis[(8E,11E)-heptadeca-8,11-dien-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-9) To a solution of intermediate-5 (670 mg, 0.0012 mol) and intermediate-6 (276 mg, 0.0015 mol) in toluene (25 mL, 37 V), PTSA monohydrate (361 mg, 0.0018 mol) was added at 25–30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The reaction was monitored by TLC, which indicated completion of the reaction. The reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 50 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by combiflush, and the product was eluted with 25–45% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4,5-bis[(8E,11E)-heptadeca-8,11-dien-1-yl]-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-9) as a light brown liquid. Yield: 454 mg (58%); 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 75 Calculation for NO2[M+H]: 614.9; HPLC purity: 98.2% 1 H NMR (400MHz, CDCl3):5.42-5.37 (8H, m), 5.03-5.00 (1H, m), 3.56-3.55 (2H, m), 2.67-2.66 (4H, m), 2.46-2.42 (2H, m), 2.26 (6H, s), 2.02-1.96 (8H, m), 1.88-1.82 (2H, m), 1.54-1.45 (6H, m), 1.39-1.24 (30H, m), 0.91-0.87 (6H, t, J= 6.8Hz)

[0076] Example 10: Synthesis of Lipid-10 [ka] To an ice-cold solution of Intermediate-1 (10 g, 0.037 mol) / DCM (200 mL, 20 V), Des-Martin periodinane (17.50 g, 0.041 mol) was added at 0-5°C. The resulting reaction mixture was brought to 25-30°C and stirred at the same temperature for 1.5 hours. The reaction was monitored by TLC, and after 1.5 hours, TLC indicated completion of the reaction. The reaction mixture was quenched at 0-5°C with 10% NaHCO3 solution (100 mL, 10 V), and the product was extracted with DCM (3 x 100 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 3-4% ethyl acetate:hexane. The pure fraction was evaporated to obtain ethyl(9Z,12Z)-octadeca-9,12-dienal (intermediate-2) as a colorless liquid. Yield: 6.8 g (68.8%)

[0077] Step 2: Synthesis of octadecanal (intermediate-4) Des-Martin periodinane (28.3 g, 0.066 mol) was added at 0-5°C to an ice-cold solution of Intermediate-3 (15 g, 0.0554 mol) / DCM (300 mL, 20 V). The reaction mixture was brought to 25-30°C and stirred at the same temperature for 1.5 hours. The progress of the reaction was monitored by TLC, and completion of the reaction was indicated by TLC after 1.5 hours. The reaction mixture was quenched at 0-5°C with 10% NaHCO3 solution (150 mL, 10 V), and the product was extracted with DCM (3 x 150 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure at 45°C to obtain the crude product. The crude product was purified by combiflush, and the compound was eluted with 3-4% ethyl acetate:hexane. The pure fraction was evaporated to obtain octadecanal (intermediate-4) as a white solid. Yield: 11.6 g (77.9%)

[0078] Step 3: Synthesis of (6Z,9Z)-hexatriaconta-6,9-diene-18,19-diol (intermediate-5) To a stirred solution of zinc powder (10.8 g, 0.166 mol) / 1,4-dioxane (120 mL, 15 V) and DCM (120 mL, 15 V), titanium chloride (132.5 mL, 0.121 mol) was added over 10 minutes at 5–10°C. The reaction mixture was stirred at 5–10°C for 30 minutes. Solutions of intermediate-2 (8 g, 0.030 mol) and intermediate-4 (8.1 g, 0.030 mol) in 1,4-dioxane (120 mL, 15 V) were added to the reaction mixture at 5–10°C. The reaction mixture was then heated to 25–30°C and stirred at that temperature for 3 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was quenched with 10% potassium carbonate solution at 0-5°C, filtered through a Celite bed plug, and the filtrate was extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried on anhydrous sodium sulfate and evaporated at 40°C to obtain the crude product. The crude product was purified by a combifling process using 5-6% ethyl acetate / hexane to obtain (6Z,9Z)-hexatriaconta-6,9-diene-18,19-diol (intermediate-5) as a white semi-solid. Yield: 600 mg (46.5%)

[0079] Step 4: Synthesis of (2-{4-[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-10) To a solution of intermediate-4 (4.5 g, 0.0084 mol) and intermediate-5 (488 mg, 0.00105 mol) in toluene (140 mL, 31 V), PTSA monohydrate (2 g, 0.0105 mol) was added at 25–30°C. The resulting reaction mixture was refluxed at 130°C for 18 hours in a Dean-Stark apparatus. The progress of the reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was evaporated to remove toluene, and the resulting residue was treated with sodium bicarbonate solution. The product was extracted with ethyl acetate (3 x 100 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to obtain the crude product. The crude product was purified by combifling, and the compound was eluted with 40–50% ethyl acetate:hexane. The pure fraction was evaporated to obtain (2-{4-[(8Z,11Z)-heptadeca-8,11-dien-1-yl]-5-heptadecyl-1,3-dioxolan-2-yl}ethyl)dimethylamine (lipid-10) as a brown liquid. Yield: 2 g (38.4%) 1 Characterization by H-NMR and LC-MS LCMS:(EI, m / z) C 41 H 79 Calculated as NO2[M+H]: 618.8%; HPLC purity: 74.09% 1 H NMR (400MHz, CDCl3):5.40~5.32 (4H, m), 5.04-5.01 (1H, t, J= 4.8Hz), 3.56-3.55 (2H, m), 2.79-2.76 (2H, t, J= 6.4Hz), 2.58-2.40 (2H, m), 2.31 (6H, s), 2.08-2.03 (4H, q, J1= 13.6Hz, J2= 13.6Hz), 1.89-1.87 (2H, t, J= 3.2Hz), 1.54-1.53 ​​(4H, m), 1.44-1.26 (44H, m), 0.91-0.86 (6H, m).

[0080] Example 11: Formulation containing mRNA and sgRNA Lipid nanoparticles (LNPs) were prepared by rapid mixing. A mixed solution containing ionizable lipids, DSPC, cholesterol, and DMG-PEG in ethanol in a molar ratio of 45:10:44:1 was mixed with an aqueous sample containing Cas9 mRNA and synthetic sgRNA in 100 mM sodium acetate. The molar fractions of each formulation were maintained when changing the ionizable lipids. Immediately after formulation, the particles were neutralized by dilution in phosphate-buffered saline (PBS). LNP size was measured using a Wyatt Plate Reader III with default settings.

[0081] The particles were functionally tested using amplicon sequencing. For testing, 20 μL of each formulation was administered to 5,000 HEK293 FT cells or Jurkat cells. After 4 days of culture, the cells were lysed, and the target gene locus was amplified using PCR primers. These PCR products were prepared for sequencing and sequenced by MiSeq. Subsequently, the sequence data was processed / analyzed using CRISPR-DAV, and the indel percentage was calculated (Table 1). For general reference, see Xuning Wang, Charles Tilford, Isaac Neuhaus, Gabe Mintier, Qi Guo, John N Feder, Stefan Kirov, CRISPR-DAV: CRISPR NGS data analysis and visualization pipeline, Bioinformatics, Volume 33, Issue 23, December 01, 2017, Pages 3811-3812. [Table 4]

Claims

1. Table 1 below: Table 1 A compound selected from or a pharmaceutically acceptable salt thereof.

2. A lipid nanoparticle formulation comprising the compound described in claim 1, and optionally comprising PEG-modified lipids.

3. The lipid nanoparticle formulation according to claim 2, further comprising cholesterol.

4. Lipid nanoparticles comprising the compound according to claim 2, further comprising a PEG-modified lipid, a non-cationic lipid, and cholesterol.

5. The formulation according to claim 4, wherein the PEG-modified lipid is DMG-PEG.

6. Noncationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleyl phosphatidylcholine (POPC), palmitoyl oleyl phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine. 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof. A formulation according to claim 4, selected from the following.

7. The formulation according to claim 4, wherein the noncationic lipid is DOPE or DSPC.

8. A composition comprising the compound described in claim 1, a PEG-modified lipid, and a messenger RNA (mRNA) encoding a protein or peptide encapsulated within lipid nanoparticles containing a noncationic lipid.

9. The composition according to claim 8, wherein the lipid nanoparticles have a size of less than about 150 nm or less than about 100 nm.

10. The composition according to claim 8, wherein the mRNA is Cas9 mRNA.

11. A method for delivering mRNA to produce a protein or peptide in vivo, comprising administering a composition containing mRNA encoding a protein or peptide to a subject, wherein the mRNA is encapsulated within lipid nanoparticles, and upon administration of the composition, the protein or peptide encoded by the mRNA is expressed, and the lipid nanoparticles comprise PEG-modified lipids, the compound described in claim 1, noncationic lipids, and cholesterol.

12. The method according to claim 11, wherein the noncationic lipid is selected from DOPE and DSPC.

13. The method according to claim 11, wherein the mRNA is Cas9 mRNA.

14. (a) A solution containing one or more mRNAs and one or more sgRNAs; and (b) A lipid solution comprising one or more compounds according to claim 1, one or more noncationic lipids, and one or more PEG-modified lipids. A method for encapsulating mRNA in lipid nanoparticles, including a step of mixing [a certain substance].

15. The method according to claim 14, wherein the lipid solution further comprises cholesterol.

16. The following table: Table 2 A composition comprising a compound selected from, a PEG-modified lipid, and a noncationic lipid, encapsulated within lipid nanoparticles containing messenger RNA (mRNA) and sgRNA encoding a protein or peptide.

17. The composition according to claim 16, wherein the lipid nanoparticles have a size of less than about 150 nm or less than about 100 nm.

18. The composition according to claim 16, wherein the mRNA is Cas9 mRNA.

19. A method for delivering mRNA to produce a protein or peptide in vivo, comprising administering a composition containing mRNA encoding a protein or peptide to a target, wherein the mRNA is encapsulated within lipid nanoparticles, and upon administration of the composition, the protein or peptide encoded by the mRNA is expressed, and the lipid nanoparticles are PEG-modified lipids, sgRNA, and the following table: Table 3 A method comprising a compound selected from, a noncationic lipid, and cholesterol.

20. The method according to claim 19, wherein the noncationic lipid is selected from DOPE and DSPC.

21. The method according to claim 19, wherein the mRNA is Cas9 mRNA.

22. (a) mRNA solution containing one or more mRNAs and sgRNAs; and (b) The following table: Table 4 A lipid solution comprising one or more compounds selected from, one or more noncationic lipids, and one or more PEG-modified lipids. A method for encapsulating mRNA in lipid nanoparticles, including a step of mixing [a certain substance].

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