Ionizable cationic lipids for RNA delivery

Abstract

To provide ionizable cationic lipids for RNA delivery. [Solution] Provided is a compound of the following formula I, or a pharmaceutically acceptable salt thereof, in which R1 is a C10-31 branched alkyl; R2 is a C2-20 linear alkyl, alkenyl, or alkynyl; L1 and L2 are the same or different and each is a C1-20 linear alkylene or a C2-20 linear alkenylene; X1 is S or O; R3 is a C1-6 linear or branched alkylene; and R4 and R5 are the same or different and each is H or a C1-6 linear or branched alkyl. TIFF2023071947000139.tif2743

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 15 / 387,067, filed December 21, 2016, the entire contents of which are incorporated herein by reference.

[0002] The entire disclosures of U.S. Patent Application No. 14 / 707,876, filed May 8, 2015 (now U.S. Patent No. 9,365,610, issued June 14, 2016), U.S. Patent Application No. 14 / 707,796, filed May 8, 2015 (now U.S. Patent No. 9,567,296, issued February 14, 2017); U.S. Patent Application No. 14 / 546,105, filed November 18, 2014 (now U.S. Patent No. 9,593,077, issued March 14, 2017); and U.S. Provisional Patent Application No. 61 / 905,724, filed November 18, 2013, are incorporated herein by reference. [Background technology]

[0003] Several different kinds of nucleic acids are currently being developed as therapeutic agents for the treatment of several diseases.As these molecules are developed, there is a need to produce them in a form that is stable, has a long shelf life, and can be easily incorporated into anhydrous organic or anhydrous polar aprotic solvents to allow encapsulation of nucleic acid without side reactions that may occur in polar aqueous solutions or non-polar solvents.

[0004] The present disclosure relates to novel lipid compositions that facilitate intracellular delivery of biologically active therapeutic molecules. The disclosure also relates to pharmaceutical compositions that include such lipid compositions and are useful for delivering therapeutically effective amounts of biologically active molecules to cells in a patient.

[0005] The delivery of therapeutic compounds to subjects is important for their therapeutic effect, and it can be usually hindered by the limited ability of compounds to reach target cells and tissues.Improvements are important for such compounds to enter target cells of tissues by various delivery means.The present description relates to novel lipids in composition and preparation methods that promote targeted intracellular delivery of bioactive molecules.

[0006] Examples of biologically active molecules for which effective targeting to patient tissues is often not achieved include many proteins, including immunoglobulin proteins, polynucleotides such as genomic DNA, cDNA, or mRNA antisense polynucleotides; and many low molecular weight compounds, whether synthetic or naturally occurring, such as peptide hormones and antibiotics.

[0007] One of the fundamental challenges currently facing physicians is that several different types of nucleic acids are currently being developed as therapeutics for the treatment of several diseases. These nucleic acids include mRNA for gene expression, DNA in gene therapy, plasmids, small interfering nucleic acids (siNA), siRNA, and microRNA (miRNA) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, and aptamers. As these nucleic acids are developed, there is a need to produce lipid formulations that are easy to make and can be easily delivered to target tissues. Summary of the Invention [Means for solving the problem]

[0008] Formula I [ka] (In the formula, R 1 is a branched alkyl group consisting of 10 to 31 carbon atoms; R 2is a straight chain alkyl, alkenyl, or alkynyl of 2 to 20 carbons; L 1 and L 2 are the same or different, each being a linear alkylene of 1 to 20 carbons or a linear alkenylene of 2 to 20 carbons; X 1 is S or O, R 3 is a linear or branched alkylene of 1 to 6 carbons; R 4 and R 5 are the same or different and each is hydrogen or a straight or branched alkyl group of 1 to 6 carbon atoms. or a pharma- ceutically acceptable salt or solvate thereof is described.

[0009] In one embodiment, the following: [ka] [ka] [ka] The compound is selected from the group consisting of compounds of formula ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88 as follows:

[0010] In one embodiment, those described herein are 1 is a branched alkyl of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 carbons; R 2is a straight chain alkyl, alkenyl, or alkynyl of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons; L 1 and L 2 are the same or different, and are respectively a linear alkylene of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons, or a linear alkenylene of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons; X 1 is S or O; R 3 is a linear or branched alkylene of 1, 2, 3, 4, 5, 6, or 6 carbons; R 4 and R 5 are the same or different and are each hydrogen, or a straight or branched alkyl of 1, 2, 3, 4, 5, or 6 carbons.

[0011] In a preferred embodiment, R 1 is -CH((CH 2 ) n CH 3 ) 2 where n is 4, 5, 6, or 7 carbons; R 2 is linear alkenyl; L 1 is a linear alkylene of 1, 2, 3, or 5 carbons; L 2 is a linear alkylene of 1, 3, or 5 carbons; X 1 is S;R 3 is a linear alkylene of 2 or 3 carbons; R 4 and R 5 are the same or different and are 1 or 2 carbons, respectively.

[0012] In one embodiment, the cationic lipid described herein is present in a pharmaceutical composition.The pharmaceutical composition preferably comprises lipid nanoparticles containing nucleic acid, preferably RNA polynucleotide.The lipid nanoparticles preferably increase the average life span of RNA in circulation.In another embodiment, upon administration of the pharmaceutical composition, the lipid nanoparticles therein deliver the nucleic acid to cells in the body.Preferably, the nucleic acid has the activity of suppressing the expression of a target gene.Alternatively, the nucleic acid has the activity of increasing the production of the protein it codes for upon expression in cells of the body.

[0013] Also described herein is a method for introducing a nucleic acid into a mammalian cell by using any of the above compositions. The cell may be any of the following: liver, lung, kidney, brain, The composition may be present in blood, spleen or bone.The composition is preferably administered intravenously, subcutaneously, intraperitoneally or intrathecally.Preferably, the composition described herein is used in a method for treating cancer or inflammatory disease.The disease may be selected from the group consisting of immune disorder, cancer, kidney disease, fibrotic disease, genetic abnormality, inflammation and cardiovascular disorder. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows the synthesis pathway of ATX-43 (RL-43A) from hexanoate (SM1), 4-aminobutanoic acid (SM2), and 4-bromobutyric acid (SM3). Intermediates (Int) 1-8 and reactions are described in Example 2.

[0015] [Diagram 2] Figure 2 shows the synthetic pathway of ATX-57 (RL-43C) from octanoate (SM1), 4-aminobutanoic acid (SM2), and 4-bromobutyric acid (SM3). Int1-8 and reactions are described in Example 3.

[0016] [Diagram 3]Figure 3 shows the synthetic route of ATX-58 (RL-43B) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-7 and reactions are described in Example 4.

[0017] [Figure 4] Figure 4 shows the synthetic route of ATX-81 (RL-48B) from SM1, SM2, and SM3, which is the same as in Figure 2. Int1-8 and reactions are described in Example 5.

[0018] [Diagram 5] Figure 5 shows the synthetic route of ATX-82 (RL-47A) from SM1, SM2, and SM3, which are identical to those in Figure 2. Int1-7 and reactions are described in Example 6.

[0019] [Figure 6] Figure 6 shows the synthetic route of ATX-86 (RL-48A) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-8 and reactions are described in Example 7.

[0020] [Figure 7] Figure 7 shows the synthetic route of ATX-87 (RL-48C) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-8 and reactions are described in Example 8.

[0021] [Figure 8] Figure 8 shows the synthetic route of ATX-88 (RL-48D) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-8 and reactions are described in Example 9.

[0022] [Figure 9] Figure 9 shows the synthetic route of ATX-83 (RL-47B) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-8 and reactions are described in Example 10.

[0023] [Figure 10] Figure 10 shows the synthetic route of ATX-84 (RL-47C) from SM1, SM2, and SM3, which are the same as those in Figure 2. Int1-8 and reactions are described in Example 11.

[0024] [Figure 11] Figure 11 shows the synthetic route of ATX-61 (RL-42D) from SM1 and SM2, which are the same as those in Figure 1. Int1-5 and reactions are described in Example 12.

[0025] [Figure 12] Figure 12 shows the synthetic route of ATX-63 (RL-42A) from SM1 and SM2, which are the same as those in Figure 1. Int1-5 and reactions are described in Example 13.

[0026] [Figure 13] Figure 13 shows the synthetic route of ATX-64 (RL-42C) from SM1 and SM2, which are the same as those in Figure 1. Int1-5 and reactions are described in Example 14.

[0027] [Figure 14] FIG. 14 shows EPO mRNA levels (ng / ml) following injection into mice of 0.03 mg / kg and 0.1 mg / kg of mRNA in nanoparticles containing ATX-2, ATX-57, ATX-81, ATX-82, ATX-83, ATX-84, ATX-85, ATX-86, or ATX-87 cationic lipids.

[0028] [Figure 15] FIG. 15 shows the anti-Factor VII knockdown activity of liposomes bearing ATX-57 and ATX-58 versus the activity of ATX-2 and the control (PBS alone).

[0029] [Figure 16]FIG. 16 shows the anti-EPO knockdown activity of liposomes carrying ATX-57 and the activity of ATX-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition "At least one" means one or more (e.g., 1-3, 1-2, or 1).

[0031] "Composition" means a product comprising the specified ingredients in the specified amounts, and any product that results directly or indirectly from combining the specified ingredients in the specified amounts.

[0032] "In combination with" refers to the administration of a compound of Formula I together with another pharmaceutical agent in the method of treatment of the invention, and means that the compound of Formula I and the other pharmaceutical agent are administered sequentially or simultaneously in separate dosage forms, or simultaneously in the same dosage form.

[0033] "Mammal" means a human or other mammalian animal, or a human being.

[0034] "Patient" means both humans and other mammals, preferably humans.

[0035] "Alkyl" is a saturation sum In various embodiments, the alkyl group has 1 to 18 carbons, i.e., C 1 ~C 18 or C 1 ~C 12 group, C 1 ~C 6 Group, or C 1 ~C 4Independently, in various embodiments, the alkyl group has zero branches (i.e., straight chain), one branch, two branches, or more than two branches. An "alkenyl" is an unsaturated alkyl that may have one double bond, two double bonds, or more than two double bonds. An "alkynyl" is an unsaturated alkyl that may have one triple bond, two triple bonds, or more than two triple bonds. The alkyl chain may be optionally substituted with one substituent (i.e., the alkyl group is monosubstituted), or one to two substituents, or one to three substituents, or one to four substituents, and the like. The substituents may be selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano, and the like. When an alkyl group incorporates one or more heteroatoms, the alkyl group is referred to herein as a heteroalkyl group. When the substituents on an alkyl group are hydrocarbons, the resulting group is simply referred to as a substituted alkyl. In various aspects, the alkyl group, including substituents, has less than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 carbons.

[0036] "Lower alkyl" means a group having 1 to 6 carbons in the chain, which can be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0037] "Alkoxy" means an alkyl-O- group, where alkyl is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and heptoxy. The bond to the parent moiety is through the ether oxygen.

[0038] "Alkoxyalkyl" means an alkoxy-alkyl group, where alkoxy and alkyl are as previously described. Preferred alkoxyalkyls contain a lower alkyl group. The bond to the parent moiety is through the alkyl.

[0039] "Alkylaryl" means an alkyl-aryl group, where alkyl and aryl are as previously described. Preferred alkylaryls contain a lower alkyl group. The bond to the parent moiety is through the aryl.

[0040] "Aminoalkyl" means an NH2-alkyl group attached to the parent moiety through an alkyl group, where alkyl is defined above.

[0041] "Carboxyalkyl" means a HOOC-alkyl group attached to the parent moiety through an alkyl group, where alkyl is as defined above.

[0042] "Commercially available chemicals" and chemicals used in the examples described herein can be obtained from standard commercial sources, such as Acros Organics (Pittsburgh, PA), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0043] "Compounds described in the chemical literature" can be identified by reference books and databases of chemical compounds and chemical reactions, as known to those skilled in the art. Suitable reference books and treatises detailing the synthesis of reactants useful in the preparation of the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, "Synthetic Organic Chemistry," John Wiley and Sons, Inc., New York, 1989. ;SR Sandler et al., "Organic Functional Group Preparations", 2nd ed., Academic Press, New York, 1983;HO House, "Modern Synthetic Reactions", 2nd ed., WA Benjamin, Inc. Menlo Park, Calif., 1972;TL Glichrist, "Heterocyclic Chemistry", 2nd ed., John Wiley and Sons, New York , 1992; J. March, “Advanced Organic Chemistry: reactions, Mechanisms and Structure, 5th ed., Wiley Interscience, New York, 2001. Specific and similar reactants may also be identified through the index of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, available at most public and university libraries, as well as through online databases (for more information, contact the American Chemical Society, Washington, DC). Chemicals that are known in catalogs but not commercially available can be prepared by custom chemical synthesis companies, and many standard chemical supply companies (such as those listed above) offer custom synthesis services.

[0044] "Halo" means a fluoro, chloro, bromo, or iodo group, with fluoro, chloro, or bromo being preferred, and fluoro and chloro being more preferred.

[0045] "Halogen" means fluorine, chlorine, bromine, or iodine, with fluorine, chlorine, and bromine being preferred.

[0046] "Heteroalkyl" means a saturated or unsaturated, straight or branched, chain containing carbon and at least one heteroatom. The heteroalkyl group can have one heteroatom, or one to two heteroatoms, or one to three heteroatoms, or one to four heteroatoms, in various embodiments. In one aspect, the heteroalkyl chain contains 1 to 18 (i.e., 1 to 18) member atoms (carbon and heteroatoms), and in various embodiments, 1 to 12, or 1 to 6, or 1 to 4 member atoms. Independently, in various embodiments, the heteroalkyl group has zero branches (i.e., straight chain), one branch, two branches, or more than two branches. Independently, in one embodiment, the heteroalkyl group is saturated. In another embodiment, the heteroalkyl group is unsaturated. In various embodiments, the unsaturated heteroalkyl group can have one double bond, two double bonds, more than two double bonds, and / or one triple bond, two triple bonds, or more than two triple bonds. The heteroalkyl chain can be substituted or unsubstituted. In one embodiment, the heteroalkyl chain is unsubstituted. In another embodiment, the heteroalkyl chain is substituted. Substituted heteroalkyl chains can have one substituent (i.e., by monosubstitution), or can have, for example, 1-2 substituents, or 1-3 substituents, or 1-4 substituents. Exemplary heteroalkyl substituents include ester (-C(O)-OR) and carbonyl (-C(O)-).

[0047] "Hydroxyalkyl" means a HO-alkyl group, where alkyl is defined above. Preferred hydroxyalkyl groups contain a lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

[0048] "Hydrate" is a compound in which the solvent molecule is H 2 O.

[0049] "Lipid" means an organic compound containing esters of fatty acids and is characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes, (2) "complex lipids," which include phospholipids and glycolipids, and (3) "derived lipids," such as steroids.

[0050] The "lipid particles" are used to deliver therapeutic nucleic acids (e.g., The term "lipid formulation" refers to a lipid formulation that can be used to deliver nucleic acids (e.g., mRNA). In a preferred embodiment, the lipid particle is a nucleic acid-lipid particle, typically formed from a cationic lipid, a non-cationic lipid (e.g., phospholipid), a conjugate lipid (e.g., PEG-lipid) that prevents particle aggregation, and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) is encapsulated in the lipid portion of the particle, which can protect it from enzymatic degradation.

[0051] The lipid particles typically have an average diameter of 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, 70nm to 80nm, or 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, and are substantially non-toxic. Furthermore, nucleic acids, when present in lipid particles of the present invention, are resistant in aqueous solution to degradation by nucleases.

[0052] "Solvate" refers to a physical association of a compound of the present disclosure with one or more solvent molecules. This physical association includes varying degrees of ionic and covalent bonds, including hydrogen bonds. In certain cases, the solvate is capable of isolation, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase solvates and isolable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0053] "Encapsulated lipid" refers to a lipid particle that provides a therapeutic nucleic acid, such as an mRNA, with complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid (e.g., the mRNA) is completely encapsulated in the lipid particle.

[0054] "Lipid conjugate" refers to a conjugated lipid that inhibits lipid particle aggregation. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, such as, for example, PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugate), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugate), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG coupled to ceramide, cationic PEG lipid, polyoxazoline (POZ)-lipid conjugate, polyamide oligomer, and mixtures thereof. PEG or POZ can be directly conjugated to lipid, or can be linked to lipid via a linker moiety. Any linker moiety suitable for coupling PEG or POZ to lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, non-ester-containing linker moieties, such as amide or carbamate, are used.

[0055] "Amphipathic lipid" refers to a substance in which the hydrophobic portion of the lipid material is oriented toward the hydrophobic phase and the hydrophilic portion is oriented toward the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0056] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also within the group designated as amphipathic lipids.In addition, the above-mentioned amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0057] "Neutral lipid" refers to a lipid species that exists in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0058] "Non-cationic lipid" means an amphipathic lipid or a neutral lipid or anionic lipid, and is described in more detail below.

[0059] "Anionic lipid" refers to a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0060] "Hydrophobic lipid" refers to a compound having apolar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic group(s).Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0061] "Cationic lipid" and "amino lipid" are used interchangeably and refer to lipids and their salts having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). Cationic lipids are typically selected from lipids and their salts that are pH-titratable, with the pK of the cationic lipid being the most important cationic lipid. a It is protonated (i.e., positively charged) at a pH below pK a The cationic lipids of the present invention are also referred to as titratable cationic lipids. In some embodiments, the cationic lipids have: a protonatable tertiary amine (e.g., pH-titrateable) head group; C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 25 -C 36 -C 26 -C 37 -C 27 -C 38 -C 39 -C 40 -C 41 -C 42 -C 43 -C 54 -C 55 -C 56 -C 67 -C 70 -C 71 -C 72 -C 73 -C 74 -C 75 -C 76 -C 77 -C 78 -C 79 -C 82 -C 83 -C 84 -C 85 -C 96 -C 97 -C 108 -C 109 -C 110 -C 120 -C 131 -C 14 -C 15 -C 16 -C 17 -C 18 -C 25 -C 26 -C 19 -C 27 -C 28 -C 39 -C 40 -C 55 -C 61 -C 72 -C 14 -C 29 -C 30 -C 41 -C 55 -C 62 -C 73 -C 84 -C 95 -C 106 -C 117 -C 125 -C 138 -C 14 -C 25 -C 18and an ether, ester, or ketal linkage between the head group and the alkyl chain. Such cationic lipids include DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-M P-DMA) (also known as 1-Bl 1).

[0062] "Substituted" means substitution with a specified group other than hydrogen, or with one or more groups, moieties or radicals which may be the same or different, eg, each independently selected.

[0063] "Antisense nucleic acid" refers to a non-enzymatic nucleic acid molecule that binds to target RNA and modifies the activity of the target RNA through RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993, Nature, vol. 365, p. 566) interactions (for review, see Stein and Cheng, 1993, Science, vol. 261, p. 1004, and Woolf et al., U.S. Pat. No. 5,849,902). Typically, an antisense molecule is complementary to a target sequence along a single continuous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to a substrate such that the substrate molecule forms a loop, and / or an antisense molecule can bind to a substrate such that the antisense molecule forms a loop. Thus, an antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences, or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence, or both. Furthermore, antisense DNA can be used to target RNA by DNA-RNA interactions, thereby activating RNase H, which digests the double-stranded target RNA. Antisense oligonucleotides can contain one or more RNAse H activating regions that are capable of activating RNAse H cleavage of the target RNA. Antisense DNA can be chemically synthesized or expressed by using a single-stranded DNA expression vector or its equivalent. An "antisense RNA" is an RNA strand that has a sequence complementary to the mRNA of a target gene, which can induce RNAi by binding to the mRNA of the target gene. An "antisense RNA" is an RNA strand that has a sequence complementary to the mRNA of a target gene and is believed to induce RNAi by binding to the mRNA of the target gene. A "sense RNA" has a sequence complementary to the antisense RNA, and is annealed to its complementary antisense RNA to form an iNA. These antisense and sense RNAs have traditionally been synthesized on an RNA synthesizer.

[0064] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses synthetic, naturally occurring, and non-naturally occurring nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages, which have similar binding properties as the reference nucleic acid and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0065] "RNA" refers to a molecule that contains at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. The term includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include, for example, the addition of non-nucleotide material to the end(s) of an interfering RNA, or internally, for example, at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the invention can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of naturally occurring RNA. As used herein In the present specification, the terms "ribonucleic acid" and "RNA" refer to molecules containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and polyvalent RNA. Ribonucleotides are nucleotides that have a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. These terms include isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA and altered RNA that differ from naturally occurring RNA by the addition, deletion, substitution, modification, alteration of one or more nucleotides. Modification of RNA includes the addition of non-nucleotide material, for example, to the end(s) of an interfering RNA or internally, for example, at one or more nucleotides of an RNA nucleotide in an RNA molecule, and may include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs.

[0066] "Nucleotide" refers to natural (standard) and modified bases as known in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified in the sugar, phosphate, and / or base moieties (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc., see, e.g., Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92 / 07065; Usman et al., International PCT Publication No. WO 93 / 15187; Uhlman and Peyman, supra, all of which are incorporated herein by reference). According to Limbach et al., Nucleic Acids Res., vol. 22, p. 2183, 1994: As summarized, there are some examples of modified nucleobases known in the art.Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include: inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), or 6-azapyrimidine, or 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, and others (Burgin et al., Biochemistry, vol. 35:14090, 1996; Uhlman and Peyman, supra)."Modified base" in this embodiment refers to nucleotide bases other than adenine, guanine, cytosine, and uracil at 1' position, or their equivalents.

[0067] "Complementary nucleotide bases" refers to a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acid hybridize to each other (i.e., are joined by hydrogen bonds). "Complementary" means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional binding modes.

[0068] "MicroRNA" (miRNA) refers to 21-23 nucleotide long single-stranded RNA molecules that regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into proteins (non-coding RNA); instead, they are processed from a primary transcript known as pri-miRNA to short stem-loop structures called pre-miRNAs and finally to functional miRNAs. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and their main function is to downregulate gene expression.

[0069] "Small interfering RNA (siRNA)" and "Short interfering RNA" and "silencing RNA" refer to a class of double-stranded RNA molecules, 16-40 nucleotides long, that play a variety of roles in biology. Most notably, siRNAs are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, for example as an antiviral mechanism, or in shaping the chromatin structure of the genome, although the complexity of these pathways is only now being unraveled.

[0070] "RNAi" refers to the RNA-dependent gene silencing process controlled by the RNA-induced silencing complex (RISC) and initiated by short double-stranded RNA molecules in cells that interact with the catalytic RISC component Argonaute. When double-stranded RNA or RNA-like iNA or siRNA is exogenous (derived from infection with a virus with an RNA genome, or from transfected iNA or siRNA), the RNA or iNA is directly imported into the cytoplasm and cleaved into short fragments by the enzyme dicer. The initiating dsRNA can also be endogenous (derived from the cell), as in the case of pre-microRNAs expressed from RNA-coding genes in the genome. Primary transcripts from such genes are first processed in the nucleus to form the characteristic stem-loop structure of pre-miRNAs, then transported to the cytoplasm to be cleaved by dicer. Thus, the two dsRNA pathways, exogenous and endogenous, converge on the RISC complex. The active components of the RNA-induced silencing complex (RISC) are endonucleases called Argonaute proteins, which cleave target mRNA strands complementary to their bound siRNAs or iNAs. Because the fragments produced by dicer are double-stranded, they can theoretically produce functional siRNAs or iNAs, respectively. However, only one of the two strands, known as the guide strand, binds to the Argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation.

[0071] Compounds of Formula I

[0072] Reference to a compound of formula I herein is understood to include reference to its salts, unless otherwise indicated. As used herein, the term "salt(s)" refers to acid salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. Furthermore, when a compound of formula I contains both a basic moiety, such as, but not limited to, pyridine or imidazole, and an acidic moiety, such as, but not limited to, carboxylic acid, zwitterions ("internal salts") may be formed and are included within the term "salt(s)" as used herein. The salts may be pharma-ceutically acceptable (i.e., non-toxic, physiologically acceptable) salts, although other salts are also useful. Salts of compounds of formula I can be formed, for example, by reacting a compound of formula I with an equivalent amount of acid or base in a medium in which the salt precipitates, or in an aqueous medium, followed by lyophilization.

[0073] Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, These include sulfonates (such as those described herein), tartrates, thiocyanates, toluenesulfonates (also known as tosylates), undecanoates, etc. Additionally, acids which are generally considered suitable for forming pharma- ceutically useful salts from basic pharmaceutical compounds are described, for example, in S. Berge et al., J. Pharmaceutical Sciences (1977) 66(1), 1982; No.) pp. 1-19; P. Gould, International J. Pharmaceutics (1986) vol. 33 , pp. 201-217; Anderson et al., The Practice of Medicinal Chemistry (199 6, Academic Press, New York; and The Orange Book (Food & Drug Administration, Washington, DC, on website, the disclosures of which are incorporated herein by reference.

[0074] Exemplary base salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as benzathine, dicyclohexylamine, hydrabamine (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and salts with amino acids such as arginine or lysine. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, diamyl sulfate), long chain halides (e.g., decyl chloride, bromide, and iodide, lauryl, myristyl, and stearyl), aryl alkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0075] All such acid and base salts are intended to be pharma- ceutically acceptable salts within the scope of this disclosure, and for purposes of this disclosure, all acid and base salts are considered equivalent to the corresponding free form of the compounds of Formula I.

[0076] Compounds of formula I can exist in unsolvated and solvated forms, including hydrated forms. In general, solvated forms with pharma- ceutically acceptable solvents such as water, ethanol, etc., are equivalent to unsolvated forms for the purposes of this disclosure.

[0077] The compounds of Formula I, and their salts, solvates, may exist in their tautomeric forms (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the disclosure.

[0078] Polymorphs of the compounds of this disclosure are also within the scope of this disclosure (ie, polymorphs of the compounds of formula I are within the scope of this disclosure).

[0079] All stereoisomers (e.g., geometric isomers, optical isomers, etc.) of the compounds, including those that may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotamer forms, atropisomers, and diastereomeric forms (including salts, solvates, and prodrugs of the compounds, as well as salts and solvates of the prodrugs), are contemplated within the scope of the present disclosure. Individual stereoisomers of the compounds of the present disclosure may, for example, be substantially free of other isomers, or may be mixed, for example, as racemates, or with all or other selected stereoisomers. The chiral centers of the compounds herein may have the S or R configuration as defined by the IUPAC 1974 Recommendations. The use of terms such as "salts", "solvates", and the like, is intended to apply equally to the enantiomers, stereoisomers, rotamers, tautomers, racemates, or salts and solvates of prodrugs of the disclosed compounds.

[0080] Classes of compounds that can be used as chemotherapeutic (antineoplastic) agents include alkylating agents, metabolic These include antagonists, natural products and their derivatives, hormones and steroids (including synthetic analogues), and synthetics. Examples of compounds within these classes are listed below.

[0081] lipid particles The compound of formula I comprises a pharma- ceutically acceptable salt thereof in a lipid composition comprising a nanoparticle or a bilayer of lipid molecules. The lipid bilayer preferably further comprises a neutral lipid or polymer. The lipid composition preferably comprises a liquid medium. The composition preferably further encapsulates a nucleic acid. The nucleic acid preferably has an activity of suppressing the expression of a target gene by utilizing RNA interference (RNAi). The lipid composition preferably further comprises a nucleic acid and a neutral lipid or polymer. The lipid composition preferably encapsulates a nucleic acid.

[0082] The description provides lipid particles that include one or more therapeutic mRNA molecules encapsulated within the lipid particle.

[0083] In some embodiments, the mRNA is fully encapsulated within the lipid portion of the lipid particle such that the mRNA in the lipid particle is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals, such as humans. The lipid particles typically have an average diameter of 30 nm to 150 nm, 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, or 70 to 90 nm. The lipid particles of the present invention also typically have a lipid:RNA ratio (mass / mass ratio) of 1:1 to 100:1, 1:1 to 50:1, 2:1 to 25:1, 3:1 to 20:1, 5:1 to 15:1, or 5:1 to 10:1, or 10:1 to 14:1, or 9:1 to 20:1. In one embodiment, the lipid particles have a lipid:RNA ratio (mass / mass ratio) of 12:1. In another embodiment, the lipid particles have a lipid:mRNA ratio (mass / mass) of 13:1.

[0084] In a preferred embodiment, the lipid particle comprises an mRNA, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid (e.g., one or more PEG-lipid conjugates) that inhibits particle aggregation.The lipid particle may also comprise cholesterol.The lipid particle may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mRNAs that express one or more polypeptides.

[0085] In nucleic acid-lipid particles, mRNA can be completely encapsulated in the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation.In a preferred embodiment, the lipid particle containing mRNA is completely encapsulated in the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation.In certain examples, the mRNA in the lipid particle is not substantially degraded after exposing the particle to nuclease at 37°C for at least 20, 30, 45, or 60 minutes.In certain other examples, the mRNA in the lipid particle is not substantially degraded after incubating the particle in serum at 37°C for at least 30, 45, or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.In other embodiments, the mRNA is complexed with the lipid portion of the particle. One advantage of the present formulations is that the nucleic acid-lipid particle compositions are substantially non-toxic to mammals, such as humans.

[0086] "Fully encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded upon exposure to serum or after nuclease assays that would significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle, more preferably less than 25% of the nucleic acid in the particle, is degraded in a process that would normally degrade 100% of the free nucleic acid. Or less than 10%, and most preferably less than 5%, of the nucleic acid in the particles is degraded. "Fully encapsulated" also means that the nucleic acid-lipid particles do not rapidly degrade into their component parts upon in vivo administration.

[0087] In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that enhances fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to the liposome formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the liposome bilayer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation is determined by E = (I 0 -I) / I 0 It can be calculated as I 0 refers to the fluorescence intensity before and after addition of detergent.

[0088] In other embodiments, the present invention provides nucleic acid-lipid particle compositions comprising a plurality of nucleic acid-lipid particles.

[0089] The lipid particles contain mRNA fully encapsulated within the lipid portion of the particle, resulting in 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 30%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95%, 80%-95%, 85%-95%, 90%-95%, 30%-90 ... 0%, 50%-90%, 60%-90%, 70%-90%, 80%-90%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any percentage or range therein) of the particles have mRNA encapsulated therein.

[0090] Depending on the intended use of the lipid particles, the ratio of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0091] Cationic lipids The description includes the synthesis of certain cationic lipid compounds.The compounds are particularly suitable for delivering polynucleotides to cells and tissues, as shown in the following sections.The lipomacrocycles described herein can be used for other purposes, as well as for example recipients and additives.

[0092] The cationic lipid compounds can be synthesized by techniques known in the art. Those skilled in the art will recognize other methods for producing these compounds, as well as other compounds described.

[0093] Cationic lipid compounds can be combined with agents to form microparticles, nanoparticles, liposomes, or micelles.The agents delivered by particles, liposomes, or micelles can be in gas, liquid, or solid form, and the agents can be polynucleotides, proteins, peptides, or small molecules.The lipomacrocycles can be combined with other cationic lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, or lipids to form particles.These particles can then be combined with pharmaceutical excipients to form pharmaceutical compositions as needed.

[0094] The present specification provides novel cationic lipid compounds and drug delivery systems based on the use of such cationic lipid compounds. The systems can be used in the pharmaceutical / drug delivery field to deliver polynucleotides, proteins, small molecules, peptides, antigens, or drugs to patients, tissues, organs, or cells. These novel compounds can also be used as coating agents. It may also be used as a substance, additive, excipient, material, or biotechnology.

[0095] The cationic lipid compounds herein provide several different uses in the field of drug delivery. The amine-containing moieties of cationic lipid compounds can be used to complex polynucleotides, thereby enhancing the delivery of polynucleotides and preventing their degradation. Cationic lipid compounds can also be used in the formation of picoparticles, nanoparticles, microparticles, liposomes, and micelles that contain the agent to be delivered. Preferably, the cationic lipid compounds are biocompatible and biodegradable, and the particles formed are also biodegradable and biocompatible and can be used to provide controlled sustained release of the agent to be delivered. These particles and their corresponding particles can also respond to pH changes, considering that they are protonated at lower pH. They can also act as proton sponges in the delivery of agents to cells to cause endosome lysis.

[0096] In certain embodiments, the cationic lipid compounds are relatively non-cytotoxic. The cationic lipid compounds may be biocompatible and biodegradable. The cationic lipids have a pK ranging from about 5.5 to about 7.5, more preferably between about 6.0 to about 7.0. a It may have a desired pK between about 3.0 and about 9.0, or between about 5.0 and about 8.0. a The cationic lipid compounds described herein are particularly attractive for drug delivery for several reasons: they contain amino groups to interact with DNA, RNA, other polynucleotides, and other negatively charged agents, to buffer pH, to cause endo-osmolysis, to protect the agent to be delivered; they can be synthesized from commercially available starting materials; and / or they are pH responsive and have a desired pK a It can be operated with.

[0097] Neutral Helper Lipids Non-limiting examples of non-cationic lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine ...DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phos Phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably C 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0098] Further examples of non-cationic lipids include steroids, such as cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0099] In some embodiments, the non-cationic lipid present in lipid particle comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In other embodiments, the non-cationic lipid present in lipid particle comprises or consists of one or more phospholipids, for example, cholesterol-free lipid particle formulations.In still other embodiments, the non-cationic lipid present in lipid particle comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid particle formulations.

[0100] Other examples of non-cationic lipids include non-phosphorus-containing lipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0101] In some embodiments, the non-cationic lipids constitute 10 mol% to 60 mol%, 20 mol% to 55 mol%, 20 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 50 mol%, 25 mol% to 45 mol%, 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, 37 mol% to 42 mol%, or 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, or 45 mol% (or any percentage or range thereof) of the total lipid present in the particle.

[0102] In embodiments in which the lipid particle contains a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may constitute up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle. In some embodiments, the phospholipid component in the mixture may comprise 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol%, or 4 mol% to 10 mol% (or any percentage or range thereof) of the total lipid present in the particle. In certain preferred embodiments, the phospholipid component in the mixture comprises 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any percentage or range thereof) of the total lipid present in the particle. In other embodiments, the cholesterol component in the mixture may comprise 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 27 mol% to 37 mol%, 25 mol% to 30 mol%, or 35 mol% to 40 mol% (or any percentage or range thereof) of the total lipid present in the particle. In certain preferred embodiments, the cholesterol component in the mixture may comprise 25 mol% to 35 mol%, 27 mol% to 35 mol%, 29 mol% to 35 mol%, 30 mol% to 35 mol%, 30 mol% to 34 mol%, 31 mol% to 33 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, or 35 mol% (or any percentage or range thereof) of the total lipid present in the particle. any percentage or range thereof. In embodiments in which the lipid particle does not contain phospholipids, cholesterol or a derivative thereof may constitute up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle.

[0103] In some embodiments, cholesterol or a derivative thereof in a phospholipid-free lipid particle formulation may constitute 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 31 mol% to 39 mol%, 32 mol% to 38 mol%, 33 mol% to 37 mol%, 35 mol% to 45 mol%, 30 mol% to 35 mol%, 35 mol% to 40 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, or 40 mol% (or any percentage or range thereof) of the total lipid present in the particle.

[0104] In other embodiments, the non-cationic lipids comprise 5 mol% to 90 mol%, 10 mol% to 85 mol%, 20 mol% to 80 mol%, 10 mol% (e.g., phospholipids only), or 60 mol% (e.g., phospholipids and cholesterol or derivatives thereof) of the total lipids present in the particle (or any percentage or range therein).

[0105] The percentage of non-cationic lipid present in the lipid particle is a target amount, and the actual amount of non-cationic lipid present in the formulation can vary, for example, by ±5 mole %.

[0106] The composition containing the cationic lipid compound may be 30-70% cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid, and 1-10% polyethylene glycol (PEG). Preferably, the composition is 30-40% cationic lipid compound, 40-50% cholesterol, and 10-20% PEG. In another preferred embodiment, the composition is 50-75% cationic lipid compound, 20-40% cholesterol, 5-10% phospholipid, and 1-10% PEG. The composition may contain 60-70% cationic lipid compound, 25-35% cholesterol, and 5-10% PEG. The composition may contain up to 90% cationic lipid compound and 2-15% helper lipid.

[0107] The formulation may be a lipid particle formulation, for example containing 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipid, and 2-10% helper lipid, or even 100% cationic lipid.

[0108] Lipid conjugates In addition to cationic, lipid particles as described herein can further comprise lipid conjugate.Conjugated lipid is useful in that it prevents particle aggregation.Suitable conjugated lipid includes but is not limited to PEG-lipid conjugate, cationic polymer-lipid conjugate, and their mixture.

[0109] In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include PEG coupled to dialkyloxypropyl (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), and PEG conjugates to ceramide. Examples of suitable conjugated PEG include, but are not limited to, PEG conjugated to cholesterol, PEG conjugated to cholesterol or its derivatives, and mixtures thereof.

[0110] PEGs are linear, water-soluble polymers of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH 2 ), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), and such compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH 2 ).

[0111] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 550 daltons to 10,000 daltons. In certain examples, the PEG moiety has an average molecular weight of 750 daltons to 5,000 daltons (e.g., 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0112] In certain examples, PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. PEG can be directly conjugated to lipids or can be linked to lipids via a linker moiety. Any linker moiety suitable for coupling PEG to lipids can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linkers include amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-0-), succinyl (-(0)CCH 2 CH 2 C(0)-), succinamidyl (-NHC(0)CH 2 CH 2 Linkers that can be used to couple PEG to lipids include, but are not limited to, C(0)NH-), ethers, disulfides, and combinations thereof, such as linkers that contain both carbamate and amide linker moieties. In a preferred embodiment, a carbamate linker is used to couple PEG to the lipid.

[0113] In other embodiments, PEG is coupled to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0114] Phosphatidylethanolamines having various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids having carbon chain lengths in the range of 0.1 to 100 are preferred. Phosphatidylethanolamines having mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE). Examples of suitable polyether ether esters include, but are not limited to, diphenyl ether (DSPE).

[0115] The term "diacylglycerol" or "DAG" refers to a diacylglycerol consisting of two fatty acyl chains, R, having between 2 and 30 carbons independently, both attached to the 1- and 2-positions of glycerol by ester linkages. 1 and R 2 The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and Icosoil (C 20 In a preferred embodiment, R1 and R 2 are identical, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), and R 1 and R 2 are both stearoyl (i.e., distearoyl).

[0116] The term "dialkyloxypropyl" or "DAA" includes compounds that have two alkyl chains, R and R, both of which independently have between 2 and 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation.

[0117] Preferably, the PEG-DAA conjugate is PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate, or PEG-distearyloxypropyl (C 18 ) conjugates. In these embodiments, the PEG preferably has an average molecular weight of 750 or 2,000 daltons. In certain embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0118] In addition to the above, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0119] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol% to 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% to 1.5 mol% (or any percentage or range therein) of the total lipid present in the particle. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0 mol% to 20 mol%, 0.5 mol% to 20 mol%, 2 mol% to 20 mol%, 1.5 mol% to 18 mol%, 2 mol% to 15 mol%, 4 mol% to 15 mol%, 2 mol% to 12 mol%, 5 mol% to 12 mol%, or 2 mol% (or any percentage or range therein) of the total lipid present in the particle.

[0120] In further embodiments, the lipid conjugate (e.g., PEG-lipid) constitutes 4 mol% to 10 mol%, 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any percentage or range thereof) of the total lipid present in the particle.

[0121] Percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid particle of the present invention is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ±2 mol %. One of skill in the art will understand that the concentration of lipid conjugate may vary depending on the lipid conjugate used and the rate at which the lipid particle becomes fusogenic.

[0122] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate is exchanged from lipid particle and therefore the rate at which lipid particle becomes fusogenic can be controlled.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which lipid particle becomes fusogenic.Other methods that can be used to control the rate at which lipid particle becomes fusogenic will be clear to those skilled in the art upon reading this disclosure.In addition, by controlling the composition and concentration of lipid conjugate, the size of lipid particle can be controlled.

[0123] Compositions and Formulations for Administration

[0124] The nucleic acid-lipid composition of the present disclosure can be administered by various routes, for example, via intravenous, parenteral, intraperitoneal, or local routes, to effect systemic delivery.In some embodiments, siRNA can be delivered intracellularly in target tissue, such as lung or liver, or intracellularly in inflamed tissue.In some embodiments, the present disclosure provides a method for delivering siRNA in vivo.The nucleic acid-lipid composition can be administered intravenously, subcutaneously, or intraperitoneally to a subject.In some embodiments, the present disclosure provides a method for in vivo delivery of interfering RNA to the lung of a mammalian subject.

[0125] In some embodiments, the present disclosure provides a method for treating disease or disorder in a mammalian subject.A therapeutically effective amount of the composition of the present disclosure, which comprises nucleic acid, cationic lipid, amphiphile, phospholipid, cholesterol, and PEG-linked cholesterol, can be administered to a subject having a disease or disorder associated with the expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition.

[0126] The compositions and methods of the present disclosure can be administered to a subject by a variety of mucosal administration modes, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or dermal delivery, or by topical delivery to the eye, ear, skin, or other mucosal surface. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically actuated, or other types of actuators.

[0127] The compositions of the present disclosure may be administered in aqueous solution as a nasal or pulmonary spray, and may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is accomplished by administering the compositions in the form of drops, particles, or sprays, which may be, for example, aerosolized, atomized, or nebulized. The particles of the composition, spray, or aerosol may be in either liquid or solid form. A preferred system for dispensing liquids as nasal sprays is disclosed in U.S. Pat. No. 4,511,069. Such formulations may be conveniently prepared by dissolving a composition according to the present disclosure in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in a multi-dose container in a sealed dispensing system, for example, as disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, edited by Y.W. Chien, Elsevier Publishers, New York, 1985; and U.S. Pat. Additional aerosol delivery forms include nebulizers, e.g., compressed air, jet, ultrasonic, and piezoelectric, which deliver a bioactive agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or mixtures thereof. It is possible.

[0128] The nasal and pulmonary spray solutions of the present disclosure typically include a drug or drugs to be delivered, optionally formulated with a surfactant, such as a non-ionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further includes a propellant. The pH of the nasal spray solution may be pH 6.8 to 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer, pH 4 to 6. Other components, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0129] In some embodiments, the present disclosure is a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0130] The dosage form of the composition of the present disclosure may be liquid, may be in the form of droplets or an emulsion, or may be in the form of an aerosol.

[0131] The dosage form of the composition of the present disclosure may be a solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0132] To formulate compositions for pulmonary delivery within the scope of the present disclosure, the bioactive agent can be combined with various pharma- ceutically acceptable additives and bases or carriers for dispersing the active agent(s). Examples of additives include pH adjusters such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonicity agents (e.g., sodium chloride, mannitol, sorbitol), anti-adsorption agents (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w / v) saline taken as the equivalent, is typically adjusted to a value that will not induce substantial, irreversible tissue damage to the mucosa at the administration site. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most frequently 3 / 4 to 1.7.

[0133] The bioactive agent may be dispersed in a base or vehicle, which may contain a hydrophilic compound capable of dispersing the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, polycarboxylic acids or their salts, copolymers of carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.); hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone; cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose; and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid; and non-toxic metal salts thereof. Often, biodegradable polymers are selected as the base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. may be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination and can impart enhanced structural integrity to the carriers by partial crystallization, ionic bonding, crosslinking, etc. Carriers are provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films, for direct application to the nasal mucosa. The use of selected carriers in this context can facilitate absorption of the bioactive agent.

[0134] Formulations for mucosal, nasal, or pulmonary delivery may contain hydrophilic low molecular weight compounds as bases or excipients. Such hydrophilic low molecular weight compounds provide a passage medium through which a water-soluble active agent, such as a bioactive peptide or protein, can diffuse through the base to a body surface where the active agent is absorbed. The hydrophilic low molecular weight compounds are If necessary, it absorbs moisture from the mucous membrane or the administration atmosphere to dissolve the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally 10,000 or less, preferably 3,000 or less. Examples of hydrophilic low molecular weight compounds include polyol compounds such as oligosaccharides, disaccharides and monosaccharides including sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiobiose, glycerin, polyethylene glycol, and mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[0135] The compositions of the present disclosure may alternatively contain, as pharma- ceutically acceptable carriers, substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof.For solid compositions, conventional non-toxic pharma-ceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0136] In certain embodiments of the present disclosure, bioactive agents can be administered in time release formulations, for example in compositions that contain slow release polymers.Active agents can be prepared with carriers that protect against rapid release, such as controlled release vehicles, for example polymers, microencapsulated delivery systems, or bioadhesive gels.Long-term delivery of active agents in various compositions of the present disclosure can be achieved by including agents that delay absorption, such as aluminum monostearate hydrogels and gelatin, in the composition.

[0137] Although the present disclosure has been described with respect to certain specific embodiments and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may be significantly modified without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications, and equivalents. In particular, the present disclosure includes any combination of the features, terms, or elements of the various exemplary components and examples. EXAMPLES

[0138] Example 1: Exemplary lipids Exemplary compounds of Formula I are presented in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0139] Example 2: Synthesis of ATX-43 FIG. 1 shows the synthetic pathway for ATX-43 (RL-43A), which is further described as follows.

[0140] ATX-43: Step 1 [ka]

[0141] In a 500 mL single-neck round-bottom flask, 25 g of hexanoic acid (SM1; 1 eq.) dissolved in dichloromethane (DCM; 200 mL) was placed, then 27.6 mL of oxalyl chloride (1.5 eq.) was added slowly at 0° C. and stirred under nitrogen atmosphere, followed by the addition of 0.5 ml of dimethylformamide (DMF; catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

[0142] In a separate 1 L two-neck round bottom flask, 31.4 g of N,O-dimethylhydroxylamine hydrochloride (1.5 eq.) in DCM (200 ml) was added 89.8 ml of triethylamine (Et 3 N, 3 eq.) was added and stirred at 0° C. To the resulting solution, after concentration under reduced pressure, the above acid chloride was dissolved in DCM (100 ml). The resulting reaction solution was stirred at room temperature under a nitrogen atmosphere for 3 hours.

[0143] The progress of the reaction was monitored by thin layer chromatography (TLC) (20% ethyl acetate (EtOAc) / hexane; Rf: 0.5). The reaction mass was diluted with water (300 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure.

[0144] The crude compound was subjected to column chromatography using (60-120 silica gel; 10% EtOAc / hexanes). Yield: 20.0 g; Yield: 58%.

[0145] ATX-43: Step 2 [ka]

[0146] To a solution of 33 g of pentylmagnesium bromide (1.5 equivalents) in tetrahydrofuran (THF; 100 ml) placed in a 500 ml two-neck round-bottom flask stirred at 0° C. under nitrogen atmosphere, a solution of 20 g of N-methoxy-N-methylhexanamide (1 equivalent) (dissolved in 200 ml of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0147] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4It was quenched with Cl solution (150 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure.

[0148] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / Hexanes). Yield: 15.0 g; Yield: 66%.

[0149] ATX-43: Step 3 [ka]

[0150] To a solution of 15 g of undecan-6-one (1 eq.) dissolved in 25 ml of methanol (MeOH) in 150 ml of THF, 4.9 g of sodium borohydride (1.5 eq.) was added at 0° C. and the resulting solution was stirred at room temperature for 2 hours.

[0151] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (100 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (150 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 14.0 g; Yield, 93%.

[0152] ATX-43: Step 4 [ka]

[0153] To a solution of 15 g of 4-aminobutanoic acid (1 eq.) dissolved in 150 ml of THF was added sequentially using an addition funnel over 15 min each, 145 ml of 1 N aqueous NaOH (1 eq.) at 0° C., followed by 43.4 ml of Boc anhydride (1.3 eq.). The resulting solution was stirred at room temperature for 4 h.

[0154] The progress of the reaction was monitored by TLC (chloroform (CHCl 3 ) in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (150 ml) and then EtOAc (100 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 68%.

[0155] ATX-43: Step 5 [ka]

[0156] To a solution of 12 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equiv.) in DCM (200 ml) cooled to below 0 °C, 14.7 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)·HCl (1.3 equiv.), 10.6 ml of Et 3 N (1.3 eq.), and 0.72 g of 4-dimethylaminopyridine (DMAP; 0.1 eq.) were added. To this resulting solution, the alcohol was added at the same temperature by dissolving it in DCM (50 ml) using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 h.

[0157] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (100 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×50 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3The solution was washed with 100 ml of ethyl acetate and then extracted with EtOAc (2×50 ml). The organic layer was concentrated under reduced pressure and carried onto the next step crude. Yield, 8.5 g; yield, 48%.

[0158] ATX-43: Step 6 [ka]

[0159] To a solution of 8.5 g of undecan-6-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 70 ml of DCM was added trifluoroacetic acid (TFA; 10 eq.) at 0° C. and stirred at room temperature under nitrogen for 4 hours.

[0160] The progress of the reaction was monitored by TLC (70% EtOAc / Hexane; Rf: 0.2). The reaction mass was concentrated under reduced pressure. The obtained crude was dissolved in saturated NaHCO 3 The organic layer was separated and concentrated under reduced pressure. was concentrated with

[0161] The crude compound was purified by HPLC using a HPLC-MS chromatography system (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine) to recover the alcohol. Yield: 5.0 g; yield: 33% (based on the alcohol).

[0162] ATX-43: Step 7 [ka]

[0163] To a solution of 14 g of 4-bromobutyric acid (1 equiv.) in DCM (100 ml) cooled to <0 °C, 21 g of EDC HCl (1.3 equiv.), 15.2 ml of Et 3N (1.3 eq.), and 1 g of DMAP (0.1 eq.) were added. To this resulting solution, 8.3 g of (Z)-non-2-en-1-ol (0.7 eq.) was added by dissolving in 50 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 16 hours.

[0164] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (50 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×50 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (100 ml) and then extracted with EtOAc (2×50 ml). The organic layer was separated and concentrated under reduced pressure.

[0165] The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. Yield, 11.0 g; Yield, 64%.

[0166] ATX-43: Step 8 [ka]

[0167] To a 250 ml round bottom flask were added 2 g of undecane-6-yl 4-aminobutanoate (1 eq.) and 2.2 g of (Z)-non-2-en-1-yl 4-bromobutanoate (1 eq.), 1.2 g of potassium carbonate (1.2 eq.) in DMF, and the resulting mixture was refluxed at 90° C. for 4 hours under nitrogen atmosphere.

[0168] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5) Ice water was added to the reaction mass, then extracted with EtOAc, dried over sodium sulfate and concentrated under reduced pressure.

[0169] The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexanes. The starting amine and bromo compound were recovered. Yield, 1.45 g; Yield, 40%.

[0170] ATX-43: Step 9 [ka]

[0171] To a solution of 1.45 g of (Z)-non-2-en-1-yl 4-((4-oxo-4-(undecan-6-yloxy)butyl)amino)butanoate (1 eq.) dissolved in dry DCM was added 1.29 ml of triethylamine (3 eq.) and 360 mg of triphosgene (0.4 eq.) at 0° C. under nitrogen atmosphere at intervals of 5 minutes. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0172] To 360 mg of sodium hydride (5.5 eq.) dissolved in dry THF (20 ml) in a two-necked 250 ml round bottom flask stirred at 0° C. under nitrogen atmosphere, 2.1 g of 2-(dimethylamino)ethane-1-thiol hydrochloride (5.5 eq.) in THF (30 ml) was added and stirring was continued for 5 min under nitrogen atmosphere. To the resulting solution, the above carbonyl chloride dissolved in THF (50 mL) was added slowly using an addition funnel for about 15 min and stirred at room temperature for 1 h.

[0173] Reaction mass was saturated with NH 4 It was quenched with Cl solution (20 ml) followed by the addition of EtOAc (20 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×20 ml). The combined organic layers were concentrated and the crude product obtained was subjected to column chromatography. The progress of the reaction was monitored by TLC (60% EtOAc / Hex; Rf: 0.5; PMA char).

[0174] Purification was carried out using silica gel (100-200 mesh; 18% EtOAc / hexanes) chromatography. Yield, 500 mg; yield, 26%. 1 The product was confirmed by 1 H NMR; HPLC; and mass spectrometry.

[0175] [ka]

[0176] Example 3: Synthesis of ATX-57 FIG. 2 shows the synthetic route for ATX-57(RL-43C), which is further described as follows.

[0177] ATX-57: Step 1: N-Methoxy-N-methyloctanamide [ka]

[0178] In a 2-liter two-necked round-bottom flask, octanoic acid (1 eq.) dissolved in DCM (300 ml) was placed, then 1.5 eq. of oxalyl chloride was added slowly at 0° C. under nitrogen atmosphere with stirring. The resulting reaction mixture was stirred at room temperature for 2 hours. In another 2-liter two-necked round-bottom flask, 2 eq. of N,O-dimethylhydroxylamine hydrochloride in DCM (200 ml) was added 3 eq. of trimethylamine using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was added dropwise using an addition funnel by dissolving in DCM (150 ml) under nitrogen atmosphere for 20 minutes. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0179] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexane). Yield, 65%. Yield, 85 g.

[0180] ATX-57: Step 2: Hexadecane-8-one [ka]

[0181] To a solution of octylmagnesium bromide in THF (100 ml) placed in a 1-liter, two-necked, round-bottom flask stirred at 0° C. under nitrogen, was added the N-methoxy-N-methyloctanamide solution (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0182] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (250 ml) followed by addition of EtOAc (350 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / Hexane). Yield, 65 g; yield, 63%.

[0183] ATX-57: Step 3: Hexadecan-8-ol [ka]

[0184] To a solution of hexadecan-8-one (1 eq) dissolved in MeOH / THF was added 1 eq of sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1.5 h.

[0185] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (75 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 60 g; yield, 91%.

[0186] ATX-57: Step 4: 4-((tert-butoxycarbonyl)amino)butanoic acid [ka]

[0187] To a solution of 4-aminobutanoic acid dissolved in THF was added sequentially over a period of 15 min using an addition funnel 1N aqueous NaOH at 0° C. followed by Boc anhydride. The resulting solution was stirred at room temperature for 4 h.

[0188] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 81%.

[0189] ATX-57: Step 5: Hexadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate [ka]

[0190] A solution of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (200 ml) cooled to <0 °C was treated with EDC·HCl, Et3 N, and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of hexadecan-8-ol alcohol was added at the same temperature by dissolving in DCM (150 ml) using an addition funnel and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0191] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (150 ml) and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield: 80 g (crude; required compound and alcohol).

[0192] ATX-57: Step 6: Hexadecan-8-yl 4-aminobutanoate [ka]

[0193] To a solution of hexadecan-8-yl-4-((tert-butoxycarbonyl)amino)butanoate in DCM was added TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours. The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (300 ml) and then extracted with EtOAc (2×200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by HPLC using a 50-mL column chromatography (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine) to recover the alcohol. Product amount: 40 g; Yield: 59% for two steps; Confirmed by Mass.

[0194] ATX-57: Step 7: (Z)-Nona-2-en-1-yl 4-bromobutanoate [ka]

[0195] A solution of 4-bromobutyric acid in DCM (400 ml) cooled to <0 °C was treated with EDC·HCl, Et 3 N, and DMAP were added. To this resulting solution, (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0196] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (300 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×150 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (200 ml) and then extracted with EtOAc (150 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. The alcohol was recovered. Yield, 51%.

[0197] ATX-57: Step 8: Hexadecan-8-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate [ka]

[0198] To a solution of hexadecan-8-yl 4-aminobutanoate, (Z)-non-2-en-1-yl 4-bromobutanoate in acetonitrile (ACN) was added potassium carbonate and the resulting mixture was refluxed at 90° C. for 4 hours under nitrogen atmosphere. The progress of the reaction was monitored by TLC (CHCl 3The reaction mass was filtered, washed with ACN (20 ml) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexane. The starting amine and bromo compounds were recovered. Yield, 20 g; 40%; confirmed by Mass.

[0199] ATX-57: Step 9 [ka]

[0200] To a solution of hexadecan-8-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in dry DCM was added trimethylamine and triphosgene at 0° C. in 5 min intervals under nitrogen atmosphere. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0201] 2-(Dimethylamino)propane-1-thiol hydrochloride was added to sodium hydride dissolved in dry THF (50 ml) in a two-necked 100 ml round bottom flask stirred at 0° C. under nitrogen atmosphere and continued to stir for 5 minutes under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (80 ml) was slowly added by syringe over about 10 minutes. The resulting solution was stirred at room temperature under nitrogen atmosphere for 6 hours.

[0202] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (75 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0203] The first purification was performed using silica gel (60-120 mesh). 22 g of crude compound was adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. 7.5 g of crude compound was adsorbed onto 18 g of neutral alumina and poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 29%; 1 Confirmed by 1 H NMR, HPLC, and Mass.

[0204] [ka]

[0205] Example 4: Synthesis of ATX-58 FIG. 3 shows the synthetic route of ATX-58 (RL-43B), which is further described as follows.

[0206] ATX-58: Step 1 [ka]

[0207] N 2 In a 500 ml two-necked round bottom flask, 30 g of 8-bromooctanoic acid (1 eq.) dissolved in 200 ml of DCM was placed under atmosphere and then slowly added to 26.7 ml of oxalyl chloride (1.5 eq.) under nitrogen atmosphere and stirring at 0° C. The resulting reaction mixture was stirred at room temperature for 2 hours.

[0208] In another 1-liter two-neck round-bottom flask, 40.5 g of N,O-dimethylhydroxylamine hydrochloride (2 eq.) in 300 ml of DCM was added with 87 ml of trimethylamine (3 eq.) stirred at 0° C. To this resulting solution, the above-mentioned acid chloride was added dropwise using an addition funnel over 15 minutes by dissolving in 500 ml of DCM after concentrating under reduced pressure. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.

[0209] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (300 ml). The organic layer was separated and the aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure.

[0210] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexanes). Yield: 28 g.

[0211] ATX-58: Step 2 [ka]

[0212] To a solution of 28 g of hexylmagnesium bromide (1 eq.) in THF (100 ml) stirred at 0° C. under a nitrogen atmosphere, 36.8 g of N-methoxy-N-methyloctanamide (1.3 eq.) in 200 ml of THF was added and the resulting reaction mixture was stirred at room temperature for 5 hours.

[0213] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.7). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure.

[0214] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% ethyl acetate / hexanes). Yield, 24 g; Yield, 77%.

[0215] ATX-58: Step 3 [ka]

[0216] To a solution of 24 g of tetradecan-7-one (1 eq.) dissolved in MeOH / THF was added 4.27 g of sodium borohydride (1 eq.) at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0217] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (50 ml). Methanol was reduced under reduced pressure. The crude was partitioned between EtOAc (200 ml) and water. The organic layer was separated and the aqueous layer was washed with EtOAc (2×80 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 21.5 g; yield, 89%.

[0218] ATX-58: Step 4 [ka]

[0219] To a solution of 20 g of 4-aminobutanoic acid dissolved in 140 ml of THF, 196 ml of 1N aqueous NaOH was added at 0° C., followed by 36.8 g of Boc anhydride using a funnel. The resulting solution was stirred at room temperature for 4 hours.

[0220] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 ml) and then EtOAc (200 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 76%.

[0221] ATX-58: Step 5 [ka]

[0222] To a solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equiv.) in DCM (150 ml) cooled to below 0 °C was added successively at 10 min intervals 12.2 g of EDC HCl (1.3 equiv.), 20.4 ml of Et 3 N (3 eq.), and 488 mg of DMAP (0.1 eq.) were added. To this resulting solution, the alcohol was added by dissolving in DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0223] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mass was quenched with water (100 ml) and the organic layer was separated. The aqueous layer was washed with DCM (2×50 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with EtOAc (100 ml) and the organic layer was separated, concentrated under reduced pressure and carried onto the next step crude. Yield: 12.7 g (crude).

[0224] ATX-58: Step 6 [ka]

[0225] To a solution of 12.5 g of tetradecan-7-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 100 ml of DCM was added 23.9 ml of TFA (10 eq.) at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0226] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The obtained crude was dissolved in saturated NaHCO 3The solution was washed with ethyl acetate (100 ml) and EtOAc (100 ml) was added. The organic layer was separated and concentrated under reduced pressure.

[0227] The crude compound was purified by HPLC using a HPLC-MS chromatography system (60-120 mesh silica gel; 4% MeOH / CHCl 3 The alcohol was recovered by column chromatography using 1,000 ml of ...

[0228] ATX-58: Step 7 [ka]

[0229] To a solution of 20 g of 4-bromobutyric acid (1 equiv.) in DCM (150 ml) cooled to 0 °C was added successively at 10 min intervals 1.5 equiv. of EDC HCl, 3 equiv. of Et 3 N, and 0.1 equivalents of DMAP were added. To this resulting solution, 0.7 equivalents of (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0230] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.7). The reaction mass was quenched with water (100 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was purified by elution with saturated NaHCO 3 The solution was washed with EtOAc (150 ml) and the organic layer was separated and concentrated under reduced pressure.

[0231] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 5% EtOAc / hexanes). Yield: 17 g; Yield: 69%. 1 Confirmed by 1 H NMR.

[0232] ATX-58: Step 8 [ka]

[0233] To a solution of 6 g of tetradecan-7-yl 4-aminobutanoate (1 eq.), 5.8 g of (Z)-non-2-en-1-yl 4-bromobutanoate (1 eq.) in ACN (125 ml) was added 2.7 g of potassium carbonate (1.2 eq.) and the resultant was refluxed at 90° C. for 3 hours under nitrogen atmosphere.

[0234] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ;Rf:0.5) The reaction mass was filtered and the filtrate was concentrated under reduced pressure.

[0235] The crude compound was subjected to column chromatography using (100-200 mesh silica gel; 15% EtOAc / hexane). Product amount: 4.5 g; Yield: 44%; confirmed by mass.

[0236] ATX-58: Step 9 [ka]

[0237] To a solution of 4.4 g of (Z)-non-2-en-1-yl 4-((4-oxo-4-(tetradecan-7-yloxy)butyl)amino)butanoate (1 eq.) dissolved in 30 ml of dry DCM was added 0.83 ml of trimethylamine (3 eq.) and 418 mg of triphosgene (0.5 eq.) at 0° C. under nitrogen atmosphere at intervals of 5 minutes. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0238] To 192 mg of sodium hydride (10 eq.) dissolved in dry THF (25 ml) in a two-necked 100 ml round bottom flask, 564 mg of 2-(dimethylamino)propane-1-thiol hydrochloride (5 eq.) was added at 0° C. and kept stirring for 5 min under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (35 ml) was slowly added by syringe for about 10 min. The resulting solution was stirred at room temperature under nitrogen atmosphere for 4 h.

[0239] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl (30 mL) and then EtOAc (100 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×50 mL). The combined organic layers were concentrated and the resulting crude product was subjected to column chromatography.

[0240] The first purification was performed using silica gel (60-120 mesh). 5.0 g of crude compound was adsorbed onto 9 g of silica gel and poured onto 90 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. 1.5 g of crude compound was adsorbed onto 4 g of neutral alumina and poured onto 40 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.2 g; Yield, 21%; 1 Confirmed by H NMR, HPLC, and Mass.

[0241] [ka]

[0242] Example 5: Synthesis of ATX-81 FIG. 4 shows the synthetic pathway for ATX-81 (RL-48B), which is further described as follows.

[0243] ATX-81: Step 1 [ka]

[0244] In a 2-liter two-necked round-bottom flask, octanoic acid dissolved in DCM (200 ml) was placed, and then 1.5 equivalents of oxalyl chloride was added slowly at 0° C. under nitrogen atmosphere while stirring. The resulting reaction mixture was stirred at room temperature for 2 hours. In another 2-liter two-necked round-bottom flask, 2 equivalents of N,O-dimethylhydroxylamine hydrochloride in DCM (200 ml) was added 3 equivalents of trimethylamine using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was added dropwise using an addition funnel by dissolving in DCM (150 ml) under nitrogen atmosphere for 20 minutes. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0245] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexane). Yield, 84%. Yield, 33 g.

[0246] ATX-81: Step 2 [ka]

[0247] To a solution of 22 g of heptylmagnesium bromide (1.5 eq.) in THF (100 ml) placed in a 1-liter two-necked round-bottom flask stirred at 0° C. under nitrogen atmosphere was added a solution of N-methoxy-N-methyloctanamide (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0248] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (250 ml) followed by addition of EtOAc (350 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / Hexane). Yield, 22 g; yield, 65%.

[0249] ATX-81: Step 3 [ka]

[0250] To a solution of 22 g of pentadecan-8-one (1 eq.) dissolved in MeOH / THF was added 1.5 eq. of sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0251] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (75 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 20 g; yield, 90%.

[0252] ATX-81: Step 4 [ka]

[0253] To a solution of 50 g of 4-aminobutanoic acid dissolved in 350 ml of THF was added sequentially over a period of 15 minutes using an addition funnel 490 ml of 1N aqueous NaOH at 0° C. followed by 140 ml of Boc anhydride. The resulting solution was stirred at room temperature for 4 hours.

[0254] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 81%.

[0255] ATX-81: Step 5 [ka]

[0256] To a solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (250 ml) cooled to <0 °C, 1.3 equivalents of EDC HCl, Et 3 N, and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of pentadecan-7-ol alcohol was added at the same temperature by dissolving in DCM (150 ml) using an addition funnel and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0257] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (150 ml) and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield: 8.5 g (crude; required compound and alcohol).

[0258] ATX-81: Step 6 [ka]

[0259] To a solution of 8.5 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate in 65 ml of DCM was added 10 equivalents of TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0260] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (300 ml) and then extracted with EtOAc (2×200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by HPLC using a 50-mL column chromatography (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine) to recover the alcohol. Product amount: 4 g for two steps; yield: 25%; confirmed by mass.

[0261] ATX-81: Step 7 [ka]

[0262] A solution of 4-bromobutyric acid in DCM (300 ml) cooled to <0 °C was treated with EDC HCl, Et 3 N, and DMAP were added. To this resulting solution, 20 g of (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere. The mixture was stirred for 24 hours.

[0263] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (300 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×150 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3The solution was washed with ethyl acetate (200 ml) and then extracted with EtOAc (150 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. The alcohol was recovered. Yield, 55%.

[0264] ATX-81: Step 8 [ka]

[0265] To a solution of 4.5 g of pentadecan-8-yl 4-aminobutanoate, 1 equivalent of (Z)-non-2-en-1-yl 4-bromobutanoate in 70 ml of acetonitrile (ACN), 1.4 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90° C. for 4 hours under a nitrogen atmosphere.

[0266] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (20 ml) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexane. The starting materials, amine and bromo compound were recovered. Yield, 2.1 g; Yield, 27%; confirmed by Mass.

[0267] ATX-81: Step 9 [ka]

[0268] To a solution of 2.1 g of pentadecan-8-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 150 ml of dry DCM was added 3 equivalents of triethylamine and triphosgene at 0° C. at 5 min intervals under nitrogen atmosphere. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0269] To 7 equivalents of sodium hydride dissolved in dry THF (80 ml) in a two-necked 100 ml round bottom flask stirred at 0°C under nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride was added and stirring was continued for 5 minutes under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (80 ml) was slowly added by syringe for about 10 minutes. The resulting solution was stirred overnight at 0°C to room temperature under nitrogen atmosphere.

[0270] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (75 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0271] The first purification of the crude compound was performed using silica gel (60-120 mesh), adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.5 g; Yield, 45%; 1 Confirmed by 1 H NMR, HPLC, and Mass.

[0272] [ka]

[0273] Example 6: Synthesis of ATX-82 FIG. 5 shows the synthetic pathway for ATX-82 (RL-47A), which is further described as follows.

[0274] ATX-82: Step 1 [ka]

[0275] In a 2-liter two-necked round-bottom flask, 30 g of octanoic acid dissolved in DCM (200 ml) was placed, and then 1.5 equivalents of oxalyl chloride was added slowly at 0° C. under nitrogen atmosphere with stirring. The resulting reaction mixture was stirred at room temperature for 2 hours. In another 2-liter two-necked round-bottom flask, 2 equivalents of N,O-dimethylhydrochloride in DCM (200 ml) was added. To the xylamine hydrochloride, 3 equivalents of trimethylamine were added using an addition funnel and stirred at 0° C. To the resulting solution, the above acid chloride, after concentration under reduced pressure, was added dropwise using an addition funnel by dissolving in DCM (150 ml) over 20 minutes under nitrogen atmosphere. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0276] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexane). Yield, 84%. Yield, 33 g.

[0277] ATX-82: Step 2 [ka]

[0278] To a solution of heptylmagnesium bromide (1.5 eq.) in THF (100 ml) placed in a 1 liter two-necked round bottom flask stirred at 0° C. under nitrogen atmosphere was added a solution of 28 g of N-methoxy-N-methyloctanamide (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0279] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (250 ml) followed by addition of EtOAc (350 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / Hexane). Yield, 22 g; yield, 65%.

[0280] ATX-82: Step 3 [ka]

[0281] To a solution of 22 g of pentadecan-8-one (1 eq.) dissolved in MeOH / THF was added 1.5 eq. of sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0282] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (75 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 20 g; yield, 90%.

[0283] ATX-82: Step 4 [ka]

[0284] To a solution of 15 g of 4-aminobutanoic acid dissolved in 120 ml of THF was added sequentially over a period of 15 minutes using an addition funnel 185 ml of 1N aqueous NaOH at 0° C. followed by 50 ml of Boc anhydride. The resulting solution was stirred at room temperature for 4 hours.

[0285] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 85%.

[0286] ATX-82: Step 5 [ka]

[0287] To a solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (250 ml) cooled to <0 °C, 1.3 equivalents of EDC HCl, Et 3 N, and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of pentadecan-7-ol alcohol was added at the same temperature by dissolving in DCM (150 ml) using an addition funnel and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0288] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (150 ml) and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield, 8 g (crude; required compound and alcohol).

[0289] ATX-82: Step 6 [ka]

[0290] To a solution of 8.0 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate in 60 ml of DCM was added 10 equivalents of TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0291] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (300 ml) and then extracted with EtOAc (2×200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by HPLC using a 50-mL column chromatography (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine) to recover the alcohol. Product amount: 4 g; Yield: 25% for two steps; Confirmed by Mass.

[0292] ATX-82: Step 7 [ka]

[0293] To a solution of 4-bromobutyric acid in DCM (400 ml) cooled to <0 °C, 1.5 equivalents of EDC HCl, 3 equivalents of Et 3 N, and DMAP were added. To this resulting solution, 20 g of (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0294] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (300 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×150 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3The solution was washed with ethyl acetate (200 ml) and then extracted with EtOAc (150 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. The alcohol was recovered. Yield, 55%.

[0295] ATX-82: Step 8 [ka]

[0296] To a solution of 4.0 g of pentadecan-8-yl 4-aminobutanoate, 1 equivalent of (Z)-non-2-en-1-yl 4-bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90° C. for 4 hours under a nitrogen atmosphere.

[0297] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (20 ml) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexane. The starting materials (amine and bromo compound) were recovered. Yield, 2.2 g; Yield, 30%; confirmed by Mass.

[0298] ATX-82: Step 9 [ka]

[0299] To a solution of 2.2 g of pentadecan-8-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 ml of dry DCM was added 3 equivalents of triethylamine and triphosgene at 0° C. at 5 min intervals under nitrogen atmosphere. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0300] To 7 equivalents of sodium hydride dissolved in dry THF (100 ml) in a two-necked 100 ml round bottom flask stirred at 0° C. under nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride were added and stirring was continued for 5 minutes under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (100 ml) was slowly added by syringe for about 10 minutes. The resulting solution was stirred overnight at 0° C. to room temperature under nitrogen atmosphere.

[0301] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (75 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0302] The first purification of the crude compound was performed using silica gel (60-120 mesh), adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.2 g; Yield, 43%; 1 Confirmed by 1 H NMR, HPLC, and Mass.

[0303] [ka]

[0304] Example 7: Synthesis of ATX-86 FIG. 6 shows the synthetic route for ATX-86 (RL-48A), which is further described as follows.

[0305] ATX-86: Step 1 [ka]

[0306] In a 2-liter two-necked round-bottom flask, 30 g of octanoic acid dissolved in DCM (200 ml) was placed, then 1.5 equivalents of oxalyl chloride was added slowly at 0° C. under nitrogen atmosphere with stirring. The resulting reaction mixture was stirred at room temperature for 2 hours. In another 2-liter two-necked round-bottom flask, 2 equivalents of N,O-dimethylhydroxylamine hydrochloride in DCM (200 ml) was added 3 equivalents of trimethylamine using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was added dropwise using an addition funnel by dissolving in DCM (150 ml) under nitrogen atmosphere for 20 minutes. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0307] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexane). Yield, 38 g; Yield, 84%; Mass confirmed.

[0308] ATX-86: Step 2 [ka]

[0309] To a solution of hexylmagnesium bromide (1.5 eq.) in THF (100 ml) placed in a 1 liter two-necked round bottom flask stirred at 0° C. under nitrogen atmosphere was added a solution of 38 g of N-methoxy-N-methyloctanamide (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0310] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (250 ml) and then EtOAc (350 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was purified by filtration (60-120 mesh silica gel). The product was subjected to column chromatography using 2% EtOAc / hexane (hexane / hexane). Yield: 65%. Confirmed by mass.

[0311] ATX-86: Step 3 [ka]

[0312] To a solution of 44 g of tridecan-7-one (1 eq.) dissolved in MeOH / THF was added 1.5 eq. of sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0313] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (75 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 40 g; Yield, 90%; confirmed by Mass.

[0314] ATX-86: Step 4 [ka]

[0315] To a solution of 50 g of 4-aminobutanoic acid dissolved in 350 ml of THF was added sequentially over a period of 15 minutes using an addition funnel 490 ml of 1N aqueous NaOH at 0° C. followed by 140 ml of Boc anhydride. The resulting solution was stirred at room temperature for 4 hours.

[0316] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 80 g; Yield, 81%; confirmed by Mass.

[0317] ATX-86: Step 5 [ka]

[0318] To a solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (250 ml) cooled to <0 °C, 1.3 equivalents of EDC HCl, Et 3 N, and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of pentadecan-7-ol alcohol was added at the same temperature by dissolving in DCM (150 ml) using an addition funnel and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0319] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3The solution was washed with ethyl acetate (150 ml) and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield, 8 g (crude; required compound and alcohol).

[0320] ATX-86: Step 6 [ka]

[0321] To a solution of 8.0 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate in 60 ml of DCM was added 10 equivalents of TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0322] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (300 ml) and then extracted with EtOAc (2×200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by HPLC using a 50-mL column chromatography (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 mL of triethylamine) to recover the alcohol. Yield: 3.5 g; Yield: 52% for two steps; Confirmed by Mass.

[0323] ATX-86: Step 7 [ka]

[0324] To a solution of 4-bromobutyric acid in DCM (400 ml) cooled to <0 °C, 1.5 equivalents of EDC HCl, 2 equivalents of Et 3N, and DMAP were added. To this resulting solution, 20 g of (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0325] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (300 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×150 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (200 ml) and then extracted with EtOAc (150 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. The alcohol was recovered. Yield, 55%.

[0326] ATX-86: Step 8 [ka]

[0327] To a solution of 4.0 g of tridecan-7-yl 4-aminobutanoate, 1 equivalent of (Z)-non-2-en-1-yl 4-bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90° C. for 4 hours under a nitrogen atmosphere.

[0328] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (20 ml) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexane. The starting materials, amine and bromo compound were recovered. Yield, 2.2 g; Yield, 30%; confirmed by Mass.

[0329] ATX-86: Step 9 [ka]

[0330] To a solution of 2.2 g of tridecan-7-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 ml of dry DCM was added 3 equivalents of triethylamine and triphosgene at 0° C. at 5 min intervals under nitrogen atmosphere. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0331] To 7 equivalents of sodium hydride dissolved in dry THF (100 ml) in a two-necked 100 ml round bottom flask stirred at 0° C. under nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride were added and stirring was continued for 5 minutes under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (100 ml) was slowly added by syringe for about 10 minutes. The resulting solution was stirred overnight at 0° C. to room temperature under nitrogen atmosphere.

[0332] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (75 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0333] The first purification of the crude compound was performed using silica gel (60-120 mesh), adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.2 g; Yield, 43%; 1Confirmed by 1 H NMR, HPLC, and Mass.

[0334] [ka]

[0335] Example 8: Synthesis of ATX-87 FIG. 7 shows a synthetic route to ATX-87 (RL-48C) which includes nine steps.

[0336] ATX-87: Step 1 [ka]

[0337] In a 2-liter two-necked round-bottom flask, 20 g of octanoic acid dissolved in DCM (200 ml) was placed, then 1.5 equivalents of oxalyl chloride was added slowly at 0° C. under nitrogen atmosphere with stirring. The resulting reaction mixture was stirred at room temperature for 2 hours. In another 2-liter two-necked round-bottom flask, 2 equivalents of N,O-dimethylhydroxylamine hydrochloride in DCM (200 ml) was added 3 equivalents of trimethylamine using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was added dropwise using an addition funnel by dissolving in DCM (150 ml) under nitrogen atmosphere for 20 minutes. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0338] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexane). Yield, 84%. Yield, 20 g.

[0339] ATX-87: Step 2 [ka]

[0340] To a solution of hexylmagnesium bromide (1.5 eq.) in THF (100 ml) placed in a 1 liter two-necked round bottom flask stirred at 0° C. under nitrogen atmosphere was added a solution of 20 g of N-methoxy-N-methyloctanamide (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0341] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (250 ml) followed by addition of EtOAc (350 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / Hexane). Yield, 25 g; yield, 65%.

[0342] ATX-87: Step 3 [ka]

[0343] To a solution of 25 g of tridecan-7-one (1 eq.) dissolved in MeOH / THF was added 1.5 eq. of sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0344] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (75 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (150 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×100 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 22 g; Yield, 90%.

[0345] ATX-87: Step 4 [ka]

[0346] To a solution of 50 g of 4-aminobutanoic acid dissolved in 350 ml of THF was added 490 ml of 1N aqueous NaOH solution at 0° C. followed by 140 ml of Boc anhydride sequentially using an addition funnel over a period of 15 minutes. The resulting solution was stirred at room temperature for 4 hours.

[0347] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 81%.

[0348] ATX-87: Step 5 [ka]

[0349] To a solution of 17 g of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (250 ml) cooled to <0 °C, 1.3 equivalents of EDC HCl, Et 3 N, and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of tridecan-7-ol was added at the same temperature by dissolving it in DCM (150 ml) using an addition funnel and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0350] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO3 The solution was washed with ethyl acetate (150 ml) and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield, 15 g (crude; required compound and alcohol).

[0351] ATX-87: Step 6 [ka]

[0352] To a solution of 15.0 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate in 80 ml of DCM was added 10 equivalents of TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0353] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (300 ml) and then extracted with EtOAc (2×200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was purified by HPLC using a 50-mL column chromatography (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 mL of triethylamine) to recover the alcohol. Product amount: 7 g; Yield: 24% for two steps; Confirmed by Mass.

[0354] ATX-87: Step 7 [ka]

[0355] To a solution of 4-bromobutyric acid in DCM (400 ml) cooled to <0 °C, 1.5 equivalents of EDC HCl, 2 equivalents of Et 3 N, and DMAP were added to the resulting solution using an addition funnel. 20 g of (Z)-non-2-en-1-ol was added by dissolving in CM and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0356] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (300 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×150 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (200 ml) and then extracted with EtOAc (150 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / Hexanes. The alcohol was recovered. Yield, 55%.

[0357] ATX-87: Step 8 [ka]

[0358] To a solution of 4.0 g of tridecan-7-yl 4-aminobutanoate, 1 equivalent of (Z)-non-2-en-1-yl 4-bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90° C. for 4 hours under a nitrogen atmosphere.

[0359] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (20 ml) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / Hexane. The starting materials, amine and bromo compound were recovered. Yield, 2.2 g; Yield, 30%; confirmed by Mass.

[0360] ATX-87: Step 9 [ka]

[0361] To a solution of 2.2 g of tridecan-7-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 ml of dry DCM was added 3 equivalents of triethylamine and triphosgene at 0° C. at 5 min intervals under nitrogen atmosphere. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0362] To 7 equivalents of sodium hydride dissolved in dry THF (100 ml) in a two-necked 100 ml round bottom flask stirred at 0° C. under nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride were added and stirring was continued for 5 minutes under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (100 ml) was slowly added by syringe for about 10 minutes. The resulting solution was stirred overnight at 0° C. to room temperature under nitrogen atmosphere.

[0363] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (75 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0364] The first purification of the crude compound was performed using silica gel (60-120 mesh), adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.2 g; Yield, 43%; 1 Confirmed by 1 H NMR, HPLC, and Mass.

[0365] [ka]

[0366] Example 9: Synthesis of ATX-88 FIG. 8 shows the synthetic route for ATX-88 (RL-48D), which is further described as follows.

[0367] ATX-88: Step 1 [ka]

[0368] N 2 In a 500 ml two-necked round bottom flask under atmosphere, 25 g of 8-bromooctanoic acid (1 eq.) dissolved in 200 ml of DCM was placed and then slowly added with stirring under nitrogen to 1.5 eq. of oxalyl chloride at 0° C. The resulting reaction mixture was stirred at room temperature for 2 hours.

[0369] In another 1-liter two-neck round-bottom flask, 2 equivalents of N,O-dimethylhydroxylamine hydrochloride in 300 ml of DCM was added with 3 equivalents of trimethylamine and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride was added dropwise using an addition funnel over 15 minutes by dissolving in 500 ml of DCM after concentrating under reduced pressure. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.

[0370] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (300 ml). The organic layer was separated and the aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure.

[0371] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / Hexanes). Yield: 21 g; Yield: 66%.

[0372] ATX-88: Step 2 [ka]

[0373] To a solution of 1.3 equivalents of octylmagnesium bromide in THF (100 ml) stirred at 0° C. under nitrogen atmosphere, 20 g of N-methoxy-N-methyloctanamide in 100 ml of THF was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0374] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.7). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (100 ml). The organic layer was separated and The layers were washed with EtOAc (2×100 ml) and the combined organic layers were concentrated under reduced pressure.

[0375] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% ethyl acetate / hexane). The yield was 17.4 g; 68%.

[0376] ATX-88: Step 3 [ka]

[0377] To a solution of 17 g hexadecan-7-one (1 eq.) dissolved in 135 ml MeOH / THF was added 1.5 eq. sodium borohydride at 0° C. and the resulting solution was stirred at room temperature for 1 h.

[0378] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mass was washed with saturated NH 4Quenched with Cl solution (50 ml). Methanol was reduced under reduced pressure. The crude was partitioned between EtOAc (200 ml) and water. The organic layer was separated and the aqueous layer was washed with EtOAc (2×80 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 14.5 g; yield, 85%.

[0379] ATX-88: Step 4 [ka]

[0380] To a solution of 50 g of 4-aminobutanoic acid dissolved in 350 ml of THF, 490 ml of 1N aqueous NaOH was added at 0° C., followed by 140 ml of Boc anhydride using a funnel. The resulting solution was stirred at room temperature for 4 hours.

[0381] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 ml) and then EtOAc (200 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 81%.

[0382] ATX-88: Step 5 [ka]

[0383] To a solution of 1 equivalent of 4-((tert-butoxycarbonyl)amino)butanoic acid in DCM (200 ml) cooled to <0 °C, 3 equivalents of EDC HCl, Et 3 N (3 eq.), and DMAP (0.1 eq.) were added. To this resulting solution, the alcohol was added by dissolving in DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0384] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mass was quenched with water (100 ml) and the organic layer was separated. The aqueous layer was washed with DCM (2×50 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with EtOAc (100 ml) and the organic layer was separated, concentrated under reduced pressure and carried onto the next step crude. Yield: 19 g (crude).

[0385] ATX-88: Step 6 [ka]

[0386] To a solution of 19 g of hexadecan-7-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 140 ml of DCM was added 10 eq. of TFA at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0387] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The obtained crude was dissolved in saturated NaHCO 3 The solution was washed with ethyl acetate (100 ml) and EtOAc (100 ml) was added. The organic layer was separated and concentrated under reduced pressure.

[0388] The crude compound was purified by HPLC using a HPLC-MS chromatography system (60-120 mesh silica gel; 4% MeOH / CHCl 3 The alcohol was recovered by column chromatography using 1,000 ml of ...

[0389] ATX-88: Step 7 [ka]

[0390] To a solution of 30 g of 4-bromobutyric acid (1 equiv.) in DCM (500 ml) cooled to 0 °C was added successively at 10 min intervals 1.5 equiv. of EDC HCl, 3 equiv. of Et 3 N, and 0.1 equivalents of DMAP were added. To this resulting solution, 0.7 equivalents of (Z)-non-2-en-1-ol was added by dissolving in 100 ml of DCM using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0391] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.7). The reaction mass was quenched with water (100 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was purified by elution with saturated NaHCO 3 The solution was washed with EtOAc (150 ml) and the organic layer was separated and concentrated under reduced pressure.

[0392] The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 5% EtOAc / hexanes). Yield: 27 g; Yield: 51%. 1 Confirmed by 1 H NMR.

[0393] ATX-88: Step 8 [ka]

[0394] To a solution of 6 g of hexadecan-8-yl 4-aminobutanoate (1 eq.), 1 eq. of 5(Z)-non-2-en-1-yl 4-bromobutanoate in ACN (70 ml) was added 1.2 eq. of potassium carbonate and the resultant was refluxed at 90° C. for 3 h under nitrogen atmosphere.

[0395] The progress of the reaction was monitored by TLC (10% MeOH / CHCl 3 ;Rf:0.5) The reaction mass was filtered and the filtrate was concentrated under reduced pressure.

[0396] The crude compound was subjected to column chromatography using (100-200 mesh silica gel; 15% EtOAc / hexane). Product amount: 4.5 g; Yield: 44%; confirmed by mass.

[0397] ATX-88: Step 9 [ka]

[0398] To a solution of 4.4 g of (Z)-non-2-en-1-yl 4-((4-oxo-4-(tetradecan-7-yloxy)butyl)amino)butanoate (1 eq.) dissolved in 30 ml of dry DCM was added 0.83 ml of trimethylamine (3 eq.) and 418 mg of triphosgene (0.5 eq.) at 0° C. under nitrogen atmosphere at intervals of 5 minutes. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0399] To 192 mg of sodium hydride (10 eq.) dissolved in dry THF (25 ml) in a two-necked 100 ml round bottom flask, 564 mg of 2-(dimethylamino)propane-1-thiol hydrochloride (5 eq.) was added at 0° C. and kept stirring for 5 min under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in THF (35 ml) was slowly added by syringe for about 10 min. The resulting solution was stirred at room temperature under nitrogen atmosphere for 4 h.

[0400] The progress of the reaction was monitored by TLC (60% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (30 ml) and then EtOAc (100 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×50 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0401] The first purification was performed using silica gel (60-120 mesh). 5.0 g of crude compound was adsorbed onto 9 g of silica gel and poured onto 90 g of silica gel in a column. The compound was eluted with 35% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. 1.5 g of crude compound was adsorbed onto 4 g of neutral alumina and poured onto 40 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / Hexanes. Yield, 1.2 g; Yield, 21%; 1 Confirmed by H NMR, HPLC, and Mass.

[0402] [ka]

[0403] Example 10: Synthesis of ATX-83 FIG. 9 shows the synthetic route for ATX-83 (RL-47B), which is further described as follows.

[0404] ATX-83: Step 1 [ka]

[0405] In a 500 ml single neck round bottom flask was placed 50 g of octanoic acid (1 eq.) dissolved in DCM (200 mL), then 44.6 ml of oxalyl chloride (1.5 eq.) was added slowly via addition funnel at 0° C. and stirred under nitrogen atmosphere, followed by addition of 1 ml of DMF (catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

[0406] In another 2-liter two-necked round bottom flask, 67.4 g of N,O-dimethylhydroxylamine hydrochloride (2 eq.) in DCM (300 ml) was added with 144 ml of triethylamine (3 eq.) using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride, after concentration under reduced pressure, was added dropwise by dissolving in DCM (350 ml) using an addition funnel over 20 minutes under nitrogen atmosphere. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0407] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was diluted with water (300 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0408] The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 10% EtOAc / Hexanes. Yield, 55.0 g; Yield, 84%.

[0409] ATX-83: Step 2 [ka]

[0410] To a solution of 55 g of heptylmagnesium bromide (1 eq.) in ether placed in a 2 L two-necked round bottom flask and stirred at 0° C. under nitrogen, a solution of 89.6 g of N-methoxy-N-methyloctanamide (1.5 eq.) dissolved in 400 ml of dry ether was added, and the resulting reaction solution was stirred at room temperature for 4 hours.

[0411] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7; PMA char). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (250 ml). The organic layer was separated and the aqueous layer was washed with ether (2×100 ml). The combined organic layers were washed with anhydrous Na 2 SO4 and concentrated under reduced pressure.

[0412] The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc / Hexanes. Yield, 50.0 g; Yield, 75%.

[0413] ATX-83: Step 3 [ka]

[0414] To a solution of 50 g of pentadecan-8-one (1 eq.) dissolved in 290 ml of MeOH / THF was added 12.5 g of sodium borohydride (1.5 eq.) at 0° C. and the resulting solution was stirred at room temperature for 2 hours.

[0415] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (80 ml). The solvent was removed under reduced pressure and the crude material obtained was partitioned between EtOAc (250 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (3×80 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 Drying at rt, concentrating under reduced pressure and drying under vacuum gave a white solid: Yield, 46.0 g; Yield, 90%.

[0416] ATX-83: Step 4 [ka]

[0417] To a solution of 50 g of 4-aminobutanoic acid (1 eq.) dissolved in THF was added 490 ml of 1N aqueous NaOH (1 eq.) at 0° C. followed by 140 ml of Boc anhydride (1.3 eq.) sequentially over a period of 15 min using an addition funnel. The resulting solution was stirred at room temperature for 15 min. The mixture was stirred for 4 hours.

[0418] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (350 ml) and then EtOAc (300 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×150 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 It was dried at 40° C. and concentrated under reduced pressure to give a sticky liquid: Yield, 77.0 g; Yield, 78%.

[0419] ATX-83: Step 5 [ka]

[0420] The synthesis was carried out in four batches. In each, a solution of 23 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equiv.) in DCM (400 ml) cooled to <0 °C was successively added, at 10 min intervals, under a nitrogen atmosphere, with 32.3 g of EDC·HCl (1.5 equiv.), 47 ml of Et 3 N (3 eq.) and 1.3 g of DMAP (0.1 eq.) were added. To this resulting solution, 20 g of pentadecan-8-ol (0.77 eq.) was added by dissolving in DCM (200 ml) using an addition funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.

[0421] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.4). The reaction mass was quenched with water (250 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The resulting crude was dissolved in saturated NaHCO 3 The organic layer was separated and washed with anhydrous Na 2 SO 4 It was dried at 40° C., concentrated under reduced pressure and then carried onto the next step crude. Yield: 105 g (crude; desired compound and alcohol).

[0422] ATX-83: Step 6 [ka]

[0423] To a solution of 105 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 450 ml of DCM was added 194 ml of TFA (10 eq.) at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0424] The progress of the reaction was monitored by TLC (CHCl 3 The reaction was monitored by elution with 10% MeOH (Rf: 0.3).

[0425] The reaction mass was concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was stirred for 10 min with 200 ml of EtOAc and then with 300 ml of EtOAc. The organic layer was separated and the aqueous layer was washed with 2×100 ml of EtOAc. The combined organic layers were washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0426] The crude compound was dissolved in 4% MeOH / CHCl 3 and subjected to column chromatography (silica gel 60-120 mesh) using 1 ml of triethylamine. Product amount: 60.0 g for two steps; yield: 54%.

[0427] ATX-83: Step 7 [ka]

[0428] The reaction was carried out in two batches, in which a solution of 20 g of 6-bromohexanoic acid (1 equiv.) in DCM (300 ml) cooled to <0 °C was successively treated with 29.3 g of EDC·HCl (1.5 equiv.), 42.8 ml of Et 3N (3 eq.), and 1.2 g of DMAP (0.1 eq.) were added. To this resulting solution, 14.5 g of (Z)-non-2-en-1-ol (1 eq.) was added using an addition funnel (by dissolving in 100 ml of DCM) and stirred at room temperature under nitrogen for 24 hours.

[0429] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (200 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (150 ml) and then extracted with EtOAc (2×150 ml). The organic layer was separated and washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0430] The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc / Hexanes. The alcohol reactant was collected. Yield, 55%. Yield, 36.0 g.

[0431] ATX-83: Step 8 [ka]

[0432] The reaction was carried out in 6 batches: in each, to a solution of 10 g of pentadecan-8-yl 4-aminobutanoate (Int 6, 1 eq.), 10.1 g of (Z)-non-2-en-1-yl 6-bromohexanoate (Int 7, 1 eq.) in 120 ml of ACN, 6.1 g of anhydrous potassium carbonate (1.4 eq.) was added, and the resulting mixture was refluxed at 90° C. for 4 hours under nitrogen atmosphere.

[0433] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (2×20 ml) and the filtrate was concentrated under reduced pressure.

[0434] The crude compound was subjected to column chromatography (silica gel 100-200 mesh) using 20-80% EtOAc / Hexanes. The starting material was recovered. Yield, 35%.

[0435] ATX-83: Step 9 [ka]

[0436] The reaction was carried out in three batches. In each, to a solution of 10 g of (Z)-non-2-en-1-yl 6-((4-oxo-4-(pentadecan-8-yloxy)butyl)amino)hexanoate (1 eq.) dissolved in 100 ml of dry DCM, 7.5 ml of triethylamine (3 eq.) and 2.68 g of triphosgene (0.5 eq.) were added at 0° C. under nitrogen atmosphere at 5 min intervals. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 h. The resulting reaction mass was concentrated under reduced pressure and kept under nitrogen atmosphere.

[0437] To a suspension of 3 g of sodium hydride (7 eq.) in dry THF (100 ml) in a two-necked 500 ml RB flask stirred at 0° C. under nitrogen atmosphere, 8.9 g of 2-(dimethylamino)ethane-1-thiol hydrochloride (3.5 eq.) was added and stirring was continued for 5 min under nitrogen atmosphere. To this resulting solution, the above carbamoyl chloride dissolved in dry THF (200 ml) was slowly added by syringe over about 10 min. The resulting solution was stirred overnight at room temperature under nitrogen atmosphere.

[0438] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (100 ml) and then EtOAc (350 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×80 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0439] The first purification was performed using neutral alumina. The crude compound dissolved in hexane was loaded on top of neutral alumina (700 g was loaded on column). The compound was eluted with 8-10% EtOAc / Hexane. The second purification was performed using silica gel (100-200 mesh). The compound dissolved in hexane was loaded on top of silica gel (500 g was loaded on column). The compound was eluted with 20-25% EtOAc / Hexane. The final compound (dissolved in hexane) was subjected to charcoal treatment (200 mg / g) and filtered (after stirring for 20 min) through a celite bed and then through a syringe end membrane filter (PTFE; 0.2 micron, 25 mm diameter). The resulting filtrate was concentrated under reduced pressure. Yield, 15.5 g; yield , 41%.

[0440] [ka]

[0441] Example 11: Synthesis of ATX-84 FIG. 10 shows the synthetic route for ATX-84 (RL-47C), which is further described as follows.

[0442] ATX-84: Step 1 [ka]

[0443] In a 500 ml single neck round bottom flask, 30 g of heptanoic acid (1 eq.) dissolved in DCM (200 mL) was placed and then 26.7 g of oxalyl chloride (1.5 eq.) was slowly added under stirring under nitrogen atmosphere at 0° C., followed by 1 ml of DMF (catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

[0444] In another 1 L two-necked round bottom flask, 40.5 g of N,O-dimethylhydroxylamine hydrochloride (2 eq.) in DCM (250 ml) was added with 86.6 ml of trimethylamine (3 eq.) using an addition funnel and stirred at 0° C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was added dropwise using an addition funnel by dissolving in DCM (100 ml) over 20 minutes under nitrogen atmosphere. The resulting reaction solution was stirred at room temperature under nitrogen atmosphere for 3 hours.

[0445] The progress of the reaction was monitored by TLC (20% EtOAc / Hexane; Rf: 0.5). The reaction mass was diluted with water (250 ml). The organic layer was separated and the aqueous layer was washed with DCM (3×100 ml). The combined organic layers were concentrated under reduced pressure.

[0446] The crude compound was subjected to column chromatography using (60-120 silica gel) with 10% EtOAc / Hexanes. Yield, 38.0 g; Yield, 84%.

[0447] ATX-84: Step 2 [ka]

[0448] To a solution of 8 g of hexylmagnesium bromide (1 eq.) in 250 ml of dry ether placed in a 1-liter two-necked round-bottom flask stirred at 0° C. under nitrogen atmosphere, 2.3 g of N-methoxy-N-methylheptanamide (0.5 eq.) dissolved in 250 ml of ether was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

[0449] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (200 ml). The organic layer was separated and the aqueous layer was washed with ether (2×100 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0450] The crude compound was subjected to column chromatography using (60-120 mesh silica gel) with 2% EtOAc / Hexanes. Yield, 30.8 g; Yield, 71%.

[0451] ATX-84: Step 3 [ka]

[0452] To a solution of 30 g of tridecan-7-one (1 eq.) dissolved in 200 ml of MeOH / THF was added 8.5 g of sodium borohydride (0.5 eq.) at 0° C. and the resulting solution was stirred at room temperature for 2 hours.

[0453] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mass was washed with saturated NH 4 Quenched with Cl solution (80 ml). The solvent was removed under reduced pressure and the crude was partitioned between EtOAc (200 ml) and water (100 ml). The organic layer was separated and the aqueous layer was washed with EtOAc (2×70 ml). The combined organic layers were concentrated under reduced pressure to give a white solid. Yield, 27.2 g; Yield, 90%.

[0454] ATX-84: Step 4 [ka]

[0455] To a solution of 5 g of 6-aminohexanoic acid (1 eq.) dissolved in 120 ml of THF was added sequentially over a period of 15 min using an addition funnel 125 ml of 1N aqueous NaOH at 0° C. followed by 34 ml of Boc anhydride (1.3 eq.) The resulting solution was stirred at room temperature for 4 h.

[0456] The progress of the reaction was monitored by TLC (CHCl 3in 10% MeOH; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 ml) and then EtOAc (150 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2×100 ml). The combined organic layers were concentrated under reduced pressure to give a viscous liquid. Yield, 22.4 g; yield, 85%.

[0457] ATX-84: Step 5 [ka]

[0458] To a solution of 10 g of 6-((tert-butoxycarbonyl)amino)hexanoic acid (1 equiv.) in DCM (200 ml) cooled to <0 °C, 10.7 g of EDC HCl (1.3 equiv.), 18 ml of Et 3 N (3 eq.), and 525 mg of DMAP (0.1 eq.) were added. Using an addition funnel, 6 g of tridecan-7-ol (Int 3, 0.7 eq.) was added at the same temperature by dissolving in DCM (50 ml) and stirred at room temperature for 24 hours under nitrogen atmosphere.

[0459] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.4). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×75 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with 100 ml of ethyl acetate and then extracted with 2×100 ml of EtOAc. The organic layer was separated, concentrated under reduced pressure and carried on crude to the next step. Yield: 8.5 g (crude; desired compound and alcohol).

[0460] ATX-84: Step 6 [ka]

[0461] To a solution of 10 g of tridecan-7-yl 6-((tert-butoxycarbonyl)amino)hexanoate (1 eq.) dissolved in 65 ml of DCM was added 18.5 ml of TFA (10 eq.) at 0° C. and stirred at room temperature under nitrogen for 3 hours.

[0462] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The crude obtained was diluted with saturated NaHCO 3 The solution was washed with ethyl acetate (100 ml) and then extracted with EtOAc (3×100 ml). The organic layer was separated and concentrated under reduced pressure.

[0463] The crude compound was purified by HPLC using a HPLC-MS chromatography system (60-120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine) to recover the alcohol starting material. Quantity, 4.5 g for two steps; Yield, 33%.

[0464] ATX-84: Step 7 [ka]

[0465] To a solution of 20 g of 6-bromohexanoic acid (1 equiv.) in DCM (300 ml) cooled to <0 °C, 29.3 g of EDC HCl (1.5 equiv.), 42.8 ml of Et 3 N (3 eq.), and 1.2 g of DMAP (0.1 eq.) were added. To this resulting solution, using an addition funnel, 14.5 g of (Z)-non-2-en-1-ol (1 eq.) was added, dissolved in 100 ml of DCM, and stirred at room temperature under nitrogen for 24 hours.

[0466] The progress of the reaction was monitored by TLC (10% EtOAc in hexanes; Rf: 0.7). The reaction mass was quenched with water (200 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2×100 ml). The combined organic layers were concentrated under reduced pressure. The crude obtained was washed with saturated NaHCO 3 The solution was washed with ethyl acetate (150 ml) and then extracted with EtOAc (2×150 ml). The organic layer was separated and washed with anhydrous Na 2 SO 4 and concentrated under reduced pressure.

[0467] The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc / Hexanes. The alcohol starting material was recovered. Yield, 55%, yield, 18.0 g.

[0468] ATX-84: Step 8 [ka]

[0469] To a solution of 4.5 g of tridecan-7-yl 6-aminohexanoate (Int 6, 1 eq.) and 4.5 g of (Z)-non-2-en-1-yl 6-bromohexanoate (Int 7, 1 eq.) in 90 ml of ACN, 2.7 g of potassium carbonate (1.4 eq.) was added and the resulting mixture was refluxed at 90° C. for 4 hours under nitrogen atmosphere.

[0470] The progress of the reaction was monitored by TLC (CHCl 3 in 10% MeOH; Rf: 0.5). The reaction mass was filtered, washed with ACN (2×20 ml) and the filtrate was concentrated under reduced pressure.

[0471] The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 20% ​​EtOAc / Hexanes. The starting material was recovered. Yield, 37%.

[0472] ATX-84: Step 9 [ka]

[0473] To a solution of 2.5 g of (Z)-non-2-en-1-yl 6-((6-oxo-6-(tridecan-7-yloxy)hexyl)amino)hexanoate (1 eq.) dissolved in 30 ml of dry DCM, 1.8 ml of triethylamine (3 eq.) and 672 mg of triphosgene (0.5 eq.) were added at 0° C. under nitrogen atmosphere at intervals of 5 minutes. The resulting solution was stirred at room temperature under nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and cooled to room temperature under nitrogen atmosphere. The mixture was maintained under atmosphere.

[0474] To a suspension of 761 mg of sodium hydride in dry THF (50 ml) in a two-necked 250 ml round bottom flask stirred at 0° C. under nitrogen atmosphere, 2.2 g of 2-(dimethylamino)ethane-1-thiol hydrochloride (3.5 eq.) was added and stirring was continued for 5 min under nitrogen atmosphere. To the resulting solution, the above carbamoyl chloride dissolved in THF (60 ml) was slowly added by syringe over about 10 min. The resulting solution was stirred overnight at room temperature under nitrogen atmosphere.

[0475] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5; PMA char). The reaction mass was washed with saturated NH 4 It was quenched with Cl solution (60 ml) and then EtOAc (130 ml) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3×40 ml). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

[0476] The first purification was performed using silica gel (100-200 mesh). 4.6 g of crude compound was adsorbed onto 10.0 g of silica gel and poured onto 90.0 g of silica gel in a column. The compound was eluted with 50% EtOAc / Hexanes. The second purification was performed using neutral alumina with HPLC grade solvents. 2.0 g of crude compound was adsorbed onto 6.0 g of neutral alumina and poured onto 40.0 g of neutral alumina in a column. The compound was eluted with 20% EtOAc / Hexanes. Yield, 1.2 g; yield, 38% (300 mg mixture).

[0477] [ka]

[0478] Example 12: Synthesis of ATX-61 FIG. 10 shows the synthetic route for ATX-61(RL-42D), which is further described as follows.

[0479] ATX-61: Step 1 [ka]

[0480] 12 g of glycine ester (1 eq.) was dissolved in THF (100 ml) and cooled to below 0° C. To this solution were added sequentially through an addition funnel 24.2 ml of triethylamine (1.5 eq.) and 38.11 g of Boc anhydride (1.5 eq.).

[0481] The progress of the reaction was monitored by TLC using 50% EtOAc / Hexane; Rf: 0.4.

[0482] After 16 hours, the reaction mass was quenched with water and EtOAc (100 ml) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2×40 ml) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0483] The crude product was subjected to 60-120 silica gel (25% EtOAc / hexanes): Yield, 20.8 g;

[0484] ATX-61: Step 2 [ka]

[0485] To a solution of 18.9 g of N-Boc glycine ester (1 eq.) dissolved in THF (130 ml) was added an aqueous solution of 5.85 g of LiOH (1.5 eq.) and the resulting solution was stirred at room temperature for 4 h.

[0486] The reaction was monitored by TLC (60% EtOAc / Hexanes; Rf: 0.3) and no SM was present.

[0487] The reaction mass was concentrated, and the crude mass was quenched with 5% HCl (pH 3), then extracted with EtOAc (4×80 ml), dried over sodium sulfate, and concentrated under reduced pressure to obtain compound. Yield: 15 g; Yield: 92%; Confirmed by Mass.

[0488] ATX-61: Step 3 [ka]

[0489] To a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (30 ml) and cooled to below 0° C., 4.5 ml of Et 3 N (1.2 equiv.) and 6.44 g of EDC·HCl (1.2 equiv.) were added to the reaction solution, and 5.12 g of heptaden-9-ol (0.7 equiv.) in 20 ml of DCM was added and stirred at room temperature overnight.

[0490] No starting material was observed by TLC (10% EtOAc / Hexanes; Rf: 0.6). The reaction mass was washed with saturated NaHCO 3The solution was diluted, the organic layer was separated and the aqueous layer was washed with DCM (2×30 ml), dried over sodium sulfate and concentrated under reduced pressure. The crude material (6.8 g; mixture of product and alcohol) was carried on to the next step.

[0491] ATX-61: Step 4 [ka]

[0492] 4 g of heptadecan-9-yl(tert-butoxycarbonyl)glycinate (Int 2, 1 eq.) was dissolved in DCM (40 ml), cooled to 0° C., 7.4 ml of TFA (10 eq.) was added and stirred at room temperature for 1 h.

[0493] The reaction was complete in 2 hours by TLC (10% EtOAc / Hexane; Rf: 0.5). I acknowledged.

[0494] The reaction mass was concentrated under reduced pressure and the residual mass was washed with saturated sodium bicarbonate solution (30 ml) and extracted with EtOAc (3×30 ml), the organic layer was dried over sodium sulfate and concentrated under reduced pressure to give Int 3.

[0495] The crude product was dissolved in 1-3% MeOH / CHCl 3 and 2 mL of Et 3 The product was subjected to column chromatography (silica, 60-120) using N. Yield: 1 g; 1 Confirmed by H-NMR and Mass.

[0496] ATX-61: Step 5 [ka]

[0497] To a solution of 4 g of bromoacetic acid (1 equiv.) dissolved in DCM (35 ml) and cooled to below 0° C., 4.7 ml of Et 3N (1.2 eq.) and 354 mg of DMAP (0.1 eq.) were added, followed by 13.23 g of HATU (1.2 eq.) To this reaction solution was added 2.88 g of (Z)-non-2-en-1-ol (0.7 eq.) in 20 ml of DCM and stirred at room temperature overnight.

[0498] The reaction was monitored by TLC (10% EtOAc / Hexanes; Rf: 0.7).

[0499] Add saturated NaHCO 3 It was diluted with ethyl acetate (80 ml), the organic layer was separated, the aqueous layer was washed with DCM (40 ml), dried over sodium sulfate and concentrated under reduced pressure. The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / Hexane). Yield, 52%.

[0500] ATX-61: Step 6 [ka]

[0501] 1 g of heptadecane-9-yl glycinate (Int 3, 1 eq.) was dissolved in THF (25 ml), 0.5 ml of TEA (1.3 eq.) and 1.08 g of (Z)-non-2-en-1-yl 2-bromoacetate derivative (Int 4, 1.3 eq.) were added and stirred at room temperature overnight.

[0502] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.4). The reaction mixture was diluted with water (30 ml) and extracted with EtOAc (20 ml×2), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0503] The residual mass was purified by column (silica gel; 100-200) chromatography (2% EtOAc / hexane). Product amount: 700 mg; Yield: 47%; Mass confirmed.

[0504] ATX-61: Step 7 [ka]

[0505] To a solution of 700 mg of heptadecan-9-yl (Z)-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycinate) (1 eq.) dissolved in 15 ml of DCM and cooled to below 5° C., 0.4 ml of Et 3 N (3 eq.) was added in small portions over a period of 10 min, followed by 209 mg of triphosgene (0.5 eq.).

[0506] The progress of the reaction mixture was monitored by TLC and the reaction was completed in 0.5 h and the reaction mass was concentrated under reduced pressure.

[0507] To a solution of 423 mg of N,N-dimethylethanethiol hydrochloride (3 eq.) in dry THF (10 ml) and DMF (3 ml) stirred at 0° C. under nitrogen atmosphere, 144 mg of sodium hydride (6 eq.) was added. After 10 minutes, the above solution was added to the reaction mass by dissolving it in THF (15 ml). The resulting solution was stirred at room temperature for 1 hour.

[0508] After 1 h, the reaction was confirmed to be complete by TLC (10% MeOH / CHCl 3 ;Rf:0.5).

[0509] Reaction mass was saturated with NH 4 The mixture was quenched with Cl solution (20 ml) and water (20 ml) and EtOAc (30 ml) were added. The aqueous layer was washed with EtOAc (2×20 ml) and the combined organic layers were washed with brine solution (20 ml). The organic layers were washed with Na 2 SO 4 and concentrated under reduced pressure.

[0510] The crude material was subjected to column chromatography using silica gel (100-200) with 15% EtOAc / hexane, followed by neutral alumina with 15% EtOAc / hexane to give the pure compound. Yield, 520 mg; Yield, 58%.1 The product was confirmed by H-NMR, HPLC, and Mass.

[0511] [ka]

[0512] Example 13: Synthesis of ATX-63 FIG. 12 shows the synthetic route for ATX-63 (RL-42A), which is further described as follows.

[0513] ATX-63: Step 1 [ka]

[0514] 12 g of glycine ester (1 eq.) was dissolved in THF (100 ml) and cooled to below 0° C. To this solution were added sequentially through an addition funnel 24.2 ml of triethylamine (1.5 eq.) and 38.11 g of Boc anhydride (1.5 eq.).

[0515] The progress of the reaction was monitored by TLC using 50% EtOAc / Hexane; Rf: 0.4.

[0516] After 16 hours, the reaction mass was quenched with water and EtOAc (100 ml) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2×40 ml) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0517] The crude product was subjected to 60-120 silica gel (25% EtOAc / hexanes): Yield, 20.8 g;

[0518] ATX-63: Step 2 [ka]

[0519] To a solution of 18.9 g of N-Boc glycine ester (1 eq.) dissolved in THF (130 ml) was added an aqueous solution of 5.85 g of LiOH (1.5 eq.) and the resulting solution was stirred at room temperature for 4 h.

[0520] The reaction was monitored by TLC (60% EtOAc / Hexanes; Rf: 0.3) and no starting material was present in the reaction product.

[0521] The reaction mass was concentrated, and the crude mass was quenched with 5% HCl (pH 3), then extracted with EtOAc (4×80 ml), dried over sodium sulfate, and concentrated under reduced pressure to obtain compound. Yield: 15 g; Yield: 92%; Confirmed by Mass.

[0522] ATX-63: Step 3 [ka]

[0523] To a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (50 ml) and cooled to below 0° C., 4.5 ml of Et 3 N (1.2 equiv.) and 6.4 g of EDC·HCl (1.2 equiv.) were added to the reaction solution, 3.4 g of undecan-6-ol (0.7 equiv.) in 20 ml of DCM was added and stirred at room temperature overnight.

[0524] No starting material was observed by TLC (15% EtOAc / Hexanes; Rf: 0.6). The reaction mass was washed with saturated NaHCO 3 It was diluted with 20 ml of water, the organic layer was separated, the aqueous layer was washed with DCM (2×40 ml), dried over sodium sulfate and concentrated under reduced pressure. After column filtration, the crude material (5.5 g; a mixture of product and alcohol) was carried on to the next step.

[0525] ATX-63: Step 4 [ka]

[0526] 3.3 g of crude undecan-6-yl(tert-butoxycarbonyl)glycinate (Int 2, 1 eq.) was dissolved in DCM (20 ml), cooled to 0° C., 7.6 ml of TFA (10 eq.) was added and stirred at room temperature for 1 h.

[0527] The reaction was completed in 2 hours by TLC (10% MeOH / DCM; Rf: 0.5). The reaction mass was concentrated under reduced pressure and the residual mass was washed with saturated sodium bicarbonate solution (50 ml) and extracted with EtOAc (3×25 ml), the organic layer was dried over sodium sulfate and concentrated under reduced pressure to give Int 3.

[0528] The crude product was dissolved in 1-3% MeOH / CHCl 3 and 2 mL of Et 3 The product was subjected to column chromatography (silica, 60-120) using N. Yield: 40%. 1 Confirmed by H-NMR and Mass.

[0529] ATX-63: Step 5 [ka]

[0530] To a solution of 4 g of bromoacetic acid (1 equiv.) dissolved in DCM (35 ml) and cooled to below 0° C., 4.7 ml of Et 3 N (1.2 eq.) was added, followed by 13.23 g of HATU (1.2 eq.) and 354 mg of DMAP (0.1 eq.) To this reaction solution was added 2.88 g of (Z)-non-2-en-1-ol (0.7 eq.) in 20 mL of DCM and stirred at room temperature overnight.

[0531] The reaction was monitored by TLC (10% EtOAc / Hexanes; Rf: 0.7).

[0532] Add saturated NaHCO 3It was diluted with ethyl acetate (80 ml), the organic layer was separated, the aqueous layer was washed with DCM (40 ml), dried over sodium sulfate and concentrated under reduced pressure. The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / Hexane). Yield, 52%.

[0533] ATX-63: Step 6 [ka]

[0534] 1.2 g of undecane-6-yl glycinate (Int 3, 1 eq.) was dissolved in 25 ml of THF, 0.9 ml of TEA (1.3 eq.) and 1.37 g of (Z)-non-2-en-1-yl 2-bromoacetate (Int 4, 1 eq.) were added and stirred at room temperature overnight.

[0535] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mixture was diluted with water (30 ml) and extracted with EtOAc (20 ml×2), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0536] The residual mass was purified by column (silica gel; 100-200) chromatography (3% EtOAc / hexane). Product amount: 800 mg; Yield: 37%; Mass confirmed.

[0537] ATX-63: Step 7 [ka]

[0538] To a solution of 800 mg of (Z)-non-2-en-1-yl(2-oxo-2-(undecane-6-yloxy)ethyl)glycinate (1 eq.) dissolved in DCM and cooled to <5° C., 0.4 ml of Et 3 N (3 eq.) was added in small portions over a period of 10 min, followed by 209 mg of triphosgene (0.5 eq.).

[0539] The progress of the reaction mixture was monitored by TLC and the reaction was completed in 1 hour and the reaction mass was concentrated under reduced pressure.

[0540] To a solution of 423 mg of N,N-dimethylethanethiol hydrochloride (3 eq.) in dry THF and DMF (10 ml and 5 ml, respectively) stirred at 0° C. under nitrogen atmosphere, 144 mg of sodium hydride (6 eq.) was added. After 10 minutes, the above solution was added to the reaction mass by dissolving it in THF. The resulting solution was stirred at room temperature for 1 hour.

[0541] After 1 h, the reaction was observed to be complete by TLC (70% EtOAc / Hexanes; Rf: 0.4). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (25 ml) and water (20 ml) and EtOAc (20 ml) were added. The aqueous layer was washed with EtOAc (2×20 ml) and the combined organic layers were washed with brine solution (20 ml). The organic layers were washed with Na 2 SO 4 and concentrated under reduced pressure.

[0542] The crude material was purified by column chromatography on silica gel (100-200) with 20% EtOAc / Hexanes, then Column chromatography using neutral alumina with 5% EtOAc / hexane at 20° C. gave the pure compound. Yield: 510 mg; Yield: 48%. 1 The product was confirmed by H-NMR, HPLC, and Mass.

[0543] [ka]

[0544] Example 14: Synthesis of ATX-64 FIG. 13 shows the synthetic route for ATX-64(RL-42C), which is further described as follows.

[0545] ATX-64: Step 1 [ka]

[0546] 12 g of ethyl glycinate (1 eq.) was dissolved in THF (100 ml) and cooled to below 0° C. To this resulting solution were added sequentially through an addition funnel 24.2 ml of triethylamine (1.5 eq.) and 38.11 g of Boc anhydride (1.5 eq.).

[0547] The progress of the reaction was monitored by TLC using 50% EtOAc / Hexane; Rf: 0.4.

[0548] After 16 hours, the reaction mass was quenched with water and EtOAc (100 ml) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2×40 ml) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0549] The crude product was subjected to 60-120 silica gel (25% EtOAc / hexanes): yield, 20.8;

[0550] ATX-64: Step 2 [ka]

[0551] To a solution of 18.9 g of N-Boc glycine ester (1 eq.) dissolved in THF (130 ml) was added an aqueous solution of 5.85 g of LiOH (1.5 eq.) and the resulting solution was stirred at room temperature for 4 h.

[0552] The reaction was monitored by TLC (60% EtOAc / Hexanes; Rf: 0.3) and no starting material was present in the reaction product.

[0553] The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH 3) then extracted with EtOAc (4×80 ml), dried over sodium sulfate and concentrated under reduced pressure to obtain compound. Amount produced: 15 g; yield: 92%; confirmed by mass.

[0554] ATX-64: Step 3 [ka]

[0555] To a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (50 ml) and cooled to below 0° C., 4.5 ml of Et 3 N (1.2 equiv.) and 6.4 g of EDC·HCl (1.2 equiv.) were added to the reaction solution, which was then stirred at room temperature overnight with 4.84 g of hexadecan-10-ol (0.7 equiv.) in 15 ml of DCM.

[0556] No starting material was observed by TLC (15% EtOAc / Hexanes; Rf: 0.6). The reaction mass was washed with saturated NaHCO 3 The solution was diluted, the organic layer separated and the aqueous layer washed with DCM (2×30 ml), dried over sodium sulfate and concentrated under reduced pressure.

[0557] After column filtration, the crude material (5.5 g; mixture of product and alcohol) was carried onto the next step.

[0558] ATX-64: Step 4 [ka]

[0559] 3.85 g of crude heptadecan-9-yl(tert-butoxycarbonyl)glycinate (Int 2, 1 eq.) was dissolved in 30 ml of DCM, cooled to 0° C., 7.4 ml of TFA (10 eq.) was added and stirred at room temperature for 1 h.

[0560] The reaction was complete in 2 hours by TLC (10% MeOH / DCM; Rf: 0.5).

[0561] The reaction mass was concentrated under reduced pressure and the residual mass was washed with saturated sodium bicarbonate solution (30 ml) and extracted with EtOAc (3×30 ml), the organic layer was dried over sodium sulfate and concentrated under reduced pressure to give Int 3.

[0562] The crude product was dissolved in 1-3% MeOH / CHCl 3 and 2 mL of Et 3 The product was subjected to column chromatography (silica, 60-120) using N. Yield: 2.2 g; 1 Confirmed by H-NMR and Mass.

[0563] ATX-64: Step 5 [ka]

[0564] To a solution of 4 g of bromoacetic acid (1 equiv.) dissolved in DCM (35 ml) and cooled to below 0° C., 4.7 ml of Et 3 N (1.2 eq.) was added, followed by 13.23 g of HATU (1.2 eq.) and 354 mg of DMAP (0.1 eq.) To this reaction solution was added 2.88 g of (Z)-non-2-en-1-ol (0.7 eq.) in 20 ml of DCM and stirred at room temperature overnight.

[0565] The reaction was monitored by TLC (10% EtOAc / Hexanes; Rf: 0.7).

[0566] Add saturated NaHCO 3 It was diluted with water (80 ml), the organic layer was separated and the aqueous layer was washed with DCM (40 ml), dried over sodium sulfate and concentrated under reduced pressure.

[0567] The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / Hexane). Yield, 52%.

[0568] ATX-64: Step 6 [ka]

[0569] 2.1 g of hexadecan-8-yl glycinate (Int 3, 1 eq.) was dissolved in 50 ml of THF, 1.2 ml of TEA (1.3 eq.) and 2.39 g of (Z)-non-2-en-1-yl 2-bromoacetate (Int 4, 1.3 eq.) were added and stirred at room temperature overnight.

[0570] The progress of the reaction was monitored by TLC (10% EtOAc / Hexane; Rf: 0.5). The reaction mixture was diluted with water (30 ml) and extracted with EtOAc (2×30 ml), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0571] The residual mass was purified by column (silica gel; 100-200) chromatography (3% EtOAc / hexane). Product amount: 2.2 g; Yield: 65%; Mass confirmed.

[0572] ATX-64: Step 7 [ka]

[0573] 2.2 g of heptadecan-9-yl (Z)-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycinate dissolved in 15 ml of DCM and cooled to below 5° C. ) (1 equiv.) in 1.6 ml of Et 3 N (3 eq.) was added in portions over a period of 10 min, followed by 678 mg of triphosgene (0.5 eq.).

[0574] The progress of the reaction mixture was monitored by TLC and the reaction was completed in 1 hour and the reaction mass was concentrated under reduced pressure.

[0575] To a solution of 3.94 g of N,N-dimethylethanethiol hydrochloride (7 eq.) in dry THF and DMF (35 ml and 15 ml, respectively) stirred at 0° C. under nitrogen atmosphere, 672 mg of sodium hydride (7 eq.) was added. After 10 minutes, the above solution was added to the reaction mass by dissolving it in THF. The resulting solution was stirred at room temperature for 1 hour.

[0576] After 1 h, the reaction was observed to be complete by TLC (70% EtOAc / Hexanes; Rf: 0.4). The reaction mass was washed with saturated NH 4 The mixture was quenched with Cl solution (25 ml) and water (20 ml) and EtOAc (20 ml) were added. The aqueous layer was washed with EtOAc (20 ml x 2) and the combined organic layers were washed with brine solution (20 ml). The organic layers were washed with Na 2 SO 4 and concentrated under reduced pressure.

[0577] The crude material was subjected to column chromatography using silica gel (100-200) with 25% EtOAc / hexane, followed by neutral alumina with 15-20% EtOAc / hexane to give the pure compound. Yield, 1.0 mg; Yield, 40%. 1 The product was confirmed by H-NMR, HPLC, and Mass.

[0578] [ka]

[0579] Example 15: pKa Values The lipids were titrated to determine their pKa values, and the results are shown in the table below. [Table 2-1] [Table 2-2]

[0580] Example 16: EPO mRNA stability in vivo The levels of mRNA in plasma were measured after injection of nanoparticles containing different cationic lipids. were compared. For the evaluation of plasma erythropoietin (EPO) levels in vivo after injection of lipid-encapsulated mouse EPO mRNA, female Balb / c mice (6-8 weeks old) were used. All formulations were administered intravenously via tail vein injection at doses of 0.03 and 0.1 mg / kg in a dosing volume of 5 ml / kg. Terminal blood collection was performed via cardiac puncture under 2% isoflurane 6 hours after formulation injection. Blood was collected in 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 min. Serum was collected and EPO mRNA levels were analyzed. The results are shown in Figure 14. The results show substantial improvement over ATX-2 for ATX-57, ATX-81, ATX-82, ATX-83, ATX-84, ATX-85, ATX-86, and ATX-87.

[0581] Example 17: Silencing of mouse Factor VII and EPO expression in vivo A liver-directed in vivo screen of liposomal libraries was used to test a series of compounds that promote high levels of siRNA-mediated gene silencing in hepatocytes, the cells that make up the liver parenchyma. Factor VII, a blood clotting factor, is a suitable target gene for assaying functional siRNA delivery to the liver. Because this factor is produced specifically in hepatocytes, gene silencing indicates successful delivery to the parenchyma, rather than to cells of the reticuloendothelial system (e.g., Kupffer cells). Furthermore, factor VII is a secreted protein that can be easily measured in serum, eliminating the need to euthanize animals. Silencing at the mRNA level can be easily determined by measuring the levels of the protein. This is because the protein has a short half-life (2-5 hours). Compositions with siRNAs targeting factor VIII were formulated with ATX-ATX-002, ATX-57, and ATX-58, as well as phosphate-buffered saline (PBS) for comparative samples. Female C57BL / 6 mice (6–8 weeks old) were used for FVII siRNA knockdown (KD) experiments.

[0582] All formulations were administered intravenously via tail vein injection at doses of 0.03 and 0.1 mg / kg with a dose volume of 5 mg / kg. Terminal blood collection was performed via cardiac puncture under 2% isoflurane 48 hours after formulation injection. Blood was collected into 0.109 M citrate buffer tubes and processed by centrifugation at 1200G for 10 minutes. Plasma was collected and factor VII protein levels were analyzed by a chromogenic assay (Biophen FVII, Aniara Corporation). Standard curves were constructed using samples from PBS-injected mice to determine relative factor VII expression by comparing treated groups to untreated PBS controls. Results showed that ATX-57 and ATX-58 were substantially more effective than ATX-002 at both 0.03 and 0.1 mg / kg (Figure 15).

[0583] For the evaluation of EPO protein expression in vivo after delivery of lipid-encapsulated mouse EPO mRNA, female Balb / c mice (6-8 weeks old) were used. All formulations were administered intravenously via tail vein injection at doses of 0.03 and 0.1 mg / kg in a dosing volume of 5 mL / kg. Terminal blood collection was performed via cardiac puncture under 2% isoflurane 6 hours after formulation injection. Blood was collected in 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 min. Serum was collected and EPO protein levels were analyzed by EPO ELISA assay (R&D systems). A standard curve was constructed using samples from PBS-injected mice to determine relative factor VII expression by comparing treated groups to untreated PBS controls. Results showed that EPO mRNA was expressed in substantially higher amounts in ATX-57 nanoparticles than in ATX-2 at 0.1 mg / ml (Figure 16).

[0584] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) Formula I: [ka] or a salt or solvate thereof, wherein R 1 is a branched alkyl group consisting of 10 to 31 carbon atoms; R 2 is a straight chain alkyl, alkenyl, or alkynyl of 2 to 20 carbons; R 3 is a linear or branched alkylene of 1 to 6 carbons; R 4 and R 5 are the same or different and each is hydrogen or a linear or branched alkyl group of 1 to 6 carbon atoms; L 1 and L 2 are the same or different, each being a linear alkylene of 1 to 20 carbons or a linear alkenylene of 2 to 20 carbons; X 1 is S or O; The compound, or a salt or solvate thereof. (Section 2) X 1 The compound according to the above item 1, wherein is S. (Section 3) R 3 The compound according to item 1, wherein is ethylene or propylene. (Section 4) R 4 and R 5 and independently are methyl or ethyl. (Section 5) L 2 is alkenyl of 1, 3, or 5 carbons. (Section 6) L 1 is alkenyl of 1, 2, 3, or 5 carbons. (Section 7) R 2 is alkenyl. (Section 8) R 2 is an alkenyl consisting of 9 carbons. (Section 9) R 1 But -CH((CH 2 ) n CH 3 ) 2 8. The compound according to claim 7, wherein n is 4, 5, 6, or 7. (Section 10) n is 5, and L 1 10. The compound according to item 9, wherein is alkenyl consisting of 1 or 3 carbons. (Section 11) n is 6, and L 1 10. The compound according to item 9, wherein is alkenyl consisting of 3 or 5 carbons. (Section 12) n is 7, and L 110. The compound according to claim 9, wherein is alkenyl of 1, 2, or 3 carbons. (Section 13) n is 8, and L 1 10. The compound according to item 9, wherein is alkenyl consisting of 1 or 3 carbons. (Section 14) R 1 But -CH((CH 2 ) n CH 3 )((CH 2 ) n+1 CH 3 8. The compound according to claim 7, wherein n is 6 or 7. (Section 15) Formulas ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88: [ka] [ka] [ka] Item 3. The compound according to item 1, selected from the group consisting of compounds represented by the formula: (Section 16) A pharmaceutical composition comprising the compound according to item 1 above. (Section 17) Item 2. The pharmaceutical composition according to item 1, comprising the compound according to item 1 in lipid nanoparticles. (Section 18) Item 18. The pharmaceutical composition according to item 17, wherein the lipid nanoparticles further comprise a neutral lipid and a conjugated lipid. (Section 19) Item 18. The pharmaceutical composition according to item 17, wherein the lipid nanoparticles encapsulate mRNA. . (Section 20) Item 18. The pharmaceutical composition according to item 17, further comprising an mRNA encoding a biologically active protein. (Section 21) Item 18. The pharmaceutical composition according to item 17, further comprising RNA comprising a nucleotide sequence homologous to an mRNA in a target cell.

Claims

1. 1. A parenteral pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid, the lipid nanoparticles comprising: Formula I: 【Chemistry 127】 or a pharma- ceutically acceptable salt or solvate thereof, During the ceremony, R １ is -CH((CH ２ ) ｎ CH ３ ) ２ (wherein n is 4, 5, 6, or 7), or -CH((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｎ＋１ CH ３ ) wherein n is 6 or 7; R ２ is a straight chain alkenyl of 8 to 10 carbons; R ３ is a linear or branched alkylene of 1 to 6 carbons; R ４ and R ５ are the same or different and each is hydrogen or a straight or branched alkyl of 1 to 6 carbons; L １ and L ２ are the same or different, each a straight chain alkylene of 2 to 5 carbons, or a straight chain alkenylene of 2 to 5 carbons, and X １ is S or O, With the proviso that the cationic lipid is not ATX-0091, ATX-0102, ATX-0095, ATX-0094, ATX-0109, ATX-0122, or ATX-0124; 【Chemistry 200-1】 【Chemistry 200-2】 Parenteral pharmaceutical compositions.

2. X １ The parenteral pharmaceutical composition of claim 1 , wherein is S.

3. R ３ The parenteral pharmaceutical composition of claim 1 , wherein is ethylene or propylene.

4. R ４ and R ５ The parenteral pharmaceutical composition of claim 1 , wherein is independently methyl or ethyl.

5. L ２ The parenteral pharmaceutical composition of claim 1 , wherein is an alkylene of 3 or 5 carbons.

6. L １ The parenteral pharmaceutical composition of claim 5, wherein is an alkylene of 2, 3, or 5 carbons.

7. R １ But -CH((CH ２ ) ｎ CH ３ ) ２ and n is 4, 5, 6, or 7.

8. n is 5 and L １ The parenteral pharmaceutical composition of claim 7, wherein is a three carbon alkylene.

9. n is 6 and L １ is an alkylene of 3 or 5 carbons.

8. The parenteral pharmaceutical composition according to claim 7.

10. n is 7 and L １ The parenteral pharmaceutical composition of claim 7, wherein is an alkylene of 2 or 3 carbons.

11. R １ But -CH((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｎ＋１ CH ３ 2. The parenteral pharmaceutical composition of claim 1 , wherein n is 6 or 7.

12. 1. A parenteral pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid, said lipid nanoparticles being selected from the group consisting of ATX-43, ATX-57, ATX-58, ATX-81, ATX-82, ATX-83, ATX-84, ATX-86, ATX-87, and ATX-88: 【Chemistry 128-1】 【Chemistry 128-2】 A parenteral pharmaceutical composition comprising a cationic lipid selected from the group consisting of:

13. 1. A parenteral pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid, said lipid nanoparticles having the formula ATX-88: 【Chemistry 128-3】 or a pharma- ceutically acceptable salt or solvate thereof.

14. 14. The parenteral pharmaceutical composition of claim 1, wherein the parenteral pharmaceutical composition is selected from an intravenous delivery composition, a subcutaneous delivery composition, and an intraperitoneal delivery composition.

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