Ionizable cationic lipid for RNA delivery

Abstract

To provide ionizable cationic lipids for RNA delivery.SOLUTION: Described herein are a compound of formula (I), in which R1 is a branched alkyl having 10 to 31 carbons; R2 is a linear alkyl, alkenyl, or alkynyl having 2 to 20 carbons; L1 and L2 are the same or different, each a linear alkylene having 1 to 20 carbons or a linear alkenylene having 2 to 20 carbons; X1 is S or O; R3 is a linear or branched alkylene having 1 to 6 carbons; and R4 and R5 are the same or different, each hydrogen or a linear or branched alkyl having 1 to 6 carbons; or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Patent Application No. 15 / 387,067, filed on December 21, 2016, the entire content of which is incorporated herein by reference.

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

Background Art

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

[0004] The description herein relates to novel lipid compositions that facilitate intracellular delivery of biologically active therapeutic molecules. The description also relates to pharmaceutical compositions that contain such lipid compositions and are useful for delivering a therapeutically effective amount of a biologically active molecule to a patient's cells.

[0005] The delivery of therapeutic compounds to a subject is important for their therapeutic effect and can often be hindered by the limited ability of the compound to reach the target cells and tissues. Improvements for getting such compounds into the target cells of tissues by various delivery means are important. The present description relates to novel lipids in compositions and preparation methods that facilitate the targeted intracellular delivery of bioactive molecules.

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

[0007] One of the fundamental problems currently faced by physicians is that several different types of nucleic acids are currently being developed as therapeutic agents 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. Since these nucleic acids are being developed, there is a need to manufacture lipid formulations that are easy to make and can be easily delivered to the target tissue.

Summary of the Invention

Means for Solving the Problems

[0008] Formula I

Chemical Formula

[0009] In one embodiment, as follows:

Chemical formula

Chemical formula

Chemical formula

[0010] In one embodiment, what is described herein is that R 1 is a branched-chain alkyl consisting 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 consisting 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 each the same or different and are a straight-chain 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 straight-chain alkenylene consisting 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 straight-chain or branched alkylene consisting of 1, 2, 3, 4, 5, 6, or 6 carbons; R 4 and R 5 are each the same or different and are each hydrogen, or a straight-chain or branched alkyl consisting of 1, 2, 3, 4, 5, or 6 carbons, and consists of a compound

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

[0012] In one embodiment, the cationic lipids described herein are present in a pharmaceutical composition. The pharmaceutical composition preferably comprises lipid nanoparticles containing a nucleic acid, preferably an RNA polynucleotide. The lipid nanoparticles preferably increase the average lifespan of the 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 encodes upon expression in the cells of the body.

[0013] Also described herein is a method for introducing a nucleic acid into mammalian cells by using any of the above compositions. The cells can be present in the liver, lung, kidney, brain, blood, spleen, or bone. The composition is preferably administered intravenously, subcutaneously, intraperitoneally, or intrathecally. Preferably, the compositions described herein are used in a method for treating cancer or an inflammatory disease. The disease can be selected from the group consisting of immune disorders, cancer, kidney diseases, fibrotic diseases, genetic abnormalities, inflammation, and cardiovascular disorders.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

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

[0031] "Composition" means a product containing specific components in specific amounts, and any product directly or indirectly resulting from combining specific components in specific amounts.

[0032] "In combination with" means administering a compound of formula I together with another medicament in the treatment method of the present invention, meaning administering the compound of formula I and another medicament sequentially or simultaneously in separate dosage forms, or simultaneously in the same dosage form.

[0033] "Mammal" means a human or other mammal, or means a human.

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

[0035] "Alkyl" means a saturated or unsaturated, straight-chain or branched hydrocarbon chain. In various embodiments, the alkyl group has 1 to 18 carbons, i.e., C 1 ~C 18 group, or a C 1 ~C 12 group, a C 1 ~C 6 group, or a C 1 ~C 4is a base. Independently, in various embodiments, the alkyl group has 0 branches (i.e., straight-chain), 1 branch, 2 branches, or more than 2 branches. "Alkenyl" is an unsaturated alkyl that may have one double bond, two double bonds, or more than two double bonds. "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 1 substituent (i.e., the alkyl group is monosubstituted), or 1 to 2 substituents, or 1 to 3 substituents, or 1 to 4 substituents, etc. The substituents may be selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano, etc. When the alkyl group incorporates one or more heteroatoms, the alkyl group is referred to herein as a heteroalkyl group. When the substituent on the alkyl group is a hydrocarbon, the resulting group is simply called a substituted alkyl. In various aspects, the alkyl group containing a substituent is having 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or less than 7 carbons.

[0036] "Lower alkyl" means a group having 1 to 6 carbons in the chain, and the chain may be straight-chain 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 as 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 described above. 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 defined above. Preferred alkylaryls contain a lower alkyl group. The attachment 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 as defined above.

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

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

[0043] "Compounds described in the chemical literature" can be identified by reference books and databases on chemical compounds and chemical reactions, as known to those skilled in the art. Appropriate reference books and treatises that detail the synthesis of reactants useful in the preparation of the compounds disclosed herein or provide references to papers describing the preparation of the compounds disclosed herein include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York ; S. R. Sandler et al., "Organic Functional Group Preparations", 2nd Edition, Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Edition, W. A. Benjamin, Inc. Menlo Park, Calif., 1972; T. L. Glichrist, "Heterocyclic Chemistry", 2nd Edition, John Wiley and Sons, New York , 1992; J. March, "Advanced Organic Chemistry: reactions, Mechanisms including "Structure", 5th Edition, Wiley Interscience, New York, 2001. Specific and similar reactants can also be identified by indexes of known chemical substances prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through online databases (for details, please contact the American Chemical Society, Washington, D.C.). Chemical substances that are known in the catalog but not commercially available can be prepared by custom chemical synthesis companies, and many standard chemical substance supply companies (such as those listed above) offer custom synthesis services.

[0044] "Halo" means a fluoro, chloro, bromo, or iodo group. Fluoro, chloro, or bromo is preferred, and fluoro and chloro are more preferred.

[0045] "Halogen" means fluorine, chlorine, bromine, or iodine. Fluorine, chlorine, and bromine are preferred.

[0046] "Heteroalkyl" means a saturated or unsaturated, straight-chain or branched chain containing carbon and at least one heteroatom. In various embodiments, a heteroalkyl group can have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms. In one aspect, the heteroalkyl chain contains 1-18 (i.e., 1 to 18) constituent atoms (carbon and heteroatoms), and in various embodiments, contains 1-12, or 1-6, or 1-4 constituent atoms. Independently, in various embodiments, the heteroalkyl group has 0 branches (i.e., straight-chain), 1 branch, 2 branches, or more than 2 branches. Independently, in one embodiment, the heteroalkyl group is saturated. In another embodiment, the heteroalkyl group is unsaturated. In various embodiments, the unsaturated heteroalkyl 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. The substituted heteroalkyl chain may 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)-O-R) and carbonyl (-C(O)-).

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

[0048] "Hydrate" means a solvate where the solvent molecule is H 2 O.

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

[0050] "Lipid particle" means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., a cell, tissue, organ, etc.). 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., a phospholipid), a conjugate lipid (e.g., a PEG-lipid) that prevents aggregation of the particles, and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) is encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

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

[0052] "Solvate" means a physical association of a compound of the present disclosure with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, a solvate can be isolated, 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" means a lipid particle that provides a therapeutic nucleic acid, such as mRNA, with complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid particle.

[0054] "Lipid conjugate" means a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as 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 conjugated to ceramide, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG or POZ to a lipid, including ester-free linker moieties and ester-containing linker moieties, can be used. In certain preferred embodiments, an ester-free linker moiety such as an amide or carbamate is used.

[0055] "Amphiphilic lipid" means a substance in which the hydrophobic part of the lipid substance is oriented in the hydrophobic phase and the hydrophilic part is oriented in the aqueous phase. The hydrophilic characteristics are derived from the presence of polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, amines, sulfhydryls, nitro groups, hydroxyl groups, and other similar groups. Hydrophobicity can be conferred by the inclusion of nonpolar groups including long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as such groups substituted by one or more aromatic, alicyclic, or heterocyclic groups (multiple possible), but not limited thereto. Examples of amphiphilic 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 lacking phosphorus such as sphingolipids, the sphingoglycolipid family, diacylglycerol, and β-acyl oxy acids are also within the group designated as amphiphilic lipids. Furthermore, the above-mentioned amphiphilic lipids can be mixed with other lipids including triglycerides and sterols.

[0057] "Neutral lipid" means a lipid species that exists in either a non-charged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

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

[0059] "Anionic lipid" means 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 linked to neutral lipids.

[0060] "Hydrophobic lipid" means a compound having a nonpolar group that includes, but is not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted by one or more aromatic, alicyclic, or heterocyclic groups (multiple possible). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacetyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0061] "Cationic lipid" and "amino lipid" are used interchangeably and mean a lipid and its 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 protonated (i.e., positively charged) at a pH below the pK a of the cationic lipid and substantially neutral at a pH above the pK a of the cationic lipid. The cationic lipids of the present invention are also referred to as titratable cationic lipids. In some embodiments, the cationic lipid has: a protonatable tertiary amine (e.g., pH-titratable) head group; each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds and is a C 18comprising an alkyl chain; and an ether, ester, or ketal linkage between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, 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 particular group other than hydrogen, or one or more groups, moieties, or radicals, which may be the same or different, for example, each independently selected.

[0063] "Antisense nucleic acid" means a non-enzymatic nucleic acid molecule that binds to a target RNA by RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993, Nature, Vol. 365, p. 566) interaction and modifies the activity of the target RNA (see, for reviews, Stein and Cheng, 1993, Science, Vol. 261, p. 1004, and Woolf et al., U.S. Patent 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, the antisense molecule can bind to a substrate such that the substrate molecule forms a loop, and / or the antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of the antisense molecule can be complementary to the target sequence, or both. Further, antisense DNA can be used to target RNA by DNA-RNA interaction, thereby activating RNase H, which digests the target RNA that is double-stranded. An antisense oligonucleotide can contain one or more RNase H activation regions capable of activating RNase H cleavage of the target RNA. Antisense DNA can be chemically synthesized or expressed by use of a single-stranded DNA expression vector or its equivalent. "Antisense RNA" is an RNA strand having a sequence complementary to the mRNA of a target gene and capable of inducing RNAi by binding to the mRNA of the target gene. "Antisense RNA" is considered to be an RNA strand having a sequence complementary to the mRNA of a target gene and capable of inducing RNAi by binding to the mRNA of the target gene. "Sense RNA" has a sequence complementary to the antisense RNA and anneals to the complementary antisense RNA to form iNA. These antisense and sense RNAs have conventionally been synthesized by an RNA synthesizer.

[0064] "Nucleic acid" means deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, whether synthetic, naturally occurring, or non-naturally occurring, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0065] "RNA" means a molecule containing at least one ribonucleotide residue. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribo-furanose moiety. The term includes isolated RNAs such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNAs that differ from naturally occurring RNAs by addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include, for example, the addition of non-nucleotide substances to the end(s) or internally of interfering RNA, e.g., at one or more nucleotides of the RNA. The nucleotides in the RNA molecules of the present 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 RNAs. As used herein 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 multivalent RNA. Ribonucleotides are nucleotides having a hydroxyl group at the 2'-position of the β-D-ribo-furanose moiety. These terms include isolated RNAs such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNAs and altered RNAs that differ from naturally occurring RNAs by the addition, deletion, substitution, modification, or alteration of one or more nucleotides. Modifications of RNA include, for example, the addition of non-nucleotide substances to the end(s) of interfering RNA or internally, e.g., at one or more nucleotides of the RNA nucleotides in an RNA molecule, and may include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs.

[0066] "Nucleotide" means natural bases (standards) and modified bases well known in the art. Such bases are generally located at the 1'-position of the nucleotide sugar moiety. Nucleotides generally include a base, a sugar, and a phosphate group. Nucleotides may or may not be modified in the sugar, phosphate, and / or base moieties (also interchangeably referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc., see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO92 / 07065; Usman et al., International PCT Publication No. WO93 / 15187; Uhlman and Peyman, supra, all of which are incorporated herein by reference). as described by Limbach et al., Nucleic Acids Res., Vol. 22, p. 2183, 1994 As summarized, there are several 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, 35:14090, 1996; Uhlman and Peyman, supra). "Modified base" in this context means a nucleotide base other than adenine, guanine, cytosine, and uracil at the 1' position or their equivalents.

[0067] "Complementary nucleotide bases" means 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 acids hybridize to each other (i.e., are linked by hydrogen bonds). "Complementary" means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional binding modes.

[0068] "MicroRNA" (miRNA) means a single-stranded RNA molecule 21 - 23 nucleotides in length that regulates gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNAs); instead, they are processed from primary transcripts known as pre-miRNAs into short stem-loop structures called pre-miRNAs and finally into 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 that are 16 to 40 nucleotides in length and play various roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway in which it interferes with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act, for example, in RNAi-related pathways as antiviral mechanisms or in shaping the chromatin structure of the genome, although the complexity of these pathways is only now being elucidated.

[0070] "RNAi" refers to an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and initiated by short double-stranded RNA molecules within the cell that interact with the catalytic RISC component, argonaute. When the double-stranded RNA or RNA-like iNA or siRNA is exogenous (derived from infection by a virus having an RNA genome, or 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-microRNA expressed from RNA-coding genes in the genome. The primary transcript from such a gene is first processed in the nucleus to form the characteristic stem-loop structure of pre-miRNA and then transported to the cytoplasm and cleaved by dicer. Thus, both the exogenous and endogenous dsRNA pathways converge on the RISC complex. The active component of the RNA-induced silencing complex (RISC) is an endonuclease called an argonaute protein, which cleaves the target mRNA strand complementary to its bound siRNA or iNA. Since the fragments produced by dicer are double-stranded, they could theoretically each produce a functional siRNA or iNA. However, only one of the two strands, known as the guide strand, binds to the argonaute protein and leads to gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation.

[0071] Compound of formula I

[0072] References to compounds of formula I in this specification are understood to include references to salts thereof, unless otherwise indicated. As used herein, the term "salt(s)" means acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. Further, 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, a carboxylic acid, a zwitterion ("inner salt") can be formed and is included within the term "salt(s)" as used herein. The salts can be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts, but 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 acid or base in an amount such as an equivalent 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, dodecylsulfate, 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, Sulfonates (such as those described herein), tartrates, thiocyanates, toluenesulfonates (also known as tosylates), undecanoates, and the like are included. Further, acids that are generally considered suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds are, for example, S. Berge et al., J. Pharmaceutical Sciences (1977) 66(1 ), pp. 1-19; P. Gould, International J. Pharmaceutics (1986) 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, D.C., website). These disclosures are incorporated herein by reference.

[0074] Exemplary basic salts include ammonium salts; alkali metal salts such as sodium salts, lithium salts, and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; salts with organic bases such as benzathine, dicyclohexylamine, hydrabamine (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine (e.g., organic amines); 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 sulfates), long-chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0075] All salts of such acids and bases are intended to be pharmaceutically acceptable salts within the scope of the present disclosure, and for the purposes of the present disclosure, all salts of acids and bases are considered equivalent to the free form of the corresponding compound of formula I.

[0076] The compounds of formula I may exist in unsolvated and solvated forms, including hydrated forms. Generally, solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. are considered equivalent to the unsolvated forms for the purposes of the present disclosure.

[0077] The compounds of formula I and their salts and solvates may exist in their tautomeric forms (e.g., as amides or imino ethers). All such tautomeric forms are contemplated herein as part of the present disclosure.

[0078] Polymorphs of the compounds of the present disclosure are also within the scope of the present disclosure (i.e., polymorphs of the compounds of formula I are within the scope of the present disclosure).

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

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

[0081] Lipid particles The compound of Formula I is included in a lipid composition comprising a nanoparticle or a bilayer of lipid molecules, together with its pharmaceutically acceptable salts. The lipid bilayer preferably further comprises neutral lipids or polymers. 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 using RNA interference (RNAi). The lipid composition preferably further comprises a nucleic acid and neutral lipids or polymers. The lipid composition preferably encapsulates a nucleic acid.

[0082] There is provided a lipid particle comprising one or more therapeutic mRNA molecules encapsulated within the lipid particle.

[0083] In some embodiments, the mRNA is completely encapsulated within the lipid moiety of the lipid particle such that the mRNA in the lipid particle is resistant to nuclease degradation in an aqueous solution. 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 particle has a lipid:RNA ratio (mass / mass ratio) of 12:1. In another embodiment, the lipid particle has a lipid:mRNA ratio (mass / mass ratio) of 13:1.

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

[0085] In the nucleic acid-lipid particle, the mRNA can be completely encapsulated within 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 within 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 for 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 forms a complex with the lipid portion of the particle. One advantage of the formulations of the present invention is that the nucleic acid-lipid particle composition is substantially non-toxic to mammals such as humans.

[0086] "Completely encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or after a nuclease assay that would significantly degrade free RNA. When completely encapsulated, in a process that would normally degrade 100% of the free nucleic acid, preferably less than 25% of the nucleic acid in the particle, more preferably Less than 10%, 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 constituent 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 using a dye that enhances fluorescence when associated with the nucleic acid. Encapsulation is determined by adding the dye to the liposome preparation, 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 can be calculated as E = (I 0 - I) / I 0 where I 0 refers to the fluorescence intensity before and after addition of the detergent.

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

[0089] The lipid particles contain mRNA that is fully encapsulated within the lipid portion of the particles, such that 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%, 40% - 90%, 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 proportion or range thereof) of the particles have the 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 lipid 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 subsequent sections. The lipomacrocyclic compounds described herein can be used for other purposes and, for example, for recipients and additives.

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

[0093] The cationic lipid compounds can be combined with agents for forming microparticles, nanoparticles, liposomes, or micelles. The agents delivered by the particles, liposomes, or micelles can be in gaseous, liquid, or solid form, and the agents can be polynucleotides, proteins, peptides, or small molecules. The lipomacrocyclic compounds 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, if necessary, to form pharmaceutical compositions.

[0094] This 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 substances, additives, excipients, substances, or in biotechnology.

[0095] The cationic lipid compounds of the present specification provide several different uses in the field of drug delivery. The amine-containing moiety of the cationic lipid compound can be used to complex polynucleotides, thereby enhancing the delivery of polynucleotides and preventing their degradation. The cationic lipid compound can also be used in the formation of picoparticles, nanoparticles, microparticles, liposomes, and micelles containing the agent to be delivered. Preferably, the cationic lipid compound is biocompatible and biodegradable, and the formed particles 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 a lower pH. They can also act as a proton sponge in the delivery of agents to cells to cause endosome lysis.

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

[0097] Neutral helper lipid ​​Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyl oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a C 10 ~C 24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0098] Further examples of non-cationic lipids include steroids such as cholesterol and its derivatives It includes cholesterol 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 the 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 the lipid particle comprises or consists of one or more phospholipids, for example, a lipid particle formulation that does not contain cholesterol. In still other embodiments, the non-cationic lipid present in the lipid particle comprises or consists of a lipid particle formulation that does not contain cholesterol or its derivatives, for example, phospholipids.

[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 polymer, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amide, dioctadecyldimethylammonium bromide, ceramide, and sphingomyelin.

[0101] In some embodiments, the non-cationic lipid constitutes 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 ratio or range thereof) of the total lipids present in the particles.

[0102] In embodiments where the lipid particles contain a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may constitute up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the particles. In some embodiments, the phospholipid component in the mixture may constitute 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 ratio or range thereof) of the total lipids present in the particles. In certain preferred embodiments, the phospholipid component in the mixture constitutes 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 ratio or range thereof) of the total lipids present in the particles. In other embodiments, the cholesterol component in the mixture can constitute 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 ratio or range thereof) of the total lipids present in the particles. In certain preferred embodiments, the cholesterol component in the mixture is 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 ratio or range thereof) of the total lipids present in the particles. In embodiments where the lipid particles do not contain phospholipids, cholesterol or its derivatives can constitute up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the particles.

[0103] In some embodiments, cholesterol or its derivatives in lipid particle formulations that do not contain phospholipids can 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 ratio or range thereof) of the total lipids present in the particles.

[0104] In other embodiments, the non-cationic lipid constitutes 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 its derivatives) (or any ratio or range thereof) of the total lipids present in the particles.

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

[0106] The composition containing the cationic lipid compound can 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 other preferred embodiments, the composition is 50 - 75% cationic lipid compound, 20 - 40% cholesterol, and 5 - 10% phospholipid, and 1 - 10% PEG. The composition can contain 60 - 70% cationic lipid compound, 25 - 35% cholesterol, and 5 - 10% PEG. The composition can contain up to 90% cationic lipid compound and 2 - 15% helper lipid.

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

[0108] Lipid conjugate In addition to being cationic, the lipid particles described herein may further contain a lipid conjugate. Conjugate lipids are useful in preventing particle aggregation. Suitable conjugate lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[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), PEG conjugated to ceramide, PEG conjugated to cholesterol or its derivatives, and mixtures thereof, but are not limited thereto.

[0110] PEG is a linear water-soluble polymer of ethylene PEG repeating units having two terminal hydroxyl groups. PEGs are classified by their molecular weights and include: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH 2 2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-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 2).

[0111] ​The PEG moiety of the PEG-lipid conjugate described herein can include an average molecular weight in the range of 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, the PEG can be optionally substituted with an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG to a lipid can be used, including ester-free linker moieties and ester-containing linker moieties. In preferred embodiments, the linker moiety is an ester-free linker moiety. Suitable ester-free linkers include amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH 2 CH 2 C(0)-), succinamidyl (-NHC(0)CH 2 CH 2 C(0)NH-), ether, disulfide, and combinations thereof (such as a linker containing both a carbamate linker moiety and an amide linker moiety), but are not limited thereto. In preferred embodiments, a carbamate linker is used to couple PEG to the lipid.

[0113] In other embodiments, an ester-containing linker moiety is used to couple PEG to a lipid. 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. C 10 ~C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids having a carbon chain length in the range of 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, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0115] The terms "diacylglycerol" or "DAG" both refer to two fatty acyl chains, R 1 and R 2 having independently between 2 and 30 carbons each bonded to the 1- and 2-positions of glycerol by ester linkages. The acyl groups may be saturated or have various degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (C 16 ), stearoyl (C 18 ), and icosoyl (C 20 ). In preferred embodiments, R1 and R 2 are the same, that is, 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 having two alkyl chains, R and R, both of which independently have between 2 and 30 carbons. The alkyl groups may be saturated or have various degrees of unsaturation.

[0117] Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryl oxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG-dipalmitoyloxypropyl (C 16 ) conjugate, or a PEG-distearyloxypropyl (C 18 ) conjugate. In these embodiments, PEG preferably has an average molecular weight of 750 or 2,000 daltons. In certain embodiments, the terminal hydroxyl group of PEG is substituted 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) constitutes 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 ratio or range thereof) of the total lipids present in the particles. In other embodiments, the lipid conjugate (e.g., PEG-lipid) constitutes 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 ratio or range thereof) of the total lipids present in the particles.

[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 ratio or range thereof) of the total lipids present in the particles.

[0121] The percentage of the lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the present invention is the target amount, and the actual amount of the lipid conjugate present in the formulation can vary, for example, by ±2 mol%. One of ordinary skill in the art will understand that the concentration of the lipid conjugate can vary depending on the lipid conjugate and lipid particles used and the rate at which they become membrane fusogenic.

[0122] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate is exchanged from the lipid particles, and thus the rate at which the lipid particles become membrane-fusogenic, can be controlled. Further, for example, other variables including pH, temperature, or ionic strength can be used to alter and / or control the rate at which the lipid particles become membrane-fusogenic. Other methods that can be used to control the rate at which the lipid particles become membrane-fusogenic will be apparent to those skilled in the art upon reading the present disclosure. Also, by controlling the composition and concentration of the lipid conjugate, the size of the lipid particles can be controlled.

[0123] Compositions and formulations for administration

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

[0125] In some embodiments, the present disclosure provides methods for treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a composition of the present disclosure containing a nucleic acid, a cationic lipid, an amphiphilic substance, a phospholipid, cholesterol, and a 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 various mucosal administration modes, including by oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or cutaneous delivery, or by topical delivery to the eye, ear, skin, or other mucosal surfaces. In some aspects of the present disclosure, the mucosal tissue layer includes an epithelial cell layer. The epithelial cells can be of the lung, trachea, bronchus, alveoli, nose, cheek, epidermis, or gastrointestinal tract. The compositions of the present disclosure can be administered using conventional actuators such as mechanical spray devices, and pressurized, electrically actuated, or other types of actuators.

[0127] The compositions of the present disclosure can be administered in aqueous solution as nasal or pulmonary sprays and can be dosed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved by administering the composition in the form of drops, particles, or sprays, which can be aerosolized, nebulized, or atomized, for example. The particles of the composition, spray, or aerosol can be in either liquid or solid form. A preferred system for dosing a liquid as a nasal spray is disclosed in U.S. Patent No. 4,511,069. Such formulations can be conveniently prepared by dissolving the composition according to the present disclosure in water to produce an aqueous solution and rendering the solution sterile. The formulation can be presented, for example, in a multi-dose container of the sealed dosing system disclosed in U.S. Patent 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. Patent No. 4,778,810. Additional aerosol delivery forms include, for example, nebulizers that deliver a bioactive agent dissolved or suspended in a pharmaceutical solvent such as water, ethanol, or a mixture thereof, such as compressed air, jet, ultrasonic, and piezoelectric. can be included.

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

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

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

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

[0132] For formulating a composition for pulmonary delivery within the scope of the present disclosure, the bioactive agent can be combined with various pharmaceutically acceptable additives and a base or carrier 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), tonicity 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, when measured with reference to the tonicity of 0.9% (w / v) physiological saline considered to be equivalent, is typically adjusted to a value that will not substantially induce 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 can be dispersed in a base or vehicle that may contain a hydrophilic compound having the ability to disperse the active agent and any desired additives. The base can be selected from a wide range of suitable carriers including polycarboxylic acids or their salts, copolymers of carboxylic acid anhydrides (e.g., maleic anhydride) and other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.); hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone; cellulose derivatives such as hydroxymethyl cellulose, hydroxypropyl cellulose; and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid; as well as their non-toxic metal salts. Often, biodegradable polymers such as polylactic acid, poly(lactic-co-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric-co-glycolic acid) copolymer, and mixtures thereof are selected as the base or carrier. Alternatively, or in addition, synthetic fatty acid esters such as polyglycerol fatty acid esters, sucrose fatty acid esters can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination and can impart enhanced structural integrity to the carrier by partial crystallization, ionic bonding, crosslinking, etc. The carrier can be provided in various forms including fluids or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of the selected carrier in this context can promote the absorption of the bioactive agent. The use of the selected carrier in this context can promote the absorption of the bioactive agent.

[0134] Formulations for mucosal, nasal, or pulmonary delivery can contain a hydrophilic low molecular weight compound as a base or excipient. Such hydrophilic low molecular weight compounds provide a passage medium through which water-soluble active agents such as physiologically active peptides or proteins can diffuse through the base to the body surface where the active agent is absorbed. The hydrophilic low molecular weight compound is If necessary, it absorbs moisture from the mucosa or the administration atmosphere and dissolves 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 the hydrophilic low-molecular-weight compound 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 the hydrophilic low-molecular-weight compound include N-methylpyrrolidone, alcohols (such as oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

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

[0136] In certain embodiments of the present disclosure, the bioactive agent can be administered in a time release formulation, for example, in a composition containing a sustained release polymer. The active agent can be prepared using a controlled release vehicle such as a carrier that protects against rapid release, such as a polymer, a microencapsulation delivery system, or a bioadhesive gel. The long-term delivery of the active agent in the various compositions of the present disclosure can be achieved by including in the composition agents that retard absorption, such as aluminum monostearate hydrogel and gelatin.

[0137] The present disclosure has been described with respect to certain embodiments, and many details have been set forth for purposes of illustration. However, 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 various exemplary components and features, terms, or elements of the 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) Figure 1 shows the synthetic route of ATX-43 (RL-43A), which is further described as follows.

[0140] ATX-43: Step 1

Chemical formula

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

[0142] In another 1 L two-necked round-bottom flask, to 31.4 g of N,O-dimethylhydroxylamine hydrochloride (1.5 eq) in DCM (200 ml), 89.8 ml of triethylamine (Et 3 N, 3 eq) was added using an addition funnel and stirred at 0 °C. To this resulting solution, the above-mentioned acid chloride after concentration under reduced pressure was dissolved in DCM (100 ml), and thereby, it was added dropwise over 20 minutes under a nitrogen atmosphere using an addition funnel. The resulting reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

[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 / hexane). Production amount, 20.0 g; Yield, 58%.

[0145] ATX-43: Step 2

Chemical formula

[0146] Into a 500 ml two-necked round-bottom flask, a solution of 33 g of pentylmagnesium bromide (1.5 eq) in tetrahydrofuran (THF; 100 ml) stirred at 0 °C under a nitrogen atmosphere was added to a solution of 20 g of N-methoxy-N-methylhexanamide (1 eq) (dissolved in 200 ml of THF), 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 hexane; Rf: 0.7). The reaction mass was saturated with 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 / hexane). The production amount was 15.0 g; the yield was 66%.

[0149] ATX - 43: Step 3

Chemical formula

[0150] To a solution of 15 g of undecan - 6 - one (1 equivalent) dissolved in 25 ml of methanol (MeOH) in 150 ml of THF, 4.9 g of sodium borohydride (1.5 equivalents) 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 hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (100 ml). The solvent was removed under reduced pressure, and the resulting crude product 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 obtain a white solid. The production amount was 14.0 g; the yield was 93%.

[0152] ATX - 43: Step 4

Chemical formula

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

[0154] The progress of the reaction was monitored by TLC (10% MeOH in chloroform (CHCl 3 ); 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 sticky liquid. Yield, 20.0 g; Yield, 68%.

[0155] ATX-43: Step 5

Chemical Structure

[0156] To a solution of 12 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equiv) dissolved 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 equiv), and 0.72 g of 4-dimethylaminopyridine (DMAP; 0.1 equiv) were added sequentially at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, alcohol was added at the same temperature by dissolving it in DCM (50 mL) using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0157] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3It was washed with the solution (100 ml), and then extracted with EtOAc (2 × 50 ml). The organic layer was concentrated under reduced pressure, and the crude product was used for the next step. Production amount, 8.5 g; Yield, 48%.

[0158] ATX-43: Step 6

Chemical formula

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

[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 product was washed with saturated NaHCO 3 solution (150 ml), and then extracted with EtOAc (2 × 100 ml). The organic layer was separated and concentrated under reduced pressure .

[0161] The crude compound was subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 and 1 ml of triethylamine), and the alcohol was recovered. Production amount, 5.0 g; Yield, 33% (based on the alcohol).

[0162] ATX-43: Step 7

Chemical formula

[0163] To a solution of 14 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (100 ml) cooled to below 0 °C, under a nitrogen atmosphere, 21 g of EDC·HCl (1.3 equivalents) and 15.2 ml of Et were sequentially added at 10-minute intervals 3N (1.3 equivalents) and 1 g of DMAP (0.1 equivalent) were added. To this resulting solution, 8.3 g of (Z)-nona-2-en-1-ol (0.7 equivalent) was added by dissolving it in 50 ml of DCM using an addition funnel, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere.

[0164] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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. 3 The resulting crude product was washed with saturated NaHCO

[0165] solution (100 ml), and then extracted with EtOAc (2 × 50 ml). The organic layer was separated and concentrated under reduced pressure.

[0166] ATX-43: Step 8

Chemical formula

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

[0168] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 ; Rf: 0.5). Ice water was added to the reaction mass, and then it was 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 / hexane. The starting amine and bromo compound were recovered. Production amount, 1.45 g; Yield, 40%.

[0170] ATX - 43: Step 9

Chemical formula

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

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

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

[0174] Purification was carried out using silica gel (100 - 200 mesh; 18% EtOAc / hexane) chromatography. Production amount, 500 mg; Yield, 26%; 1 Confirmed by 1H NMR; HPLC; and mass spectrometry (Mass).

[0175]

Chemical formula

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

[0177] ATX - 57: Step 1: N - methoxy - N - methyloctanamide

Chemical formula

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

[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). Amount produced, 85 g; Yield, 65%.

[0180] ATX - 57: Step 2: Hexadecan - 8 - one

Chem.

[0181] Into a 1 - liter two - necked round - bottom flask, a solution of octylmagnesium bromide in THF (100 ml) stirred at 0 °C under a nitrogen atmosphere was added to a solution of N - methoxy - N - methyloctanamide (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 hexane; Rf: 0.7). The reaction mass was quenched with saturated NH 4 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 subjected to column chromatography using (60 - 120 mesh silica gel; 2% EtOAc / hexane). Amount produced, 65 g; Yield, 63%.

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

Chem.

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

[0185] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (75 ml). The solvent was removed under reduced pressure, and the resulting crude product 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

Chem.

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

[0188] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; 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 sticky liquid. Yield, 80 g; yield, 81%.

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

Chem.

[0190] To a solution of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (200 ml) cooled to below 0 °C, EDC·HCl, Et were added sequentially at 10-minute intervals under a nitrogen atmosphere3 N and 4-dimethylaminopyridine (DMAP) were added. To this resulting solution, 1 equivalent of hexadecane-8-ol was added at the same temperature by dissolving it in DCM (150 ml) using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0191] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (150 ml), and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure, and the crude product was used to proceed to the next step. Yield, 80 g (crude; required compound and alcohol).

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

Chemical formula

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

[0194] ATX-57: Step 7: (Z)-Non-2-en-1-yl 4-bromobutanoate [Chemical formula]

[0195] To a solution of 4-bromobutyric acid dissolved in DCM (400 ml) cooled to below 0 °C, EDC·HCl, Et 3 N, and DMAP were added sequentially at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, (Z)-non-2-en-1-ol was added by dissolving it in 100 ml of DCM using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0196] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (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 / hexane. The alcohol was recovered. Production amount, 27 g; Yield, 51%.

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

[0198] Potassium carbonate was added to a solution of hexadec-8-yl 4-aminobutanoate and (Z)-non-2-en-1-yl 4-bromobutanoate in acetonitrile (ACN), and the resulting mixture was refluxed at 90 °C for 4 hours under a nitrogen atmosphere. The progress of the reaction was monitored by TLC (CHCl 3Monitored by TLC (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, 20 g; Yield, 40%; Confirmed by Mass.

[0199] ATX - 57: Step 9 [Chemical formula]

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

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

[0202] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 Cl solution (75 ml), 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 product was subjected to column chromatography.

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

[0204] [Chemical formula]

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

[0206] ATX - 58: Step 1 [Chemical formula]

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

[0208] In another 1-L two-necked round-bottom flask, 87 mL of trimethylamine (3 eq) stirred at 0 °C was added to 40.5 g of N,O-dimethylhydroxylamine hydrochloride (2 eq) in 300 mL of DCM. After concentrating the resulting solution under reduced pressure and dissolving it in 500 mL of DCM, the above acid chloride was added dropwise using an addition funnel over 15 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under a 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 / hexane). Amount produced, 28 g.

[0211] ATX-58: Step 2

Chemical formula

[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 quenched with saturated NH 4 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 / hexane). Amount produced, 24 g; yield, 77%.

[0215] ATX-58: Step 3

Chemical formula

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

[0217] The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (50 ml). Methanol was reduced under reduced pressure. The resulting crude product 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

Chemical formula

[0219] To a solution of 20 g of 4-aminobutyric acid dissolved in 140 ml of THF, 196 ml of 1N aqueous NaOH solution was added at 0 °C, followed by the addition of 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 sticky liquid. Yield, 30 g; Yield, 76%.

[0221] ATX-58: Step 5

Chem.

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

[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 resulting crude product was washed with saturated NaHCO 3 solution, and EtOAc (100 ml) was added. The organic layer was separated, concentrated under reduced pressure, and the crude product was used for the next step. Yield, 12.7 g (crude).

[0224] ATX-58: Step 6

Chem.

[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, 23.9 ml of TFA (10 eq) was added at 0 °C, and the mixture was stirred at room temperature for 3 hours under a nitrogen atmosphere.

[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 resulting crude product was saturated NaHCO 3It was washed with a solution (100 ml), and EtOAc (100 ml) was added. The organic layer was separated and concentrated under reduced pressure.

[0227] The crude compound was subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 ) to recover the alcohol. The production amount was 7 g for two steps; the yield was 47%; confirmed by Mass.

[0228] ATX - 58: Step 7

Chemical formula

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

[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 resulting crude product was washed with a saturated NaHCO 3 solution and EtOAc (150 ml) was added. 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 / hexane). The production amount was 17 g; the yield was 69%; 1 confirmed by 1H NMR.

[0232] ATX - 58: Step 8

Chemical formula

[0233] To a solution of 6 g of tetradecan-7-yl 4-aminobutanoate (1 eq) and 5.8 g of (Z)-nona-2-en-1-yl 4-bromobutanoate (1 eq) in ACN (125 ml), 2.7 g of potassium carbonate (1.2 eq) was added, and the resulting mixture was refluxed at 90 °C for 3 h under a 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). The amount produced was 4.5 g; the yield was 44%; confirmed by Mass.

[0236] ATX-58: Step 9

Chemical formula

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

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

[0239] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 carried out using silica gel (60 - 120 mesh). 5.0 g of the crude compound was adsorbed onto 9 g of silica gel and poured onto 90 g of silica gel in the column. The compound was eluted with 35% EtOAc / hexane. The second purification was carried out using neutral alumina together with HPLC grade solvents. 1.5 g of the crude compound was adsorbed onto 4 g of neutral alumina and poured onto 40 g of neutral alumina in the column. The compound was eluted with 10% EtOAc / hexane. Amount produced, 1.2 g; Yield, 21%; 1 Confirmed by H NMR, HPLC, and Mass.

[0241]

Chemical Structure

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

[0243] ATX-81: Step 1

Chem.

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

[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). The production amount was 33 g; the yield was 84%.

[0246] ATX-81: Step 2

Chem.

[0247] A solution of 22 g of heptylmagnesium bromide (1.5 equivalents) in THF (100 ml) was placed in a 1-liter two-necked round-bottom flask and stirred at 0 °C under a nitrogen atmosphere. A solution of N-methoxy-N-methyloctanamide (1 equivalent) (dissolved in 200 ml of THF) was added, 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 quenched with saturated NH 4 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 subjected to column chromatography using (60 - 120 mesh silica gel; 2% EtOAc / hexanes). Amount produced, 22 g; yield, 65%.

[0249] ATX - 81: Step 3

Chem.

[0250] To a solution of 22 g of pentadecan - 8 - one (1 equiv) dissolved in MeOH / THF, 1.5 equiv of sodium borohydride was added 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 quenched with saturated NH 4 Cl solution (75 mL). The solvent was removed under reduced pressure, and the resulting crude product 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. Amount produced, 20 g; yield, 90%.

[0252] ATX - 81: Step 4

Chem.

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

[0254] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; 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 sticky liquid. Yield, 80 g; Yield, 81%.

[0255] ATX-81: Step 5

Chemical formula

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

[0257] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (150 ml), then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure, and the crude product was used for the next step. Yield, 8.5 g (crude; required compound and alcohol).

[0258] ATX-81: Step 6

Chemical formula

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

[0260] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO 3 solution (300 ml), and then extracted with EtOAc (2 × 200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 3 and 1 ml of triethylamine), and the alcohol was recovered. The amount produced was 4 g for 2 steps; the yield was 25%; confirmed by Mass.

[0261] ATX-81: Step 7

Chemical Structure

[0262] To a solution of 4-bromobutyric acid dissolved in DCM (300 ml) cooled to below 0 °C, EDC·HCl, Et 3 N, and DMAP were sequentially added at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, 20 g of (Z)-nona-2-en-1-ol was added by dissolving it in 100 ml of DCM using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0263] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3It was washed with a solution (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 / hexane. The alcohol was recovered. Production amount, 19 g; Yield, 55%.

[0264] ATX - 81: Step 8

Chemical formula

[0265] To a solution of 4.5 g of pentadec - 8 - yl 4 - aminobutanoate and 1 equivalent of (Z) - non - 2 - en - 1 - yl 4 - bromobutanoate in 70 ml of acetonitrile (ACN), 1.4 equivalents of potassium carbonate was 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 (10% MeOH in CHCl 3 3; 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, the amine and the bromo - compound, were recovered. Production amount, 2.1 g; Yield, 27%; Confirmed by Mass.

[0267] ATX - 81: Step 9

Chemical formula

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

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

[0270] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0271] The first purification of the crude compound was carried out using silica gel (60 - 120 mesh), adsorbed on 60 g of silica gel, and poured onto 500 g of silica gel packed in a column. The compound was eluted with 35% EtOAc / hexane. The second purification was carried out using neutral alumina together with HPLC-grade solvent. The crude compound was adsorbed on 18 g of neutral alumina, and the resulting material was poured onto 130 g of neutral alumina packed in a column. The compound was eluted with 10% EtOAc / hexane. Production amount, 1.5 g; Yield, 45%; 1 Confirmed by H NMR, HPLC, and Mass.

[0272]

Chemical formula

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

[0274] ATX-82: Step 1 [Chem.]

[0275] Place 30 g of octanoic acid dissolved in DCM (200 ml) into a 2-liter two-necked round-bottom flask, and then slowly add 1.5 equivalents of oxalyl chloride dropwise at 0 °C while stirring under a nitrogen atmosphere. 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-dimethylhydro xylylamine hydrochloride in DCM (200 ml) was added with 3 equivalents of trimethylamine using an addition funnel and stirred at 0 °C. The above-mentioned acid chloride after concentration under reduced pressure was dissolved in DCM (150 ml), and then added dropwise over 20 minutes under a nitrogen atmosphere using an addition funnel to this obtained solution. The resulting reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

[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). Production amount, 33 g; yield, 84%.

[0277] ATX-82: Step 2 [Chem.]

[0278] Place in a 1-liter two-necked round-bottom flask, and add a solution of 28 g of N-methoxy-N-methyloctanamide (1 equivalent) dissolved in 200 ml of THF to a solution of heptylmagnesium bromide (1.5 equivalents) in THF (100 ml) stirred at 0 °C under a nitrogen atmosphere. 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 hexane; Rf: 0.7). The reaction mass was quenched with saturated NH 4 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 subjected to column chromatography using (60 - 120 mesh silica gel; 2% EtOAc / hexane). Amount produced, 22 g; yield, 65%.

[0280] ATX - 82: Step 3

Chemical formula

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

[0282] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (75 ml). The solvent was removed under reduced pressure, and the resulting crude product 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. Amount produced, 20 g; yield, 90%.

[0283] ATX - 82: Step 4

Chemical formula

[0284] To a solution of 15 g of 4 - aminobutanoic acid dissolved in 120 ml of THF, 185 ml of 1N aqueous NaOH solution was sequentially added dropwise at 0 °C over 15 minutes using an addition funnel, 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 (10% MeOH in CHCl 3 3; 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 sticky liquid. Yield, 27 g; Yield, 85%.

[0286] ATX-82: Step 5

Chemical Structure

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

[0288] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (150 ml), and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure, and the crude product was used for the next step. Yield, 8 g (crude; required compound and alcohol).

[0289] ATX-82: Step 6

Chemical Structure

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

[0291] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO 3 solution (300 ml), and then extracted with EtOAc (2 × 200 ml). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 3 and 1 ml of triethylamine), and the alcohol was recovered. The amount produced was 4 g; the yield was 25% over 2 steps; confirmed by Mass.

[0292] ATX-82: Step 7

Chemical Structure

[0293] To a solution of 4-bromobutyric acid dissolved in DCM (400 ml) cooled to below 0 °C, 1.5 equivalents of EDC·HCl, 3 equivalents of Et 3 3N, and DMAP were sequentially added at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, 20 g of (Z)-nona-2-en-1-ol was added by dissolving it in 100 ml of DCM using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0294] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3It was washed with a solution (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 / hexane. The alcohol was recovered. Production amount, 18 g; Yield, 55%.

[0295] ATX - 82: Step 8

Chemical formula

[0296] To a solution of 4.0 g of pentadec - 8 - yl 4 - aminobutanoate and 1 equivalent of (Z) - non - 2 - en - 1 - yl 4 - bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate was 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 (10% MeOH in CHCl 3 3; 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. Production amount, 2.2 g; Yield, 30%; Confirmed by Mass.

[0298] ATX - 82: Step 9

Chemical formula

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

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

[0301] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0302] The first purification of the crude compound was carried out using silica gel (60 - 120 mesh), adsorbed on 60 g of silica gel, and poured onto 500 g of silica gel placed in a column. The compound was eluted with 35% EtOAc / hexane. The second purification was carried out using neutral alumina together with HPLC-grade solvent. The crude compound was adsorbed on 18 g of neutral alumina, and the resulting material was poured onto 130 g of neutral alumina placed in a column. The compound was eluted with 10% EtOAc / hexane. Production amount, 1.2 g; Yield, 43%; 1 Confirmed by H NMR, HPLC, and Mass.

[0303]

Chemical formula

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

[0305] ATX-86: Step 1 [Chem.]

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

[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). The amount produced was 38 g; the yield was 84%; confirmed by Mass.

[0308] ATX-86: Step 2 [Chem.]

[0309] A solution of 38 g of N-methoxy-N-methyloctanamide (1 equivalent) dissolved in 200 ml of THF was added to a solution of hexylmagnesium bromide (1.5 equivalents) in THF (100 ml) placed in a 1-liter two-necked round-bottom flask and stirred at 0 °C under a nitrogen atmosphere. 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 hexane; Rf: 0.7). The reaction mass was quenched with saturated NH 4 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 subjected to column chromatography using (60 - 120 mesh silica gel ; 2% EtOAc / hexane). The amount produced was 44 g; the yield was 65%; confirmed by Mass.

[0311] ATX - 86: Step 3

Chemical formula

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

[0313] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (75 ml). The solvent was removed under reduced pressure, and the resulting crude product 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 obtain a white solid. The amount produced was 40 g; the yield was 90%; confirmed by Mass.

[0314] ATX - 86: Step 4

Chemical formula

[0315] To a solution of 50 g of 4-aminobutanoic acid dissolved in 350 ml of THF, 490 ml of 1N aqueous NaOH was added dropwise at 0 °C over 15 minutes using an addition funnel, 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 (10% MeOH in CHCl 3 3; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml), 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 sticky liquid. Yield, 80 g; 81%; confirmed by Mass.

[0317] ATX-86: Step 5

Chemical formula

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

[0319] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5) The reaction mass was quenched with water (150 ml), 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 product was washed with saturated NaHCO 3Washed with solution (150 ml), then added EtOAc (200 ml). The organic layer was separated, concentrated under reduced pressure, and the crude product was used to proceed to the next step. Yield, 8 g (crude; required compound and alcohol).

[0320] ATX-86: Step 6

Chem.

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

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

[0323] ATX-86: Step 7

Chem.

[0324] To a solution of 4-bromobutyric acid dissolved in DCM (400 ml) cooled to below 0 °C, 1.5 equivalents of EDC·HCl and 2 equivalents of Et were sequentially added at 10-minute intervals under a nitrogen atmosphere. 3N and DMAP were added. To this resulting solution, 20 g of (Z)-nona-2-en-1-ol was added by dissolving it in 100 ml of DCM using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0325] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (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 / hexane. The alcohol was recovered. The amount produced was 18 g; the yield was 55%.

[0326] ATX-86: Step 8

Chemical formula

[0327] To a solution of 4.0 g of tridecan-7-yl 4-aminobutanoate and 1 equivalent of (Z)-nona-2-en-1-yl 4-bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate was 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 (10% MeOH in CHCl 3 ; 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 amine and bromo compound were recovered. The amount produced was 2.2 g; the yield was 30%; confirmed by Mass.

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

[0330] To a solution of 2.2 g of tridecan-7-yl (Z)-4-((4-(nona-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 ml of dry DCM, 3 equivalents of triethylamine and triphosgene were added at 0 °C at 5-minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 hour under a nitrogen atmosphere. The resulting reaction mass was concentrated under reduced pressure and maintained under a 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 a nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride were added, and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution, the above-mentioned carbamoyl chloride dissolved in THF (100 ml) was slowly added dropwise with a syringe over about 10 minutes. The resulting solution was stirred overnight at 0 °C to room temperature under a nitrogen atmosphere.

[0332] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0333] The first purification of the crude compound was carried out using silica gel (60 - 120 mesh), adsorbed onto 60 g of silica gel, and poured onto 500 g of silica gel packed in a column. The compound was eluted with 35% EtOAc / hexane. The second purification was carried out using neutral alumina together with HPLC-grade solvent. The crude compound was adsorbed onto 18 g of neutral alumina, and the resulting material was poured onto 130 g of neutral alumina packed in a column. The compound was eluted with 10% EtOAc / hexane. Production amount, 1.2 g; Yield, 43%; 1Confirmed by \(^1\)H NMR, HPLC, and Mass.

[0334] [Chemical formula]

[0335] (Example 8: Synthesis of ATX - 87) Figure 7 shows the synthetic route of ATX - 87 (RL - 48C) including nine steps.

[0336] ATX - 87: Step 1 [Chemical formula]

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

[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). Amount produced, 20 g; Yield, 84%.

[0339] ATX - 87: Step 2 [Chemical formula]

[0340] It was placed in a 1 - liter two - necked round - bottom flask and a solution of hexylmagnesium bromide (1.5 equivalents) in THF (100 ml) stirred at 0 °C under a nitrogen atmosphere was added dropwise with a solution of 20 g of N - methoxy - N - methyloctanamide (1 equivalent) (dissolved in 200 ml of THF). 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 hexane; Rf: 0.7). The reaction mass was quenched with saturated NH 4 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 subjected to column chromatography using (60 - 120 mesh silica gel; 2% EtOAc / hexane). The amount produced was 25 g; the yield was 65%.

[0342] ATX - 87: Step 3

Chemical formula

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

[0344] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4 Cl solution (75 ml). The solvent was removed under reduced pressure, and the resulting crude product 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. The amount produced was 22 g; the yield was 90%.

[0345] ATX-87: Step 4

Chem.

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

[0347] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; Rf: 0.5). The reaction mass was quenched with 5% HCl (250 ml), 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 sticky liquid. Yield, 80 g; Yield, 81%.

[0348] ATX-87: Step 5

Chem.

[0349] To a solution of 17 g of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (250 ml) cooled to below 0 °C, 1.3 equivalents of EDC·HCl, Et 3 N, and 4-dimethylaminopyridine (DMAP) were added sequentially at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, 1 equivalent of tridecane-7-ol was added at the same temperature by dissolving it in DCM (150 ml) using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0350] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 ml), 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 product was washed with saturated NaHCO3 It was washed with the solution (150 ml), and then EtOAc (200 ml) was added. The organic layer was separated, concentrated under reduced pressure, and the crude product was used for the next step. Yield: 15 g (crude; the required compound and alcohol).

[0351] ATX-87: Step 6

Chemical formula

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

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

[0354] ATX-87: Step 7

Chemical formula

[0355] To a solution of 4-bromobutyric acid dissolved in DCM (400 ml) cooled to below 0 °C, 1.5 equivalents of EDC·HCl, 2 equivalents of Et 3 N, and DMAP were sequentially added at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, using an addition funnel, 100 ml of D By dissolving in CM, 20 g of (Z)-nona-2-en-1-ol was added, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0356] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 resulting crude product was washed with saturated NaHCO 3 solution (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 / hexane. The alcohol was recovered. Amount produced, 19 g; Yield, 55%.

[0357] ATX - 87: Step 8

Chemical formula

[0358] To a solution of 4.0 g of tridecan - 7 - yl 4 - aminobutanoate and 1 equivalent of (Z)-nona - 2 - en - 1 - yl 4 - bromobutanoate in 90 ml of ACN, 1.4 equivalents of potassium carbonate was 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 (10% MeOH in CHCl 3 ; 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, the amine and the bromo compound, were recovered. Amount produced, 2.2 g; Yield, 30%; Confirmed by Mass.

[0360] ATX - 87: Step 9

Chemical formula

[0361] To a solution of 2.2 g of tridecan-7-yl (Z)-4-((4-(nona-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 ml of dry DCM, 3 equivalents of triethylamine and triphosgene were added at 0 °C at 5-minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 hour under a nitrogen atmosphere. The resulting reaction mass was concentrated under reduced pressure and maintained under a 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 a nitrogen atmosphere, 3.5 equivalents of 2-(dimethylamino)propane-1-thiol hydrochloride were added and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution, the above-mentioned carbamoyl chloride dissolved in THF (100 ml) was slowly added dropwise with a syringe over about 10 minutes. The resulting solution was stirred overnight at 0 °C to room temperature under a nitrogen atmosphere.

[0363] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0364] The first purification of the crude compound was carried out 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 / hexane. The second purification was carried out using neutral alumina together with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and the resulting material was poured onto 130 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / hexane. Amount produced, 1.2 g; Yield, 43%; 1 Confirmed by H NMR, HPLC, and Mass.

[0365]

Chem.

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

[0367] ATX-88: Step 1

Chem.

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

[0369] In another 1 liter two-necked round-bottom flask, 3 equivalents of trimethylamine was added to 2 equivalents of N,O-dimethylhydroxylamine hydrochloride in 300 ml of DCM and stirred at 0 °C. The resulting solution was concentrated under reduced pressure and then dissolved in 500 ml of DCM. Using an addition funnel, the above acid chloride was added dropwise over 15 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under a 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 / hexane). Production amount, 21 g; Yield, 66%.

[0372] ATX-88: Step 2

Chem.

[0373] A solution of 1.3 equivalents of octylmagnesium bromide in THF (100 ml), stirred at 0 °C under a nitrogen atmosphere, was added to 20 g of N-methoxy-N-methyloctanamide in 100 ml of THF, 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 quenched with saturated NH 4 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.

[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

Chem.

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

[0378] The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with saturated NH 4It was quenched with Cl solution (50 ml). Methanol was reduced under reduced pressure. The obtained crude product 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 obtain a white solid. Production amount, 14.5 g; yield, 85%.

[0379] ATX-88: Step 4

Chemical formula

[0380] To a solution of 50 g of 4-aminobutyric acid dissolved in 350 ml of THF, 490 ml of 1N aqueous NaOH solution was added at 0 °C, and then 140 ml of Boc anhydride was added 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 obtain a sticky liquid. Production amount, 80 g; yield, 81%.

[0382] ATX-88: Step 5

Chemical formula

[0383] To a solution of 1 equivalent of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (200 ml) cooled to below 0 °C, 3 equivalents of EDC·HCl, Et 3 N (3 equivalents), and DMAP (0.1 equivalent) were sequentially added at 10-minute intervals. To this resulting solution, alcohol was added by dissolving it in DCM using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[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 obtained crude product was washed with saturated NaHCO 3 solution, and EtOAc (100 ml) was added. The organic layer was separated and concentrated under reduced pressure, and the next step was carried out using the crude product. Production amount, 19 g (crude).

[0385] ATX-88: Step 6

Chemical formula

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

[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 product was washed with saturated NaHCO 3 solution (100 ml), and EtOAc (100 ml) was added. The organic layer was separated and concentrated under reduced pressure.

[0388] The crude compound was subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 ) to recover the alcohol. Production amount, 9.4 g; Yield, 50% for 2 steps; Confirmed by Mass.

[0389] ATX-88: Step 7

Chemical formula

[0390] A solution of 30 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (500 ml) cooled to 0 °C was sequentially added with 1.5 equivalents of EDC·HCl, 3 equivalents of Et 3 N, and 0.1 equivalent of DMAP at 10-minute intervals. To this resulting solution, 0.7 equivalent of (Z)-nona-2-en-1-ol was added by dissolving it in 100 ml of DCM using a funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[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 resulting crude product was washed with saturated NaHCO 3 solution, and EtOAc (150 ml) was added. 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 / hexane). The production amount was 27 g; the yield was 51%; 1 confirmed by 1H NMR.

[0393] ATX-88: Step 8

Chemical formula

[0394] To a solution of 6 g of hexadecane-8-yl 4-aminobutanoate (1 equivalent) and 1 equivalent of 5(Z)-nona-2-en-1-yl 4-bromobutanoate in ACN (70 ml), 1.2 equivalents of potassium carbonate was added, and the resulting mixture was refluxed at 90 °C for 3 hours under a 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). The amount produced was 4.5 g; the yield was 44%; confirmed by Mass.

[0397] ATX - 88: Step 9

Chemical formula

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

[0399] To 192 mg of sodium hydride (10 equivalents) 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 equivalents) was added at 0 °C and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution, the above - mentioned carbamoyl chloride dissolved in THF (35 ml) was slowly added dropwise with a syringe over about 10 minutes. The resulting solution was stirred at room temperature for 4 hours under a nitrogen atmosphere.

[0400] The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0401] The first purification was carried out using silica gel (60 - 120 mesh). 5.0 g of the 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 / hexane. The second purification was carried out using neutral alumina together with HPLC grade solvents. 1.5 g of the crude compound was adsorbed onto 4 g of neutral alumina and the resulting material was poured onto 40 g of neutral alumina in a column. The compound was eluted with 10% EtOAc / hexane. Amount produced, 1.2 g; Yield, 21%; 1 Confirmed by H NMR, HPLC, and Mass.

[0402] [Chemical formula]

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

[0404] ATX - 83: Step 1 [Chemical formula]

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

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

[0407] The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5; PMA charring). 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 dried over 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 / hexane. Amount produced, 55.0 g; Yield, 84%.

[0409] ATX-83: Step 2

Chemical formula

[0410] Into a 2-liter two-necked round-bottom flask, a solution of 89.6 g of N-methoxy-N-methyloctanamide (1.5 equivalents) dissolved in 400 ml of dry ether was added to a solution of 55 g of heptylmagnesium bromide (1 equivalent) in ether stirred at 0 °C under a nitrogen atmosphere, 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 hexane; Rf: 0.7; PMA charring). The reaction mass was quenched with saturated NH 4 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 dried over anhydrous Na 2 SO4 It was dried and concentrated under reduced pressure.

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

[0413] ATX - 83: Step 3

Chemical formula

[0414] To a solution of 50 g of pentadecan - 8 - one (1 equivalent) dissolved in 290 ml of MeOH / THF, 12.5 g of sodium borohydride (1.5 equivalents) was added 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 hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 Cl solution (80 ml). The solvent was removed under reduced pressure, and the resulting crude product 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 dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure and dried under vacuum to obtain a white solid. Amount produced, 46.0 g; Yield, 90%.

[0416] ATX - 83: Step 4

Chemical formula

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

[0418] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; 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 dried over anhydrous Na 2 SO 4 4 and concentrated under reduced pressure to give a sticky liquid. Yield, 77.0 g; Yield, 78%.

[0419] ATX-83: Step 5

Chemical formula

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

[0421] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 product was washed with saturated NaHCO 3 3 solution (150 ml), and EtOAc (250 ml) was added. The organic layer was separated, dried over anhydrous Na 2 SO 4 4, concentrated under reduced pressure, and then the crude product was used to proceed to the next step. Yield, 105 g (crude; required compound and alcohol).

[0422] ATX-83: Step 6

Chem.

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

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

[0425] The reaction mass was concentrated under reduced pressure. The resulting crude product was stirred with saturated NaHCO 3 solution (200 ml) for 10 minutes and then with EtOAc (300 ml). The organic layer was separated, and the aqueous layer was washed with EtOAc (2 × 100 ml). The combined organic layers were dried over anhydrous Na 2 SO 4 4 and concentrated under reduced pressure.

[0426] The crude compound was subjected to column chromatography (silica gel 60 - 120 mesh) using 4% MeOH / CHCl 3 3 and 1 ml of triethylamine. The amount produced was 60.0 g for 2 steps; the yield was 54%.

[0427] ATX-83: Step 7

Chem.

[0428] The reaction was carried out in 2 batches. In each batch, to a solution of 20 g of 6-bromohexanoic acid (1 eq) dissolved in DCM (300 ml) cooled to below 0 °C, 29.3 g of EDC·HCl (1.5 eq) and 42.8 ml of Et 3N (3 equivalents) and 1.2 g of DMAP (0.1 equivalent) were added. To this resulting solution, 14.5 g of (Z)-nona-2-en-1-ol (1 equivalent), dissolved in 100 ml of DCM, was added using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[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. 3 The resulting crude product was washed with saturated NaHCO 2 solution (150 ml), and then extracted with EtOAc (2 × 150 ml). The organic layer was separated, dried over anhydrous Na 4 SO

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

[0431] ATX - 83: Step 8

Chemical formula

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

[0433] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 ; 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 / hexane. The starting material was recovered. Production amount, 36.9 g; Yield, 35%.

[0435] ATX - 83: Step 9

Chemical formula

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

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

[0438] The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.

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

[0440]

Chemical formula

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

[0442] ATX - 84: Step 1

Chemical formula

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

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

[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 / hexane. Amount produced, 38.0 g; Yield, 84%.

[0447] ATX-84: Step 2

Chemical Structure

[0448] 8 g of hexylmagnesium bromide (1 equivalent) in 250 mL of dry ether, which was placed in a 1 L two-necked round-bottom flask and stirred at 0 °C under a nitrogen atmosphere, was added to 2.3 g of N-methoxy-N-methylheptanamide (0.5 equivalent) dissolved in 250 mL of ether, 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 hexane; Rf: 0.7). The reaction mass was quenched with saturated NH 4 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 dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.

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

[0451] ATX - 84: Step 3

Chemical formula

[0452] To a solution of 30 g of tridecan - 7 - one (1 equivalent) dissolved in 200 ml of MeOH / THF, 8.5 g of sodium borohydride (0.5 equivalent) was added 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 quenched with saturated NH 4 Cl solution (80 ml). The solvent was removed under reduced pressure, and the resulting crude product 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. Amount produced, 27.2 g; Yield, 90%.

[0454] ATX - 84: Step 4

Chemical formula

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

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

[0457] ATX-84: Step 5

Chem.

[0458] To a solution of 10 g of 6-((tert-butoxycarbonyl)amino)hexanoic acid (1 equiv) dissolved in DCM (200 mL) cooled to below 0 °C, 10.7 g of EDC·HCl (1.3 equiv), 18 mL of Et 3 N (3 equiv), and 525 mg of DMAP (0.1 equiv) were added sequentially at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, 6 g of tridecan-7-ol (Int 3, 0.7 equiv) was added at the same temperature by dissolving in DCM (50 mL) using an addition funnel, and the mixture was stirred at room temperature for 24 h under a nitrogen atmosphere. 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), then the organic layer was separated. The aqueous layer was washed with DCM (2 x 75 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO

[0459] solution (100 mL), then extracted by adding EtOAc (2 x 100 mL). The organic layer was separated, concentrated under reduced pressure, and the crude product was used for the next step. Yield, 8.5 g (crude; required compound and alcohol). 3

[0460] ATX-84: Step 6

Chem.

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

[0462] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 3; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO 3 solution (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 subjected to column chromatography using (60 - 120 mesh silica gel; 4% MeOH / CHCl 3 3 and 1 ml of triethylamine) to recover the alcohol starting material. Quantity, 4.5 g for 2 steps; Yield, 33%.

[0464] ATX-84: Step 7

Chemical formula

[0465] To a solution of 20 g of 6-bromohexanoic acid (1 eq) dissolved in 300 ml of DCM cooled to below 0 °C, 29.3 g of EDC·HCl (1.5 eq), 42.8 ml of Et 3 N (3 eq), and 1.2 g of DMAP (0.1 eq) were sequentially added at 10-minute intervals under a nitrogen atmosphere. To this resulting solution, 14.5 g of (Z)-nona-2-en-1-ol (1 eq) dissolved in 100 ml of DCM was added using an addition funnel, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere.

[0466] The progress of the reaction was monitored by TLC (10% EtOAc in hexane; 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 obtained crude product was washed with saturated NaHCO 3 solution (150 ml), and then extracted with EtOAc (2 × 150 ml). The organic layer was separated and dried over 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 / hexane. The alcohol starting material was recovered. Amount produced, 18.0 g; Yield, 55%.

[0468] ATX - 84: Step 8

Chem.

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

[0470] The progress of the reaction was monitored by TLC (10% MeOH in CHCl 3 ; 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 / hexane. The starting materials were recovered. Amount produced, 3.0 g; Yield, 37%.

[0472] ATX - 84: Step 9

Chem.

[0473] To a solution of 2.5 g of (Z)-nona-2-en-1-yl 6-((6-oxo-6-(tridec-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 at 5-minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 hour under a nitrogen atmosphere. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere. atmosphere.

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

[0475] The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with saturated NH 4 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 product was subjected to column chromatography.

[0476] The first purification was carried out using silica gel (100 - 200 mesh). 4.6 g of the 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 / hexane. The second purification was carried out using neutral alumina together with HPLC - grade solvents. 2.0 g of the crude compound was adsorbed onto 6.0 g of neutral alumina and the resulting material was poured onto 40.0 g of neutral alumina in a column. The compound was eluted with 20% EtOAc / hexane. Amount produced, 1.2 g; yield, 38% (from 300 mg of the mixture).

[0477] [Chemical formula]

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

[0479] ATX - 61: Step 1 [Chemical formula]

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

[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), 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 / hexane). Amount produced, 20.8 g; Yield, 88%.

[0484] ATX - 61: Step 2

Chem.

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

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

[0487] The reaction mass was concentrated, 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 the compound. Amount produced, 15 g; Yield, 92%; Confirmed by Mass.

[0488] ATX - 61: Step 3

Chem.

[0489] To a solution of 5 g of N - Boc - glycine ester (Int 1, 1 equiv) 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 this reaction solution, 5.12 g of heptadecen - 9 - ol (0.7 equiv) in 20 ml of DCM was added, and the mixture was stirred at room temperature overnight.

[0490] The starting material was observed to be absent by TLC (10% EtOAc / hexane; Rf: 0.6). The reaction mass was saturated with NaHCO 3It was diluted with a solution, the organic layer was separated, the aqueous layer was washed with DCM (2×30 ml), dried over sodium sulfate, and concentrated under reduced pressure. The crude product (6.8 g; a mixture of the product and alcohol) was used to proceed to the next step.

[0491] ATX-61: Step 4

Chemical formula

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

[0493] The completion of the reaction was confirmed by TLC (10% EtOAc / hexane; Rf: 0.5) in 2 hours. confirmed.

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

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

[0496] ATX-61: Step 5

Chemical formula

[0497] A solution of 4 g of bromoacetic acid (1 equivalent) dissolved in DCM (35 ml) and cooled to below 0 °C was added with 4.7 ml of Et 31.2 equivalents of N and 354 mg of DMAP (0.1 equivalent) were added, followed by 13.23 g of HATU (1.2 equivalents). To this reaction solution, 2.88 g of (Z)-nona-2-en-1-ol (0.7 equivalent) in 20 ml of DCM was added and stirred overnight at room temperature.

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

[0499] The reaction mass was diluted with saturated NaHCO 3 solution (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 residue was purified by silica gel (60 - 120) column chromatography (1.5% EtOAc / hexane). Amount produced, 4 g; Yield, 52%.

[0500] ATX-61: Step 6

Chemical formula

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

[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), extracted with EtOAc (20 ml × 2), the combined organic layers were dried over sodium sulfate, and concentrated under reduced pressure.

[0503] The residue was purified by column (silica gel; 100 - 200) chromatography (2% EtOAc / hexane). Amount produced, 700 mg; Yield, 47%; Confirmed by Mass.

[0504] ATX-61: Step 7 [Chem.]

[0505] A solution of 700 mg of heptadec-9-yl (Z)-(2-(nona-2-en-1-yloxy)-2-oxoethyl) glycinate) (1 equiv), dissolved in 15 ml of DCM and cooled to below 5 °C, was treated with 0.4 ml of Et 3 N (3 equiv), followed by 209 mg of triphosgene (0.5 equiv) added portionwise over 10 minutes.

[0506] The progress of the reaction mixture was monitored by TLC and the reaction was allowed to proceed for 0.5 h and the reaction mass was concentrated under reduced pressure.

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

[0508] After 1 h, completion of the reaction was observed by TLC (10% MeOH / CHCl 3 ; Rf: 0.5).

[0509] The reaction mass was quenched with saturated NH 4 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 layer was dried over Na 2 SO 4 and concentrated under reduced pressure.

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

[0511] [Chemical formula]

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

[0513] ATX-63: Step 1 [Chemical formula]

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

[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), 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 / hexane). Amount produced, 20.8 g; yield, 88%.

[0518] ATX-63: Step 2 [Chemical formula]

[0519] A solution of 18.9 g of N-Boc glycine ester (1 equivalent) dissolved in THF (130 ml) was added dropwise with an aqueous solution of 5.85 g of LiOH (1.5 equivalents), and the resulting solution was stirred at room temperature for 4 hours.

[0520] The reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.3), and the starting material was not present in the reaction product.

[0521] The reaction mass was concentrated, 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 the compound. Yield, 15 g; Yield, 92%; Confirmed by Mass.

[0522] ATX-63: Step 3

Chemical formula

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

[0524] The starting material was observed to be absent by TLC (15% EtOAc / hexane; Rf: 0.6). The reaction mass was diluted with saturated NaHCO 3 solution (20 ml), 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 product (5.5 g; a mixture of product and alcohol) was used for the next step.

[0525] ATX-63: Step 4

Chemical formula

[0526] Dissolve 3.3 g of crude undecan-6-yl (tert-butoxycarbonyl) glycinate (Int 2, 1 equivalent) in DCM (20 ml), cool to 0 °C, add 7.6 ml of TFA (10 equivalents), and stir at room temperature for 1 hour.

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

[0528] The crude product was subjected to column chromatography (silica, 60 - 120) using 1 - 3% MeOH / CHCl 3 and 2 mL of Et 3 N. The amount produced was 1.2 g; the yield was 40%; 1 confirmed by 1H-NMR and Mass.

[0529] ATX-63: Step 5

Chemical formula

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

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

[0532] The reaction mass was saturated with NaHCO 3It was diluted with a solution (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 residue was purified by silica gel (60 - 120) column chromatography (1.5% EtOAc / hexane). Production amount, 4 g; Yield, 52%.

[0533] ATX - 63: Step 6

Chemical formula

[0534] 1.2 g of undecan - 6 - yl glycinate (Int 3, 1 equivalent) was dissolved in 25 ml of THF, 0.9 ml of TEA (1.3 equivalents) and 1.37 g of (Z) - non - 2 - en - 1 - yl 2 - bromoacetate (Int 4, 1 equivalent) were added, and the mixture was 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), extracted with EtOAc (20 ml × 2), the combined organic layers were dried over sodium sulfate, and concentrated under reduced pressure.

[0536] The residue was purified by column (silica gel; 100 - 200) chromatography (3% EtOAc / hexane). Production amount, 800 mg; Yield, 37%; Confirmed by Mass.

[0537] ATX - 63: Step 7

Chemical formula

[0538] A solution of 800 mg of (Z) - non - 2 - en - 1 - yl (2 - oxo - 2 - (undecan - 6 - yloxy) ethyl) glycinate (1 equivalent) dissolved in DCM and cooled to below 5 °C was added portionwise with 0.4 ml of Et 3 N (3 equivalents), followed by 209 mg of triphosgene (0.5 equivalent) added in small portions over 10 minutes.

[0539] The progress of the reaction mixture was monitored by TLC, and the reaction was allowed to proceed for 1 hour to completion. The reaction mass was concentrated under reduced pressure.

[0540] 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 a nitrogen atmosphere was treated with 144 mg of sodium hydride (6 eq). After 10 minutes, the above solution was added to this reaction mass by dissolving it in THF. The resulting solution was stirred at room temperature for 1 hour.

[0541] After 1 hour, completion of the reaction was observed by TLC (70% EtOAc / hexane; Rf: 0.4). The reaction mass was quenched with saturated NH 4 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 layer was dried over Na 2 SO 4 and concentrated under reduced pressure.

[0542] The crude product was subjected to column chromatography using silica gel (100 - 200) with 20% EtOAc / hexane and then neutral alumina with 5% EtOAc / hexane to afford the pure compound. Amount produced, 510 mg; yield, 48%; 1 confirmed by 1H-NMR, HPLC, and Mass.

[0543]

Chemical Structure

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

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

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

[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), 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 / hexane). Amount produced, 20.8; Yield, 88%.

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

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

[0552] The reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.3) and the starting material was not present in the reaction product.

[0553] The reaction mass was concentrated, 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 the compound. Production amount, 15 g; Yield, 92%; Confirmed by Mass.

[0554] ATX-64: Step 3

Chem.

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

[0556] The starting material was observed to be absent by TLC (15% EtOAc / hexane; Rf: 0.6). The reaction mass was diluted with saturated NaHCO 3 solution, the organic layer was separated, the aqueous layer was washed with DCM (2×30 ml), dried over sodium sulfate and concentrated under reduced pressure.

[0557] After column filtration, the crude product (5.5 g; a mixture of product and alcohol) was used for the next step.

[0558] ATX-64: Step 4

Chem.

[0559] 3.85 g of crude heptadecane-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 hour.

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

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

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

[0563] ATX-64: Step 5

Chemical Structure

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

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

[0566] The reaction mass was diluted with saturated NaHCO 3 solution (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.

[0567] The residual mass was purified by silica gel (60 - 120) column chromatography (1.5% EtOAc / hexane). Yield, 4 g; Yield, 52%.

[0568] ATX-64: Step 6

Chemical Structure

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

[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), extracted with EtOAc (2 × 30 ml), the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

[0571] The residue was purified by column (silica gel; 100 - 200) chromatography (3% EtOAc / hexane). Yield, 2.2 g; Yield, 65%; Confirmed by Mass.

[0572] ATX-64: Step 7

Chemical formula

[0573] A solution of 2.2 g of heptadec-9-yl (Z)-(2-(nona-2-en-1-yloxy)-2-oxoethyl) glycinate )(1 equiv) dissolved in 15 ml of DCM and cooled to below 5 °C was added with 1.6 ml of Et 3 N (3 equiv), followed by 678 mg of triphosgene (0.5 equiv) added portionwise over 10 minutes.

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

[0575] 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 a nitrogen atmosphere, was added 672 mg of sodium hydride (7 eq). After 10 minutes, the above solution was added to this reaction mass by dissolving it in THF. The resulting solution was stirred at room temperature for 1 hour.

[0576] After 1 hour, the completion of the reaction was observed by TLC (70% EtOAc / hexane; Rf: 0.4). The reaction mass was quenched with saturated NH 4 Cl solution (25 ml), and water (20 ml) and EtOAc (20 ml) were added. The aqueous layer was washed with EtOAc (20 ml × 2), and the combined organic layers were washed with brine solution (20 ml). The organic layer was dried over Na 2 SO 4 and concentrated under reduced pressure.

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

[0578]

Chemical formula

[0579] (Example 15: pKa value) Lipids were titrated to measure their pKa values. The results are shown in the following table.

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, and compared. Female Balb / c mice (6 - 8 weeks old) were used for the evaluation of in vivo plasma erythropoietin (epo) levels after injection of mouse epo mRNA encapsulated in lipids. All formulations were administered intravenously via tail vein injection at doses of 0.03 and 0.1 mg / kg with a dosing volume of 5 ml / kg. Six hours after formulation injection, terminal blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected into 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 minutes. 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: Mouse Factor VII Silencing and EPO Expression in vivo) Using in vivo liver-directed screening of a liposome library, a series of compounds that promote high-level siRNA-mediated gene silencing in hepatocytes, the cells that make up the liver parenchyma, were tested. Factor VII, a blood coagulation factor, is an appropriate target gene for assaying functional siRNA delivery to the liver. Since this factor is specifically produced in hepatocytes, gene silencing indicates successful delivery to the parenchyma rather than delivery 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 the animals. Silencing at the mRNA level can be easily determined by measuring the protein level. This is because the half-life of the protein is short (2 - 5 hours). Compositions having siRNA targeting Factor VIII were formulated using ATX-ATX-002, ATX-57, and ATX-58, as well as the comparative example sample of phosphate buffered saline (PBS). Female C57BL / 6 mice (6 - 8 weeks old) were used for the FVII siRNA knockdown (KD) experiment.

[0582] All formulations were administered intravenously via tail vein injection at doses of 0.03 and 0.1 mg / kg at a dosing volume of 5 mg / kg. Forty-eight hours after formulation injection, terminal blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected into 0.109 M citrate buffer tubes and processed by centrifugation at 1200 G for 10 minutes. Plasma was collected and Factor VII protein levels were analyzed by a chromogenic assay (Biophen FVII, Aniara Corporation). A standard curve was constructed using samples from PBS-injected mice, and relative Factor VII expression was determined by comparing the treatment groups to untreated PBS controls. The 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 in vivo epo protein expression after delivery of lipid-encapsulated mouse epo mRNA, female Balb / c mice (6 - 8 weeks old) were used. All formulations were intravenously administered via tail vein injection at doses of 0.03 and 0.1 mg / kg with a dosing volume of 5 mL / kg. Six hours after formulation injection, terminal blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected into 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 minutes. 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, and relative factor VII expression was determined by comparing the treatment groups to untreated PBS controls. The results showed that epo mRNA was expressed at 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 are provided. (Item 1) Formula I:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

[Claim 1] The invention as depicted in the drawings.

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