BAD REFERENCE TO RELATED APPLICATIONS
This application is a continuation of US patent application no. No. 15/387,067, filed December 21, 2016, now U.S. Patent No. 10,383,952, issued August 20, 2019. Disclosure of U.S. Patent Application. Ser 14/707,876, filed May 8, 2015, now U.S. Patent No. 9,365,610, issued June 14, 2016, U.S. Patent Application No. 14/707,796, filed May 8, 2015, now US Patent U.S. No. 9,567,296, issued February 14, 2017, U.S. Patent Application. Ser No. 14/546,105, filed November 18, 2014, now U.S. Patent No. 9,593,077, issued March 14, 2017 and US Provisional Application. 61/905,724, filed November 18, 2013, are hereby incorporated by reference in their entirety.
RECORD
Various types of nucleic acids are currently being developed as therapeutic agents to treat a variety of diseases. As these molecules have evolved, there has been an increasing need to produce them in a form that is stable, durable, and can be easily incorporated into an anhydrous polar anhydrous or aprotic organic solvent to allow encapsulation of the nucleic acid without the side reactions that can occur. occur in a polar aqueous solution or in nonpolar solvents.
The disclosure herein relates to novel lipid compositions that facilitate the intracellular delivery of biologically active and therapeutic molecules. The disclosure also relates to pharmaceutical compositions comprising such lipid compositions useful for delivering therapeutically effective amounts of biologically active molecules to patient cells.
Administration of a therapeutic compound to an individual is important for its therapeutic effects and can generally be hampered by the compound's limited ability to reach target cells and tissues. Improving the penetration of such compounds into target tissue cells through various routes of administration is of crucial importance. The disclosure herein relates to novel lipid compositions and manufacturing methods that allow targeted intracellular delivery of biologically active molecules.
Examples of biologically active molecules that often fail to efficiently target tissue in a patient include: many proteins, including immunoglobulin proteins, polynucleotides such as genomic DNA, cDNA, or antisense mRNA polynucleotides. and many low molecular weight compounds, both synthetic and natural, such as peptide hormones and antibiotics.
One of the fundamental challenges for physicians today is that a large number of different types of nucleic acids are being developed as therapeutic agents to treat certain 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, antagonists, and aptamers. As these nucleic acids are developed, there is a need to produce lipid compositions that are simple to prepare and that can be easily delivered to a target tissue.
SUMMARY
A compound of formula I is described
Em
-
- R1is a branched alkyl consisting of 10 to 31 carbon atoms,
- R2is a linear alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms,
- grande1Me too2are the same or different and each is a linear alkylene or alkenylene of 2 to 20 carbon atoms;
- X1is S or O,
- R3is a linear or branched alkylene consisting of 1 to 6 carbon atoms and
- R4and R.S5are the same or different and are each hydrogen or a linear or branched alkyl having 1 to 6 carbon atoms.
or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, it is selected from the group consisting of a combination of ATX-43, ATX-57, ATX-58, ATX-61, ATX-63, ATX-64, ATX-81, ATX-82, ATX-83. ATX-84, ATX-86, ATX-87 and ATX-88 as follows.
In one embodiment, what is described herein is a compound in which R1is a branched 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 carbons ; R2is a linear alkyl, alkenyl or alkynyl group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms of carbon . big1Me too2are identical or different, each a linear alkylene or alkenylene consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons. X1is it S or O? R3is a straight or branched alkylene consisting of 1, 2, 3, 4, 5, 6 or 6 carbon atoms. and R4and R.S5are the same or different and are each hydrogen or a linear or branched alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms. In a preferred embodiment, R is1is -CH((CH2)NCH3)2ή -CH((CH).2)NCH3)((CH2)n-1CH3), where n is 4, 5, 6, 7 or 8. R2is a linear alkenyl. big1is a linear alkylene with 1, 2 or 3 carbon atoms. big2is a linear alkylene of 1-5 carbon atoms. X1is S? R3is a linear alkylene with 2 or 3 carbon atoms. and R4and R.S5are the same or different, each has 1 or 2 carbon atoms.
In one embodiment, the cationic lipids described herein are in a pharmaceutical composition. The pharmaceutical composition preferably comprises a lipid nanoparticle comprising a nucleic acid, preferably an RNA polynucleotide. The lipid nanoparticle preferentially increases the lifetime of circulating RNA. In another embodiment, after administering the pharmaceutical composition, the lipid nanoparticle contained therein releases the nucleic acid into the body's cells. Preferably, the nucleic acid has activity to suppress expression of a target gene. Alternatively, the nucleic acid has the activity of increasing the production of a protein it encodes when expressed in cells of the body.
Also described herein is a method of introducing a nucleic acid into a mammalian cell using any of the above compositions. The cell can be 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 of treating cancer or inflammatory diseases. The disease may be selected from the group consisting of an immune disorder, cancer, a kidney disease, a fibrotic disease, a genetic abnormality, an inflammation and a cardiovascular disorder.
DETAILED DESCRIPTION OF EXPLANATORY INCLUDES The definition
"At least one" means one or more (eg 1-3, 1-2 or 1).
"Composition" means a product containing the specified ingredients in the specified amounts and any product resulting directly or indirectly from a combination of the specified ingredients in the specified amounts.
"In combination with" means administering a compound of formula I with other drugs in the treatment methods of this invention means that the compounds of formula I and the other drugs are administered sequentially or simultaneously in separate dosage forms or simultaneously in the same dosage form.
“Mammal” means a human or other mammal or human.
"Patient" means humans and other mammals, preferably humans.
"Alkyl" means straight or branched, saturated or unsaturated hydrocarbon chain. In various embodiments, the alkyl group has from 1 to 18 carbons, as well as a C group1- AGAIN18Group or is C1- AGAIN12team, a c1- AGAIN6group or a C1- AGAIN4Association. Regardless, in various embodiments, the alkyl group has zero branches (i.e., it is a straight chain), one branch, two branches, or more than two branches. "Alkenyl" is an unsaturated alkyl that can have one double bond, two double bonds or more than two double bonds. "Alkynyl" is an unsaturated alkyl that can have one triple bond, two triple bonds, or more than two triple bonds. The alkyl chains can be optionally substituted by 1 substituent (ie, the alkyl group is monosubstituted), or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, and so on. Substituents may be selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano and the like. When the alkyl group contains one or more heteroatoms, the alkyl group is referred to herein as a heteroalkyl group. When the substituents of an alkyl group are hydrocarbons, the resulting group is simply referred to as a substituted alkyl. In various aspects, the alkyl group, including substituents, has less than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 carbons.
"Lower alkyl" means a group having from one to six carbon atoms in the chain, which chain may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl and hexyl.
"Alkoxy" means an alkyl-O- group, where alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy and heptoxy. Connection with the mother part occurs through etheric oxygen.
"Alkoxyalkyl" means an alkoxy-alkyl group where alkoxy and alkyl have the meaning described above. Preferred alkoxyalkyl includes a lower alkyl group. The bond to the parent moiety is through the alkyl.
"Alkylaryl" means an alkylaryl group where alkyl and aryl have the meanings described above. Preferred alkylaryls include a lower alkyl group. Attachment to the parental moiety is through the aril.
"Aminoalkyl" means an NH 2 -alkyl group, where the alkyl is as defined above and is attached to the parent group through the alkyl group.
"Carboxyalkyl" means a HOOC-alkyl group, where the alkyl is as defined above and is attached to the parent group through the alkyl group.
"Commercially available chemicals" and chemicals used in the examples presented herein can be obtained from standard commercial sources, such sources include, for example, Acros Organics (Pittsburgh, Pennsylvania), Sigma-Adrich Chemical (Milwaukee, Wisconsin), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, N.C.), Combi-Blocks (San Diego, California), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N.Y.), Fisher Scientific Co. (Pittsburgh, Pennsylvania), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, California), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co., Illinois), Riedel de Haen (Hannover , Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland, Oregon) and Wako Chemicals USA, Inc.
"Compounds described in the chemical literature" may be identified through reference works and databases relating to chemical compounds and chemical reactions as known to those skilled in the art. Suitable reference works and treatises detailing the synthesis of reagents useful in preparing the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, Synthetic Organic Chemistry, John Wiley and Sons, Inc. New York; S.R. Sandler et al., "Organic Functional Group Preparations", 2nd edition, Academic Press, New York, 1983; HO House, "Modern Synthetic Reactions", 2nd edition, WA Benjamin, Inc. Menlo Park, California, 1972; TL Glichrst, "Heterocyclic Chemistry", 2nd ed. John Wiley and Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure," 5th Edition, Wiley Interscience, New York, 2001; Specific reagents and analogues can also be identified from the inventories of known chemicals prepared by the Chemical Abstracts Service of the American Chemical Society, which are available in most public and university libraries and online databases (the American Chemical Society, Washington, D.C. can be consulted for Chemicals that are well known but not commercially available in catalogs can be manufactured by specialist chemical compounding companies, with many of the standard chemical suppliers (such as those listed above) offering custom compounding services.
"Halo" means fluorine, chlorine, bromine or iodine groups. Fluorine, chlorine or bromine are preferred, and fluorine and chlorine are more preferred.
"Halo" means fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are preferred.
"Heteroalkyl" means saturated or unsaturated, straight or branched chain containing carbon and at least one heteroatom. In various embodiments, the 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 to 18 (i.e., 1-18) member atoms (carbon and heteroatoms) and in various embodiments contains 1-12 or 1-6 or 1-4 member atoms. Regardless, in various embodiments, the heteroalkyl group has no branches (i.e., it is a straight chain), one branch, two branches, or more than two branches. Regardless, in one embodiment, the heteroalkyl group is saturated. In another embodiment, the heteroalkyl group is unsaturated. In various embodiments, the unsaturated heterocycle 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. Heteroalkyl chains can be substituted or unsubstituted. In one embodiment, the heteroalkyl chain is unsubstituted. In another embodiment, the heteroalkyl chain is substituted. A substituted heteroalkyl chain may have one substituent (i.e. mono-substituted) or e.g. B. 1-2 substituents, 1-3 substituents or 1-4 substituents. Examples of heteroalkyl substituents include esters (-C(O)-O-R) and carbonyls (-C(O)-).
"Hydroxyalkyl" means an OH-alkyl group, where alkyl is previously defined. Preferred hydroxyalkyls include lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.
The term "hydrate" denotes a solvent in which the solvent molecule is H2O.
"Lipid" means an organic compound that includes a fatty acid ester and is characterized by being insoluble in water but soluble in many organic solvents. Lipids are commonly classified into at least three categories: (1) “simple lipids,” which include fats, oils, and waxes; (2) “complex lipids” which include phospholipids and glycolipids. and (3) "derived lipids" such as steroids.
"Lipid particle" means a lipid composition that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). In preferred embodiments, the lipid particle is a nucleic acid lipid particle typically composed of a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a conjugated lipid that prevents aggregation of the particle (e.g., a PEG lipid) and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.
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, 0 nm to 7.0 nm to 80 nm to 100 nm nm 90nm to 100nm, from 70 to 90nm, from 80nm to 90nm, from 70nm to 80nm or 30nm, 35nm, 40nm, 40nm, 40nm, 40nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80n m, 85nm, 90nm, 95nm , 100nm, 105nm, 110nm, 115nm, 20nm, 115nm, 5nm, 10nm, 5nm, 140nm, 145nm or 150nm and are essentially non-toxic. Furthermore, when present in the lipid particles of the present invention, the nucleic acids are resistant to degradation by a nuclease in aqueous solution.
"Solvent" means a physical association of a compound of this disclosure with one or more solvent molecules. This physical bonding includes varying degrees of ionic and covalent bonds, including hydrogen bonds. In some cases, the solvated complex can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. Solvate includes solution-phase solutions and isolatable solutions. Non-limiting examples of suitable solvents include ethanol, methanol and the like.
"Encapsulated lipid" means a lipid particle that provides a therapeutic nucleic acid, such as fully encapsulated mRNA, partially encapsulated mRNA, or both. In a preferred embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid particle.
"Conjugated lipid" means a conjugated lipid that inhibits the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as e.g. B. PEG conjugated to dialkyloxypropylene (eg, PEG-DAA conjugate), PEG conjugated to diacylglycerols (eg, PEG-DAG conjugates), PEG-conjugated cholesterol, PEG-conjugated to phosphatidylethanolamines, and PEG-conjugated to ceramides, 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 through a linker moiety. Any linker moiety suitable for conjugating PEG or POZ to a lipid can be used including e.g. B. non-ester linker moieties and ester linker moieties. In some preferred embodiments, no ester-containing linker moieties, such as amides or carbamates, are used.
"Amphipathic lipid" refers to material in which the hydrophobic portion of the lipid material is oriented in a hydrophobic phase while the hydrophilic portion is oriented in the aqueous phase. Hydrophilic properties arise from the presence of polar or charged groups such as carbohydrate, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl and other similar groups. The hydrophobicity can be attributed to the inclusion of polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and those groups substituted by one or more aromatic, cycloaliphatic or heterocyclic groups. Examples of amphipathic compounds include phospholipids, aminolipids, and sphingolipids, among others.
Representative examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyleoylphosphatidylcholine, lysophosphatidylleoylaminophyllositolylcholine, dieloylphosphatidylcholine, distearoylphosphatidylcholine, atidylcholine , and dilinoleoylphosphatidylcholine, among others. Other compounds without phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols and β-acyloxyacids also belong to the group of amphipathic lipids. Furthermore, the amphipathic lipids described above can be mixed with other lipids, including triglycerides and sterols.
By "neutral lipid" is meant a lipid species that exists in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
"Noncationic lipid" means an amphipathic lipid or a neutral lipid or anionic lipid and is described in more detail below.
By "anionic lipid" is meant a lipid that is negatively charged at normal pH. These lipids include, but are not limited to, phosphatidilglycerol, cardiolipins, diacilphosphathydilserins, diacilphosphatic acids, n-dodecanoilphosphatidylelamines, n-cucinillphosphatidilenelamines, n-gluturiliaglycano-glycanoglycaliaglycidilates, olphospactidydilglycerol (Popgs ) and other anionic modifying groups that bind to neutral lipids.
"Hydrophobic lipids" means compounds having non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and those groups optionally substituted by one or more aromatic, cycloaliphatic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-dialkyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.
"Cationic lipid" and "aminolipid" are used interchangeably to mean lipids and their salts that contain one, two, three or more fatty acids or fatty alkyl chains and a pH-titter amino head group (e.g., a group alkylamino or dialkylamino). The cationic lipid is typically protonated (ie, positively charged) at pH below the pK.asof the cationic lipid and is essentially neutral at pH above the pKas. Cationic lipids according to the invention may also be referred to as titratable cationic lipids. In some embodiments, the cationic lipids include: a primary tertiary amine head group (eg, pH titratable); To do18Alkyl chains, each alkyl chain independently having 0 to 3 (e.g. 0, 1, 2 or 3) double bonds. and ether, ester, or ketal linkages between the head group and the alkyl chains. These 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-DM A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (aka MC2), DLin-M - C3-DMA (also known as MC3) and (DLin-MP-DMA) (also known as 1-Bl 1).
"Substituted" means replacement with certain groups other than hydrogen, or with one or more groups, fractions or fractions, which may be the same or different, each being, for example, independently selected.
"Antisense nucleic acid" means a non-enzymatic nucleic acid molecule that binds and modifies target RNA through RNA-RNA or RNA-DNA or RNA-PNA interactions (protein nucleic acid; Egholm et al., 1993 Nature 365 , 566) target RNA activity (for reviews see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., US Patent No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in some embodiments, an antisense molecule can bind to the substrate such that the substrate molecule forms a loop and/or an 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 segments of an antisense molecule can be complementary to a target sequence, or both. Furthermore, antisense DNA can be used to target RNA through DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense oligonucleotides may comprise one or more RNAse H activation domains capable of activating RNAse H cleavage of a target RNA. The antisense DNA can be chemically synthesized or expressed using a single-stranded DNA expression vector or equivalent. . "Antisense RNA" is a strand of RNA that has a sequence complementary to a target gene mRNA and is believed to induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense RNA and combines with the complementary antisense RNA to form iNA. These antisense and sense RNAs were synthesized conventionally with an RNA synthesizer.
"Nucleic acid" means deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids that contain known nucleotide analogues or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to the reference nucleic acid, and are metabolized similarly to the nucleotide. of reference. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).
"RNA" means a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. Terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from native RNA by addition, deletion, substitution, etc. ./or alteration of one or several nucleotides. Such alterations may include the addition of non-nucleotide material, for example, at the end(s) of an interfering RNA or internally, for example, 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 may be referred to as analogues or naturally occurring RNA analogues. As used herein, the terms "ribonucleic acid" and "RNA" refer to a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, noncoding RNA, and polyvalent RNA . A ribonucleotide is a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. These terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified and altered RNA that differs from natural RNA by addition, deletion, substitution, modification. and/or Alteration in one or more nucleotides. Alterations to an RNA can include the addition of non-nucleotide material, for example, at the end(s) of an interfering RNA or internally, for example, to one or more nucleotides of an RNA. Nucleotides in an RNA molecule include nonstandard nucleotides, such as nucleotides that do not occur naturally or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be called analogues.
"Nucleotides" means natural (standard) bases and modified bases known in the art. Such bases are usually located at the 1' position of a sugar nucleotide moiety. Nucleotides usually include a base, a sugar and a phosphate group. The nucleotides can be unmodified or modified in the sugar, phosphate and/or base moiety (alternatively referred to as nucleotide analogues, modified nucleotides, unnatural nucleotides, non-standard nucleotides and others; see, for example, Usman and McSwiggen, supra). Eckstein et al., PCT International Publication No. WO 92/07065, Usman et al., PCT International Publication No. WO 93/15187, Uhlman & Peyman, supra, all incorporated herein by reference). There are many examples of modified nucleic acid bases known in the art, as summarized by Limbach et al., Nucleic Acids Res. 22:2183, 1994. 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-alurimidine, 6-bromidine (e.g., or 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, and others ( Burgin et al., Biochemistry 35:14090, 1996 , Uhlman & Peyman, supra.) By "modified bases" herein is meant nucleotide bases other than adenine, guanine, and cytosine and uracil at the 1' position or their equivalents.
“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 acid that hybridize (ie, hydrogen bond) to each other. By "complementary" is meant that a nucleic acid can form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick linkage or other non-traditional types of linkage.
“MicroRNAs” (miRNA) are single-stranded RNA molecules 21 to 23 nucleotides long that regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into proteins (non-coding RNA). Instead, they are processed from primary transcripts, called pri-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.
"Small interfering RNA (siRNA)" and "small interfering RNA" and "silent RNA" refer to a class of double-stranded RNA molecules from 16 to 40 nucleotides in length that play a variety of roles in biology. More specifically, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in RNAi signaling, siRNAs also act in RNAi-related signaling pathways, e.g. as an antiviral mechanism or in designing the chromatin structure of a genome. The complexity of these pathways is only now being clarified.
"RNAi" denotes an RNA-dependent gene silencing process controlled by the RNA-induced silencing complex (RISC) and initiated by short double-stranded RNA molecules in a cell, where they combine with the argonaut of the RISC catalytic component to interact . When the RNA-like double-stranded RNA or iNA is exogenous (originating from viral infection with a transfected RNA or iNA or siRNA genome), the RNA or iNA is directly imported into the cytoplasm and cleaved into small fragments by the enzymatic fragment. The initial dsRNA can also be endogenous (cell-derived), as in pro-microRNAs expressed from RNA-encoding genes in the genome. The primary transcripts of such genes are first processed to form the characteristic pre-miRNA stem-loop structure in the nucleus and then exported to the cytoplasm, where they are cleaved via tableting. Thus, the two dsRNA pathways, exogenous and endogenous, converge in the RISC complex. The active components of an RNA-induced silencing complex (RISC) are endonucleases, called Argonaute proteins, which cleave the target mRNA strand complementary to its bound siRNA or iNA. As the fragments produced by Dicer are double-stranded, they could theoretically produce a functional siRNA or iNA. However, only one of the two chains, the so-called leader chain, binds to the Argonaute protein and controls gene silencing. The other anti-driver or passenger branch is affected during RISC activation.
Union Type I
Reference herein to a compound of formula I also includes reference to its salts, unless otherwise specified. The term "salt(s)" as used herein means acid salts formed with inorganic and/or organic acids and base salts formed with inorganic and/or organic bases. Furthermore, when the compound of formula I contains both a basic moiety, such as a pyridine or imidazole, and an acidic moiety, such as a carboxylic acid, zwitterions ("inner salts") are formed. ) can form and are included in the term "salt(s)" as used herein. The salts may be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts, although other salts are also useful. Salts of a compound of formula I can be formed, for example, by reacting a compound of formula I with an amount of acid or base, for example an equivalent amount, in a medium, for example one in which the salt precipitates, or in an aqueous medium. medium, followed by lyophilization.
Examples of acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, glycofuranphosphates, glycofurosulfates, ethanefurosulfates, heptanoic acid, hexanoates , hydrochlorides. , hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, picrates, propionates, propionates, propiones, valylsulfonates (as those listed mentioned herein), tartrates, thiocyanates, toluenesulfonates (also known as tosylates), undecanoate, and the like. Furthermore, acids which are generally considered suitable for the formation of pharmaceutically useful salts from a basic pharmaceutical compound are described, for example, by S. Berge et al. discussed.J Pharmaceutical Sciences(1977) 66(1)1-19; P. Gould, InternationalJ. Pharmaceuticals(1986) 33 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and the Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are expressly incorporated herein by reference.
Examples of base salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g. organic amines ), such as benzathines, dicyclohexylamines, hydrabins (formed). with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine and salts with amino acids such as arginine or lysine. Nitrogen-containing backbone 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, and dimethyl sulfates) . long chain halides (eg decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), arylalkyl halides (eg benzyl and phenethyl bromides) and others.
All such acidic and basic salts are intended to be pharmaceutically acceptable salts within the scope of the disclosure and all acidic and basic salts are considered equivalent to the free forms of the corresponding compound of formula I for purposes of the disclosure.
The compound of formula I can exist in unsolvated and solvated forms, including hydrated forms. In general, forms dissolved with pharmaceutically acceptable solvents, such as water, ethanol and the like, are synonymous with undissolved forms for purposes of this disclosure.
The compound of formula I and its salts and solvates may exist in their tautomeric form (for example as an amide or iminoether). All such tautomeric forms are herein considered part of the present disclosure.
Also within the scope of the present disclosure are polymorphs of the compound of this disclosure (i.e., polymorphs of the compound of formula I are within the scope of this disclosure).
All stereoisomers (e.g., geometric isomers, optical isomers and the like) of the present compound (including the salts, solvates and prodrugs of the compound and the salts and solvates of the prodrugs) that may exist, for example, due to carbons asymmetric in different substituents, including enantiomeric forms (which can also exist in the absence of asymmetric carbons), rotameric forms, atropisomers and diastereomeric forms are contemplated within the scope of this disclosure. The individual stereoisomers of the compound of this disclosure can be, for example, essentially free of other isomers, or they can be, for example, racemates or mixed with any other or selected stereoisomers. The chiral centers of the compound herein may have the S or R configuration as defined in the 1974 IUPAC Recommendations. The use of the terms "salt", "solution" and the like are intended to apply equally to salt and solvated complex enantiomers, stereoisomers, rotamers, tautomers, racemates or prodrugs of the disclosed compound.
Classes of compounds that can be used as chemotherapeutic (antineoplastic) agents include: alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogues), and synthetics. Examples of connections in these categories are listed below.
lipid particles
A compound of formula I comprises a pharmaceutically acceptable salt thereof in a lipid composition comprising a nanoparticle or bilayer of lipid molecules. The lipid bilayer preferably further comprises a neutral lipid or polymer. The lipid composition preferably comprises a liquid medium. The composition further preferably encapsulates a nucleic acid. The nucleic acid preferably has an activity to suppress target gene expression through RNA interference (RNAi). The lipid composition preferably further comprises a nucleic acid and a neutral lipid or polymer. The lipid composition preferably encapsulates the nucleic acid.
The disclosure provides lipid particles comprising one or more therapeutic mRNA molecules encapsulated in the lipid particles.
In some embodiments, the mRNA is completely encapsulated in the lipid portion of the lipid particle such that the mRNA in the lipid particle is resistant to nuclease degradation in aqueous solution. In other embodiments, the lipid particles described herein are essentially non-toxic to mammals, such as humans. 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 at 9 or 0 nm. ratio (mass/mass ratio) from 1:1 to 100:1, from 1:1 to 50:1, from 2:1 to 25:1, from 3:1 to 20:1, from 5:1 to 15 : 1, or from 5:1 to 10:1, or from 10:1 to 14:1, or from 9:1 to 20:1. In one embodiment, the lipid particles have a lipid:RNA ratio (mass-to-mass ratio) of 12:1. In another embodiment, the lipid particles have a lipid:mRNA ratio (mass-to-mass ratio) of 13:1.
In preferred embodiments, the lipid particles comprise an mRNA, a cationic lipid (e.g., one or more cationic lipids described herein or salts thereof), a phospholipid, and a conjugated lipid that inhibits particle aggregation (e.g., one or more PEG -lipid). conjugates). Lipid particles can also contain cholesterol. The lipid particles can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mRNAs that express one or more polypeptides.
In lipid nucleic acid particles, the mRNA can be completely encapsulated in the lipid portion of the particle, protecting the nucleic acid from degradation by nucleases. In preferred embodiments, a lipid particle comprising an mRNA is completely encapsulated in the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In some cases, the mRNA in the lipid particle is not significantly degraded after the particle is exposed to nuclease at 37°C for at least 20, 30, 45 or 60 minutes. In some other cases, the mRNA in the lipid particle is not significantly degraded after incubating the particle in serum at 37°C for at least 30, 45, or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours. In other embodiments, the mRNA is complexed with the lipid portion of the particle. One of the advantages of the formulations of the present invention is that the nucleic acid-lipid particle compositions are essentially non-toxic to mammals, such as humans.
"Fully encapsulated" means that the nucleic acid (eg, mRNA) in the lipid nucleic acid particle is not significantly degraded upon exposure to serum or a nuclease assay that would significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of the free nucleic acid, more preferably less than 10% and even more preferably less than 5% of the nucleic acid in the particle is degraded. "Fully encapsulated" also means that the lipid nucleic acid particles do not readily break down into their components when administered in vivo.
In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay using a dye that exhibits increased fluorescence when bound to nucleic acid. Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence and comparing it to the fluorescence observed when a small amount of non-ionic detergent is added. Detergent-mediated destruction of the liposomal 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)/I0, where/me too0refers to the fluorescence intensities before and after addition of detergent.
In other embodiments, the present invention provides a nucleic acid lipid particle composition comprising a plurality of nucleic acid lipid particles.
The lipid particle comprises mRNA completely encapsulated in the lipid portion of the particle such that from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to ..., 100%, 80% to 100%, 90% to 100%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95 %, from 80% to 95%, from 85% to 95%, from 90% to 95%, from 30% to 90%, from 40% to 90%, from 50% to 90%, from 60% to 90% , from 70% to 90%, from 80% to 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 fraction thereof or region thereof) of particles have the mRNA encapsulated in them .
Depending on the intended use of the lipid particles, the proportions of ingredients can be varied and the delivery effectiveness of a particular composition can be measured using tests known in the art.
cationic lipids
The description includes the synthesis of certain cationic lipid compounds. The compounds are particularly useful for delivering polynucleotides to cells and tissues, as will be demonstrated in the following sections. The lipomacrocyclic compound described herein can be used for other purposes, such as, for example, as receptors and additives.
Synthetic methods for cationic lipid compounds can be developed by those skilled in the art. Those skilled in the art will recognize other methods for preparing these compounds, as well as for preparing the other compounds of the specification.
Cationic lipid compounds can be combined with a drug to form microparticles, nanoparticles, liposomes or micelles. The active agent to be released from the particles, liposomes or micelles can be in the form of a gas, liquid or solid, and the active agent can be a polynucleotide, protein, peptide or small molecule. Lipomacrocyclic compounds can be combined with other cationic lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins or lipids to form the particles. These particles can then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.
The present disclosure provides novel cationic lipid compounds and drug delivery systems based on the use of such cationic lipid compounds. The system can be used in pharmaceutical/drug delivery technology to deliver polynucleotides, proteins, small molecules, peptides, antigens or drugs to a patient, tissue, organ or cell. These new compounds can also be used as materials for coatings, additives, adjuvants, materials or biotechnology.
The cationic lipid compounds of the present disclosure have many different uses in drug delivery technology. The amine-containing portion of cationic lipid compounds can be used to complex polynucleotides, thereby enhancing the distribution of polynucleotides and preventing their degradation. Cationic lipid compounds can also be used to form picoparticles, nanoparticles, microparticles, liposomes and micelles containing the drug to be administered. Preferably, the cationic lipid compounds are biocompatible and biodegradable and the formed particles are also biodegradable and biocompatible and can be used to provide controlled and sustained release of the active ingredient to be released. These and their counterparts can also respond to changes in pH as they become protonated at lower pH. They can also act as proton sponges, delivering an agent to a cell to induce endosome lysis.
In some embodiments, the cationic lipid compounds are relatively non-cytotoxic. Cationic lipid compounds can be biocompatible and biodegradable. The cationic lipid can have a pKasranging from about 5.5 to about 7.5, more preferably between about 6.0 and about 7.0. It can be designed to have a desired pK valueasbetween about 3.0 and about 9.0 or between about 5.0 and about 8.0. The cationic lipid compounds described here are particularly attractive for drug delivery for several reasons: they contain amino groups to interact with DNA, RNA, other polynucleotides and other negatively charged drugs, they regulate pH, induce endoosmosis and protect the drug. commercially available starting materials. and/or are pH responsive and can be designed with a desired pK valueas.
Additional neutral lipids
Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, augolipomelin encephalosides, diacetyl phosphate, distearoyl phosphatidyl inium (DSPC), dicoleleoyl phosphatidylcholine (DOPC), dipalmitoyl idylcholine phosphate (DPPC) , di-oleoylphosphatidylglycerol (DOPG) DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dipalmitoylphosphatidylethanolaminesulfatidylethanolamine (DEPE), stearoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine and mixtures thereof. Other phospholipids diacylphosphatidylcholine and diacylphosphatidylethanolamine can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids C10- AGAIN24Kohlenstoffketten, z.B. Lauroil, Miristoil, Palmitoil, Estearoil ou Oleoil.
Other examples of non-cationic lipids are sterols such as cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5α-coprostanol, cholesteryl (2'-hydroxy) ethyl ether, cholesteryl (4'-hydroxy) butyl ether and 6-ketocholestanol. non-polar analogues such as 5α-cholestan, cholestenone, 5α-cholestanone, 5α-cholestanone and cholesterol decanoate. and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analogue such as cholesteryl (4'-hydroxy)butyl ether.
In some embodiments, the noncationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of one or more phospholipids, for example. B. a cholesterol-free lipid particle formulation. In yet other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of cholesterol or a derivative thereof, e.g. B. a phospholipid-free lipid particle formulation.
Other examples of non-cationic lipids include lipids that do not contain phosphorus, such as. B. stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric polyethylaminaryl lauromer acrylates, amphoteric polyaminaryl acrylates, acetylfurlates, acetyl ester polymers. ylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide and sphingomyelin.
In some embodiments, the non-cationic lipid comprises 10 mol % to 60 mol %, 20 mol % to 55 mol %, 20 mol % to 45 mol %, 20 mol % to 40 mol % and 25 % in mol. up to 50% by mol, from 25% by mol to 45% by mol, from 30% by mol to 50% by mol, from 30% by mol to 45% by mol, from 30% by mol to 40% by mol, from 35% by mol to 45% by mol from 37% by mol to 42% by mol or 35% by mol, 36% by mol, 37% by mol, 38% by mol, 39% by mol, 40% by mol , 41% by mol, 42% by mol, 43% by mol, 44% by mol or 45 mol% (or any fraction or range thereof) of the total lipid present in the particle.
In applications where the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture can comprise up to 40 mol%, 45 mol%, 50 mol%, 55 mol% or 60 mol% of the total of lipids contained therein. to the particle.
In some embodiments, the phospholipid component in the mixture can be from 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol% mol or less constituting 4 mol%. to 10 mol% (or any fraction or range thereof) of the total lipid present in the particle. In some preferred embodiments, the phospholipid component in the mixture comprises 5 mol % to 10 mol %, 5 mol % to 9 mol %, 5 mol % to 8 mol %, 6 mol % to 9 mol % and 6 mol% to 8 mol%. to 8 mole % or 5 mole %, 6 mole %, 7 mole %, 8 mole %, 9 mole % or 10 mole % (or any fraction or range thereof) of the total lipid present in the particle.
In other embodiments, the cholesterol component in the mixture can be from 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol to 40% in mol and 27% in mol includes % in mol. to 37 mole %, from 25 mole % to 30 mole %, or from 35 mole % to 40 mole % (or any fraction or range thereof) of the total lipid present in the particle. In some preferred embodiments, the cholesterol component in the mixture comprises 25 mol % to 35 mol %, 27 mol % to 35 mol %, 29 mol % to 35 mol %, 30 mol % to 35 mol %. mol and 30% mol to 35% mol. to 34 mol %, from 31 mol % to 33 mol % or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol % or 35 mol % (or any fraction or range thereof). ) of the total lipid present in the particle.
In embodiments in which the lipid particles are free of phospholipids, cholesterol or its derivative may be up to 25% in mol, 30% in mol, 35% in mol, 40% in mol, 45% in mol, 50% in mol , 55% by mol or 60% by mol. % constitute molar % of the total lipid present in the particle.
In some embodiments, the cholesterol or its derivative in the composition of lipid particles without phospholipids can comprise 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol% and 30% in mol, 40% in mol, from 31% in mol to 39% in mol, from 32% in mol to 38% in mol, from 33% in mol to 37% in mol, from 35% in mol to 45% in mol mol%, from 30% mol to 35% mol, from 35% mol to 40% mol or 30% mol, 31% mol, 32% mol, 33% mol, 34% mol, 35 % in mol, 36% in mol, 37% in mol, 38% in mol, 39% in mol or 40% in mol (or any fraction or range thereof) of the total lipid present in the particle.
In other embodiments, the non-cationic lipid comprises 5 mol % to 90 mol %, 10 mol % to 85 mol %, 20 mol % to 80 mol %, 10 mol % (e.g., phospholipids only) or 60 mol% (eg, phospholipid and cholesterol or a derivative thereof) (or any fraction or region thereof) of the total lipid present in the particle.
The percentage of non-cationic lipid present in the lipid particles is a target amount and the actual amount of non-cationic lipid present in the composition can vary, for example, by ±5 mol%.
A composition containing a cationic lipid compound may consist of 30-70% cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid, and 1-10% polyethylene glycol (PEG). Preferably, the composition consists of 30-40% cationic lipid compound, 40-50% cholesterol and 10-20% PEG. In other preferred embodiments, the composition consists of 50-75% cationic lipid compound, 20-40% cholesterol and 5-10% phospholipid and 1-10% PEG. The composition may contain 60-70% cationic lipid compound, 25-35% cholesterol and 5-10% PEG. The composition may contain up to 90% cationic lipid compound and 2-15% auxiliary lipid.
The formulation may be a lipid particle formulation containing, for example, 8-30% compound, 5-30% accessory lipid, and 0-20% cholesterol. 4-25% cationic lipids, 4-25% accessory lipids, 2-25% cholesterol, 10-35% PEG-cholesterol and 5% amine-cholesterol. or 2-30% cationic lipid, 2-30% accessory lipid, 1-15% cholesterol, 2-35% PEG-cholesterol, and 1-20% amine-cholesterol. or up to 90% cationic lipids and 2-10% accessory lipids or even 100% cationic lipids.
lipid conjugates
In addition to being cationic, the lipid particles described herein may also comprise a lipid conjugate. The conjugated lipid is useful because it prevents particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.
In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG lipids include PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceram, Mit PEG conjugated to cholesterol or one thereof derivative and mixtures thereof.
PEG is a water-soluble linear polymer of repeating ethylene-PEG units terminated by two hydroxyl groups. PEGs are classified based on their molecular weight. and include the following: Monomethoxy Polyethylene Glycol (Mepeg-OH), Monomethoxy Polyethylene Glycol Sopoate (Mepeg-S), Monomethoxy Polyethylene Glycol Succinimidyl (Mepeg-S-nhs), Glycolic Acid (MePEG-TRES), Monomethoxy Polyethylene Glycol Imidazolylcarbonyl (MePEG -IM), as well as those compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e.g. HO-PEG-S, HO-PEG-S-NHS HO-PEG-NH2).
The PEG portion of the PEG-lipid conjugates described herein can have an average molecular weight ranging from 550 daltons to 10,000 daltons. In some cases, 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, 0 ton, 750 to 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of 2000 daltons or 750 daltons.
In some cases, the PEG may optionally be replaced with an alkyl, alkoxy, acyl or aryl group. PEG can be conjugated directly to the lipid or linked to the lipid through a linker moiety. Any linker moiety suitable for conjugating PEG to a lipid can be used including e.g. B. non-ester linker moieties and ester linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester linkage moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-) and carbamate (-NHC(O ) ) O-), urea ( -NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), Succinamidil (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (such as a linker containing a carbamate linker portion and an amido linker portion). In a preferred embodiment, a carbamate linker is used to conjugate the PEG to the lipid.
In other embodiments, an ester-containing linker moiety is used to conjugate the PEG to the lipid. Suitable ester-containing linker moieties include e.g. B. carbonates (-OC(O)O-), succinates, phosphates (-O-(O)POH-O-), sulfonic acid esters, and combinations thereof.
Phosphatidylethanolamines having a variety of acyl chain groups with different chain lengths and degrees of saturation can be conjugated with PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the C range10in G20are favoured. It is also possible to use phosphatidylethanolamines with mono or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids. Suitable phosphatidyl ethanolamines include, but are not limited to, dimyristoyl phosphatidyl ethanolamine (DMPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dioleoyl phosphatidyl ethanolamine (DOPE) and distearoyl phosphatidyl ethanolamine (PEE).
The term "diacylglycerol" or "DAG" encompasses a compound with two fatty acyl chains, R1and R.S2Both independently have between 2 and 30 carbon atoms attached to the 1 and 2 positions of glycerol through ester linkages. Acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), Miristoil (C14), Palmitoil (C16), Stearoyl (C18) e icosoil (C20). In preferred embodiments, R is1and R.S2are equal, that is, R1and R.S2sind beide Myristoyl (d. h. Dimyristoyl), R1and R.S2and also Estearoil (i.e. Distearoil).
The term "dialkyloxypropyl" or "DAN" embraces a compound having two alkyl chains, R and R, each independently having between 2 and 30 carbon atoms. Alkyl groups can be saturated or have varying degrees of unsaturation.
Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10)-Conjugate, a PEG-Dilauryloxypropyl (C12)-Conjugate, a PEG-Dimyristyloxypropyl (C14)-Conjugate, um PEG-Dipalmitiloxipropyl (C16) conjugate or a distearyloxypropyl PEG (C18) coupled. In these embodiments, the PEG preferably has an average molecular weight of 750 or 2000 daltons. In certain embodiments, the terminal hydroxyl group of PEG is replaced with a methyl group.
In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used in place of PEG include polyvinyl pyrolidone, polymethyl oxazoline, polyethylxazoline, polyhydropropyl metastlamide, polymetariamid and polycarpopulin. Hydrocellular Hydrocellulose or Factored Hydrocytes.
In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0.1% mol to 2% mol, 0.5% mol to 2% mol, 1% mol to 2% mol, 0.6% mol to 1.9 mol%. from 0.7% by mol to 1.8% by mol, from 0.8% by mol to 1.7% by mol, from 0.9% by mol to 1.6% by mol, from 0.9% in mol to 1.8% in mol, from 1% in mol to 1.8% in mol, from 1% in mol to 1.7% in mol, from 1.2% in mol to 1.8% in mol , from 1.2% by mol to 1.7% by mol, from 1.3% by mol to 1.6% by mol, or from 1.4% by mol to 1.5% by mol (or any fraction or portion thereof) of the total lipid present in the particle. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 0 mol % to 20 mol %, 0.5 mol % to 20 mol %, 2 mol % to 20 mol %, 1, 5% mol to 18% mol, 2% mol to 15% mol, 4% mol to 15% mol, 2% mol to 12% mol, 5% mol to 12 % in mol, or 2% in mol (or any fraction or range thereof) of the total lipid present in the particle.
In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises 4 mol % to 10 mol %, 5 mol % to 10 mol %, 5 mol % to 9 mol %, 5 mol % at 8% by 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 fraction thereof or range within) of the total lipid present in the particle.
The percentage of conjugated lipid (e.g., PEG-lipid) present in the lipid particles of the invention is a target amount, and the actual amount of conjugated lipid present in the formulation can vary, for example, ±2 mol%. Those skilled in the art will recognize that the concentration of the lipid conjugate can vary depending on the lipid conjugate used and the rate at which the lipid particle is desired to be fosfogenic.
By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate is exchanged from the lipid particle and, in turn, the rate at which the lipid particle becomes fusionogenic. In addition, other variables including e.g. for example, pH, temperature or ionic strength, can be used to alter and/or control the rate at which the lipid particle becomes fusionogenic. Other methods that can be used to control the rate at which the lipid particle becomes fusible will become apparent to those skilled in the art after reading this disclosure. By controlling the composition and concentration of the lipid conjugate, the size of the lipid particles can also be controlled.
compositions and preparations for administration
The lipid nucleic acid compositions of this disclosure can be administered in a variety of ways, for example, to achieve systemic administration via intravenous, parenteral, intraperitoneal or topical routes. In some embodiments, an siRNA can be administered intracellularly, for example, to cells in a target tissue, such as lung or liver, or to inflamed tissue. In some embodiments, this disclosure provides a method for delivering siRNA in vivo. A lipid nucleic acid composition can be administered to a subject intravenously, subcutaneously or intraperitoneally. In some embodiments, the disclosure provides methods for in vivo delivery of interfering RNA to the lungs of a mammal.
In some embodiments, this disclosure provides a method of treating a disease or disorder in a mammal. A therapeutically effective amount of a composition of this disclosure containing a nucleic acid, a cationic lipid, an amphiphile, a phospholipid, cholesterol and a PEG-linked cholesterol can be administered to a patient suffering from a disease or disorder related to gene expression or overexpression. . suffers .that can be reduced, diminished, diminished or muted from the composition.
The compositions and methods of disclosure can be administered to individuals via a variety of mucosal routes of administration, including oral, rectal, vaginal, intranasal, intrapulmonary or transdermal or dermal administration, or by topical administration to the eyes, ears, skin or other surfaces. mucous membranes. In some aspects of this disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal or gastrointestinal. The compositions of this disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically powered, or other types of actuators.
The compositions of this disclosure can be administered in an aqueous solution as a nasal or lung spray and can be administered in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this disclosure is accomplished by administering the composition in the form of drops, particles or sprays which may be, for example, aerosolized, sprayed or nebulized. The composition, spray or aerosol particles can be in liquid or solid form. Preferred systems for delivering liquids such as nasal sprays are described in U.S. Pat. No. 4,511,069. Such compositions can be easily prepared by dissolving compositions in accordance with the present disclosure in water to produce an aqueous solution and rendering that solution sterile. The formulations can be presented in multidose containers, such as the sealed delivery system described in US Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, Y.W. Chien ed., Elsevier Publishers, New York, 1985; and playing in the USA. No. 4,778,810. For other forms of aerosol delivery, z. B. Compressed air, jet, ultrasonic, and piezoelectric nebulizers that dissolved or suspended the biologically active agent in a pharmaceutical solvent, z. B. water, ethanol or mixtures thereof.
Nasal and lung spray solutions of the present disclosure typically comprise the medicament or drug to be administered, optionally formulated with a surfactant, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray also includes a propellant. The pH of the nasal spray solution can range from 6.8 to 7.2. Slightly acidic aqueous buffers with a pH of 4-6 can also be used as pharmaceutical solvents. Other ingredients may be added to improve or maintain chemical stability, including preservatives, surfactants, dispersants or gases.
In some embodiments, this disclosure is a pharmaceutical product comprising a solution containing a composition of this disclosure and an activator for a pulmonary, mucosal, or intranasal spray or aerosol.
A dosage form of the composition of this disclosure can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol.
A dosage form of the composition of this disclosure can be a solid that can be converted to a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet or gel.
To form compositions for pulmonary administration within the scope of the present disclosure, the biologically active agent can be combined with various pharmaceutically acceptable additives and a base or vehicle for dispersing the active agent(s). Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid and mixtures thereof. Other additives are local anesthetics (e.g. benzyl alcohol), isotonic agents (e.g. sodium chloride, mannitol, sorbitol), absorption inhibitors (e.g. Tween 80), solubility enhancers (e.g. cyclodextrins and their derivatives ), serum albumin stabilizers. and reducing agents (eg, glutathione). When the composition for transmucosal administration is liquid, the tonicity of the formulation, as measured by the tonicity of 0.9% normal saline (w/v), taken as a unit, is typically adjusted to a level at which no significant amounts are present. . Irreversible damage to mucosal tissue occurs at the site of administration. Generally, the tonicity of the solution is set to a value of ⅓ to 3, more typically ½ to 2, and most often 3/4 to 1.7.
The biologically active agent can be dispersed in a base or vehicle, which can comprise a hydrophilic compound capable of dispersing the active agent and any desired excipients. The base can be selected from a wide range of suitable carriers including, but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic acid anhydrides (e.g. maleic anhydride) with other monomers (e.g. methyl, acrylic acid, etc.). ). etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc. and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, hyaluronic acid. . A biodegradable polymer is often chosen as the base or carrier, for example polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid copolymer, poly(hydroxybutyric acid-glycolic acid) and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerol fatty acid esters, sucrose fatty acid esters, etc. they can also be used as vehicles. Hydrophilic polymers and other supports can be used singly or in combination, and improved structural integrity can be imparted to the support through partial crystallization, ionic bonding, crosslinking, and the like. The vehicle can be supplied in a variety of forms, including liquid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a vehicle selected in this context can have the effect of promoting the absorption of the biologically active agent.
Formulations for mucosal, nasal or pulmonary administration may contain a low molecular weight hydrophilic compound as a base or excipient. Such low molecular weight hydrophilic compounds provide a transit medium through which a water-soluble drug, such as a physiologically active peptide or protein, can diffuse through the bed to the body surface where the drug is absorbed. The low molecular weight hydrophilic compound optionally absorbs moisture from the mucosa or administration atmosphere and dissolves the water-soluble active peptide. The molecular weight of the low molecular weight hydrophilic compound is generally not greater than 10,000 and preferably not greater than 3,000. Examples of low molecular weight hydrophilic compounds include polyol compounds such as oligo-, di- and monosaccharides such as sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol and mixtures thereof. Other examples of low molecular weight hydrophilic compounds include N-methylpyrrolidone, alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.) and mixtures thereof.
The compositions of this disclosure may alternatively contain as pharmaceutically acceptable carrier substances necessary to approximate physiological conditions, such as pH adjusters and buffering agents, tonicity adjusters and wetting agents, for example sodium acetate, sodium lactate, sodium chloride , potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and mixtures thereof. For solid formulations, conventional non-toxic pharmaceutically acceptable carriers may be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like.
In some embodiments of the disclosure, the biologically active agent may be administered in a sustained release formulation, for example, a formulation comprising a slow release polymer. The active ingredient may be formulated with vehicles that protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Sustained release of the active ingredient can be achieved in various compositions of the disclosure by including in the composition agents which delay absorption, for example, monosteric aluminum hydrogels and gelatin.
While this disclosure has been described in terms of specific embodiments and many details are presented for purposes of illustration, it will be apparent to those skilled in the art that this disclosure encompasses additional embodiments and that some of the details described herein can vary significantly. without departing from this disclosure. This disclosure includes such additional embodiments, modifications and equivalents. In particular, this disclosure covers any combination of the features, terms or elements of the various figures and examples.
EXAMPLES Example 1: Exemplary Lipids
Examples of compounds of formula I are listed in Table 1.
Example 2: Synthesis of ATX-43
ATX-43: Stage 1
In a 500 mL, single-neck, round-bottom flask, 25 g of hexanoic acid (1 equivalent) dissolved in dichloromethane (DCM, 200 mL) were placed, followed by 27.6 mL of oxalyl chloride (1, 5 equivalent) were added slowly at 0°. C. Add with stirring under a nitrogen atmosphere and then 0.5 ml of dimethylformamide (DMF, catalyst) is added. The resulting reaction mixture was stirred at room temperature for 2 hours.
In a separate 1 L two-necked flask, to 31.4 g of N,O-dimethylhydroxylamine hydrochloride (1.5 equiv.) in DCM (200 mL) was added 89.8 mL of triethylamine (Et3N, 3 equivalents) was stirred at 0°C using a dropping funnel. To this resulting solution, after concentration under reduced pressure under nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (100 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (silica gel 60-120, 10% EtOAc/hexane). Production quantity: 20.0 g; Yield: 58%.
ATX-43: Stage 2
To a solution of 33 g of pentylmagnesium bromide (1.5 equivalents) in tetrahydrofuran (THF, 100 mL) placed in a 500 mL two-neck round bottom flask and stirred at 0°C under a nitrogen atmosphere were added 20 g of N-Methoxy-N-methylhexanamide (1 eq.) solution (dissolved in 200 mL of THF) and the resulting reaction mixture were stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (150 mL) was added followed by EtOAc (300 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). Quantity produced: 15.0 g; Yield: 66%.
ATX-43: Stage 3
To a solution of 15 g of undecan-6-one (1 equivalent) dissolved in 25 ml of methanol (MeOH) in 150 ml of THF was added 4.9 g of sodium borohydride (1.5 equivalent) at 0°C and the resulting solution was added and stirred at room temperature for 2 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production quantity: 14.0 g; Yield: 93%.
ATX-43: Stage 4
To a solution of 15 g of 4-aminobutyric acid (1 equivalent) dissolved in 150 mL of THF was added 145 mL of 1N aqueous NaOH solution (1 equivalent) at 0 °C, followed by 43.4 mL of Boc anhydride (1.3 equivalent). . .), successively through another funnel, at intervals of 15 minutes. The resulting solution was stirred at room temperature for 4 hours.
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 washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity: 20.0 g; Yield: 68%.
ATX-43: Stage 5
A solution of 12 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equivalent) dissolved in DCM (200 ml) was cooled below 0°C. Added 14.7 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), HCl (1.3 equiv), 10.6 mL of Et3N (1.3 equiv) and 0.72 g 4-dimethylaminopyridine (DMAP, 0.1 equiv) sequentially under nitrogen at 10 min intervals. To this resulting solution was added alcohol at the same temperature, dissolved in DCM (50 mL) using an additional funnel and stirred at room temperature for 24 hours under a nitrogen atmosphere.
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 (2x50ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (100 mL) and then extracted with EtOAc (2x50 mL). The organic layer was concentrated under reduced pressure and proceeded to the next step with crude oil. Production quantity: 8.5g; Yield: 48%.
ATX-43: Stage 6
To a solution of 8.5 g of undecan-6-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 equivalent) dissolved in 70 mL of DCM was added trifluoroacetic acid (TFA; 10 equivalents) at 0°C and stirred at room temperature for 4 hours under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (70% EtOAc/hexane, Rf: 0.2). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (150 mL) and then extracted with EtOAc (2x100 mL). The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. Production quantity: 5.0 g; Yield: 33% (based on alcohol).
ATX-43: Stage 7
To a solution of 14 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (100 mL) cooled below 0 °C were added 21 g of EDC•HCl (1.3 equivalents), 15.2 mL of Et3N (1.3 equivalents) and 1 g DMAP (0.1 equivalents) sequentially under nitrogen at 10 minute intervals. To this resulting solution was added 8.3 g of (Z)-non-2-en-1-ol (0.7 equiv.) dissolved in 50 ml of DCM using an additional funnel and stirred for 16 hours at room temperature under a stirred atmosphere of nitrogen. .
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 (2x50ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (100 mL) and then extracted with EtOAc (2x50 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. Production quantity: 11.0 g; Yield: 64%.
ATX-43: Stage 8
To a 250 mL round bottom flask are added 2 g of undecan-6-yl-4-aminobutanoate (1 eq.) and 2.2 g of (Z)-en-2-en-1-yl-4- bromobutanoate (1 eq.) was added DMF, 1.2 g of potassium carbonate (1.2 equivalents) was added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH in CHCl).3; Rf: 0.5). Ice water was added to the reaction mass and then extracted with EtOAc, dried over sodium sulfate and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. Original amines and bromine compounds were recovered. Production Quantity: 1.45g; Yield: 40%.
ATX-43: Stage 9
Dissolved in a solution of 1.45 g of (Z)-meth-2-en-1-yl 4-((4-oxo-4-(undecan-6-yloxy)butyl)amino)butanoate (1 equivalent). in dry DCM, 1.29 mL of triethylamine (3 equivalents) and 360 mg of triphosgene (0.4 equivalents) were added at 5 minute intervals at 0°C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To 360 mg of sodium hydride (5.5 equiv) dissolved in dry THF (20 mL) in a 250 mL two-necked round bottom flask stirred at 0°C under a nitrogen atmosphere was added 2.1 g of 2-(dimethylamino). Ethane, -1-thiol hydrochloride (5.5 equivalents) in THF (30 ml) and stirring continued for 5 minutes under a nitrogen atmosphere. To this resulting solution, the above carbonyl chloride dissolved in THF (50 mL) was added slowly over about 15 minutes using an additional funnel, added to this resulting solution and stirred at room temperature for 1 hour.
The reaction mass was quenched with saturated NH 34Cl (20 mL) was added followed by EtOAc (20 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 20 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography. Reaction progress was monitored by TLC (60% EtOAc/Hex, Rf: 0.5, PMA decarbonization).
Purification was by silica gel chromatography (100-200 mesh, 18% EtOAc/hexane). Quantity produced: 500 mg; Yield: 26%; confirmed by11H NMR; HPLC; and operation.
Example 3: Synthesis of ATX-57
ATX-57: Stage 1: N-Methoxy-N-methyloctanamide
To a 2 L two neck round bottom flask was added octanoic acid (1 eq.) dissolved in DCM (300 mL) and then 1.5 eq. Oxalyl chloride was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2 liter double-necked round bottom flask, in 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in DCM (200 mL) added 3 equiv. Trimethylamine was added using a dropping funnel and stirred at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (150 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity, 85g; Yield: 65%.
ATX-57: Step 2: Hexane-8-one
To a solution of octylmagnesium bromide in THF (100 mL) placed in a 1 L two-necked round bottom flask and stirred at 0 °C under a nitrogen atmosphere was added 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.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (250 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). Production quantity, 65g; Yield: 63%.
ATX-57: Stage 3: Hexahexan-8-ol
In a solution of hexadecan-8-one (1 equiv.) dissolved in MeOH/THF, 1 equiv. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1.5 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. production quantity, 60 g; Yield: 91%.
ATX-57: Step 4: 4-((tert-Butoxycarbonyl)amino)butanoic acid
To a solution of 4-aminobutyric acid dissolved in THF was added 1N aqueous NaOH solution at 0°C, followed by Boc anhydride sequentially using an addition funnel over a period of 15 minutes. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity, 80g; Yield: 81%.
ATX-57: Step 5: Hexadecane-8-il-4-((terc-butoxicarbonil)amino)butanoato
In a solution of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (200 mL) cooled below 0°C. added EDC.HCl, Et3N and 4-dimethylaminopyridine (DMAP) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution is added 1 equiv. At the same temperature, decahexan-8-ol alcohol was added, dissolved in DCM (150 mL) using an additional funnel and stirred at room temperature under nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Added solution (150 mL) and then EtOAc (200 mL). The organic layer was separated, concentrated under reduced pressure and proceeded to the next step with crude oil. Quantity produced: 80 g (crude oil, necessary compound and alcohol).
ATX-57: Step 6: Hexadecan-8-yl-4-aminobutanoate
To a solution of hexadecen-8-yl 4-((tert-butoxycarbonyl)amino)butanoate dissolved in DCM, TFA was added at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere. Reaction progress 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 NaHCO33solution (300 mL) and then extracted with EtOAc (2x200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. production quantity, 40 g; Yield: 59% for two steps. confirmed by music.
ATX-57: Step 7: (Z)-Me-2-en-1-yl-4-bromobutanoato
In a solution of 4-bromobutyric acid dissolved in DCM (400 mL) cooled below 0°C. added to EDC.HCl, Et3N and DMAP sequentially under a nitrogen atmosphere at 10 minute intervals. To this resulting solution was added (Z)-non-2-en-1-ol, dissolved in 100 mL of DCM using an additional funnel and stirred at room temperature for 24 hours under a nitrogen atmosphere.
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 (2x150ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (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. Alcohol recovered. Production quantity: 27g; Yield: 51%.
ATX-57: Step 8: Hexadecan-8-yl (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate.
To a solution of hexadecen-8-yl-4-aminobutanoate, (Z)-non-2-en-1-yl-4-bromobutanoate in acetonitrile (ACN) was added potassium carbonate and the resulting mixture was refluxed at 90° W. . for 4 hours under a nitrogen atmosphere. Reaction progress 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 concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. SMs (amines and bromine compounds) were recovered. production quantity, 20 g; Yield: 40%; confirmed by music.
ATX-57: Stage 9
To a solution of hexadecan-8-yl(Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in dry DCM were added trimethylamine and triphosgene. Leave for 5 minutes at 0°C under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To sodium hydride dissolved in dry THF (50 mL) in a 100 mL two-necked round bottom flask was 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 carbamoyl chloride dissolved in THF (80 mL) was slowly added via syringe over a period of about 10 minutes. The resulting solution was stirred at room temperature for 6 hours under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (75 mL) was added followed by EtOAc (150 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
The first purification was done with silica gel (60-120 mesh). 22 g of the crude compound were adsorbed onto 60 g of silica gel and poured onto 500 g of silica gel obtained from the column. The compound was eluted in 35% EtOAc/hexane. The second purification was done using neutral alumina with HPLC grade solvents. 7.5 g of crude compound was adsorbed onto 18 g of neutral alumina and the resultant was poured onto 130 g of neutral alumina taken from the column. The compound was eluted in 10% EtOAc/hexane. Yield: 29%; confirmed by NMR, HPLC and mass.
ATX-57/RL-43C: (PPM, 400 MHz, CDCl3): δ=5,63 (m, 1), 5,51 (m, 1), 4,68 (m, 1), 4,83 (d, J=7,0, 2), 3,19 ( brs, 4), 3,22 (m, 2), 2,52 (m, 2), 2,23-2,37 (4), 2,18 (s, 6), 2,08 (m, 2 ), 1,84–1,93 (4), 1,46–1,54 (4), 1,20–1,40 (30), 0,83–0,91 (9).
Example 4: Synthesis of ATX-58
ATX-58: Stage 1
In a 500 ml two-necked bottle under N2Atmosphere, 30 g of 8-bromooctanoic acid (1 equivalent) were taken dissolved in 200 mL of DCM and then slowly added to 26.7 mL of oxalyl chloride (1.5 equivalent) at 0°C with stirring under a nitrogen atmosphere . The resulting reaction mixture was stirred at room temperature for 2 hours.
In a separate 1 L round bottom round bottom flask, 40.5 g of N,O-dimethylhydroxylamine hydrochloride (2 equivalents) in 300 mL of DCM was added with 87 mL of trimethylamine (3 equivalents) and stirred at 0 °C. To this resulting solution, after concentration under reduced pressure by dissolving in 500 ml of DCM, the above hydrochloric acid was added dropwise using a dropping funnel over 15 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (2x100ml). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity, 28 g.
ATX-58: Stage 2
To a solution of 28 g of hexylmagnesium bromide (1 equivalent) in THF (100 mL) stirred at 0 °C under a nitrogen atmosphere was added 36.8 g of N-methoxy-N-methyloctanamide (1.3 equivalent) in 200 mL of THF. and the resulting reaction mixture was stirred at room temperature for 5 hours.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl (100mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 2% ethyl acetate/hexane). production amount, 24 g; Yield: 77%.
ATX-58: Stage 3
To a solution of 24 g of tetradecan-7-one (1 equivalent) dissolved in MeOH/THF was added 4.27 g of sodium borohydride (1 equivalent) at 0°C and the resulting solution was stirred at room temperature for 1 hour. .
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl (50mL). 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 washed with EtOAc (2 x 80 mL). The combined organic layer was concentrated under reduced pressure to obtain a white solid. Production Quantity: 21.5g; Yield: 89%.
ATX-58: Stage 4
To a solution of 20 g of 4-aminobutyric acid dissolved in 140 mL of THF was added, using a funnel, 196 mL of 1N aqueous NaOH solution at 0°C, followed by 36.8 g of Boc anhydride. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress 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 washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. production quantity, 30 g; Yield: 76%.
ATX-58: Stage 5
A solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid (1 equiv) dissolved in DCM (150 mL) was cooled below 0°C. Added 12.2 g of EDC.HCl (1.3 eq.), 20.4 mL of Et3N (3 equivalents) and 488 mg DMAP (0.1 equivalents) sequentially at 10 minute intervals. To this resulting solution was added alcohol dissolved in DCM using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mass was quenched with water (100ml) and the organic layer separated. The aqueous layer was washed with DCM (2x50ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution and EtOAc (100 mL) was added. The organic layer was separated and concentrated under reduced pressure and proceeded to the next step with crude oil. Production Quantity: 12.7 g (raw).
ATX-58: Stage 6
To a solution of 12.5 g of tetradecan-7-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 100 mL of DCM was added 23.9 mL of TFA (10 eq.) at 0 °C added. and stirred at 0°C to room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH/CHCl).3; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Solution (100 mL) and EtOAc (100 mL) were added. The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3) and alcohol was found. production amount, 7 g; Yield: 47% for two steps. confirmed by music.
ATX-58: Stage 7
A solution of 20 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (150 ml) was cooled to 0°C. 1.5 equiv. added. EDC.HCl, 3 equiv. etc.3N is 0.1 equiv. DMAP consecutively with an interval of 10 minutes. In this resulting solution, 0.7 equiv. (Z)-Me-2-en-1-ol was added, dissolved in 100 mL of DCM with a funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution and EtOAc (150 mL) was added. The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 5% EtOAc/hexane). Production quantity: 17g; Yield: 69%; confirmed by1H-RMN.
ATX-58: Stage 8
To a solution of 6 g of tetradecan-7-yl-4-aminobutanoate (1 equivalent) is added 5.8 g of (Z)-non-2-en-1-yl 4-bromobutanoate (1 equivalent) in ACN ( 125 mL) given. 2.7 g of potassium carbonate (1.2 equivalents) were added and the resultant was refluxed at 90°C for 3 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH/CHCl).3; Rf: 0.5). The reaction mass was filtered and the filtrate was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (100-200 mesh silica gel, 15% EtOAc/hexane). Production quantity: 4.5g; Yield: 44%; confirmed by music.
ATX-58: Stage 9
In a solution of 4.4 g of (Z)-non-2-en-1-yl-4-((4-oxo-4-(tetradecan-7-yloxy)butyl)amino)butanoate (1 eq.) , dissolved in 30 ml of dry DCM, 0.83 ml of trimethylamine (3 equivalents) and 418 mg of triphosgene (0.5 equivalents) were added for 5 minutes at 0°C under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To 192 mg of sodium hydride (10 equivalents) dissolved in dry THF (25 mL) in a 100 mL two-necked round bottom flask was added 564 mg of 2-(dimethylamino)propane-1-thiol hydrochloride (5 equivalents) at 0 °C and dissolved stirring continued for 5 minutes under a nitrogen atmosphere. To this resulting solution was added the above carbamoyl chloride dissolved in THF (35 mL) slowly over about 10 minutes via syringe. The resulting solution was stirred at room temperature for 4 hours under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (30 mL) was added followed by EtOAc (100 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 50 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
A first purification was done with silica gel (60-120 mesh). 5.0 g of crude compound was adsorbed onto 9 g of silica gel and poured onto 90 g of silica gel obtained from the column. The compound was eluted in 35% EtOAc/hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 1.5g of crude compound was adsorbed onto 4g of neutral alumina and the resultant was poured onto 40g of neutral alumina removed from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.2g; Yield: 21%; confirmed by1H-NMR; HPLC; Mass.
ATX-58/RL-43B: III-RMN (PPM, 400 MHz, CDCl3): δ=5,65 (m, 1), 5,52 (m, 1), 4,86 (m, 1), 4,63 (d, J=7,0, 2), 3, 37 (brs, 4), 3,02 (t, J = 6,0, 2), 2,53 (t, J = 6,0, 2), 2,27-2,36 (4), 2, 27 (s, 6), 2,09 (m, 2), 1,83-1,96 (4), 1,46-1,54 (4), 1,20-1,40 (26) , 0 ,84-0,91 (9).
Example 5: Synthesis of ATX-81
ATX-81: Stage 1
Octanoic acid dissolved in DCM (200 mL) and then 1.5 eq. Oxalyl chloride was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2 liter double-necked round bottom flask, in 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in DCM (200 mL) added 3 equiv. Trimethylamine was added using a dropping funnel and stirred at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (150 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity: 33g; Yield: 84%.
ATX-81: Stage 2
To a solution of 22 g of heptylmagnesium bromide (1.5 equivalents) in THF (100 mL) placed in a 1 liter two neck round bottom flask and stirred at 0°C under nitrogen was added N-methoxy-N. -methyloctanamide solution (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (250 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). production amount, 22 g; Yield: 65%.
ATX-81: Stage 3
In a solution of 22 g of pentadecan-8-one (1 eq.) dissolved in MeOH/THF, 1.5 eq. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1 hour.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. production quantity, 20 g; Yield: 90%.
ATX-81: Stage 4
To a solution of 50 g of 4-aminobutyric acid dissolved in 350 mL of THF was added 490 mL of a 1N aqueous NaOH solution at 0°C, followed by 140 mL of Boc anhydride sequentially using an addition funnel over a period of 15 minutes time. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity, 80g; Yield: 81%.
ATX-81: Stage 5
A solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (250 ml) was cooled below 0°C. 1.3 equiv. added. EDC.HCl, Et.3N and 4-dimethylaminopyridine (DMAP) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution is added 1 equiv. Alcohol pentadecan-7-ol was added at the same temperature, dissolved in DCM (150 mL) using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Added solution (150 mL) and then EtOAc (200 mL). The organic layer was separated, concentrated under reduced pressure and proceeded to the next step with crude oil. Production quantity: 8.5 g (crude oil, necessary compound and alcohol).
ATX-81: Stage 6
10 equiv. TFA dissolved at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 NaHCO33solution (300 mL) and then extracted with EtOAc (2x200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. production amount, 4 g; Yield: 25% for two steps. confirmed by music.
ATX-81: Stage 7
In a solution of 4-bromobutyric acid dissolved in DCM (300 mL) cooled below 0°C. added to EDC.HCl, Et3N and DMAP sequentially under a nitrogen atmosphere at 10 minute intervals. To this resulting solution, 20 g of (Z)-non-2-en-1-ol dissolved in 100 mL of DCM was added using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x150ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (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. Alcohol recovered. Production quantity: 19g; Yield: 55%.
ATX-81: Stage 8
In a solution of 4.5 g of pentadecan-8-yl-4-aminobutanoate, 1 equiv. (Z)-en-2-en-1-yl-4-bromobutanoate in 70 mL acetonitrile (ACN), 1.4 equiv. Potassium carbonate was added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress 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 concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. SMs (amines and bromine compounds) were recovered. Production quantity: 2.1g; Yield: 27%; confirmed by music.
ATX-81: Stage 9
To a solution of 2.1 g of pentadecan-8-yl(Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 150 mL of dry DCM , became 3 equiv. added. Triethylamine and triphosgene with 5 minutes interval at 0°C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
In 7 equiv. Sodium hydride dissolved in dry THF (80 mL) in a 100 mL two-neck round bottom flask with stirring at 0°C. under nitrogen atmosphere 3.5 equiv. 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 carbamoyl chloride dissolved in THF (80 mL) was slowly added via syringe over a period of about 10 minutes. The resulting solution was stirred overnight at 0°C and room temperature under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (75 mL) was added followed by EtOAc (150 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
The first purification was carried out using silica gel (60-120 mesh) of the crude compound adsorbed on 60 g of silica gel and poured onto 500 g of silica gel on the column. The compound was eluted in 35% EtOAc/hexane. The second purification was done using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and the resultant was poured onto 130 g of neutral alumina taken from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.5g; Yield: 45%. confirmed by1H RMN, HPLC e massa.
ATX-81/RL-48B:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,52 (m, 1), 4,86 (m, 1), 4,63 (d, J=7,0, 2), 3, 31- 3,44 (4), 3,02 (t, J=7,0, 2), 2,52 (t, J=7,0, 2), 2,26-2,36 (4), 2, 27 (s, 6), 2,10 (m, 2), 1,84-1,95 (4), 1,46-1,54 (4), 1,20-1,40 (26), 0 ,85-0,94 (9).
Example 6: Synthesis of ATX-82
ATX-82: Stage 1
To a 2-liter, two-necked, round-bottom flask was added 30 g of octanoic acid dissolved in DCM (200 mL) and then 1.5 equiv. Oxalyl chloride was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2 liter double-necked round bottom flask, in 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in DCM (200 mL) added 3 equiv. Trimethylamine was added using a dropping funnel and stirred at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (150 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity: 33g; Yield: 84%.
ATX-82: Stage 2
To a solution of heptyl magnesium 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 was added 28 g of N- methoxy-N. -methyloctanamide solution (1 eq.) (dissolved in 200 ml of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (250 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). production amount, 22 g; Yield: 65%.
ATX-82: Stage 3
In a solution of 22 g of pentadecan-8-one (1 eq.) dissolved in MeOH/THF, 1.5 eq. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1 hour.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. production quantity, 20 g; Yield: 90%.
ATX-82: Stage 4
To a solution of 15 g of 4-aminobutyric acid dissolved in 120 mL of THF at 0 °C was added 185 mL of 1N aqueous NaOH solution followed by 50 mL of Boc anhydride using sequentially through another funnel of an additional funnel 15 min . the resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity: 27g; Yield: 85%.
ATX-82: Stage 5
A solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (250 ml) was cooled below 0°C. 1.3 equiv. added. EDC.HCl, Et.3N and 4-dimethylaminopyridine (DMAP) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution is added 1 equiv. Alcohol pentadecan-7-ol was added at the same temperature, dissolved in DCM (150 mL) using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Added solution (150 mL) and then EtOAc (200 mL). The organic layer was separated, concentrated under reduced pressure and proceeded to the next step with crude oil. Amount produced: 8 g (crude oil, necessary compound and alcohol).
ATX-82: Stage 6
10 equiv. TFA dissolved at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 NaHCO33solution (300 mL) and then extracted with EtOAc (2x200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. production amount, 4 g; Yield: 25% for two steps. confirmed by music.
ATX-82: Stage 7
In a solution of 4-bromobutyric acid dissolved in DCM (400 mL) cooled below 0°C. to 1.5 equiv. added. EDC.HCl, 3 equiv. etc.3N and DMAP sequentially under a nitrogen atmosphere at 10 minute intervals. To this resulting solution, 20 g of (Z)-non-2-en-1-ol dissolved in 100 mL of DCM was added using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x150ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (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. Alcohol recovered. Production quantity: 18g; Yield: 55%.
ATX-82: Stage 8
In a solution of 4.0 g of pentadecan-8-yl-4-aminobutanoate, 1 equiv. (Z)-Me-2-en-1-yl-4-bromobutanoate in 90 mL ACN, 1.4 eq. Potassium carbonate was added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress 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 concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. The starting materials (amines and bromine compounds) were recovered. Production quantity: 2.2g; Yield: 30%; confirmed by music.
ATX-82: Stage 9
To a solution of 2.2 g of pentadecan-8-yl(Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)butanoate dissolved in 25 mL of dry DCM , became 3 equiv. added. Triethylamine and triphosgene with 5 minutes interval at 0°C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
In 7 equiv. Sodium hydride dissolved in dry THF (100 mL) in a 100 mL two-neck round bottom flask with stirring at 0°C. under nitrogen atmosphere 3.5 equiv. 2-(Dimethylamino)propane-1-thiol hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution was added the above carbamoyl chloride dissolved in THF (100 mL) slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at 0°C and room temperature under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (75 mL) was added followed by EtOAc (150 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
The first purification was carried out using silica gel (60-120 mesh) of the crude compound adsorbed on 60 g of silica gel and poured onto 500 g of silica gel on the column. The compound was eluted in 35% EtOAc/hexane. The second purification was done using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and the resultant was poured onto 130 g of neutral alumina taken from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.2g; Yield: 43%; confirmed by1H RMN, HPLC e massa.
ATX-82/RL-47A:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,52 (m, 1), 4,87 (m, 1), 4,62 (d, J=7,0, 2), 3,61 ( t, J=7,0, 2), 3,28-3,37 (2), 3,02 (t, J=7,0, 2), 2,61 (m, 2), 2,52 ( t, J = 7,0, 2), 2,31 (m, 2), 2,27 (s, 6), 2,10 (m, 2), 1,62-1,70 (6), 1 ,21-1,40 (32), 0,85-0, 91 (9).
Example 7: Synthesis of ATX-86
ATX-86: Stage 1
To a 2-liter, two-necked, round-bottom flask was added 30 g of octanoic acid dissolved in DCM (200 mL) and then 1.5 equiv. Oxalyl chloride was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2 liter double-necked round bottom flask, in 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in DCM (200 mL) added 3 equiv. Trimethylamine was added using a dropping funnel and stirred at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (150 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity: 38g; Yield: 84%; confirmed by music.
ATX-86: Stage 2
To a solution of magnesium hexyl 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 was added 38 g of N-methoxy-N.-methyloctanamide solution (1 eq.) (dissolved in 200 mL of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (250 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). Production quantity, 44g; Yield: 65%; confirmed by music.
ATX-86: Stage 3
In a solution of 44 g of tridecan-7-one (1 eq.), dissolved in MeOH/THF, 1.5 eq. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1 hour.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. production quantity, 40 g; Efficiency: 90%; confirmed by music.
ATX-86: Stage 4
To a solution of 50 g of 4-aminobutyric acid dissolved in 350 mL of THF was added 490 mL of a 1N aqueous NaOH solution at 0°C, followed by 140 mL of Boc anhydride sequentially using an addition funnel over a period of 15 minutes time. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity, 80g; Yield: 81%; confirmed by music.
ATX-86: Stage 5
A solution of 10 g of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (250 ml) was cooled below 0°C. 1.3 equiv. added. EDC.HCl, Et.3N and 4-dimethylaminopyridine (DMAP) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution is added 1 equiv. Alcohol pentadecan-7-ol was added at the same temperature, dissolved in DCM (150 mL) using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Added solution (150 mL) and then EtOAc (200 mL). The organic layer was separated, concentrated under reduced pressure and proceeded to the next step with crude oil. Amount produced: 8 g (crude oil, necessary compound and alcohol).
ATX-86: Stage 6
10 equiv. TFA dissolved at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 NaHCO33solution (300 mL) and then extracted with EtOAc (2x200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. Production quantity: 3.5g; Yield: 52% for two steps. confirmed by music.
ATX-86: Stage 7
In a solution of 4-bromobutyric acid dissolved in DCM (400 mL) cooled below 0°C. to 1.5 equiv. added. EDC.HCl, 2 equiv. etc.3N and DMAP sequentially under a nitrogen atmosphere at 10 minute intervals. To this resulting solution, 20 g of (Z)-non-2-en-1-ol dissolved in 100 mL of DCM was added using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x150ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (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. Alcohol recovered. Production quantity: 18g; Yield: 55%.
ATX-86: Stage 8
In a solution of 4.0 g of tridecan-7-yl-4-aminobutanoate, 1 equiv. (Z)-Me-2-en-1-yl-4-bromobutanoate in 90 mL ACN, 1.4 eq. Potassium carbonate was added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress 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 concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. SMs (amines and bromine compounds) were recovered. Production quantity: 2.2g; Yield: 30%; confirmed by music.
ATX-86: Stage 9
To a solution of 2.2 g of (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)tridecan-7-ylbutanoate dissolved in 25 mL of dry DCM was added 3 equiv. added. Triethylamine and triphosgene with 5 minutes interval at 0°C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
In 7 equiv. Sodium hydride dissolved in dry THF (100 mL) in a 100 mL two-neck round bottom flask with stirring at 0°C. under nitrogen atmosphere 3.5 equiv. 2-(Dimethylamino)propane-1-thiol hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution was added the above carbamoyl chloride dissolved in THF (100 mL) slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at 0°C and room temperature under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (75 mL) was added followed by EtOAc (150 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
The first purification was carried out using silica gel (60-120 mesh) of the crude compound adsorbed on 60 g of silica gel and poured onto 500 g of silica gel on the column. The compound was eluted in 35% EtOAc/hexane. The second purification was done using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and the resultant was poured onto 130 g of neutral alumina taken from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.2g; Yield: 43%; confirmed by1H RMN, HPLC e massa.
ATX-86/RL-48A:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,51 (m, 10, 4,87 (m, 1), 4,63 (d, J=7,0, 2), 3,30-3 ,44 (4), 3,02 (t, J=7,0, 2), 2,52 (t, J=7,0, 2), 2,26-2,36 (4), 2,27 (s, 6), 2,09 (m, 2), 1,82-1,96 (4), 1,46-1,54 (4), 1,21 -1,40 (24), 0, 84-0,91 (9).
Example 8: Synthesis of ATX-87
ATX-87: Stage 1
To a two-liter, two-necked round bottom flask was added 20 g of octanoic acid dissolved in DCM (200 mL) and then 1.5 equiv. Oxalyl chloride was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2 liter double-necked round bottom flask, in 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in DCM (200 mL) added 3 equiv. Trimethylamine was added using a dropping funnel and stirred at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (150 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). production quantity, 20 g; Yield: 84%.
ATX-87: Stage 2
To a solution of magnesium hexyl 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 was added 20 g of N-methoxy-N.-methyloctanamide solution (1 eq.) (dissolved in 200 mL of THF) and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (250 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 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). production quantity, 25 g; Yield: 65%.
ATX-87: Stage 3
In a solution of 25 g of tridecan-7-one (1 eq.) dissolved in MeOH/THF, 1.5 eq. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1 hour.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. production amount, 22 g; Yield: 90%.
ATX-87: Stage 4
To a solution of 50 g of 4-aminobutyric acid dissolved in 350 mL of THF was added 490 mL of a 1N aqueous NaOH solution at 0°C, followed by 140 mL of Boc anhydride sequentially using an addition funnel over a period of 15 minutes time. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity, 80g; Yield: 81%.
ATX-87: Stage 5
A solution of 17 g of 4-((tert-butoxycarbonyl)amino)butanoic acid dissolved in DCM (250 ml) was cooled below 0°C. 1.3 equiv. added. EDC.HCl, Et.3N and 4-dimethylaminopyridine (DMAP) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution is added 1 equiv. Tridecan-7-ol was added at the same temperature, dissolved in DCM (150 mL) using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Added solution (150 mL) and then EtOAc (200 mL). The organic layer was separated, concentrated under reduced pressure and proceeded to the next step with crude oil. Quantity produced: 15 g (crude oil, necessary compound and alcohol).
ATX-87: Stage 6
To a solution of 15.0 g of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate dissolved in 80 mL of DCM, add 10 equiv. TFA dissolved at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 NaHCO33solution (300 mL) and then extracted with EtOAc (2x200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and alcohol. production amount, 7 g; Yield: 24% for two steps. confirmed by music.
ATX-87: Stage 7
In a solution of 4-bromobutyric acid dissolved in DCM (400 mL) cooled below 0°C. to 1.5 equiv. added. EDC.HCl, 2 equiv. etc.3N and DMAP sequentially under a nitrogen atmosphere at 10 minute intervals. To this resulting solution, 20 g of (Z)-non-2-en-1-ol dissolved in 100 ml of DCM was added using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x150ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (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. Alcohol recovered. Production quantity: 19g; Yield: 55%.
ATX-87: 8 stages
In a solution of 4.0 g of tridecan-7-yl-4-aminobutanoate, 1 equiv. (Z)-Me-2-en-1-yl-4-bromobutanoate in 90 mL ACN, 1.4 eq. Potassium carbonate was added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress 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 concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc/hexane. SMs (amines and bromine compounds) were recovered. Production quantity: 2.2g; Yield: 30%; confirmed by music.
ATX-87: Stage 9
To a solution of 2.2 g of (Z)-4-((4-(non-2-en-1-yloxy)-4-oxobutyl)amino)tridecan-7-ylbutanoate dissolved in 25 mL of dry DCM was added 3 equiv. added. Triethylamine and triphosgene with 5 minutes interval at 0°C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
In 7 equiv. Sodium hydride dissolved in dry THF (100 mL) in a 100 mL two-neck round bottom flask with stirring at 0°C. under nitrogen atmosphere 3.5 equiv. 2-(Dimethylamino)propane-1-thiol hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution was added the above carbamoyl chloride dissolved in THF (100 mL) slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at 0°C and room temperature under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (75 mL) was added followed by EtOAc (150 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
The first purification was carried out using silica gel (60-120 mesh) of the crude compound adsorbed on 60 g of silica gel and poured onto 500 g of silica gel on the column. The compound was eluted in 35% EtOAc/hexane. The second purification was done using neutral alumina with HPLC grade solvents. The crude compound was adsorbed onto 18 g of neutral alumina and the resultant was poured onto 130 g of neutral alumina taken from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.2g; Yield: 43%; confirmed by1H RMN, HPLC e massa.
ATX-87/RL-48C:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,52 (m, 1), 4,87 (m, 1), 4,63 (d, J=7,0, 2), 3,30- 3,44 (4), 3,02 (t, J=7,0, 2), 2,52 (t, J=7,0, 2), 2,26-2,36 (4), 2, 27 (s, 6), 2,09 (m, 2), 1,83-1,96 (4), 1,46-1,54 (4), 1,21-1,40 (32), 0 ,85-0,90 (9).
Example 9: Synthesis of ATX-88
ATX-88: Stage 1
In a 500 ml two-necked bottle under N2Atmosphere 25 g of 8-bromooctanoic acid (1 equivalent) was taken dissolved in 200 ml of DCM and then oxalyl chloride, 1.5 equivalents, was added slowly at 0°C with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours.
In a separate 1 liter bottle, round bottom and double neck, 2 equiv. N,O-Dimethylhydroxylamine hydrochloride in 300 mL of DCM was added 3 equiv. added. Trimethylamine was added and stirred at 0°C. To this resulting solution, after concentration under reduced pressure by dissolving in 500 ml of DCM, the above acid chloride was added dropwise using a dropping funnel over 15 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (2x100ml). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 10% EtOAc/hexane). Production quantity: 21g; Yield: 66%.
ATX-88: Stage 2
In a solution of 1.3 equiv. Octyl magnesium bromide in THF (100 mL) stirred at 0°C. Under nitrogen atmosphere, 20 g of N-methoxy-N-methyloctanamide was added to 100 ml of THF and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl (100mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 2% ethyl acetate/hexane). The quantitative yield was 17.4 g. 68%
ATX-88: Stage 3
In a solution of 17 g of hexadecan-7-one (1 eq.) dissolved in 135 ml of MeOH/THF, 1.5 eq. Sodium borohydride was added at 0°C and the resulting solution was stirred at room temperature for 1 hour.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl (50mL). 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 washed with EtOAc (2 x 80 mL). The combined organic layer was concentrated under reduced pressure to obtain a white solid. Production Quantity: 14.5g; Yield: 85%.
ATX-88: Stage 4
To a solution of 50 g of 4-aminobutyric acid dissolved in 350 mL of THF was added, using a funnel, 490 mL of 1N aqueous NaOH solution at 0°C, followed by 140 mL of Boc anhydride. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress 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 washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production quantity, 80g; Yield: 81%.
ATX-88: Stage 5
In a solution of 1 equiv. 4-((tert-Butoxycarbonyl)amino)butanoic acid dissolved in DCM (200 mL) cooled below 0°C. 3 equiv. added. EDC.HCl, Et.3N (3 eq.) and DMAP (0.1 eq.) sequentially at 10 minute intervals. To this resulting solution was added alcohol dissolved in DCM using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mass was quenched with water (100ml) and the organic layer separated. The aqueous layer was washed with DCM (2x50ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution and EtOAc (100 mL) was added. The organic layer was separated and concentrated under reduced pressure and proceeded to the next step with crude oil. Production quantity, 19 g (raw).
ATX-88: Stage 6
To a solution of 19 g of hexadecan-7-yl 4-((tert-butoxycarbonyl)amino)butanoate (1 eq.) dissolved in 140 ml of DCM, 10 eq. TFA dissolved at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH/CHCl).3; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33Solution (100 mL) and EtOAc (100 mL) were added. The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3) and alcohol was found. Production Quantity: 9.4g; Efficiency: 50% for two steps. confirmed by music.
ATX-88: Stage 7
In a solution of 30 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (500 mL) cooled to 0°C. 1.5 equiv. added. EDC.HCl, 3 equiv. etc.3N is 0.1 equiv. DMAP consecutively with an interval of 10 minutes. In this resulting solution, 0.7 equiv. (Z)-Me-2-en-1-ol was added, dissolved in 100 mL of DCM with a funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution and EtOAc (150 mL) was added. The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel, 5% EtOAc/hexane). Production quantity: 27g; Yield: 51%; confirmed by1H-RMN.
ATX-88: Stage 8
In a solution of 6 g of hexadecan-8-yl-4-aminobutanoate (1 eq.) 1 eq. 5(Z)-enone-2-en-1-yl-4-bromobutanoate in ACN (70 mL), 1.2 equiv. Potassium carbonate was added and the resultant was refluxed at 90°C for 3 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH/CHCl).3; Rf: 0.5). The reaction mass was filtered and the filtrate was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (100-200 mesh silica gel, 15% EtOAc/hexane). Production quantity: 4.5g; Yield: 44%; confirmed by music.
ATX-88: Stage 9
In a solution of 4.4 g of (Z)-non-2-en-1-yl-4-((4-oxo-4-(tetradecan-7-yloxy)butyl)amino)butanoate (1 eq.) , dissolved in 30 ml of dry DCM, 0.83 ml of trimethylamine (3 equivalents) and 418 mg of triphosgene (0.5 equivalents) were added for 5 minutes at 0°C under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To 192 mg of sodium hydride (10 equivalents) dissolved in dry THF (25 mL) in a 100 mL two-necked round bottom flask was added 564 mg of 2-(diethylamino)propane-1-thiol hydrochloride (5 equivalents) at 0°C and stirring was continued for 5 minutes under a nitrogen atmosphere. To this resulting solution was added the above carbamoyl chloride dissolved in THF (35 mL) slowly over about 10 minutes via syringe. The resulting solution was stirred at room temperature for 4 hours under a nitrogen atmosphere.
The progress of the reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.5, PMA carbon). The reaction mass was quenched with saturated NH 34Cl (30 mL) was added followed by EtOAc (100 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 50 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
A first purification was done with silica gel (60-120 mesh). 5.0 g of crude compound was adsorbed onto 9 g of silica gel and poured onto 90 g of silica gel obtained from the column. The compound was eluted in 35% EtOAc/hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 1.5g of crude compound was adsorbed onto 4g of neutral alumina and the resultant was poured onto 40g of neutral alumina removed from the column. The compound was eluted in 10% EtOAc/hexane. Production quantity: 1.2g; Yield: 21%; confirmed by1H-NMR; HPLC; Mass.
ATX-88/RL-48D:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,51 (m, 1), 4,87 (m, 1), 4,63 (d, J=7,0, 2), 3,30- 3,44 (4), 2,90 (t, J=7,0 , 2), 2,46-2,55 (6), 2,26-2,37 (4), 2,09 (m, 2), 1,71-1,80 (4), 1,46-1,55 (4), 1,21-1,41 (32), 1,01 (t, J=7,0, 6) , 00,85-0,91 (9).
Example 10: Synthesis of ATX-83
ATX-83: Stage 1
Into a 500 mL single neck round bottom flask was placed 50 g of octanoic acid (1 equivalent) dissolved in DCM (200 mL) and then 44.6 mL of oxalyl chloride (1.5 equivalent) was slowly added to 0°C. Additional funnel, stirring under nitrogen and then 1 ml of DMF (catalyst) was added. The resulting reaction mixture was stirred at room temperature for 2 hours.
To a separate 2 L, two neck, round bottom flask, 67.4 g of N,O-dimethylhydroxylamine hydrochloride (2 equivalents) in DCM (300 mL) was added using an additional funnel with 144 mL of triethylamine (3 equivalents) with stirring at 0°C. To this resulting solution, after concentration under reduced pressure under a nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (350 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
The progress of the reaction was monitored by TLC (20% EtOAc/hexane, Rf: 0.5, PMA decarburization). The reaction mass was diluted with water (300 mL). The organic layer was separated and the aqueous layer washed with DCM (3 x 100ml). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 10% EtOAc/hexane. Production quantity: 55.0 g; Yield: 84%
ATX-83: Stage 2
To a solution of 55 g of heptylmagnesium bromide (1 equiv.) in ether placed in a 21 round bottom flask and stirred at 0°C under a nitrogen atmosphere was added 89.6 g of a solution of N- Methoxy-N-methyloctanamide (1.5 eq.) was dissolved in 400 ml of dry ether and the resulting reaction solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% EtOAc in hexane, Rf: 0.7, PMA decarbonization). The reaction mass was quenched with saturated NH 34Cl solution (250 mL). The organic layer was separated and the aqueous layer was washed with ether (2 x 100 ml). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc/hexane. Quantity produced: 50.0 g; Yield: 75%.
ATX-83: Stage 3
To a solution of 50 g of pentadecan-8-one (1 equivalent) dissolved in 290 mL of MeOH/THF was added 12.5 g of sodium borohydride (1.5 equivalent) at 0°C and the resulting solution was stirred at room temperature for 2 hours. .
Reaction progress was monitored by TLC (10% EtOAc in hexane, Rf: 0.5, PMA decarbonization). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (3 x 80 mL). The combined organic layer was dried over anhydrous Na2ALSO4Concentrated under reduced pressure and dried in vacuo to give a white solid. Production quantity: 46.0 g; Yield: 90%.
ATX-83: Stage 4
To a solution of 50 g of 4-aminobutyric acid (1 equivalent) dissolved in THF was added 490 mL of 1N aqueous NaOH solution (1 equivalent) at 0°C, followed by 140 mL of Boc anhydride (1.3 equivalent ). successively. With an additional funnel for 15 minutes. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).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 washed with EtOAc (3 x 150 mL). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated to a viscous liquid under reduced pressure. Production quantity: 77.0 g; Yield: 78%.
ATX-83: Stage 5
The composition was made in 4 batches. To each solution of 23 g of 4-((tert-butoxycarbonyl)amino)butyric acid (1 eq.) in DCM (400 mL) cooled below 0°C was added 32.3 g of EDC•HCl (1.5 ) eq.), 47 mL Et3N (3 eq.) and 1.3 g of DMAP (0.1 eq.) successively under a nitrogen atmosphere with an interval of 10 min. Into this resulting solution was dissolved 20 g of pentadecan-8-ol (0.77 eq.). in DCM (200 mL), added using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. This resulting crude product was washed with saturated NaHCO33Solution (150 mL) and EtOAc (250 mL) were added. The organic layer was separated and dried over anhydrous Na2ALSO4and concentrated under reduced pressure and then proceeded to the next step with argon. Amount produced, 105 g (crude oil, required compound and alcohol)
ATX-83: Stage 6
To a solution of pentadecan-8-yl 4-((tert-butoxycarbonyl)amino)butanoate 105 g (1 eq.) dissolved in 450 ml of DCM was added 194 ml of TFA (10 eq.) at 0°C and touched . at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 stirred with saturated NaHCO33solution (200 mL) for 10 minutes, then EtOAc (300 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 100 mL). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% MeOH/CHCl3and 1 ml of triethylamine. Production quantity: 60.0 g; Yield: 54% for two steps
ATX-83: Stage 7
The reaction was carried out in two batches, each in a solution of 20 g of 6-bromohexanoic acid (1 equiv.) dissolved in DCM (300 ml) cooled to below 0°C. Added 29.3 g of EDC.HCl (1.5 equiv), 42.8 mL of Et3N (3 equivalents) and 1.2 g DMAP (0.1 equivalent) sequentially under nitrogen at 10 minute intervals. To this resulting solution was added 14.5 g of (Z)-non-2-en-1-ol (1 eq.) (dissolved in 100 mL of DCM) using an addition funnel and stirred at room temperature under an atmosphere of nitrogen for 24 hours. hours.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (150 mL) and then extracted with EtOAc (2x150 mL). The organic layer was separated and dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc/hexane. Alcohol recovered. Production quantity: 36.0 g; Yield: 55%.
ATX-83: Stage 8
The reaction was carried out in six batches. In each of a solution of 10 g of pentadecan-8-yl-4-aminobutanoate (Int 6, 1 eq.), 10.1 g of (Z)-non-2-en-1-yl-6-bromohexanoate (Int 7, 1 eq.) In 120 mL of ACN was added 6.1 g of anhydrous potassium carbonate (1.4 eq.) and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH in CHCl).3; Rf: 0.5). The reaction mass was filtered, washed with ACN (2 x 20 ml) and the filtrate concentrated under reduced pressure.
The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 20-80% EtOAc/hexane. The original materials have been restored. Production Quantity: 36.9g; Yield: 35%.
ATX-83: Stage 9
The reaction was carried out in three batches. In each of a solution of 10 g of (Z)-non-2-en-1-yl 6-((4-oxo-4-(pentadecan-8-yloxy)butyl)amino)hexanoate (1 eq.) To 100 ml of dry DCM, 7.5 ml of triethylamine (3 equivalents) and 2.68 g of triphosgene (0.5 equivalents) were added at 5 minute intervals at 0°C under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To a suspension of 3 g of sodium hydride (7 equivalents) in dry THF (100 mL) in a 500 mL two-necked RB flask stirred at 0°C under a nitrogen atmosphere was added 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 was added the above carbamoyl chloride dissolved in dry THF (200 mL) slowly over about 10 minutes via syringe. The resulting solution was stirred overnight at room temperature under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% EtOAc/hexane, Rf: 0.5, PMA decarburization). The reaction mass was quenched with saturated NH 34Cl (100 mL) was added followed by EtOAc (350 mL). The organic layer was separated and the aqueous layer washed with EtOAc (2 x 80 mL). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
A first cleaning was performed with neutral aluminum oxide. The crude compound dissolved in hexane was loaded onto neutral alumina (700g was loaded onto the column). The compound was eluted in 8-10% EtOAc/hexane. A second purification was performed with silica gel (100-200 mesh). The compound dissolved in hexane was applied to silica gel (500 g was applied to the column). The compound was eluted in 20-25% EtOAc/hexane. The final compound (dissolved in hexane) was treated with activated charcoal (200 mg/g) and filtered through a celite pad (after stirring for 20 min) and then through a syringe-tipped membrane filter (PTFE, 0 .2 micron, diameter 25 mm).directed). The resulting filtrate was concentrated under reduced pressure. Production quantity: 15.5g; Yield: 41%.
ATX-83/RL-47B:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,52 (m, 1), 4,87 (m, 1), 4,62 (d, J=7,0, 2), 3,24- 3,42 (4), 3,02 (t, J=7,0, 2), 2,53 (t, J=7,0, 2), 2,26-2,34 (4), 2, 26 (s, 6), 2,10 (m, 2), 1,45-1,70 (6), 1,20-1,41 (34), 0,84-0,92 (9).
Example 11: Synthesis of ATX-84
ATX-84: Stage 1
Into a 500 mL one-neck flask were placed 30 g of heptanoic acid (1 equivalent) dissolved in DCM (200 mL) and then 26.7 g of oxalyl chloride (1.5 equivalent) were added slowly at 0°C with 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.
In a separate 1 L round bottom flask, 40.5 g of N,O-dimethylhydroxylamine hydrochloride (2 equivalents) in DCM (250 mL) was added with stirring using an additional funnel, 86.6 mL of trimethylamine (3 equivalents) at 0°C. To this resulting solution, after concentration under reduced pressure under nitrogen atmosphere, the above acid chloride was added dropwise by dissolving it in DCM (100 ml) using a dropping funnel for 20 minutes. The resulting reaction solution was stirred at room temperature for 3 hours under nitrogen atmosphere.
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 washed with DCM (3 x 100ml). The combined organic layer was concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (silica gel 60-120) using 10% EtOAc/hexane. Production quantity: 38.0 g; Yield: 84%.
ATX-84: Stage 2
To a solution of 8 g of hexylmanesium bromide (1 equiv.) in 250 mL of dry ether placed in a 1 L round bottom flask and stirred at 0°C under a nitrogen atmosphere were added 2.3 g of N-methoxy-N.-Methylheptanamide (0.5 eq.) was dissolved in 250 mL of ether and the resulting reaction mixture was stirred at room temperature for 4 hours.
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.7). The reaction mass was quenched with saturated NH 34Cl solution (200 mL). The organic layer was separated and the aqueous layer was washed with ether (2 x 100 ml). The combined organic layer was dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using (60-120 mesh silica gel) using 2% EtOAc/hexane. Production Quantity: 30.8g; Yield: 71%.
ATX-84: Stage 3
To a solution of 30 g of tridecan-7-one (1 equivalent) dissolved in 200 mL of MeOH/THF was added 8.5 g of sodium borohydride (0.5 equivalent) at 0°C and the resulting solution was stirred at room temperature for 2 hours.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mass was quenched with saturated NH 34Cl 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 washed with EtOAc (2 x 70 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production Quantity: 27.2g; Yield: 90%.
ATX-84: Stage 4
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 were added and then successively 34 ml of Boc anhydride (1.3 equivalent) at 0° C using an additional funnel over a period of 15 minutes. The resulting solution was stirred at room temperature for 4 hours.
Reaction progress was monitored by TLC (10% MeOH in CHCl).3; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer washed with EtOAc (2 x 100 mL). The combined organic layer was concentrated under reduced pressure to obtain a sticky liquid. Production Quantity: 22.4g; Yield: 85%.
ATX-84: Stage 5
A solution of 10 g of 6-((tert-butoxycarbonyl)amino)hexanoic acid (1 equivalent) dissolved in DCM (200 ml) was cooled below 0°C. Added 10.7 g of EDC.HCl (1.3 eq.), 18 mL of Et3N (3 eq.) and 525 mg of DMAP (0.1 eq.) successively under a nitrogen atmosphere with an interval of 10 min. To this resulting solution was added at the same temperature 6 g of tridecan-7-ol (Int 3, 0.7 equivalents), dissolved in DCM (50 mL), using an additional funnel and stirred at room temperature under a nitrogen atmosphere for 24 hours. .
The progress of the reaction was monitored by TLC (10% EtOAc in hexane, Rf: 0.4). The reaction mass was quenched with water (150 ml) and then the organic layer was separated. The aqueous layer was washed with DCM (2 x 75 mL). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33The solution (100 mL) was added and then extracted with EtOAc (2 x 100 mL). The organic layer was separated and concentrated under reduced pressure and proceeded to the next step with crude oil. Amount produced: 8.5 g (crude oil, required compound and alcohol)
ATX-84: Stage 6
To a solution of 10 g of tridecan-7-yl 6-((tert-butoxycarbonyl)amino)hexanoate (1 equivalent) dissolved in 65 ml of DCM was added 18.5 ml of TFA (10 equivalents) at 0°C and stirred at room temperature for 3 hours under a nitrogen atmosphere.
Reaction progress 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 NaHCO33solution (100 mL) and then extracted with EtOAc (3x100 mL). The organic layer was separated and concentrated under reduced pressure.
The crude compound was subjected to column chromatography using silica gel (60-120 mesh, 4% MeOH/CHCl).3and 1 ml of triethylamine) and the initial alcohol was recovered. Quantity: 4.5g; Yield: 33% in two steps.
ATX-84: Stage 7
To a solution of 20 g of 6-bromohexanoic acid (1 equivalent) dissolved in DCM (300 mL) cooled below 0 °C were added 29.3 g of EDC·HCl (1.5 equivalents), 42.8 mL of et3N (3 eq.) and 1.2 g DMAP (0.1 eq.) sequentially under a nitrogen atmosphere with 10 minute intervals. To this resulting solution was added 14.5 g of (Z)-non-2-en-1-ol (1 equiv.) dissolved in 100 mL of DCM, added using an additional funnel and stirred for 24 h at room temperature under nitrogen atmosphere.
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 (2x100ml). The combined organic layer was concentrated under reduced pressure. The resulting crude product was washed with saturated NaHCO33solution (150 mL) and then extracted with EtOAc (2x150 mL). The organic layer was separated and dried over anhydrous Na2ALSO4and concentrated under reduced pressure.
The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc/hexane. Recovered raw alcohol. Production quantity: 18.0 g; Yield: 55%.
ATX-84: Stage 8
In a solution of 4.5 g of tridecan-7-yl-6-aminohexanoate (Int 6, 1 eq.) and 4.5 g of (Z)-non-2-en-1-yl-6-bromohexanoate ( Int 7.1 eq.) To 90 ml of ACN and 2.7 g of potassium carbonate (1.4 equivalents) were added and the resulting mixture was refluxed at 90°C for 4 hours under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% MeOH in CHCl).3; Rf: 0.5). The reaction mass was filtered, washed with ACN (2 x 20 ml) and the filtrate concentrated under reduced pressure.
The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 20% EtOAc/hexane. The original materials have been restored. Production quantity: 3.0 g; Yield: 37%.
ATX-84: Stage 9
In a solution of 2.5 g of (Z)-meth-2-en-1-yl 6-((6-oxo-6-(tridecan-7-yloxy)hexyl)amino)hexanoate (1 eq.), dissolved in 30 ml of dry DCM, 1.8 ml of triethylamine (3 equivalents) and 672 mg of triphosgene (0.5 equivalents) were added during 5 minutes at 0°C under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.
To a suspension of 761 mg of sodium hydride in dry THF (50 mL) in a 250 mL two-necked round bottom flask stirred at 0°C under a nitrogen atmosphere was added 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. To this resulting solution was added the above carbamoyl chloride dissolved in THF (60 mL) slowly over about 10 minutes via syringe. The resulting solution was stirred overnight at room temperature under a nitrogen atmosphere.
Reaction progress was monitored by TLC (10% EtOAc/hexane, Rf: 0.5, PMA decarburization). The reaction mass was quenched with saturated NH 34Cl (60 mL) was added followed by EtOAc (130 mL). The organic layer was separated and the aqueous layer washed with EtOAc (3 x 40 mL). The combined organic layer was concentrated and the resulting crude product subjected to column chromatography.
A first purification was done with silica gel (100-200 mesh). 4.6 g of crude compound was adsorbed onto 10.0 g of silica gel and poured onto 90.0 g of silica gel obtained from the column. The compound was eluted in 50% EtOAc/hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 2.0 g of crude compound was adsorbed onto 6.0 g of neutral alumina and the resultant was poured onto 40.0 g of neutral alumina taken from the column. The compound was eluted in 20% EtOAc/hexane. Production quantity: 1.2g; Yield: 38% (300 mg mixture).
ATX-84/RL-47C:1RMN de 1H (PPM, 500 MHz, CDCl3): δ=5,64 (m, 1), 5,52 (m, 1), 4,86 (m, 1), 4,62 (d, J=7,0, 2), 3, 22- 3,35 (4), 3,01 (t, J=7,0, 2), 2,53 (t, J=7,0, 2), 2,25-2,34 (4), 2, 27 (s, 6), 2,10 (m, 2), 1,45-1-73 (10), 1,20-1,40 (30), 00,84-0,91 (9) .
Example 12: Synthesis of ATX-61
ATX-61: Stage 1
12 g of glycine ester (1 equivalent) was dissolved in THF (100 ml) and cooled below 0°C. Funnels were added one by one.
Reaction progress was monitored by TLC using 50% EtOAc/hexane. Rf: 0.4.
The reaction mass was quenched with water and after 16 hours EtOAc (100 mL) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2 x 40 mL) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.
The crude product was run on silica gel 60-120 (25% EtOAc/hexane). Production quantity: 20.8; Yield: 88%.
ATX-61: Stage 2
To a solution of 18.9 g of glycine N-Boc ester (1 equivalent) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 equivalent) and the resulting solution was dissolved . Stirred at room temperature for 4 hours.
The reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.3), SM was absent.
The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH=3) and then extracted with EtOAc (4 x 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. production quantity, 15 g; Efficiency: 92%; confirmed by music.
ATX-61: Stage 3
In a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (30 mL) cooled below 0°C. 4.5 mL of Et was added3N (1.2 eq.) and 6.44 g of EDC.HCl (1.2 eq.). 5.12 g of heptaden-9-ol (0.7 equiv.) in 20 ml of DCM was added to this reaction solution, and the mixture was stirred at room temperature overnight.
No starting material was found by TLC (10% EtOAc/hexane, Rf: 0.6). The reaction mass was diluted with saturated NaHCO33The organic layer was separated, the aqueous layer was washed with DCM (2x30 ml), dried over sodium sulfate and concentrated under reduced pressure. Proceed to the next step with crude oil (6.8 g, mixture of product and alcohol).
ATX-61: Stage 4
4 g of heptadecan-9-yl (tert-butoxycarbonyl) glycinate (Int 2, 1 eq.) were dissolved in DCM (40 ml) and cooled to 0°C, 7.4 ml of TFA (10 eq.) were added and at room temperature stirred for 1 hour.
Completion of the reaction was verified by TLC (10% EtOAc/hexane, Rf: 0.5) within 2 hours.
The reaction mass was concentrated under reduced pressure, the remaining mass was washed with saturated sodium bicarbonate solution (30 mL) and extracted with EtOAc (3 x 30 mL), the organic layer was dried over sodium sulfate and concentrated under pressure. reduced to give Int. . surrender 3.
The crude product was subjected to column chromatography (silica, 60-120) using 1-3% MeOH/CHCl3and 2 ml of Et3N. Quantity produced: 1 g; confirmed by1H RMN e mass.
ATX-61: Stage 5
In a solution of 4 g of bromoacetic acid (1 equivalent) dissolved in DCM (35 mL) cooled below 0°C. 4.7 mL of Et was added3N (1.2 equiv) and 354 mg DMAP (0.1 equiv) followed by 13.23 g HATU (1.2 equiv). 2.88 g of (Z)-non-2-en-1-ol (0.7 eq.) in 20 ml of DCM were added to this reaction solution and the mixture was stirred at room temperature overnight.
The reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.7).
The reaction mass was diluted with saturated NaHCO33HCl (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 column chromatography on silica gel (60-120) (1.5% EtOAc/hexane). production amount, 4 g; Yield: 52%.
ATX-61: Stage 6
1 g of heptadecan-9-ylglycinate (Int 3, 1 eq.) was dissolved in THF (25 ml), 0.5 ml of TEA (1.3 eq.) and 1.08 g of (Z)-non- 2-ene-1-yl resolved. 2-Bromoacetic acid ester (Int 4, 1.3 equiv) was added and stirred at room temperature overnight.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.4). The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (20 mL x 2), the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel, 100-200) (2% EtOAc/hexane). Production quantity: 700 mg; Yield: 47%; confirmed by music.
ATX-61: Stage 7
In a solution of 700 mg of heptadecan-9-yl (Z)-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycinate) (1 eq.) dissolved in 15 ml of DCM, cooled under 5 °C 0.4 ml of Et was added3N (3 equivalents) followed by 209 mg of triphosgene (0.5 equivalents) in portions over 10 minutes.
The progress of the reaction mixture was monitored by TLC, the reaction was completed in 0.5 hour, the reaction mass was concentrated under reduced pressure.
A solution of 423 mg of N,N-dimethylethanethiol hydrochloride (3 equivalents) in dry THF (10 mL) and DMF (3 mL) was stirred at 0°C. Under nitrogen atmosphere, 144 mg of sodium hydride (6 eq.) were added. After 10 minutes, the above solution was added to this reaction mass and dissolved in THF (15 mL). The resulting solution was stirred at room temperature for 1 hour.
Completion of the reaction was monitored by TLC (10% MeOH/CHCl).3; Rf: 0.5), after 1 hour.
The reaction mass was quenched with saturated NH 34Cl (20 mL), water (20 mL) and EtOAc (30 mL) were added. The aqueous layer was washed with EtOAc (20 mL x 2) and the combined organic layer was washed with brine (20 mL). The organic layer was dried over Na2ALSO4and concentrated under reduced pressure.
The crude product was subjected to column chromatography using silica gel (100-200) with 15% EtOAc/hexane followed by neutral alumina with 15% EtOAc/hexane to obtain a pure compound. Production amount: 520 mg; Yield: 58%. confirmed by1H RMN, HPLC e massa.
ATX-61/RL-42D:1RMN de 1H (PPM, 400 MHz, CDCl3): δ=5,67 (m, 1), 5,51 (m, 1), 4,92 (m, 1), 4,70 (m, 2), 4,16-4,27 (4) , 3,07 (m, 2), 2,53 (m, 2. 2), 2,27 (s, 6), 2,10 (m, 2), 1-47-1,57 (4), 1,19-1,40 (32), 0,83-0,92 (9).
Example 13: Synthesis of ATX-63
ATX-63: Stage 1
12 g of glycine ester (1 equivalent) was dissolved in THF (100 ml) and cooled below 0°C. To this solution was added 24.2 mL of triethylamine (1.5 equivalents) and 38.11 g of Boc anhydride (1.5 equivalents) and an additional funnel was added sequentially.
Reaction progress was monitored by TLC using 50% EtOAc/hexane. Rf: 0.4.
The reaction mass was quenched with water and after 16 hours EtOAc (100 mL) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2 x 40 mL) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.
The crude product was run on silica gel 60-120 (25% EtOAc/hexane). Production quantity: 20.8; Yield: 88%.
ATX-63: Stage 2
To a solution of 18.9 g of glycine N-Boc ester (1 equiv) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 equiv) and the resulting solution was stirred at room temperature for 4 hours.
The reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.3), SM was absent.
The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH 3) and then extracted with EtOAc (4 x 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. production quantity, 15 g; Efficiency: 92%; confirmed by music.
ATX-63: Stage 3
In a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (50 mL) cooled below 0°C. 4.5 mL of Et was added3N (1.2 eq.) and 6.4 g of EDC.HCl (1.2 eq.). 3.4 g of undec-6-ol (0.7 equivalents) in 20 ml of DCM was added to this reaction solution, and the mixture was stirred at room temperature overnight.
No starting material was found by TLC (15% EtOAc/hexane, Rf: 0.6). The reaction mass was diluted with saturated NaHCO33HCl (20 mL), the organic layer was separated, the aqueous layer was washed with DCM (2 x 40 mL), dried over sodium sulfate and concentrated under reduced pressure. Proceed to the next step with crude oil (5.5 g, mixture of product and alcohol) after the filtered column.
ATX-63: Stage 4
3.3 g crude undecan-6-yl(tert-butoxycarbonyl)glycinate (Int 2, 1 equivalent) was dissolved in DCM (20 mL) and cooled to 10°C, 7.6 mL TFA (10 equivalents) was added and at room temperature stirred for 1 hour.
Completion of the reaction was verified by TLC (10% MeOH/DCM, Rf: 0.5) within 2 hours. The reaction mass was concentrated under reduced pressure, the remaining mass was washed with saturated sodium bicarbonate solution (50 mL) and extracted with EtOAc (3 x 25 mL), the organic layer was dried over sodium sulfate and concentrated under pressure. reduced to give Int. . surrender 3.
The crude product was subjected to column chromatography (silica, 60-120) using 1-3% MeOH/CHCl3and 2 ml of Et3N. Quantity produced: 1.2 g; Yield: 40%; confirmed by1H RMN e mass.
ATX-63: Stage 5
To a solution of 4 g of bromoacetic acid (1 equiv.) dissolved in DCM (35 mL) cooled below 0 °C was added 4.7 mL of Et3N (1.2 eq.), followed by 13.23 g of HATU (1.2 eq.) and 354 mg of DMAP (0.1 eq.). 2.88 g of (Z)-non-2-en-1-ol (0.7 equivalents) in 20 ml of DCM was added to this reaction solution, and the mixture was stirred at room temperature overnight.
The reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.7).
The reaction mass was diluted with saturated NaHCO33HCl (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 column chromatography on silica gel (60-120) (1.5% EtOAc/hexane). production amount, 4 g; Yield: 52%.
ATX-63: Stage 6
1.2 g of undecan-6-ylglycinate (Int 3, 1 eq.) was dissolved in 25 ml of THF, 0.9 ml of TEA (1.3 eq.) and 1.37 g of (Z)-non -2-ene- Added dissolved 1-yl 2 - bromoacetic acid ester (Int 4, 1eq.) and stirred overnight at room temperature.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (20 mL x 2), the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel, 100-200) (3% EtOAc/hexane). Product Quantity: 800 mg; Yield: 37%; confirmed by music.
ATX-63: Stage 7
In a solution of 800 mg of (Z)-non-2-en-1-yl-(2-oxo-2-(undecan-6-yloxy)ethyl)glycinate (1 eq.) dissolved in DCM cooled below 5 °C. 0.4 mL of Et was added3N (3 equivalents) followed by 209 mg of triphosgene (0.5 equivalents) in portions over 10 minutes.
The progress of the reaction mixture was monitored by TLC, the reaction was stopped for 1 hour, the reaction mass was concentrated under reduced pressure.
A solution of 423 mg of N,N-dimethylethanethiol hydrochloride (3 equivalents) in dry THF and DMF (10 ml and 5 ml, respectively) was stirred at 0°C under an atmosphere of nitrogen and 144 mg of sodium hydride (6) were added (eq.). After 10 minutes, the above solution dissolved in THF was added to this reaction mass. The resulting solution was stirred at room temperature for 1 hour.
Completion of the reaction was observed by TLC (70% EtOAc/hexane, Rf: 0.4) after 1 hour. The reaction mass was quenched with saturated NH 34Cl (25 mL), water (20 mL) and EtOAc (20 mL) were added. The aqueous layer was washed with EtOAc (20 mL x 2) and the combined organic layer was washed with brine (20 mL). The organic layer was dried over Na2ALSO4and concentrated under reduced pressure.
The crude product was subjected to column chromatography using silica gel (100-200) with 20% EtOAc/hexane followed by neutral alumina with 5% EtOAc/hexane to obtain a pure compound. Production amount: 510 mg; Yield: 48%; confirmed by1H RMN, HPLC e massa.
ATX-63/RL-42A:1RMN de 1H (PPM, 400 MHz, CDCl3): δ=5,67 (m, 1), 5,52 (m, 1), 4,92 (m, 1), 4,70 (m, 2), 4,15-4,27 (4) , 3,06 (m, 2), 2,53 (m, 2), 2), 2,27 (s, 6), 2,09 (m, 2), 1,47-1,57 (4) , 1,20–1,41 (20), 0,82–0,92 (9).
Example 14: Synthesis of ATX-64
ATX-64: Stage 1
12 g of ethyl glycinate (1 equivalent) was dissolved in THF (100 ml) and cooled below 0°C. To this resulting solution was added 24.2 mL of triethylamine (1.5 equivalents) and 38.11 g of Boc anhydride (1.5 equivalents). through an additional funnel were added sequentially.
Reaction progress was monitored by TLC using 50% EtOAc/hexane. Rf: 0.4.
The reaction mass was quenched with water and after 16 hours EtOAc (100 mL) was added. The organic layer was separated, the aqueous layer was washed with EtOAc (2 x 40 mL) and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.
The crude product was run on silica gel 60-120 (25% EtOAc/hexane). Production quantity: 20.8; Yield: 88%.
ATX-64: Stage 2
To a solution of 18.9 g of glycine N-Boc ester (1 equiv) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 equiv) and the resulting solution was stirred at room temperature for 4 hours.
The reaction was monitored by TLC (60% EtOAc/hexane, Rf: 0.3), SM was absent.
The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH=3) and then extracted with EtOAc (4 x 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. production quantity, 15 g; Efficiency: 92%; confirmed by music.
ATX-64: Stage 3
In a solution of 5 g of N-Boc-glycine ester (Int 1, 1 eq.) dissolved in DCM (50 mL) cooled below 0°C. 4.5 mL of Et was added3N (1.2 eq.) and 6.4 g of EDC.HCl (1.2 eq.). 4.84 g of hexadecan-10-ol (0.7 eq.) in 15 ml of DCM was added to this reaction solution, and the mixture was stirred at room temperature overnight.
No starting material was found by TLC (15% EtOAc/hexane, Rf: 0.6). The reaction mass was diluted with saturated NaHCO33The organic layer was separated, the aqueous layer was washed with DCM (2x30 ml), dried over sodium sulfate and concentrated under reduced pressure.
After column filtration with the crude product (5.5 g, mixture of product and alcohol), proceed to the next step.
ATX-64: Stage 4
3.85 g crude heptadecan-9-yl-(tert-butoxycarbonyl)glycinate (Int 2, 1 eq.) was dissolved in 30 ml DCM and cooled to 0°C, 7.4 ml TFA (10 eq. ) were added and stirred at room temperature. temperature for 1 hour.
Completion of the reaction was verified by TLC (10% MeOH/DCM, Rf: 0.5) within 2 hours.
The reaction mass was concentrated under reduced pressure, the residual mass was washed with saturated sodium bicarbonate solution (30 mL) and extracted with EtOAc (3 x 30 mL), the organic layer was dried over sodium sulfate and concentrated under pressure reduced to give Int. 3.
The crude product was subjected to column chromatography (silica, 60-120) using 1-3% MeOH/CHCl3and 2 ml of Et3N. Quantity produced: 2.2 g; confirmed by1H RMN e mass.
ATX-64: Stage 5
To a solution of 4 g of bromoacetic acid (1 equiv.) dissolved in DCM (35 mL) cooled below 0 °C was added 4.7 mL of Et3N (1.2 eq.), followed by 13.23 g of HATU (1.2 eq.) and 354 mg of DMAP (0.1 eq.). 2.88 g of (Z)-non-2-en-1-ol (0.7 eq.) in 20 ml of DCM were added to this reaction solution and the mixture was stirred at room temperature overnight.
The reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.7).
The reaction mass was diluted with saturated NaHCO33HCl (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 column chromatography on silica gel (60-120) (1.5% EtOAc/hexane). production amount, 4 g; Yield: 52%.
ATX-64: Stage 6
2.1 g of hexadecan-8-ylglycinate (Int 3, 1 equivalent) were dissolved in 50 mL of THF, 1.2 mL of TEA (1.3 equivalent) and 2.39 g of (Z)-non-2 dissolved -en-1-yl 2 - bromoacetic acid ester (Int 4, 1.3 equiv.) and stirred overnight at room temperature.
The progress of the reaction was monitored by TLC (10% EtOAc/hexane, Rf: 0.5). The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (30 mL x 2), the combined organic layer was dried over sodium sulfate and concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel, 100-200) (3% EtOAc/hexane). Production quantity: 2.2g; Yield: 65%; confirmed by music.
ATX-64: Stage 7
A solution of 2.2 g of heptadecan-9-yl(Z)-(2-(non-2-en-1-yloxy)-2-oxoethyl)glycinate) (1 equivalent) dissolved in 15 mL DCM was cooled below 5°C, 1.6 mL of Et was added3N (3 equivalents) followed by 678 mg of triphosgene (0.5 equivalents) in portions over 10 minutes.
The progress of the reaction mixture was monitored by TLC, the reaction was stopped for 1 hour, the reaction mass was concentrated under reduced pressure.
672 mg of sodium hydride (7 Ex.). After 10 minutes, the above solution dissolved in THF was added to this reaction mass. The resulting solution was stirred at room temperature for 1 hour.
Completion of the reaction was observed by TLC (70% EtOAc/hexane, Rf: 0.4) after 1 hour. The reaction mass was quenched with saturated NH 34Cl (25 mL), water (20 mL) and EtOAc (20 mL) were added. The aqueous layer was washed with EtOAc (20 mL x 2) and the combined organic layer was washed with brine (20 mL). The organic layer was dried over Na2ALSO4and concentrated under reduced pressure.
The crude product was subjected to column chromatography using silica gel (100-200) with 25% EtOAc/hexane followed by neutral alumina with 15-20% EtOAc/hexane to give the pure compound. Amount produced: 1.0 mg; Yield: 40%; confirmed by1H RMN, HPLC e massa.
ATX-64/RL-42C: (PPM, 400 MHz, CDCl3): δ=5,67 (m, 1), 5,50 (m, 1), 4,92 (m, 1), 4,70 (t, J=7,0, 2), 3,06 ( m, 2), 2,53 (m, 2), 2,27 (s, 6), 1,47-1,57 (4), 1,17-1,40 (30), 0,82-0 ,93 (9).
Example 15: pKasValues
Lipids were titrated to measure their pKasValues. The results are shown in the table below.
Example 16: Stability of EPO mRNA in vivo
Plasma mRNA levels were measured and compared after injection of nanoparticles containing different cationic lipids. Female Balb/c mice (6-8 weeks old) were used to determine in vivo plasma erythropoietin (Epo) levels following injection of lipid-encapsulated mouse Epo mRNA. All formulations were administered intravenously by injection into the tail vein at a dose of 0.03 and 0.1 mg/kg in a dose volume of 5 ml/kg. The final blood collection was by cardiac puncture under 2% isoflurane, 6 hours after the injections of the formulation. Blood was collected in a tube containing 0.109 M citrate buffer and processed by centrifugation at 5,000 rpm for 10 minutes. Serum was collected and Epo mRNA levels analyzed. The results are shown in
Example 17: In vivo mouse factor VII silencing and EPO expression
Using liver-directed in vivo screening of liposome libraries, we tested several compounds that enable high levels of siRNA-mediated gene silencing in hepatocytes, the cells that make up the liver parenchyma. Factor VII, a blood clotting factor, is a suitable target gene to determine the functional delivery of siRNA to the liver. As this factor is produced specifically in hepatocytes, gene silencing indicates successful delivery to the parenchyma, as opposed to delivery to cells of the reticuloendothelial system (eg, 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 is easily determined by measuring protein levels. This is due to the short half-life of the protein (2 to 5 hours). Factor VII-targeted siRNA constructs were formulated with ATX-002, ATX-57, and ATX-58 and a phosphate buffered saline (PBS) control. female C57BL/6 mice (6-8 weeks old) were used for siRNA FVII (KD) knockdown experiments. All formulations were administered intravenously by injection into the tail vein at doses of 0.03 and 0.1 mg/kg in a dose volume of 5 mg/kg. Final blood collection was by cardiac puncture under 2% isoflurane 48 hours after formulation injections. Blood was collected in a tube containing 0.109 M citrate buffer and processed by centrifugation at 1200 G for 10 minutes. Plasma was collected and Factor VII protein levels analyzed by a chromogenic assay (Biophen FVII, Aniara Corporation). A standard curve was constructed using samples from mice injected with PBS and the relative expression of Factor VII was determined by comparing treated groups with untreated PBS control. Results showed that ATX-57 and ATX-58 were significantly more effective than ATX-002 at 0.03 and 0.1 mg/kg (
Female Balb/c mice (6-8 weeks old) were used to assess in vivo Epo protein expression after delivery of lipid-encapsulated mouse epo mRNA. All formulations were administered intravenously by injection into the tail vein at a dose of 0.03 and 0.1 mg/kg in a dose volume of 5 ml/kg. The final blood collection was by cardiac puncture under 2% isoflurane, 6 hours after the injections of the formulation. Blood was collected in a tube containing 0.109 M citrate buffer and processed by centrifugation at 5,000 rpm for 10 minutes. Serum was collected and Epo protein levels analyzed using the Epo ELISA assay (R&D Systems). A standard curve was constructed using samples from mice injected with PBS and relative Epo expression was determined by comparing treated groups with an untreated PBS control. The results showed that Epo mRNA is expressed in ATX-57 nanoparticles at significantly higher levels than ATX-2 at 0.1 mg/mL (