Lipid conjugate for delivery of therapeutic agents to the central nervous system tissue

Abstract

Disclosed herein are compounds that include a lipid PK / PD modulator for in vivo delivery of an oligonucleotide-based agent, such as a double-stranded RNAi agent, to a specific cell type, such as CNS cells. The PK / PD modulators disclosed herein, when combined with an oligonucleotide-based therapeutic or diagnostic agent, such as an RNAi agent, can enhance delivery of the composition to a specific target cell and promote inhibition of gene expression in those cells.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 495,505, filed on April 11, 2023, and U.S. Provisional Patent Application No. 63 / 352,485, filed on June 15, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted in XML format, the entire contents of which are incorporated herein by reference. The name of this XML copy is 30707 - WO_ST26_SeqListing.xml, created on June 13, 2023, and having a size of 1519 kb.

[0003] Field of the Invention The present disclosure relates to lipid conjugates for delivering oligonucleotide - based agents, such as double - stranded RNAi agents, in vivo to specific central nervous system (CNS) cell types to inhibit genes expressed in such cell types.

Background Art

[0004] Oligonucleotide - based agents, such as antisense agents and double - stranded RNA interference (RNAi) agents, have great promise, hold the potential to revolutionize the medical field, and provide powerful treatment options for patients. However, the effective delivery of oligonucleotide - based agents, particularly double - stranded therapeutic RNAi agents, has long been a challenge in the development of practical therapeutic pharmaceuticals. This is especially true when attempting to specifically and selectively deliver oligonucleotide - based agents to extra - hepatic (i.e., non - hepatocyte) cells.

[0005] Over the past few years, various attempts have been made to deliver oligonucleotide-based drugs to specific extrahepatic cell types, including central nervous system cells, adipocytes, cardiomyocytes, etc., using, for example, cholesterol conjugates (which are non-specific and have the known drawback of distributing to various undesirable tissues and organs) or lipid nanoparticles (LNPs), which are frequently reported to have concerns about toxicity. However, no appropriate delivery has been achieved to date. As a result, there remains a need for delivery vehicles for delivering oligonucleotide-based drugs, particularly RNAi agents, to cell types other than hepatocytes.

Summary of the Invention

[0006] Disclosed herein are compounds comprising lipids conjugated (or attached) to oligonucleotide-based drugs for delivery to the central nervous system tissue. Lipid PK / PD modulator precursors are also disclosed herein.

[0007] One aspect of the invention provides a double-stranded oligonucleotide in which a lipid is conjugated to one of the terminal nucleotides of one strand. In some embodiments, the lipid is conjugated to the 5'-terminal nucleotide of one strand. In some embodiments, the lipid is conjugated to the 3'-terminal nucleotide of one strand. In some embodiments, the lipid conjugated to the terminal nucleotide of one strand is saturated. In some embodiments, the lipid is unsaturated. In some embodiments, the lipid is a sterol. In some embodiments, the lipid is a saturated lipid having 12 to 30 carbon atoms. In some embodiments, the lipid is a straight-chain lipid having 16 carbon atoms. In some embodiments, the lipid contains a hydroxyl moiety. In some embodiments, the lipid is cholesteryl.

[0008] In another aspect, the present invention provides a compound comprising an oligonucleotide in which a hydroxy lipid is bound to an internal nucleotide. In some embodiments, the hydroxy lipid comprises an aliphatic chain containing one or more hydroxyl (-OH) functional groups. In some embodiments, the hydroxy group is bound to a distal carbon of the aliphatic chain relative to the internal nucleotide (i.e., the carbon atom farthest from the internal nucleotide). In some embodiments, the hydroxy lipid is bound to the 2'-carbon of the internal nucleotide. In some embodiments, the hydroxy lipid consists of 12 to 24 carbon atoms. In some embodiments, the hydroxy lipid consists of 16 carbon atoms.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0010] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] DETAILED DESCRIPTION

[0012] Lipid PK / PD Modulators

[0013] This specification describes compounds comprising a PK / PD modulator conjugated to an oligonucleotide-based agent for delivering a payload, such as an RNA interference (RNAi) agent, to cells in vivo. Without being bound to a particular theory, it is believed that the compounds described herein modulate the pharmacokinetic and / or pharmacodynamic properties of the corresponding delivery vehicle, thereby enhancing the RNAi-mediated knockdown of target genes within cells. The compounds described herein may facilitate delivery to certain cell types, including but not limited to CNS cell types such as neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells.

[0014] The present invention provides a lipid delivery platform for oligonucleotides, methods of using the lipid delivery platform, and methods of manufacturing the lipid delivery platform.

[0015] As used herein and as would be understood by one of ordinary skill in the art, polyethylene glycol (PEG) units refer to repeating units of the formula -(CH2CH2O)-. It will be understood that in the chemical structures disclosed herein, PEG units may be represented as -(CH2CH2O)-, -(OCH2CH2)-, or -(CH2OCH2)-. It will also be understood that numbers indicating the number of repeating PEG units may be placed on either side of the parentheses representing the PEG units.

[0016] Another aspect of the present invention provides a method of manufacturing a compound comprising an RNAi agent and a lipid moiety.

[0017] In some embodiments, the method comprises conjugating an oligonucleotide-based agent comprising a first reactive moiety with a compound comprising a lipid and a second reactive moiety to produce a compound comprising an RNAi agent and a lipid moiety.

[0018] In some embodiments, the first reactive moiety is selected from the group consisting of hydroxy and amine reactive groups. In some embodiments, the first reactive moiety is an amine. In some embodiments, the first reactive moiety is a hydroxy group.

[0019] In some embodiments, the second reactive moiety is selected from the group consisting of esters (including activated esters such as, but not limited to, tetrafluorophenoxy esters and paranitrophenoxy esters), sulfones (including, but not limited to, sulfonyl halides), and phosphoramidites. In some embodiments, the second reactive moiety is an ester. In some embodiments, the second reactive moiety is a sulfone. In some embodiments, the second reactive moiety is a phosphoramidite.

[0020] Shown in Table 1 below and described herein, the formulas LP-128p, LP-132p, LP-183p, LP-183 phosphoramidite, LP-183r-p, LP-200p, LP-232p, LP-233p, LP-242p, LP-243p, LP-245p, LP-249p, LP-257p, LP-259p, LP-260p, LP-262p, LP-269p, LP-273p, LP-274p, LP-276p, LP-283p, LP-286p, LP-287p, LP-289p, LP-290p, LP-293p, LP-296p, LP-300p, LP-303p, LP-304p, LP-310p, LP-383p, LP-395p, LP-396p, LP-409p, LP-429p, LP-430p, LP-431p, LP-435p, LP-439p, LP-440p, LP-441p, LP-456p, LP-462p, LP-463p, LP-464p, LP-465p, LP-466p, LP-493p (2'-internal), (2C8C12) phosphoramidite, (2C6C10) phosphoramidite, HO-C16 phosphoramidite, C16 phosphoramidite, and C22 phosphoramidite may be referred to as "pharmacokinetic and / or pharmacodynamic modulator precursors (hereinafter, "PK / PD modulator precursors"). It will also be understood that some of the said compounds may be referred to as "pharmacokinetic and / or pharmacodynamic modulators" (hereinafter, "PK / PD modulators").When referring to the portions of the compounds of Formulae LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b(2' internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c(2' internal) shown in Tables 3 and 3a below, the term "PK / PD modulator" refers to the portion of the compound excluding R (i.e., the oligonucleotide-based agent).

[0021] The PK / PD modulator is linked to an oligonucleotide-based agent such as an RNAi agent to facilitate delivery of the RNAi agent to a target cell or tissue. A PK / PD modulator precursor having a reactive moiety including, but not limited to, an activating ester group and phosphoramidite that facilitate binding to one or more linking groups on the RNAi agent can be synthesized. Chemical reaction synthesis of linking such a PK / PD modulator precursor to an RNAi agent is known in the art. The terms "PK / PD modulator" and "lipid PK / PD modulator" may be used interchangeably herein.

[0022] PK / PD modulator precursors selected from the group consisting of the reference LP-128p, LP-132p, LP-183p, LP-183 phosphoramidite, LP-183r-p, LP-200p, LP-232p, LP-233p, LP-242p, LP-243p, LP-245p, LP-249p, LP-257p, LP-259p, LP-260p, LP-262p, LP-269p, LP-273p, LP-274p, LP-276p, LP-283p, LP-286p, LP-287p, LP-289p, LP-290p, LP-293p, LP-296p, LP-300p, LP-303p, LP-304p, LP-310p, LP-383p, LP-395p, LP-396p, LP-409p, LP-429p, LP-430p, LP-431p, LP-435p, LP-439p, LP-440p, LP-441p, LP-456p, LP-462p, LP-463p, LP-464p, LP-465p, LP-466p, LP-493p (2´ internal), (2C8C12) phosphoramidite, (2C6C10) phosphoramidite, HO-C16 phosphoramidite, C16 phosphoramidite, and C22 phosphoramidite shown in Table 1 can be used as starting materials for linking to an RNAi agent. The PK / PD modulator precursor can be covalently bound to the RNAi agent using any method known in the art. For example, in some embodiments, the activated ester PK / PD modulator precursor can react with the amine-containing moiety at the 5' end of the sense strand.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

[0023] In some embodiments, one or more PK / PD modulators can be conjugated to the RNAi agents described herein. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or more PK / PD modulators can be conjugated to the RNAi agents described herein.

[0024] The PK / PD modulator precursor can be conjugated to the RNAi agent using any method known in the art. In some embodiments, a PK / PD modulator precursor comprising an ester moiety can react with an RNAi agent comprising an amine to produce a compound comprising a PK / PD modulator conjugated to the RNAi agent. In some embodiments, the amine may be at the 5'- or 3'-terminus of the RNAi agent. In some embodiments, the amine may be at the 5'-terminus of the RNAi agent. In some embodiments, the amine may be at the 3'-terminus of the RNAi agent. In some embodiments, a PK / PD modulator precursor comprising a sulfonyl moiety can react with an RNAi agent comprising an amine moiety to produce a compound comprising a PK / PD modulator conjugated to the RNAi agent. In some embodiments, the amine moiety may be at the 5'- or 3'-terminus of the RNAi agent. In some embodiments, the amine moiety may be at the 5'-terminus of the RNAi agent. In some embodiments, the amine moiety may be at the 3'-terminus of the RNAi agent. In some embodiments, a PK / PD modulator precursor comprising a phosphoramidite moiety can react with an RNAi agent comprising a hydroxyl moiety to produce a compound comprising a PK / PD modulator conjugated to the RNAi agent. In some embodiments, the hydroxyl moiety may be at the 5'- or 3'-terminus of the RNAi agent. In some embodiments, the hydroxyl moiety may be at the 5'-terminus of the RNAi agent. In some embodiments, the hydroxyl moiety may be at the 3'-terminus of the RNAi agent.

[0025] In some embodiments, the PK / PD modulator can be conjugated to the 5'-terminus of the sense or antisense strand, the 3'-terminus of the sense or antisense strand, or an internal nucleotide of the RNAi agent. In some embodiments, the RNAi agent is synthesized to have a disulfide-containing moiety at the 3'-terminus of the sense strand, and the PK / PD modulator precursor can be conjugated to the 3'-terminus of the sense strand using any of the appropriate general synthetic schemes shown above.

[0026] In some embodiments, the lipid PK / PD modulator comprises the compounds shown in Table 2.

[0027]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Chemical formula

[0028] In some embodiments, the lipid PK / PD modulator is represented by a compound having the formula shown in Table 3.

[0029]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

[0030] In some embodiments, the lipid PK / PD modulator includes an aliphatic linker between the lipid component and the oligonucleotide. Examples of PK / PD modulators are represented by compounds having the formulas shown in Table 3a.

[0031]

Table 3a-1

Table 3a-2

Table 3a-3

Table 3a-4

Table 3a-5

Table 3a-6

[0032] Definitions

[0033] As used herein, the terms “oligonucleotide” and “polynucleotide” mean polymers of linked nucleosides, each of which can independently be modified or unmodified.

[0034] As used herein, the term "RNAi agent" (also referred to as "RNAi trigger") refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can sequence-specifically degrade or inhibit the translation of messenger RNA (mRNA) transcripts of a target mRNA (e.g., degrade or inhibit under appropriate conditions). The RNAi agents used herein can act via the RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex, i.e., RISC)), or via any alternative mechanism or pathway. Although the term RNAi agent as used herein is believed to act primarily via the RNA interference mechanism, the disclosed RNAi agents are not restricted or limited to a particular pathway or mechanism of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi agent described herein is at least partially complementary to the target mRNA. The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0035] As used herein, the term "lipid" refers to moieties and molecules soluble in nonpolar solvents. The term "lipid" includes amphiphilic molecules containing a polar, water-soluble head group and a hydrophobic tail. Lipids can be of natural or synthetic origin. Non-limiting examples of lipids include fatty acids (e.g., saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids), glycerolipids (e.g., monoacylglycerol, diacylglycerol, and triacylglycerol), phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine), sphingolipids (e.g., sphingomyelin), and cholesterol esters. As used herein, the term "saturated lipid" refers to a lipid that does not contain unsaturation. As used herein, the term "unsaturated lipid" refers to a lipid that contains at least one degree of unsaturation. As used herein, the term "branched lipid" refers to a lipid containing two or more straight chains, each straight chain being covalently bonded to at least one other straight chain. As used herein, the term "straight-chain lipid" refers to a lipid that does not contain branching.

[0036] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knocking down" when referring to the expression of a particular gene mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA, in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, is decreased in a cell, cell population, tissue, organ, or subject treated with the RNAi agent described herein as compared to a second cell, cell population, tissue, organ, or subject that has not been so treated.

[0037] As used herein, the terms "sequence" and "nucleotide sequence" mean a consecutive or ordered series of nucleobases or nucleotides described as a series of letters using standard nomenclature.

[0038] As used herein, the terms "base", "nucleotide base", or "nucleic acid base" are heterocyclic pyrimidine or purine compounds that are components of nucleotides, and include the major purine bases adenine and guanine, and the major pyrimidine bases cytosine, thymine, and uracil. Nucleic acid bases may further be modified to include, but are not limited to, universal bases, hydrophobic bases, degenerate bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleic acid bases (including phosphoramidite compounds containing modified nucleic acid bases) is known in the art.

[0039] As used herein, and unless otherwise specified, the term "complementary" when describing a first nucleic acid base or nucleotide sequence (e.g., the sense strand of an RNAi agent or a target mRNA) in relation to a second nucleic acid base or nucleotide sequence (e.g., the antisense strand of an RNAi agent or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) with an oligonucleotide or polynucleotide containing the second nucleotide sequence and form a duplex or double helix structure under certain standard conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics as long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications. For example, a and Af as defined herein are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0040] As used herein, "perfectly complementary" or "fully complementary" means that in a hybridized pair of nucleobases or nucleotide sequence molecules, all (100%) of the bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0041] As used herein, "partially complementary" means that in a hybridized pair of nucleobases or nucleotide sequence molecules, at least 70% (but not all) of the bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0042] As used herein, "substantially complementary" means that in a hybridized pair of nucleobases or nucleotide sequence molecules, at least 85% (but not all) of the bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0043] The terms "complementary", "perfectly complementary", "partially complementary", and "substantially complementary" as used herein are used with respect to the matching of nucleobases or nucleotides between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of a target mRNA.

[0044] The terms "substantially identical" or "substantial identity" as applied to a nucleic acid sequence used herein means that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or more sequence identity, or more, e.g., at least 90%, at least 95%, or at least 99% identity, when compared to a reference sequence. The percentage of sequence identity is determined by comparing two sequences that are optimally aligned over a comparison window. This percentage is calculated by determining the number of positions at which the same type of nucleobase is present in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to calculate the percentage of sequence identity. The inventions disclosed herein include nucleotide sequences that are substantially identical to those disclosed herein.

[0045] As used herein, the terms "treat", "treatment", etc. mean a method or process taken to reduce or alleviate the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, the terms "treat" and "treatment" can include preventive treatment, management, prophylactic treatment, and / or suppression or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0046] As used herein, the term "introduce into a cell", when referring to an RNAi agent, means functionally delivering the RNAi agent into the cell. The term "functional delivery" means delivering an RNAi agent into a cell in a manner that enables the RNAi agent to have the expected biological activity (e.g., sequence-specific inhibition of gene expression).

[0047] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereomers", and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four different substituents is called a "chiral center".

[0048] As used herein, unless specifically identified as a structure having a particular conformation, for each structure that has an asymmetric center and thus gives rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including optically pure forms and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as single stereoisomers.

[0049] As used in the claims herein, the term "consisting of" excludes elements, steps, or components not specified in the claim. As used in the claims herein, the term "consisting essentially of" limits the scope of the claim to the specified material or step and materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention.

[0050] One of ordinary skill in the art will readily understand and recognize that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein assume that certain functional groups such as OH, SH, or NH, etc. may be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of the protonation state based on the environment (e.g., pH), as will be readily understood by one of ordinary skill in the art.

[0051] As used herein when referring to the connection between two compounds or molecules, the terms "linked" or "bonded" mean that the two molecules are joined by a covalent bond or associated via a non-covalent bond (e.g., hydrogen bond or ionic bond). In some examples, when the terms "linked" or "bonded" refer to the bond between two molecules via a non-covalent bond, the bond between the two different molecules has a K -4 less than 1×10 -5 M (e.g., less than 1×10 -6 M, less than 1×10 -7 M, or less than 1×10 D . Unless otherwise specified, the terms "linked" and "bonded" as used herein may refer to the connection between a first compound and a second compound, regardless of the presence or absence of intervening atoms or groups of atoms.

[0052] A linking group as used herein is one or more atoms that connect one molecule or part of a molecule to another molecule or part of a molecule, or to a second molecule or a second part of a molecule. Similarly, the term "scaffold" as used in the art may be used interchangeably with a linking group. A linking group may contain any number of atoms or functional groups. In some embodiments, a linking group may not promote a biological or pharmaceutical response and may simply serve to link two biologically active molecules.

[0053] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group containing 1 to 12 (e.g., 1 to 8, 1 to 6, 1 to 4, or 1 to 3) carbon atoms. The alkyl group can be straight-chain or branched-chain. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl.

[0054] Unless otherwise specified, the symbol

Chemical formula

[0055] As used herein, the term "comprising" means the term "including but not limited to", and is used interchangeably therewith. The term "or" as used herein means the term "and / or" as used herein and is used interchangeably therewith, unless the context clearly indicates otherwise.

[0056] As used in the claims herein, the term "consisting of" excludes any element, step, or component not specified in the claim. The term "consisting essentially of" as used in the claims herein limits the scope of the claim to the specified materials or steps and materials or steps that do not substantially affect the basic and novel characteristics of the claimed invention.

[0057] Oligonucleotide-based drugs containing RNAi agents

[0058] As used herein, the "oligonucleotide-based agent" refers to about 10 to 50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32,~28~30, 30~50, 30~48, 30~46, 30~44, 30~42, 30~40, 30~38, 30~36, 30~34, 30~32, 32~50, 32~48, 32~46, 32~44, 32~42, 32~40, 32~38, 32~36, 32~34, 34~50, 34~48, 34~46, 34~44, 34~42, 34~40, 34~38, 34~36, 36~50, 36~48, 36~46, 36~44, 36~42, 36~40, 36~38, 38~50, 38~48, 38~46, 38~44, 38~42, 38~40, 40~50, 40~48, 40~46, 40~44, 40~42, 42~50, 42~48, 42~46, 42~44, 44~50, 44~48, 44~46, 46~50, 46~48, or 48~50) oligonucleotides or a nucleotide sequence comprising nucleotide base pairs. In some embodiments, the oligonucleotide-based agent has a nucleobase sequence that is at least partially complementary to the coding sequence of the target nucleic acid or target gene expressed intracellularly. In some embodiments, the oligonucleotide-based agent, when delivered to a cell that expresses a gene, can inhibit the expression of the underlying gene and is herein referred to as an "expression-inhibiting oligonucleotide-based agent". Gene expression can be inhibited in vitro or in vivo.,

[0059] The "oligonucleotide-based agent" includes, but is not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligonucleotide-based agent is a single-stranded oligonucleotide such as an antisense oligonucleotide. In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide that is an RNAi agent.,

[0060] In some embodiments, the oligonucleotide-based agent is an "RNAi agent", which, as defined herein, is an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can sequence specifically degrade or inhibit the translation of a messenger RNA (mRNA) transcript of a target mRNA. The RNAi agents used herein are compositions that act via an RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or can act via any alternative mechanism or pathway. Although the term RNAi agent as used herein is thought to act primarily via the RNA interference mechanism, the disclosed RNAi agents are not restricted or limited to a particular pathway or mechanism of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the target mRNA. The RNAi agent may contain one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0061] Generally, an RNAi agent is composed of at least a sense strand (also called a passenger strand) containing a first sequence and an antisense strand (also called a guide strand) containing a second sequence. The lengths of the sense strand and the antisense strand of the RNAi agent are each 16 to 49 nucleotides. In some embodiments, the sense strand and the antisense strand of the RNAi agent are independently 17 to 26 nucleotides. In some embodiments, the sense strand and the antisense strand are independently 19 to 26 nucleotides. In some embodiments, the sense strand and the antisense strand are independently 21 to 26 nucleotides. In some embodiments, the sense strand and the antisense strand are independently 21 to 24 nucleotides. The sense strand and the antisense strand may be of the same length or different lengths. The RNAi agent contains an antisense strand sequence that is at least partially complementary to a sequence within the target gene, and when delivered to a cell that expresses the target, the RNAi agent can inhibit the expression of one or more target genes in vivo or in vitro.

[0062] Oligonucleotide-based agents can generally, and RNAi agents in particular, be composed of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, "modified nucleotide" refers to a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. Modified nucleotides as used herein include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3'-3' linked (inverted) nucleotides, nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimics (locked nucleic acid base analogs, locked nucleotides, 3'-O-methoxy (2' nucleoside linked) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2'-position of the five-membered sugar) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides, 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-aminonucleotides, and 2'-alkyl nucleotides.

[0063] Furthermore, one or more nucleotides of an oligonucleotide-based agent (e.g., an RNAi agent) can be linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages can be phosphate-free covalent internucleoside linkages. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkyl phosphonates (e.g., methyl phosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkyl phosphoramidate, or thionophosphoramidate), thionoalkyl phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates having a normal 3'-5' linkage, 2'-5' linkage analogs of boranophosphates, or boranophosphates having inverted polarity, where adjacent pairs of nucleoside units are from linked 3'-5' to 5'-3', or from 2'-5' to 5'-2'.

[0064] Not all positions of a given compound need to be uniformly modified. Conversely, multiple modifications may be incorporated into one oligonucleotide-based agent or one nucleotide thereof.

[0065] The sense and antisense strands of an RNAi agent can be synthesized and / or modified by methods known in the art. Additional disclosures related to RNAi agents can be found, for example, in the disclosure of modifications, for example, in International Patent Application No. PCT / US2017 / 045446 (WO2018027106) of Arrowhead Pharmaceuticals, Inc., which is also incorporated herein by reference in its entirety.

[0066] Modified nucleotide

[0067] In some embodiments, the RNAi agent comprises one or more modified nucleotides. As used herein, "modified nucleotide" refers to a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides (represented herein as invdN, invN, invn), nucleotides containing modified nucleobases, bridged nucleotides, peptide nucleic acids (PNA), 2',3'-seco nucleotide mimics (locked nucleic acid base analogs, represented herein as N UNA or NUNA), locked nucleotides (represented herein as N LNA or NLNA), 3'-O-methoxy (2' internucleoside linkage) nucleotides (represented herein as 3'-OMen), 2'-F-arabinonucleotides (represented herein as NfANA or Nf ANA5'-Me, 2'-fluoro nucleotides (referred to herein as 5Me-Nf), morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (referred to herein as vpdN), vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides (cPrpN). 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2'-position of the 5-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (referred to herein as lowercase "n" in the nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (also referred to herein as 2'-fluoro nucleotides and referred to herein as Nf), 2'-deoxy nucleotides (referred to herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also referred to herein as 2'-MOE and referred to herein as NM), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions of a given compound to be uniformly modified. Conversely, even one target RNAi agent or one of its nucleotides can incorporate multiple modifications. The sense and antisense strands of the target RNAi agent can be synthesized and / or modified by methods known in the art. The modification of one nucleotide is independent of the modification of another nucleotide.

[0068] Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines, N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0069] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense and antisense strands that are ribonucleotides (i.e., unmodified). As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand that are unmodified ribonucleotides. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand that are unmodified ribonucleotides. In some embodiments, one or more of the nucleotides of the RNAi agent are unmodified ribonucleotides.

[0070] Modified nucleoside internucleoside linkages

[0071] In some embodiments, one or more nucleotides of the RNAi agent are joined by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein by the lower case "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkyl phosphonates (e.g., methylphosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkyl phosphoramidate, or thionophosphoramidate), thionoalkyl phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates having a normal 3'-5' linkage, 2'-5' linkage analogs of boranophosphates, or boranophosphates having an inverted polarity in which pairs of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkages or backbones lack a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar linkages, or one or more short chain heteroatom or heterocyclic sugar linkages. In some embodiments, the modified internucleoside backbone includes, but is not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having a mixed N, O, S, and CH2 components.

[0072] In some embodiments, the sense strand of the RNAi agent can include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of the RNAi agent can include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand can independently include 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of the RNAi agent can include 1, 2, 3, or 4 phosphorothioate bonds, the antisense strand of the RNAi agent can include 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand can independently include 1, 2, 3, or 4 phosphorothioate linkages.

[0073] In some embodiments, the sense strand of the RNAi agent includes at least two phosphorothioate internucleoside linkages. In some embodiments, the at least two phosphorothioate internucleoside linkages are between nucleotides at positions 1 to 3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5' end of the sense strand and another phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, two phosphorothioate internucleoside linkages are located at the 5' end of the sense strand and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand does not include phosphorothioate internucleoside linkages between nucleotides, but includes 1, 2, or 3 phosphorothioate linkages between the terminal nucleotides at both the 5' and 3' ends and an optionally present inverted abasic residue terminal cap. In some embodiments, the target ligand is linked to the sense strand via a phosphorothioate linkage.

[0074] In some embodiments, the RNAi agent antisense strand comprises four phosphorothioate internucleotide linkages. In some embodiments, the four phosphorothioate internucleotide linkages are between nucleotides at positions 1 to 3 from the 5' end of the antisense strand and between nucleotides at positions 19 to 21, 20 to 22, 21 to 23, 22 to 24, 23 to 25, or 24 to 26 from the 5' end. In some embodiments, the three phosphorothioate internucleotide linkages are located between positions 1 to 4 from the 5' end of the antisense strand, and the fourth phosphorothioate internucleotide linkage is located between positions 20 to 21 from the 5' end of the antisense strand. In some embodiments, the RNAi agent comprises at least three or four phosphorothioate internucleotide linkages in the antisense strand.

[0075] In some embodiments, the RNAi agent comprises one or more modified nucleotides and one or more modified internucleotide linkages. In some embodiments, the 2'-modified nucleosides are combined with the modified internucleotide linkages.

[0076] Linking groups and delivery agents

[0077] In some embodiments, the oligonucleotide-based agents such as the RNAi agents described herein include, but are not limited to, one or more non-nucleotide groups including a linking group or a delivery agent, or are attached thereto. The non-nucleotide group can enhance the targeting, delivery, or attachment of the RNAi agent. Examples of linking groups are shown in Table 4. The non-nucleotide group can be covalently linked to either the 3'-end and / or 5'-end of either the sense strand and / or the antisense strand. In some embodiments, the RNAi agent includes a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the sense strand of the RNAi agent. The non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0078] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or in vivo distribution properties of the RNAi agent or conjugate to which it is attached and improves the cell-specific or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, the non-nucleotide group enhances the endocytosis of the RNAi agent.

[0079] The RNAi agents described herein can be synthesized to have a reactive group such as an amino group (also referred to herein as an amine) at the 5'-end and / or 3'-end. The reactive group can then be used to attach a targeting moiety using methods common in the art.

[0080] For example, in some embodiments, the RNAi agents disclosed herein are synthesized to have an NH2-C6 group at the 5' end of the sense strand of the RNAi agent. The terminal amino group can then react, for example, to form a conjugate with a group containing a compound having an affinity for one or more integrins (i.e., integrin target ligands) or a PK enhancer. In some embodiments, the RNAi agents disclosed herein are synthesized to have one or more alkyne groups at the 5' end of the sense strand of the RNAi agent. The terminal alkyne group can then react, for example, with a group containing a target ligand to form a conjugate.

[0081] In some embodiments, the RNAi agent is synthesized to have a linking group that facilitates covalent linkage of the RNAi agent to a target ligand, a target group, a PK / PD modulator, or another type of delivery agent. The linking group can be linked to the 3' end and / or 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is linked to the 5' end or 3' end of the RNAi agent sense strand. In some embodiments, the linking group is linked to the 5' end of the RNAi agent sense strand. Examples of linking groups include, but are not limited to, C6-SS-Alk-Me, reactive groups such as primary amines and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, trialkyne functional groups, ribitol, and / or PEG groups.

[0082] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest via one or more covalent bonds to another chemical group (such as a target ligand, target group, PK / PD modulator, or delivery agent) or segment of interest. A labile linkage includes a labile bond. The linkage can optionally include a spacer that increases the distance between the two atoms being joined. The spacer can further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups, each of which can include one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art, and the foregoing list is not meant to be limiting.

[0083] In some embodiments, the target group is linked to the RNAi agent without using an additional linker. In some embodiments, the target group is designed such that a linker can readily be present to facilitate linkage to the RNAi agent. In some embodiments, when the composition includes two or more RNAi agents, the two or more RNAi agents can each be linked to their respective target groups using the same linker. In some embodiments, when the composition includes two or more RNAi agents, the two or more RNAi agents are linked to their respective target groups using different linkers.

[0084] RNAi agents, whether modified or unmodified, may contain 3' and / or 5' target groups, linking groups, and / or be bound to a PK / PD modulator or contain a PK / PD modulator. Any RNAi agent sequence described herein that contains a 3' or 5' target ligand, target group, PK / PD modulator, or linking group may also not contain a 3' or 5' target ligand, target group, linking group, or PK / PD modulator, or may contain different 3' or 5' target ligands, target groups, linking groups, or PK / PD modulators including, but not limited to, those shown in Tables 2 and 3. The RNAi agent duplexes listed in Table A may further contain a target ligand, target group, linking group, or PK / PD modulator, whether modified or unmodified, and the target group or linking group may be bound to the 3' or 5' end of either the sense strand or the antisense strand of the RNAi agent duplex.

[0085] In some embodiments, the linking group can be synthetically bound to the 5' or 3' end of the sense strand of the RNAi agent described herein. In some embodiments, the linking group is synthetically bound to the 5' end of the sense strand of the RNAi agent. In some embodiments, the linking group bound to the RNAi agent may be a trialkyne linking group.

[0086] Examples of specific modified nucleotides, capping moieties, and linking groups are shown in Table 4.

[0087]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

[0088] Alternatively, other linking groups known in the art may be used.

[0089] In addition to or instead of linking the RNAi agent to one or more target ligands, target groups, and / or PK / PD modulators, in some embodiments, a delivery agent may be used to deliver the RNAi agent to a cell or tissue. A delivery agent is a compound that can improve the delivery of an RNAi agent to a cell or tissue and includes, but is not limited to, polymers such as amphiphilic polymers, membrane-active polymers, peptides, melittin peptides, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines, or may be composed of these.

[0090] In some embodiments, the RNAi agent can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPC, or other delivery systems available in the art. The RNAi agent can also be chemically conjugated to a target group, lipids (including, but not limited to, cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPC (see, for example, WO2000 / 053722, WO2008 / 022309, WO2011 / 104169, and WO2012 / 083185, WO2013 / 032829, WO2013 / 158141, each of which is incorporated herein by reference), or other delivery systems available in the art.

[0091] Pharmaceutical composition

[0092] In some embodiments, the present disclosure LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c,Provide a pharmaceutical composition comprising, consisting of, or consisting essentially of one or more compounds of LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2' internal).

[0093] As used herein, "pharmaceutical composition" includes a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product) that are intentionally included in the drug delivery system. Excipients do not exert a therapeutic effect or are not intended to do so at the intended dosage. Excipients may serve roles such as a) assisting in the processing of the drug delivery system during manufacture, b) protecting, supporting, or enhancing the stability, bioavailability, or patient acceptability of the API, c) assisting in the identification of the product, and / or d) enhancing other attributes of the overall safety and effectiveness of API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0094] Excipients include, but are not limited to, absorption enhancers, anti-adhesion agents, anti-foaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, flow promoters, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water-repellent agents, and wetting agents.

[0095] The pharmaceutical compositions described herein may contain other additional components commonly included in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active substance. Pharmaceutically active substances include, but are not limited to, anti-itch agents, astringents, local anesthetics, anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0096] The pharmaceutical composition may also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts that change osmotic pressure, buffers, coating agents, or antioxidants. These may also include other agents having known therapeutic effects.

[0097] The pharmaceutical composition can be administered in various ways depending on whether local or systemic treatment is desired and the site to be treated. Administration can be by any method known in the art, for example, but not limited to, topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powder or aerosol including nebulizer, intratracheal, intranasal), epidermal, transdermal, oral, or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous (e.g., by implantable device), intracranial, intrasubstantial, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions, or suspensions. Administration can also be, for example, rectal administration using suppositories, topical or transdermal administration using, for example, ointments, creams, gels, or solutions, or parenteral administration using, for example, injection solutions.

[0098] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water-soluble), dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline. This needs to be stable under the manufacturing and storage conditions and protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersion, and the use of surfactants. In many cases, it is preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. The prolongation of the absorption of the injectable composition is brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0099] Sterile injectable solutions can be prepared, if necessary, by incorporating the required amount of the active compound in a suitable solvent into one or a combination of the ingredients listed above and then filtering to sterility. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying, whereby powders of the active ingredient and any additional desired ingredients are obtained from a previously sterile-filtered solution.

[0100] Formulations suitable for intra-articular administration may be in the form of sterile aqueous preparations of any of the ligands described herein, which may be in the form of microcrystals, for example, an aqueous microcrystalline suspension. Also, liposomal formulations or biodegradable polymer systems can be used to present any of the ligands described herein for both intra-articular and intra-ocular administration.

[0101] The active compound can be prepared together with a carrier that prevents the rapid excretion of the compound from the body, such as in a controlled release formulation including an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0102] The pharmaceutical composition can include other additional ingredients commonly included in pharmaceutical compositions. Such additional ingredients include, but are not limited to, anti-itch agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of a pharmacologically active agent that produces a pharmacological, therapeutic, or prophylactic result.

[0103] Formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c,Pharmaceuticals containing the compounds of LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal) are also the subject of the present invention, and methods for manufacturing such pharmaceuticals are also the subject of the present invention. The methods include LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b,One or more compounds of LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal), and, if desired, one or more other substances having a known therapeutic effect, into a pharmaceutically acceptable form.

[0104] The formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c,Compounds of LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal), and LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b,Pharmaceutical compositions containing compounds of LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-439c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be packaged or included in kits, containers, packs, or dispensers. Compounds of the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a,Compounds of C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal), and compounds of the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a,LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c,Pharmaceutical compositions containing the compounds of LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2' internal) can be packaged in a pre-filled syringe or vial.

[0105] Therapeutic methods and inhibition of expression

[0106] The formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c,The compounds of LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of such compounds. In some embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b,The compounds of LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be used to treat a subject (e.g., a human) who would benefit from a reduction and / or inhibition of the expression at the target mRNA and / or protein level, e.g., a subject diagnosed with or suffering from symptoms associated with a CNS disease or disorder.

[0107] In some embodiments, the subject is administered with the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c,A therapeutically effective amount of one or more compounds of LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) is administered. Treatment of a subject can include therapeutic and / or prophylactic treatment. The subject is a compound of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b,A therapeutically effective amount of one or more compounds of LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) is administered. The subject can be a human, a patient, or a human patient. The subject can be an adult, an adolescent, a child, or an infant. The pharmaceutical composition described herein can be administered to a human or an animal.,

[0108] The formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c,The compounds of LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be used to treat at least one symptom in a subject having a disease or disorder related to a target gene or having a disease or disorder mediated at least in part by the expression of a target gene. In some embodiments, the compounds of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b,The compounds of LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be used to treat or manage the clinical symptoms of a subject having a disease or disorder that would benefit, at least in part, from or be mediated by reduction of the mRNA of a target gene. The subject is of the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, described herein.LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466cA therapeutically effective amount of one or more compounds or compositions of LP-493c (2' internal) is administered. In some embodiments, the methods disclosed herein are the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a(2' internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b(2' internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c,LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, L Administering a composition comprising the compounds of P-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) to a subject to be treated. In some embodiments, the subject is of the formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b,One or more prophylactically effective amounts of a compound described in LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) are administered, whereby the subject is treated by preventing or inhibiting at least one symptom.,

[0109] In some embodiments, the present disclosure provides a method for treating a disease, disorder, condition, or pathological condition in a patient in need thereof, at least partially mediated by the expression of a target gene, wherein the method comprises administering to the patient a compound of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a(2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b(2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c,Administering any of the compounds of LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (inside 2´).

[0110] In some embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, described herein...In a subject administered with the compounds of LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2' internal), the gene expression level and / or mRNA level of the target gene is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 99% or more compared to the subject before administration of the compound or a subject not administered with the compound. The gene expression level and / or mRNA level in the subject may be reduced in the cells, cell populations, and / or tissues of the subject.,

[0111] In some embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, as described herein...The levels of the target protein in a subject administered with the compounds of LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (internal of 2´) are reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% or more compared to the subject before administration of the compound, or a subject not administered with the compound. The protein levels in the subject may be reduced in the cells, cell populations, tissues, blood, and / or other fluids of the subject.

[0112] The decrease in the target mRNA level and / or the target protein level can be evaluated by any method known in the art. As used herein, the decrease or reduction in the target mRNA level and / or protein level is collectively referred to herein as the decrease or reduction in the target gene and / or protein level, or the inhibition or decrease in the expression of the target gene.

[0113] In some embodiments, the compounds of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, as described herein...The compounds of LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal) can be used in the preparation of pharmaceutical compositions for use in the treatment of diseases, disorders, or conditions that are at least partially mediated by the expression of a target gene. In some embodiments, the disease, disorder, or condition that is at least partially mediated by the expression of a target gene is a CNS disease or disorder.,

[0114] In some embodiments, the method of treating a subject depends on the subject's weight. In some embodiments, the compounds of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c,The compounds of LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal) can be administered at a dosage of about 0.05 mg / kg to about 40.0 mg / kg based on the body weight of the subject. In other embodiments, the compounds of formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b,The compounds of LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be administered at a dose of about 5 mg / kg to about 20.0 mg / kg relative to the body weight of the subject.,

[0115] In some embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c,The compounds of LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal) may be administered in divided doses, which means that two administrations are administered to the subject in a short period (e.g., less than 24 hours). In some embodiments, about half of the desired daily dose is administered in the first administration, and about the remaining half of the desired daily dose is administered about 4 hours after the first administration.,

[0116] In some embodiments, the compounds described herein having the formulae LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c,The compounds of LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) can be administered once a week (i.e., weekly). In other embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b,The compounds of LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´ internal) can be administered every two weeks (once every two weeks).

[0117] In some embodiments, the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, as described herein...Compounds of LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2´-internal), or compositions containing such compounds, can be used for the treatment of diseases, disorders, or symptoms that are at least partially mediated by the expression of a target gene. In some embodiments, the diseases, disorders, or symptoms that are at least partially mediated by the expression of a target gene are CNS diseases or disorders.

[0118] Another aspect of the present invention provides a method for reducing the in vivo expression of a target gene, the method comprising introducing into a cell a formula LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c,Introducing the compounds of LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (internal to 2') into cells, wherein the compounds comprise at least substantially an RNAi agent complementary to a target gene. In some embodiments, the cells are CNS cells. In some embodiments, the cells are within a subject. In some embodiments, the subject is diagnosed with a disease or disorder that is treated, prevented, or ameliorated by reducing the expression of the target gene. In some embodiments, the disease or disorder is a CNS disease or disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and Lewy body disease.,

[0119] Another aspect of the invention provides for using any one of the lipid PK / PD modulators conjugated to the oligonucleotide-based agents described herein for the treatment, prevention, or amelioration of a disease or disorder. In some embodiments, the disease or disorder is a CNS disease or disorder selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and Lewy body disease.,

[0120] Cells, Tissues, and Non-Human Organisms

[0121] The formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2´ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b, LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2´ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c,Cells, tissues, and non-human organisms are contemplated that contain at least one of the compounds of LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal). The cells, tissues, and non-human organisms are of the formulas LP-128a, LP-132a, LP-183a, LP-183r-a, LP-200a, LP-232a, LP-233a, LP-242a, LP-243a, LP-245a, LP-249a, LP-257a, LP-259a, LP-260a, LP-262a, LP-269a, LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2'-internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303b, LP-304b, LP-310b, LP-383b, LP-395b,Compounds of LP-396b, LP-409b, LP-429b, LP-430b, LP-431b, LP-435b, LP-439b, LP-440b, LP-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2'-internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP-430c, LP-431c, LP-435c, LP-440c, LP-441c, LP-456c, LP-462c, LP-463c, LP-464c, LP-465c, LP-466c, and LP-493c (2'-internal) are created by delivering them to cells, tissues, and non-human organisms by any means available in the art. In some embodiments, the cells are mammalian cells including, but not limited to, human cells. In some embodiments, the cells are CNS cells.,

[0122] The above embodiments and items are illustrated using the following non-limiting examples.

Example

[0123] Example

[0124] The following examples are not limiting and are intended to illustrate specific embodiments disclosed herein.

[0125] Unless otherwise expressly stated, the numbers used to refer to the compounds of a particular example refer only to that particular example and not to other examples disclosed herein. For example, Compound 1 in "Synthesis of LP-183 Phosphoramidite" of Example 2 is different from and does not refer to Compound 1 in "Synthesis of LP-232p" of Example 2. Similarly, it will be understood that the particular compounds disclosed herein may be identified by different numbers in different examples. Compounds disclosed in various tables throughout the detailed description (i.e., LPXXa, LPXXb, and LPXX-p, where XX is a number) are consistently referred to throughout the examples of this specification.

[0126] Unless otherwise expressly stated, it will be understood that the use of the term "EDC" in the examples herein refers to commercially available EDC hydrochloride.

[0127] Example 1. Synthesis of RNAi Agents and Compositions.

[0128] The following describes the general procedures for the synthesis of specific RNAi agents and their conjugates exemplified in the non-limiting examples described herein.

[0129] Synthesis of RNAi agents. RNAi agents can be synthesized using methods known in the art. In the synthesis of the RNAi agents exemplified in the examples described herein, the sense strand and the antisense strand of the RNAi agent were synthesized according to the solid-phase phosphoramidite technology used in oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or Oligopilot 100 (GE Healthcare) was used. The synthesis was carried out on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA) or polystyrene (obtained from Kinovate, Oceanside, CA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or Hongene Biotech (Morrisville, NC, USA). Specifically, the 2'-O-methyl phosphoramidites used included the following: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-N 2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidite and 2'-O-propargyl phosphoramidite had the same protecting groups as the 2'-O-methyl phosphoramidite. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research (Virginia). Inverted abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidites were purchased from ChemGenes. UNA phosphoramidites used included: 5′-(4,4′-dimethoxytrityl)-N6-(benzoyl)-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4′-dimethoxytrityl)-N-acetyl-2′,3′-seco-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, The phosphorothioate linkages were introduced using 100 mM solutions of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile or 200 mM solutions of xanthan hydride (TCI America, Portland, OR, USA) in pyridine.

[0130] TFA amino link phosphoramidite, a commercial product (ThermoFisher), was purchased and a (NH2-C6) reactive group linker was introduced. TFA amino link phosphoramidite was dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2′O-Me), and 60 seconds (2′F). Trialkyne-containing phosphoramidites were synthesized and each (TriAlk#) linker was introduced. When used in combination with the RNAi agents presented in the specific examples herein, the trialkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2′O-Me), and 60 seconds (2′F).

[0131] In some RNAi agents, linkers such as C6-SS-C6 or 6-SS-6 groups were introduced at the 3′ end of the sense strand. Resins pre-packed with each linker are commercially available. Alternatively, for some sense strands, dT resin was used and each linker was added by standard phosphoramidite synthesis.

[0132] Cleavage and deprotection of the oligomer bound to the support. After completion of solid-phase synthesis, the dried solid support was treated at 30 °C for 1.5 hours in a 1:1 volume solution of 40 wt% aqueous methylamine and 28% - 31% aqueous ammonium hydroxide solution (Aldrich). The solution was evaporated and the solid residue was reconstituted with water (see below).

[0133] Purification. The crude oligomers were purified by anion-exchange HPLC using a TSKgel® SuperQ-5PW 13 μm column (manufactured by Tosoh Biosciences) and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% acetonitrile, and buffer B was the same as buffer A with 1.5 M sodium chloride added. A UV trace at 260 nm was recorded. Appropriate fractions were pooled and size-exclusion HPLC was performed using a GE Healthcare XK16 / 40 column packed with Sephadex® G-25 fine (available from Sigma Aldrich) with a running buffer of 100 mM ammonium bicarbonate, pH 6.7, 20% acetonitrile, or filtered water.

[0134] Annealing. Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) with 1× PBS (phosphate-buffered saline, 1×, Corning, Cellgro) to generate RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25 °C. The double-strand concentration was determined by measuring the absorbance of the solution with a UV-Vis spectrometer in 1× PBS. Next, the double-strand concentration was determined by multiplying the absorbance of the solution at 260 nm by the conversion factor and dilution factor. The conversion factor used was 0.037 mg / (mL·cm) or calculated from the experimentally determined extinction coefficient.

[0135] Example 2. Synthesis of Lipid PK / PD Modulator Precursors

[0136] Synthesis of LP-183 Phosphoramidite

Chemical Structure

[0137] To a solution of Compound 2 (2.00 g) in DCM, TEA (2.27 mL) was added, followed by dropwise addition of Compound 1 (4.931 g) at room temperature. Next, the mixture was stirred at room temperature for 2 hours. Then, the mixture was filtered. The white solid was dried overnight. The product was a white solid, with a yield of 4.267 g and 74%. LC-MS: calculated value [M+H] 356.35, measured value 356.63

Chem.

[0138] To a mixture of Compound 1 (2.54 g) in 120 mL of DCM, Compound 3 (0.61 g) was added, followed by dropwise addition of Compound 2 (5.37 g) at room temperature. Next, the mixture was stirred at room temperature overnight. 5 mL of TFA was added, followed by Celite. After removing the solvent under vacuum, the residue was packed onto a 40 g column by the dry method. The product was purified using a gradient from hexane (2% TEA) to 50% ethyl acetate (2% TEA) in hexane (2% TEA). The product was a white waxy solid, with a yield of 3.462 g and 87%. LC-MS: calculated value [M+H] 556.46, measured value 556.64.

[0139] Synthesis of LP-183r-p

Chem.

[0140] To a solution of Compound 1 (312 mg) in 10 mL of DCM, Compound 2 (299 mg) and EDC (498 mg) were added at room temperature. The reaction mixture was stirred at room temperature for 1 hour. After removing the solvent under vacuum, the residue was dry-packed onto a 12 g column. Ethyl acetate was used as the mobile phase starting from hexane. The product was a transparent oil, with a yield of 408 mg and a yield of 75%. LC-MS: calculated value [M+H] 230.10, measured value 230.34.

Chem.

[0141] To the compound 1 (408 mg) in 20 mL of DCM were added compound 2 (516 mg) and TEA (0.745 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was recrystallized from methanol. The product was a white solid, 555 mg, in 88% yield. LC-MS: calculated value [M+H] 356.35, measured value 356.45.

Chem.

[0142] To a mixture of compound 1 (200 mg) in 10 mL of DCM was added compound 3 (33.2 mg), followed by dropwise addition of compound 2 (339 mg) at room temperature. Next, the mixture was stirred at room temperature overnight. 1 mL of TFA was added, followed by Celite®. After removing the solvent under vacuum, the residue was dry-loaded onto a 4 g column. A gradient from hexane (2% TEA) to 50% ethyl acetate (2% TEA) in hexane (2% TEA) was used as the mobile phase. The product was a white waxy solid, 95 mg, in 30% yield. LC-MS: calculated value [M+H] 556.46, measured value 556.82.

[0143] Synthesis of LP-232p

Chem.

[0144] Enyl palmitoyl (100 mg) was stirred into a solution of cis-4-(boc-amino) cyclohexylamine (0.0819 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, the organics were extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness and the crude product was purified by column (from hexane to ethyl acetate). The product was 52 mg, 31%.

Chem.

[0145] To 1 (0.0520 g) was added 2 mL of dioxane:HCl (4N), and the reaction was continued until the boc deprotection was complete. After removing the solvent under vacuum, the residue was stirred in a solution of 2 (0.0316 g), DIPEA (0.0445 g), and COMU (0.0620 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column (from DCM to 20% MeOH in DCM). The product was 45 mg, 65%.

Chemical formula

[0146] To 1 (0.0449 g) was added 2 mL of dioxane:HCl (4N), and the reaction was continued until the OtBu deprotection was complete. After removing the solvent under vacuum, the residue was stirred in a solution of 2 (0.0217 g), DIPEA (0.039 mL), and COMU (0.0425 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column (from DCM to 20% MeOH in DCM). The product was 30 mg, 58%.

[0147] Synthesis of LP-233p

Chemical formula

[0148] Palmitic acid 1 (0.100 g) was stirred in a solution of 2 (0.0693 g), COMU (0.166 g), and DIPEA (0.16 mL) in 5 mL of DCM. After stirring the suspension overnight (heated at 40 °C), water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column (from hexane to ethyl acetate). The product was 96 mg, 69%.

Chemical formula

[0149] To 1 (0.0955 g), 2 mL of dioxane:HCl (4N) was added and added until the boc deprotection was complete. After removing the solvent under vacuum, the residue was stirred with a solution of 2 (0.0581 g), DIPEA (0.11 mL), and COMU (0.114 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column (from DCM to 20% MeOH in DCM). The product was 68 mg, 54%.

Chem.

[0150] To 1 (0.068 g), 2 mL of dioxane:HCl (4N) was added and added until the deprotection of otBu was complete. After removing the solvent under vacuum, the residue was stirred in a solution of tetrafluorophenol (0.021 g), DIPEA (0.059 mL), and COMU (0.064 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column (from DCM to 20% MeOH in DCM). The product was 22 mg, 28%.

[0151] Synthesis of LP-242p

Chem.

[0152] Palmitic acid (0.100 g) was stirred with a solution of tBu-3,9 diazaspiro[5,5]undecane-3-carboxylate HCl (0.073 g), COMU (0.166 g), DIPEA (0.16 mL) in 5 mL of DCM. After stirring the suspension overnight (heated at 40 °C), water was added, the organic matter was extracted with DCM, and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

Chem.

[0153] 1 (0.017 g) was treated with HCl:dioxane, and after 1 hour, the crude reaction product was dried in vacuo. To this, a solution of 2 (0.0095 g), COMU (0.0186 g), and DIPEA (0.0134 g) in 5 mL of DCM was added. After stirring the suspension, water was added, and the organic matter was extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

Chemical formula

[0154] 2 mL of dioxane:HCl (4 N) was added to 1 (0.121 g) until the otBu deprotection was complete. After removing the solvent under vacuum, the crude product 1 was stirred in a solution of tetrafluorophenol (0.0585 g), DIPEA (0.11 mL), and COMU (0.115 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, and the organic matter was extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

[0155] Synthesis of LP-243p

Chemical formula

[0156] Palmitic acid (0.100 g) was stirred in a solution of tBu-3,9 diazaspiro[5,5]undecane-3-carboxylate HCl (0.0732 g), COMU (0.166 g), and DIPEA (0.161 mL) in 5 mL of DCM. After stirring the suspension overnight (heated at 40 °C), water was added, and the organic matter was extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

Chemical formula

[0157] 1 (0.0200 g) was treated with HCl:dioxane. After 1 hour, the crude reaction product was dried in vacuo. To this, a solution of 2 (0.0119 g), COMU (0.0232 g), and DIPEA (0.022 mL) in 5 mL of DCM was added. After stirring the suspension, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

Chem.

[0158] To 1 (0.121 g), 2 mL of dioxane:HCl (4 N) was added until the deprotection of otBu was complete. After removing the solvent under vacuum, crude 1 was stirred in a solution of tetrafluorophenol (0.0363 g), DIPEA (0.104 mL), and COMU (0.112 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

[0159] Synthesis of LP-245p

Chem.

[0160] TEA was added to a mixture of 1 (2.08 g) and 2 (1.98 g) in 50 mL of toluene at room temperature. The reaction mixture was stirred at 90 °C overnight. After cooling to room temperature, ethyl acetate and water were added for workup. Purification was carried out on a 40 g column. Purification was performed using a gradient from hexane to 30% ethyl acetate in hexane. The product was a pale yellow oil, 1388 mg, 51%. LC-MS: calculated [M+H] 339.21, found 339.62.

Chem.

[0161] To a mixture of 1 (0.241 g) in MeOH / THF (4 mL / 4 mL), 1 N NaOH (6 mL) was added at room temperature. The reaction mixture was stirred at 60 °C for 1 h. After removing the organic solvents under vacuum, 1 N HCl was added to adjust the pH of the mixture to about 1. Next, NaHCO3 was added to adjust the pH to 7 - 8. DCM was added for workup. After removing DCM under vacuum, the residue was placed under high vacuum for 2 h. The residue was diluted with DCM, and DIPEA (0.248 mL), COMU (0.336 g), and 2 (0.166 g) were added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was washed with 1 N HCl, NaHCO3, and brine. Purification was performed on a 12 g column. Purification was carried out using a gradient from hexane to ethyl acetate. The product was a brown oil, 285 mg, 74%. LC-MS: calculated [M+H] 540.34, found 541.07. [Chemical formula]

[0162] To a mixture of 1 (0.0740 g) and Pd / C in ethyl acetate, H2 (1 atm) was charged at room temperature. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was filtered through a Celite® pad. After removing ethyl acetate under vacuum, the residue was placed under high vacuum for 1 h. The residue was dissolved in 3 mL of DCM, and 2 (0.166 mL) and TEA (0.115 mL) were added at room temperature. The mixture was stirred at room temperature for 2 h. Water was added for workup. Purification was performed on a 12 g column. Purification was carried out using a gradient from DCM to 20% MeOH in DCM. The product was a clear oil, 43 mg, 37%. LC-MS: calculated [M+H] 836.71, found 837.68. [Chemical formula]

[0163] A solution of 1 (0.0430 g) in 4N HCl / dioxane (3 mL) was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. The residue was dissolved in 3 mL of DMF, and then DIPEA (0.027 g), COMU (0.0660 g), and 2 (0.017 g) were added. The mixture was stirred at room temperature for 2 hours. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. It was purified using a gradient from DCM to 20% MeOH in DCM. The product was a pale yellow oil, 34 mg, 37%. LC-MS: calculated [M+H] 928.64, found 929.59.

[0164] Synthesis of LP-249p

Chemical Structure

[0165] To a mixture of 1 (0.0600 g) and 2 (0.161 mL) in 4 mL of DCM, TEA (0.111 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 2 hours. Water was added for workup. Purification was carried out on a 4 g column. It was purified using a gradient from hexane to ethyl acetate. The product was a white solid, 74 mg, 60%. LC-MS: calculated [M+H] 465.41, found 465.91.

Chemical Structure

[0166] To a solution of 1 (0.0740 g) in DCM, TFA (50% in DCM) was added at room temperature. The reaction mixture was stirred at room temperature for 0.5 hour. The solvent was removed under vacuum, and the residue was placed under high vacuum for 2 hours. The residue was dissolved in DMF, and then 2 (0.0420 g), DIPEA (0.084 mL), and COMU (0.102 g) were added at room temperature. The mixture was stirred at room temperature for 2 hours. The solvent was removed under vacuum. Purification was carried out on a 12 g column. It was purified using a gradient from DCM to 20% MeOH in DCM. The product was a white solid, 56 mg, 58%. LC-MS: calculated [M+H] 609.48, found 610.29. [Chemistry]

[0167] 1 (0.0560 g) in 4N HCl / dioxane (3 mL) was stirred overnight at room temperature. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. The residue was dissolved in 2 mL of DMF, and then DIPEA (0.048 mL), COMU (0.118 g), and 2 (0.031 g) were added. The mixture was stirred at room temperature for 2 hours. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. It was purified using a gradient from DCM to 20% MeOH in DCM. The product was an off-white solid, 16 mg, 25%. LC-MS: calculated [M+H] 701.42, found 702.20.

[0168] Synthesis of LP-257p [Chemistry]

[0169] To 1 (0.100 g) in 3 mL of DCM, 2 (0.331 mL) and TEA (0.304 mL) were added at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Ethyl acetate was added to dilute, and then the mixture was washed with 1N HCl, NaHCO3, and brine. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. It was purified using a gradient from hexane to ethyl acetate. The product was a white solid, 134 mg, 58%. LC-MS: calculated [M+H]: 422.36, found 422.79. [Chemistry]

[0170] A solution of 1 (0.134 g) in 4N HCl / dioxane (8 mL) was stirred overnight at room temperature. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. The product was a white solid, 118 mg, which was used in the next step without further purification. LC-MS: calculated [M+H] 366.30, found 366.62. [Chemistry]

[0171] To a solution of 1 (0.0490 g) in 3 mL of DMF, COMU (0.086 g), DIPEA (0.047 mL), and 2 (0.045 g) were added at room temperature. The mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate and then washed with 1N HCl, NaHCO3, and brine. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. Purification was carried out using a gradient from hexane to ethyl acetate. The product was a white solid, 23 mg, 33%. LC-MS: calculated [M+H] 514.29, found 514.79.

[0172] Synthesis of LP-259p [Chemistry]

[0173] To a solution of 1 (0.100 g) in 3 mL of DCM, 2 (0.366 mL) and TEA (0.337 mL) were added at room temperature. The reaction was stirred at room temperature for 1 hour. The reaction mixture was loaded onto a 12 g column. Purification was carried out using a gradient from hexane to ethyl acetate. The product was a white solid, 183 mg, 82%. LC-MS: calculated [M+H]: 368.32, found 368.60. [Chemistry] A solution of 1 (0.0900 g) in MeOH / THF / 1N NaOH (3 mL / 3 mL / 3 mL) was stirred at 60 °C for 1 hour. After cooling to room temperature, MeOH / THF was removed under vacuum. The pH was adjusted to approximately 1 with 1N HCl. Ethyl acetate and water were added for workup. After removing ethyl acetate under vacuum, the residue was placed under high vacuum for 3 hours. The residue was dissolved in 3 mL of DMF, and then COMU (0.136 g), DIPEA (0.085 mL), and 2 (0.053 g) were added at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Ethyl acetate was added to dilute the reaction mixture. The reaction mixture was washed with 1N HCl, NaHCO3, and brine. After removing ethyl acetate under vacuum, the residue was loaded onto a 12 g column. Purification was carried out using a gradient from hexane to ethyl acetate. The product was a white solid, 87 mg, 71%. LC-MS: calculated [M+H]: 502.29, found 502.72.

[0174] Synthesis of LP-260p

Chemical formula

[0175] To a solution of 1 (0.100 g) in DDC, 2 (0.354 mL) and TEA (0.326 mL) were added at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was loaded onto a 12 g column. Purification was carried out using a gradient from hexane to ethyl acetate. The product was a white solid, 208 mg, 87%. LC-MS: calculated [M+H]: 410.36, found 410.73.

Chemical formula

[0176] A solution of 1 (0.208 g) in 4N HCl / dioxane (8 mL) was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. The product was a white solid, 179 mg, which will be used in the next step without further purification. LC-MS: calculated [M+H] 354.30, found 354.65.

Chemical formula

[0177] Synthesis of LP-262p

Chemical Structure

[0178] To a solution of 1 (0.0220 g), 2 (0.100 g), and DIPEA (0.017 mL) in 2 mL of DMF was added COMU (0.0240 g) at room temperature. The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM. Next, it was washed with 1N HCl, saturated NaHCO3, and brine. Purification was performed on a 4 g column. Purification was carried out using a gradient from DCM to 20% MeOH in DCM. The product was a transparent solid, 77 mg, 65%. LC-MS: calculated [M+2H]+H2O: 1294.76, found 1295.29; calculated [M+3H]+H2O: 869.51, found 869.45; calculated [M+4H]: 638.88, found 638.54.

Chemical Structure

[0179] A solution of 1 (0.077 g) in DMF / piperidine (0.8 mL / 0.2 mL) was stirred at room temperature for 1 h. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL of DMF, then 2 (0.016 g) and TEA (0.013 mL) were added at room temperature. The reaction mixture was stirred at room temperature for 1.5 h. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. It was purified using a gradient from DCM to 20% MeOH in DCM. The product was a white solid, 61 mg, 78%. LC-MS: calculated [M + 2H]+H2O: 1302.84, found 1303.81; calculated [M + 4H]: 642.92, found 642.62.

Chem.

[0180] A solution of 1 (0.0610 g) in 4N HCl / dioxane (5 mL) was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL of DMF, then COMU (0.0152 g), DIPEA (0.009 mL), and 2 (0.0060 g) were added at room temperature. The reaction mixture was stirred at room temperature for 1.5 h. After removing the solvent under vacuum, the residue was loaded onto a 4 g column. It was purified using a gradient from DCM to 20% MeOH in DCM. The product was a white solid, 13 mg, 21%. LC-MS: calculated [M + 2H]+H2O: 1348.80, found 1348.94. Calculated [M + 3H]+H2O: 905.54, found 905.09.

[0181] Synthesis of LP-269p

Chem.

[0182] A solution of 1 (88.6 mg, 0.500 mmol, 1.0 equiv) and 2 (93.7 mg, 0.600 mmol, 1.20 equiv) in 20 mL of DCM was added with TEA (0.418 mL, 3.000 mmol, 6.0 equiv) under ambient conditions. The reaction mixture was stirred at room temperature for 3 h, followed by the addition of COMU (257 mg, 0.600 mmol, 1.20 equiv), and then 4-nitrophenol (166.1 mg, 1.000 mmol, 2.0 equiv) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was washed with 1 N HCl and then with brine. The mixture was dried over Na2SO4 and concentrated. The residue was purified by CombiFlash® using a gradient of EA from Hex (0 - 100%) as the stationary phase on silica gel to give 72 mg of the product (19% yield).

[0183] Synthesis of LP-273p

Chemical Structure

[0184] To a solution of compound 1 (0.200 g), NEt3 (0.255 mL), and COMU (0.261 g) in DCM, 2 (0.152 g) was added under ambient conditions. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was concentrated directly for isolation. The residue was purified by CombiFlash® using a gradient from hexane to 100% ethyl acetate via a DCM liquid load onto a 12 g column, and the product was eluted at 28% B. The product was concentrated under vacuum to give a clear pale yellow oil. MS m / z: calculated [M + H]+ 477.23 m / z, found 477.52 m / z.

[0185] Synthesis of LP-274p

Chemical Structure

[0186] To a solution of EPA1 (60.5 mg, 0.200 mmol, 1 equiv) and 2 (36.5 mg, 0.220 mmol, 1.10 equiv) in 20 mL of DCM, COMU (94.2 mg, 0.220 mmol, 1.10 equiv) was added, and then TEA (0.084 mL, 0.600 mmol, 3.0 equiv) was added under ambient conditions. The reaction mixture was stirred until complete conversion was confirmed by LC-MS. The reaction mixture was washed with 1N HCl and then with brine. The mixture was then dried over Na2SO4 and concentrated. The reaction mixture was purified by CombiFlash® with a gradient from EA to Hex 0 - 50% using silica gel as the stationary phase. 69 mg of the product was obtained (76% yield).

[0187] Synthesis of LP-283p

Chemical Structure

[0188] To a solution of Compound 1 (49 mg), NEt3 (0.068 mL), and COMU (76.8 mg) in DMF, Compound 2 (29.8 mg) was added under ambient conditions. The reaction mixture was stirred until complete conversion was observed by LC-MS. Since conversion could not be clearly observed by LC-MS, instead, the reaction mixture was stirred for 30 minutes until a bright yellow (before addition of Compound 2) changed to a honey orange color and all substances were observed to be mainly dissolved. The reaction mixture was washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® with a gradient from hexane to 100% ethyl acetate via a DCM liquid load onto a 12 g column, and the product was eluted at 31% B. The product was concentrated under vacuum to obtain a white solid residue and confirmed by 1H NMR in CDCl3.

[0189] Synthesis of LP-286p

Chemical Structure

[0190] A solution of 1 (78.5 mg, 0.200 mmol, 1 equiv) and 2 (36.5 mg, 0.220 mmol, 1.10 equiv) in 20 mL of DCM was added with COMU (94.2 mg, 0.220 mmol, 1.10 equiv), and then TEA (0.084 mL, 0.600 mmol, 3.0 equiv) was added under ambient conditions. The reaction mixture was stirred until complete conversion was observed by LC-MS. The reaction mixture was washed with 1 N HCl and then with brine. The mixture was dried over Na2SO4 and concentrated. The reaction mixture was purified by CombiFlash® on silica gel with a gradient from EA to Hex 0 - 50% as the stationary phase. 69 mg of the product was obtained (57% yield).

[0191] Synthesis of LP-287p

Chemical Structure

[0192] To a solution of 1 (43.3 mg, 0.200 mmol, 1 equiv) and 2 (36.5 mg, 0.220 mmol, 1.10 equiv) in 20 mL of DCM was added COMU (94.2 mg, 0.220 mmol, 1.10 equiv), and then TEA (0.084 mL, 0.600 mmol, 3.0 equiv) was added under ambient conditions. The reaction mixture was stirred until complete conversion was confirmed by LC-MS. The reaction mixture was washed with 1 N HCl and then with brine. The mixture was dried over Na2SO4 and concentrated. The reaction mixture was purified by CombiFlash® on silica gel with a gradient from EA to Hex 0 - 50% as the stationary phase. 52 mg of the product was obtained (71% yield).

[0193] Synthesis of LP-290p

Chemical Structure

[0194] To a solution of Compound 1 (0.0540 g), NEt3 (0.075 mL), and COMU (0.084 g) in DMF, Compound 2 (0.0327 g) was added under ambient conditions. The reaction mixture was stirred for 30 minutes until the bright yellow (before addition of 2) changed to honey orange and it was observed that all substances were almost dissolved. The reaction mixture was washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® via a gradient from hexane to 100% ethyl acetate with a DCM liquid load on a 12 g column, and the product was eluted at 31% B. The product was concentrated under vacuum to give a white solid residue, which was confirmed by 1H NMR in CDCl3. LC-MS: calculated [M+H]+ 428.14 m / z, found 428.46 m / z.

[0195] Synthesis of LP-293p

Chemical Structure

[0196] To a solution of Compound 1 (73 mg), NEt3 (0.112 mL), and COMU (126 mg) in DMF, Compound 2 (48.9 mg) was added under ambient conditions. The reaction mixture was stirred until complete conversion was observed by LC-MS. Since conversion could not be clearly observed by LC-MS, instead, the reaction mixture was stirred for 30 minutes until the bright yellow (before addition of Compound 2) changed to honey orange and it was observed that all substances were mainly dissolved. Next, the reaction mixture was washed with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by CombiFlash® via a gradient from hexane to 100% ethyl acetate with a DCM liquid load on a 12 g column, and the product was eluted at 30% B. The product was concentrated under vacuum to give a white solid residue, which was confirmed by 1H NMR in CDCl3.

[0197] Synthesis of LP-296p

Chemical Structure

[0198] To a solution of Compound 1 (0.0344 g), NEt3 (0.0117 g), and COMU (0.0182 g) in DCM, 2 (0.0071 g) was added under ambient conditions. The reaction mixture was stirred for 30 minutes until the bright yellow (pre-addition of 2) changed to honey orange and all substances were observed to be mainly dissolved. The reaction mixture was concentrated directly for isolation. The residue was purified by CombiFlash® via DCM liquid loading onto a 4 g column with 20% MeOH / DCM (from 0% B to 20% B, 40% B, 50% B, and then 100% B), and the product was eluted at 23% B. The product was concentrated under vacuum to give a clear, colorless oil, which was confirmed by 1H NMR in CDCl3. MS m / z: calculated [M+H]+ 1039.67 m / z, found 1040.36, 671.78 m / z.

[0199] Synthesis of LP-300p

Chemical Structure

[0200] To a solution of 2 (5.29 g) in 100 mL of toluene, TEA (8.4 mL) was added at room temperature, and then 1 (5.20 g) was added dropwise. The reaction mixture was stirred at 90 °C for 16 h. After cooling to room temperature, ethyl acetate and water were added for workup. Purification was carried out on a 120 g column. Purification was performed using a gradient from hexane to 30% ethyl acetate in hexane. The product was a pale yellow oil, 3658 mg, 54%. LC-MS: calculated [M+H] 339.21, found 339.17.

Chemical Structure

[0201] A mixture of 1 (0.113 g) and 10% Pd / C (0.0036 g) in 10 mL of ethyl acetate was charged with H2 (ca. 45 psi). The reaction mixture was stirred at room temperature for 4 h. After filtration, the solvent was removed under vacuum. Next, the residue was placed under high vacuum for 1 h. The residue was dissolved in 10 mL of DCM, and TEA (0.279 mL) and 2 (0.405 mL) were added at room temperature. The reaction mixture was stirred at room temperature for 1 h. Purification was carried out on a 12 g column. Purification was carried out using a gradient from hexane to 50% ethyl acetate in hexane. The product was a white solid, 141 mg, 66%. LC-MS: calcd [M+H] 635.57, found 635.95. [Chemical formula]

[0202] To a solution of 1 (0.141 g) in MeOH / THF (3 mL / 3 mL) was added 1 N NaOH (3 mL) at room temperature. The mixture was stirred at room temperature for 2 h. After removal of the organic solvent under vacuum, the residue was acidified to pH ca. 1 with concentrated hydrochloric acid. Ethyl acetate was added to extract the product. After removal of the solvent under vacuum, the residue was placed under high vacuum for 3 h. The residue was dissolved in DMF / DCM (5 mL / 5 mL), and then DIPEA (0.077 mL), COMU (0.143 g), and 2 (0.074 g) were added. The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and then washed with 1 N HCl and brine. After removal of the solvent under vacuum, the residue was loaded onto a 12 g column. Purification was carried out using a gradient from hexane to 30% ethyl acetate in hexane. The product was a white solid, 80 mg, 47%. LC-MS: calcd [M+H] 769.55, found 769.98.

[0203] Synthesis of LP-303p [Chemical formula]

[0204] A solution of vitamin D1 (185 mg, 0.500 mmol, 1 equiv) and 2 (111 mg, 0.550 mmol, 1.10 equiv) in 30 mL of DCM was added with TEA (0.139 mL, 1.00 mmol, 2.0 equiv) under ambient conditions. The reactants were stirred at room temperature for 8 h. The reaction mixture was washed with 1 N HCl and then with brine. The mixture was dried over Na2SO4 and concentrated. The residue was purified by CombiFlash® on silica gel with a gradient from EA to Hex 0 - 100% as the stationary phase. 95 mg of the product was obtained (yield 35%).

[0205] Synthesis of LP - 304p

Chemical Structure

[0206] 1 (200 mg, 0.377 mmol, 1.0 equiv) was hydrolyzed with LiOH (151 mg, 3.77 mmol, 10.0 equiv) in MeOH / TFH / H2O (1:1:1, 90 mL). After removing all the organic solvents, the aqueous phase was acidified to pH = 3 with 1 N HCl. The reaction mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried over Na2SO4 and concentrated to obtain the crude acid.

[0207] COMU (194 mg, 0.453 mmol, 1.20 equiv) was added to a solution of the above - mentioned crude acid and tetrafluorophenol 4 (68.9 mg, 0.415 mmol, 1.10 equiv) in 30 mL of DCM, and then TEA (0.158 mL, 1.13 mmol, 3.0 equiv) was added under ambient conditions. The reactants were stirred until complete conversion was observed by LC - MS. The reaction mixture was washed with 1 N HCl and then with brine. It was dried over Na2SO4 and concentrated. The reaction mixture was purified by CombiFlash® on silica gel with a gradient from EA to Hex 0 - 100% as the stationary phase. 170 mg of the product was obtained (yield 85%).

[0208] Synthesis of LP - 310p

Chemical Structure

[0209] To a solution of 1 in DCM (0.057 mL of DIPEA, 0.077 g of COMU, and 2 (0.0300 g)) were added at room temperature. After stirring at room temperature for 2 hours, the reaction was stopped with 0.1 N HCl. The organic layer was washed with brine. After removing the solvent, the residue was loaded onto a 4 g column. It was purified using a gradient from hexane to 50% hexane in ethyl acetate. The product was a white solid, 46 mg, 44%. LC-MS: calculated [M+H] 422.36, found 422.61.

Chemical formula

[0210] A solution of 1 (0.046 g) in 4N HCl / dioxane (2 mL) was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was placed under high vacuum for 3 hours. Next, the residue was dissolved in DCM at room temperature, and then COMU (0.0700 g), DIPEA (0.038 mL), and 2 (0.036 g) were added at room temperature. After stirring at room temperature for 2 hours, the solvent was removed under vacuum. The residue was loaded onto a 4 g column. It was purified using a gradient from hexane to 50% hexane in ethyl acetate. The product was a white solid, 21 mg, 38%. LC-MS: calculated [M+H] 514.29, found 514.61.

[0211] Synthesis of LP-383p

Chemical formula

[0212] To a solution of compound 1 (0.050 g) in 5 mL of DCM, compound 2 (0.023 g) and EDC (0.039 g) were added at room temperature. The mixture was stirred at room temperature for 1 hour. After removing the solvent under vacuum, the residue was dry-packed onto a 4 g column. The product was purified using hexane to 50% ethyl acetate in hexane. The product was a white solid with a yield of 29 mg. LC-MS: calculated [M+H+H2O] 388.27, found 388.03.

[0213] Synthesis of LP-409p

Chemical Structure

[0214] Compound 1 (1.40 g) and 2 (0.613 g) were dissolved in 100 mL of THF, and then TEA (2.01 mL) was added. The reaction was stirred at 60 °C until complete conversion was confirmed by LC-MS (2 - 3 hours). The reaction was cooled to room temperature. The resulting product was a white precipitate, which was filtered and washed with acetone (20 mL). The structure of the compound was 1 proven using 13 H and

Chemical Structure

[0215] Compound 1 (1.9 g), 2 (0.846 g), and 3 (2.98 g) were dissolved in 100 mL of DCM and then heated to 40 °C. The reaction was stirred until the solution became clear. The reaction was cooled to room temperature and stirred overnight. After removing all of the DCM, the product was dry-packed onto a 24 g column. The product was obtained as a white solid using 0 - 50% (ethyl acetate / hexane, with 1% TEA added) as the mobile phase.

[0216] Synthesis of LP-429p

Chemical Structure

[0217] 17-Hydroxyhexadecanoic acid (6) (3.53 g, 12.3 mmol) was added to a 500 mL round-bottom flask. The flask was purged with nitrogen, then DCM (150 mL) was added, followed by acetic anhydride (18.6 mL, 197 mmol) and pyridine (30.8 mL, 382 mmol). The reaction mixture was stirred overnight. The reaction mixture was concentrated and azeotroped three times with toluene to remove residual pyridine, acetic acid, and acetic anhydride. Next, the residue was stirred in 100 mL of a 1:1 THF / aqueous NaHCO3 mixture for 24 hours. Approximately half of the THF was removed on a rotary evaporator, the mixture was diluted with water, and acidified to pH 1 with 3 M HCl. The mixture foamed very vigorously during acidification. The product was collected by filtration and dried in vacuo to give 3.22 g (80% yield) of compound 5 as a white solid. The product was not further purified.

Chemical formula

[0218] Compound 5 (3.47 g, 10.6 mmol) was dissolved in THF (55 mL) and cooled to -15 to -20 °C in a methanol / ice bath. After cooling, N-methylmorpholine (1.4 mL, 12.7 mmol) and ethyl chloroformate (1.2 mL, 12.7 mmol) were added. The reaction mixture was stirred at -15 °C for 30 minutes. After 30 minutes, an aqueous solution (6.6 mL) of sodium azide (1.72 g, 26.4 mmol) was added, and the reaction mixture was stirred at -5 to 0 °C for 30 minutes in a water / salt / ice bath. The reaction mixture was diluted with ethyl acetate (20 mL) and water (20 mL). The layers were separated, the aqueous layer was extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were washed with water (50 mL) and brine (50 mL), dried over sodium sulfate, and concentrated to give a white solid. Proton NMR showed that no starting material remained based on the proton at the alpha position of the carbonyl. The solid was dissolved in toluene (55 mL) and heated to 65 °C until gas evolution ceased (about 30 minutes). The reaction mixture was cooled to room temperature, N-hydroxysuccinimide (1.22 g, 10.5 mmol) was added, followed by pyridine (0.85 mL, 10.5 mmol). Proton NMR showed that not all of the isocyanate had been consumed after 2 hours, so an additional 2 equivalents of N-hydroxysuccinimide (2.43 g, 21.1 mmol) were added. The reaction mixture was stirred overnight. After stirring overnight, according to proton NMR, no isocyanate remained. The reaction mixture was concentrated, and the resulting white powder was dissolved in ethyl acetate (100 mL) and poured into 300 mL of hexane. The precipitate was collected by filtration. Proton NMR of the product showed residual N-hydroxysuccinimide. The product was dissolved in DCM and purified by silica gel chromatography (from 65:35 hexane:ethyl acetate to 0:100 hexane:ethyl acetate). The product began to elute with 50% ethyl acetate and was pulled into the column. The fractions containing the product were combined to give 2.25 g (48% yield) of compound 7 as a white solid.

Chemical formula

[0219] Compound 7 (1.00 g, 2.27 mmol) was added to a solution of 6-amino-1-hexanol (0.266 g, 2.27 mmol) and NEt3 (0.95 mL, 6.81 mmol) in DCM (50 mL). A white precipitate formed. After 18 hours, no SM remained by LC-MS. The reaction mixture was concentrated on a rotary evaporator, and the residue was dissolved in approximately 8 mL of ethyl acetate and cooled to -20 °C in the freezer. A precipitate formed and settled to the bottom of the flask. The ethyl acetate was decanted twice, and the precipitate was collected and dried under vacuum to give 0.95 g (94% yield) of Compound 8 as a white powder.

Chemical Structure

[0220] In a 100 mL round-bottom flask, Compound 8 (0.95 g, 2.14 mmol) was dried by three consecutive evaporations with toluene. Diisopropylammonium tetrazolide (0.146 g, 0.86 mmol) and 4 Å molecular sieves were added to the flask. The flask was purged with nitrogen and refilled three times, and the solid was dissolved in DCM (50 mL). The mixture was stirred for 30 minutes. After 30 minutes, 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (0.98 g, 3.25 mmol) was added, and the reaction mixture was stirred for 18 hours. After 18 hours, LC-MS indicated that no starting alcohol remained. The reaction mixture was transferred to a separatory funnel and washed with saturated aqueous NaHCO3 (2 × 40 mL), water (40 mL), and brine (40 mL), dried over magnesium sulfate, and concentrated to dryness. Hexane was added to the flask, and the residue was stirred in hexane for 2 hours to obtain a white precipitate. The white solid was collected by filtration, washed with hexane (2 × 20 mL), and dried under vacuum to give 1.2 g (87% yield) of Compound 9 as a white solid.

[0221] Synthesis of LP-430p

Chemical Structure

[0222] A solution of 1,6 - hexanediol (1 equiv) and TEA (2 equiv) in DCM (5 mL) was added to a round - bottom flask containing a solution of hexadecyl isocyanate (1 equiv) in DCM (5 mL). The mixture was stirred at room temperature for 2 h. Next, the mixture was concentrated under reduced pressure and purified by CombiFlash chromatography using 2% MeOH in DCM to afford Compound 1 as an off - white solid in 20% yield. LC - MS [M + H] + 386.3634 m / z, found 386.3642 m / z.

[0223] Compound 1 (1 equiv) was evaporated to dryness twice with toluene. Next, it was dissolved in anhydrous DCM (10 mL), diisopropylammonium tetrazolide (1.4 equiv) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at room temperature for 30 min under nitrogen gas. Next, 2 - cyanoethyl N,N,N',N' - tetraisopropyl phosphorodiamidite (1.6 equiv) was added and stirring was continued at room temperature for 12 h. Next, 0.3 mL of TEA was added to stop the reaction and the mixture was directly loaded onto Celite. The pure product was obtained as a waxy off - white solid in 41.7% yield by CombiFlash chromatography using hexane:ethyl acetate + 1% TEA (70:30). LC - MS [M + H] + 586.4713 m / z, found 586.4720 m / z.

[0224] Synthesis of LP - 431p [Chemical formula]

[0225] A solution of hexadecyl chloroformate (1 equiv) was added to a round - bottom flask containing 6 - amino - 1 - hexanol (1.2 equiv) and TEA (2 equiv) in DCM (5 mL). The reaction mixture was stirred at room temperature for 2 h. Next, the mixture was concentrated under reduced pressure and purified by CombiFlash chromatography using 2% MeOH in DCM to afford Compound 1 as an off - white solid in 20% yield. LC - MS [M + H]+ 386.3634 m / z, measured value 386.3638 m / z.

[0226] Compound 1 (1 equivalent) was dried by evaporation of toluene twice. Next, this was dissolved in anhydrous DCM (10 mL), diisopropylammonium tetrazolide (1.4 equivalents) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at room temperature for 30 minutes under nitrogen gas. Next, 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.6 equivalents) was added and stirring was continued at room temperature for 12 hours. Next, 0.3 mL of TEA was added to stop the reaction and the mixture was directly loaded onto Celite. By CombiFlash chromatography, using hexane:ethyl acetate + 1% TEA (70:30), the pure product was obtained as a waxy off-white solid in 82.3% yield. LC-MS [M+H] + 586.4713 m / z, measured value 586.4705 m / z.

[0227] Synthesis of LP-435p

Chemical formula

[0228] Undecanoic acid (2.0 g, 10.7 mmol) was dissolved in toluene (30 mL), and triethylamine (3.0 mL, 21.5 mmol) and diphenylphosphoryl azide (3.84 g, 14.0 mmol) were added. The reaction mixture was stirred overnight. The acyl azide was observed by mass spectrometry under basic conditions. The mixture was concentrated and the crude product was purified by silica gel chromatography (0:100 ethyl acetate:hexane to 20:80 ethyl acetate:hexane). The product was eluted with 10% ethyl acetate. The fractions containing the product were concentrated to give 0.975 g (43% yield) of Compound 21 as a clear liquid.

Chemical formula

[0229] Compound 21 (0.975, 5.2 mmol) was dissolved in toluene (40 mL) and heated at 65 °C for 1 hour. Gas evolution was observed when 65 °C was reached and ceased after about 30 minutes. The reaction mixture was cooled to room temperature. In a separate flask, 1-amino-12-dodecanol (1.05 g, 5.2 mmol) was dissolved in THF (20 mL) and pyridine (0.85 mL, 10.5 mmol). When the toluene solution was added to the THF solution, a white precipitate formed immediately. The reaction was stirred overnight. The reaction mixture was concentrated and the crude product was recrystallized from isopropanol to give 1.5558 g (77% yield) of Compound 22 as a white solid. [Chemical formula]

[0230] In a 100 mL round-bottom flask, Compound 22 (1.55 g, 4.0 mmol) was dried by evaporating with toluene twice in succession. Diisopropylammonium tetrazolide (0.277 g, 1.6 mmol) and 4 Å molecular sieves were added to the flask. The flask was purged with nitrogen and refilled three times, and the solids were suspended in DCM (20 mL). The solids only dissolved partially. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.88 g, 6.2 mmol) was added to the mixture and the reaction was stirred for 18 hours. LC-MS indicated that no starting alcohol remained. The reaction was transferred to a separatory funnel and washed with saturated aqueous NaHCO3 (2 × 40 mL), water (40 mL), brine (40 mL), dried over sodium sulfate, and concentrated to dryness. Hexane was added to the flask and the residue was stirred in hexane for 1 hour to obtain a white precipitate. The white solid was collected by filtration, washed with hexane (2 × 20 mL), and dried under vacuum to give 1.103 g of a white powder. Proton NMR indicated that a large amount of water remained and a significant amount of the substance was insoluble in chloroform and DCM. The mixture was suspended in DCM, dried over magnesium sulfate, filtered through an additional magnesium sulfate pad, and concentrated to give 0.46 g (19% yield) of Compound LP-435p as an off-white powder.

[0231] Synthesis of LP-439p

Chem.

[0232] (3-Aminobicyclo[1.1.1]pentan-1-yl)methanol (2) (0.20 g, 1.77 mmol) and 2,5-dioxopyrrolidin-1-yl hexadecylcarbamate (3) (0.67 g, 1.75 mmol) were dissolved in DCM (40 mL), and then triethylamine (0.72 mL, 5.3 mmol) was added. The reaction mixture was stirred overnight. After 18 h, a precipitate was observed. The precipitate was collected by filtration and washed with DCM (2×10 mL). The precipitate was dried under vacuum to give 0.325 g (48% yield) of a white solid. Proton NMR analysis was consistent with the product, and the crude material was of acceptable purity to proceed to the next step.

Chem.

[0233] Compound 1 (0.3 g, 0.79 mmol) was dried by evaporating with toluene four times in succession, and then diisopropylammonium tetrazolide (0.054 g, 0.315 mmol) was added to the flask. The flask was purged with nitrogen and refilled three times, and the solid was suspended in DCM (20 mL), 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.39 mL, 1.214 mmol) was added, and the reaction mixture was stirred for 18 h. LC-MS analysis showed that no starting alcohol remained after 18 h. The reaction mixture was transferred to a separatory funnel, washed with saturated aqueous NaHCO3 (2×40 mL) and water (40 mL), and concentrated to dryness. Hexane was added to the residue, and the mixture was stirred for 1 h to give a white precipitate. The precipitate was collected by filtration, washed with hexane, and dried under vacuum to give 0.395 g (86% yield) of LP-439p as a white solid.

[0234] Synthesis of LP-440p

Chem.

[0235] Anhydrous MeOH (8 mL) was cooled to 0 °C, potassium hydroxide (3 equivalents) was added, and the solution was stirred for 30 minutes. Next, a solution of 16-bromohexadecanoic acid (1 equivalent) in anhydrous MeOH (7 mL) was added via syringe. The reaction mixture was heated to reflux temperature and stirred overnight. After cooling to room temperature, MeOH was removed under vacuum, and the resulting crude mixture was reconstituted with 1 N HCl (25 mL) and diethyl ether (5 mL). The crude product was extracted using diethyl ether (4 × 30 mL), the combined organic layers were washed with brine (30 mL), dried over Na2SO4, and then the solvent was removed under vacuum. The product was purified by column chromatography on silica gel using hexane:ethyl acetate (85:15) to give Compound 1 as an oil in 86% yield. LC-MS [M+H] + 287.2586 m / z, found 287.2590.

[0236] To a solution of Compound 1 (1 equivalent) in DCM (50 mL) were added COMU (1.2 equivalents) and DIPEA (2 equivalents). The mixture was stirred at room temperature for 30 minutes. Next, 6-amino-1-hexanol (1.2 equivalents) was added, and the reaction mixture was stirred at room temperature for 12 hours. The mixture was then washed three times with 1 M HCl (3 × 50 mL) and once with brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure. ACN (100 mL) was added to the crude product, and the mixture was carefully heated using a heat gun until all the solids had dissolved. The mixture was then left at room temperature, and white crystals formed. The precipitate was then collected by vacuum filtration and washed several times with ACN to remove the remaining pink color. Compound 2 was obtained as a white solid in 74% yield. LC-MS [M+H] + 386.3634 m / z, found 386.3626.

[0237] Compound 3 (1 equivalent) was dried by evaporation of toluene twice. Next, it was dissolved in anhydrous DCM (10 mL), diisopropylammonium tetrazolide (0.4 equivalent) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at room temperature under nitrogen gas for 30 minutes. Next, 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.5 equivalents) was added and stirring was continued at room temperature for 12 hours. Next, 0.3 mL of TFA was added to stop the reaction and the mixture was directly loaded onto Celite. Pure product was obtained in 86% yield as a waxy off-white solid using hexane:ethyl acetate + 1% TEA (70:30) by CombiFlash chromatography. LC-MS [M+H] + 586.4713 m / z, found 586.4705.

[0238] Synthesis of LP-441p

Chem.

[0239] To a round-bottom flask containing 6-amino-1-hexanol (2 equivalents) in EtOH (20 mL), 1-bromohexadecane (1 equivalent) and TEA (1.1 equivalents) were added. The mixture was refluxed for 12 hours. Next, the solution was cooled to room temperature and the solvent was removed under vacuum. Next, the crude product was dissolved in H2O (20 mL) and extracted 3 times with CH3Cl (3 × 25 mL). The combined organics were washed once with brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by CombiFlash chromatography using 10% MeOH in DCM + 1% TEA to give Compound 1 as an oil in 44% yield. LC-MS [M+H] + 342.3736 m / z, found 342.3728.

[0240] Ethyl trifluoroacetate (5 eq) and DIPEA (2 eq) were added to a round-bottom flask containing compound 1 (1 eq) in MeOH (25 mL). The reaction mixture was stirred at 40 °C for 12 h. Next, the solvent was removed under reduced pressure and the crude product was purified by CombiFlash chromatography using 4% - 6% MeOH in DCM to give compound 2 as an oil in 73% yield. LC-MS [M+H] + 438.3559 m / z, found 438.3551.

[0241] Compound 2 (1 eq) was evaporated twice with toluene and dried. Next, it was dissolved in anhydrous DCM (10 mL), diisopropylammonium tetrazolide (0.4 eq) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at room temperature for 30 min under nitrogen gas. Next, 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.5 eq) was added and stirring was continued at room temperature for 12 h. Next, 0.3 mL of TEA was added to stop the reaction and the mixture was directly loaded onto Celite. Pure product was obtained by CombiFlash chromatography using hexane:ethyl acetate + 1% TEA (70:30) as a waxy off-white solid in 56% yield. LC-MS [M+H] + 638.4637 m / z, found 638.4629.

[0242] Synthesis of LP-456p

Chemical Structure

[0243] A 1 M solution of borane-tetrahydrofuran complex in tetrahydrofuran (1.5 eq) was added dropwise to a solution of 16-(tert-r (1 eq) in anhydrous tetrahydrofuran (20 mL) at 0 °C under a nitrogen atmosphere. The resulting solution was stirred at 0 °C for 2 h, then the cooling bath was removed and the mixture was stirred at room temperature overnight. A saturated aqueous solution of sodium bicarbonate (50 mL) was added to stop the reaction. Next, the mixture was diluted with water (50 mL) and extracted 3 times (3 × 50 mL) with DCM. The combined organics were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by CombiFlash chromatography using hexane:ethyl acetate (80:20) to give Compound 1 as an oil in 82% yield. LC-MS [M+H] + 329.3056 m / z, found 329.5060.

[0244] A mixture of Compound 1 (1 eq), silver carbonate (3 eq), and a catalytic amount of iodine in DCM (5 mL) was stirred with molecular sieves for 15 min. To the mixture was added 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1.5 eq) in DCM (5 mL) (which was also stirred with molecular sieves for 15 min). The resulting mixture was covered with aluminum foil and stirred at room temperature for 48 h, then filtered through Celite and washed with ethyl acetate. The filtrate was concentrated and the crude product was purified by CombiFlash column chromatography using hexane:ethyl acetate (80:20) to give Compound 2 as an oil in 33% yield. LC-MS: [M+H2O] + 676.4034 m / z, found 676.4041.

[0245] TFA (15 mL) was added to a solution of Compound 2 in DCM (5 mL). The solution was stirred at room temperature for 2 h. Next, the mixture was carefully poured into 100 mL of saturated aqueous NaHCO3. After neutralization, the aqueous phase was extracted 3 times (3 × 100 mL) with DCM. The combined organics were dried over Na2SO4 and concentrated under reduced pressure to give Compound 3 as a white solid in 97% yield. LC-MS: [M+H] + 603.3381 m / z, found 603.3388.

[0246] To a solution of Compound 3 (1 equivalent) in DCM (10 mL) were added COMU (1.2 equivalents) and DIPEA (2 equivalents). The mixture was stirred at room temperature for 30 minutes. Next, 6-amino-1-hexanol (1.2 equivalents) was added and the reaction mixture was stirred at room temperature for 12 hours. The mixture was then washed three times with 1 M HCl (3 × 10 mL) and once with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by CombiFlash chromatography using 0 - 100% hexane:ethyl acetate over 40 minutes to give Compound 4 as an oil in 83% yield. LC-MS [M+H] + 702.4429 m / z, found 702.4421.

[0247] Compound 4 (1 equivalent) was concentrated twice with toluene on a rotary evaporator and then anhydrous DCM (10 mL) was charged into the reaction flask. The suspension was stirred at ambient temperature under nitrogen at 900 RPM using a molecular sieve. 2-Cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (1.5 equivalents) was added to the suspension, followed by diisopropylammonium tetrazolide (0.4 equivalents). After 12 hours, TEA (300 μL) was added and the reaction mixture was dry-packed onto Celite. The product was purified using hexane:ethyl acetate + 1% TEA (60:40) to give LP-456p as an oil in 64% yield. LC-MS [M+H] + 902.5507 m / z, found 902.5517.

[0248] Synthesis of LP-462p

Chemical Structure

[0249] Anhydrous THF (30 mL) was added to a round-bottom flask containing 2099-117 (1 equivalent), and the solution was cooled to -20 °C. Ethyl chloroformate (1.2 equivalents) and N-methylmorpholine (1.2 equivalents) were added to the solution, and the solution was stirred at -20 °C to -10 °C for 30 minutes. A solution of sodium azide (2.5 equivalents) in 1.5 mL of water was added to the reaction mixture, and the reaction mixture was stirred at -7 °C for 90 minutes. The reaction mixture was diluted with ethyl acetate. The aqueous layer was separated and extracted twice more with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to a clear liquid. This liquid was dissolved in toluene (30 mL) and heated at 65 °C for 1 hour, but no evolution of nitrogen gas was observed. Next, the solution was concentrated under reduced pressure and then dissolved in 30 mL of anhydrous DCM. 6-Amino-1-hexanol (3 equivalents) and pyridine (1 equivalent) were added to the reaction mixture, and stirring was continued for 12 hours. The mixture was concentrated under reduced pressure on Celite and purified by CombiFlash chromatography using 5% methanol in 95% DCM to give Compound 1 as an oil in 51% yield. LC-MS [M+H2O] + 717.4538 m / z, found 717.4530.

[0250] Compound 1 (1 equivalent) was rotary evaporated twice with toluene, and then anhydrous DCM (10 mL) was charged into the reaction flask. The suspension was stirred at room temperature at 900 RPM under a nitrogen atmosphere using a molecular sieve. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.5 equivalents) was added to the suspension, followed by diisopropylammonium tetrazolide (0.4 equivalents). After 12 hours, TEA (300 μL) was added, and the reaction mixture was dry-packed onto Celite. The product was purified using hexane:ethyl acetate + 1% TEA (60:40) to give LP-462p as an oil in 64% yield. LC-MS [M+H] + 916.5538 m / z, found 916.5543.

[0251] Synthesis of LP-463p

Chemical formula

[0252] To a solution of 16-hydroxyhexadecanoic acid (1.5 g, 5.5 mmol) in DCM (60 mL) was added acetic anhydride (8.3 mL, 88 mmol), followed by pyridine (13.75 mL, 171 mmol) at room temperature. The mixture was stirred at room temperature overnight. After removing the solvent under vacuum, the residue was redissolved in DCM and dry-packed onto an 80 g column. Purification was carried out using from hexane to 50% ethyl acetate in hexane. Compound 24 was obtained as a white solid, 1.22 g, 62%. LC-MS: calculated [M+H+H2O] 375.27, found 374.80.

Chemical formula

[0253] A suspension of compound 24 (1.22 g, 3.4 mmol) in ACN (40 mL) and saturated aqueous NaHCO3 (10 mL) was stirred at room temperature overnight. The pH was adjusted to 1 with 1N HCl. The precipitate was collected by suction filtration, washed with H2O, and air-dried to give 1.15 g (yield 107%) of compound 25 as a white solid. 1 As measured by 1H NMR, the yield exceeded 100% due to residual water. LC-MS: calculated [M+H] 315.25, found 315.59.

Chemical formula

[0254] To a solution of compound 25 (1.15 g, 3.66 mmol) and diisopropylethylamine (1.28 mL, 7.3 mmol) in DCM (40 mL) was added COMU (1.8 g, 4.4 mmol) and tert-butyl 3-aminobicyclo[1.1.1]pentane-1-carboxylate (0.81 g, 4.4 mmol) at room temperature. The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated on silica gel and purified by column chromatography (from 100% hexane:0% ethyl acetate to 0% hexane:100% ethyl acetate). The fractions containing the product were combined and the solvent was removed by rotary evaporator to give 1.66 g (94% yield) of compound 26 as a brown solid. LC-MS: calcd [M+H] 480.37, found 480.76.

Chemical formula

[0255] TFA (10 mL) was added to a solution of compound 26 in DCM (10 mL) and the reaction was stirred at room temperature for 1.5 h. After the solvent was removed under vacuum, the residue was dried under high vacuum for 2 h. The residue was dissolved in DCM (30 mL) and diisopropylethylamine (1.2 mL, 6.9 mmol). After the residue was dissolved, COMU (1.77 g, 4.1 mmol) and 6-amino-1-hexanol (0.49 g, 4.1 mmol) were added at room temperature. The mixture was stirred at room temperature for 2.5 h. After removing a part of the solvent under vacuum, the residue was recrystallized from ACN. The product was collected by suction filtration and dried under vacuum to give 1.48 g (82% yield) of compound 27 as an off-white solid. LC-MS: calcd [M+H] 523.41, found 524.06.

Chemical formula

[0256] To a mixture of compound 27 (0.3 g, 0.57 mmol) in DCM (20 mL), diisopropylammonium tetrazolide (0.039 g, 0.23 mmol) was added, followed by dropwise addition of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (0.277 g, 0.92 mmol) at room temperature. Next, the mixture was refluxed for 2 hours. After cooling to room temperature, the mixture was washed twice with saturated NaHCO3 (aqueous solution) and then with H2O. After removing almost all the solvent under vacuum, the residue was added to stirred hexane, and a white gel precipitate formed. After filtration, the white solid was collected by suction filtration and washed twice with hexane. The white solid was dried under high vacuum to give 0.305 g (73% yield) of compound LP-463p as a white solid. LC-MS: calculated [M+H] 723.52, found 724.23.

[0257] Synthesis of LP-464p [Chemical formula]

[0258] To a solution of 16-amino-hexadecanoic acid (1 equiv) in anhydrous MeOH (20 mL), ethyl trifluoroacetate (1.5 equiv) and TEA (1.1 equiv) were added. The reaction mixture was stirred at 50 °C for 12 hours under a nitrogen atmosphere. Next, the mixture was concentrated under reduced pressure, diluted with ethyl acetate (30 mL), washed twice with saturated KHSO4 (15 mL) and once with brine (15 mL), dried over Na2SO4, and concentrated under reduced pressure to give compound 1 as a white solid in 79% yield. LC-MS [M+H] + 368.2412 m / z, found 368.2419.

[0259] To a solution of Compound 1 (1 equiv) in DCM (30 mL) were added COMU (1.2 equiv) and DIPEA (2 equiv). The mixture was stirred at room temperature for 30 minutes. Next, 6-amino-1-hexanol (1.2 equiv) was added and the reaction mixture was stirred at room temperature for 12 hours. The mixture was then washed three times with 1 M HCl (3×15 mL) and once with brine (15 mL), dried over Na2SO4, and concentrated under reduced pressure. ACN (100 mL) was added to the crude product and the mixture was carefully heated using a heat gun until all the solids had dissolved. The mixture was then left at room temperature and white crystals formed. The precipitate was then collected by vacuum filtration and washed several times with ACN to remove the remaining pink color. Compound 2 was obtained as a white solid in 82% yield. LC-MS [M+H] + 467.3461 m / z, found 467.3457.

[0260] Compound 2 (1 equiv) was concentrated twice using toluene on a rotary evaporator and then anhydrous DCM (10 mL) was charged into the reaction flask. The suspension was stirred at ambient temperature at 900 RPM under a nitrogen atmosphere using a molecular sieve. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.5 equiv) was added to the suspension, followed by diisopropylammonium tetrazolide (0.4 equiv). After 12 hours, TEA (300 μL) was added and the reaction mixture was dry-packed onto Celite. The product was purified using hexane:ethyl acetate + 1% TEA (60:40) and LP-464p was obtained as a waxy solid in 77% yield. LC-MS [M+H] + 667.4539 m / z, found 667.4544.

[0261] Synthesis of LP-465p [Chemical formula]

[0262] 17-Methoxy-17-oxohexadecanoic acid (1.0 g, 3.2 mmol) was dissolved in THF (50 mL), and triethylamine (0.89 mL, 6.4 mmol) and DPPA (0.75 mL, 3.5 mmol) were added. The reaction mixture was stirred overnight. The reaction mixture was concentrated, and the crude product was purified by silica gel chromatography (from 20:80 ethyl acetate:hexane to 100:0 ethyl acetate:hexane). The product was eluted with 10% ethyl acetate. Fractions 1 - 4 containing the product were concentrated to give 0.60 g (yield 56%) of Compound 17 as a white solid.

Chemical formula

[0263] Compound 17 (0.58 g, 1.7 mmol) was dissolved in toluene (20 mL) and heated at 65 °C until no gas evolution was observed (30 minutes). After the solution was cooled to room temperature, it was added to a solution of 6-amino-1-hexanol (0.2 g, 1.7 mmol) and pyridine (0.14 g, 1.7 mmol) in THF (20 mL). The reaction mixture was diluted with acetonitrile, and the precipitate was collected by suction filtration, washed with acetonitrile and hexane, and dried in vacuo to give 0.614 g (yield 84%) of Compound 19 as a white solid.

Chemical formula

[0264] In a 100 mL round-bottom flask, compound 19 (0.60 g, 1.4 mmol) was dried by three consecutive evaporations of toluene. Diisopropylammonium tetrazolide (0.096 g, 0.56 mmol) and 4 Å molecular sieves were added to the flask. The flask was purged and refilled with nitrogen three times, and the solid was suspended in DCM (40 mL). The solid only partially dissolved. To the mixture was added 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (0.65 g, 2.2 mmol), and the reaction was stirred for 18 hours. LC-MS after 18 hours showed that no starting alcohol remained. The reaction was transferred to a separatory funnel, washed with saturated aqueous NaHCO3 (2 × 40 mL) and water (40 mL), and concentrated to dryness. Hexane was added to the flask, and the residue was stirred in hexane for 2 hours to obtain a white precipitate. The white solid was collected by filtration, washed with hexane (2 × 20 mL), and dried in vacuo to give 0.678 g (77% yield) of LP-465p as a white solid.

[0265] Synthesis of LP-466p

Chemical formula

[0266] Compound 7 (0.22 g, 0.50 mmol) and tert-butyl 3-aminobicyclo[1.1.1]pentane-1-carboxylate (0.0915 g, 0.50 mmol) were dissolved in DCM (10 mL), and triethylamine (0.21 mL, 1.5 mmol) was added. After 18 hours, by LC-MS, the remaining amount of the starting NHS ester was less than 2%. The reaction mixture was concentrated and purified by direct loading onto a silica gel column. The product was purified by column chromatography from 0% ethyl acetate / 100% hexane to 50% ethyl acetate / 50% hexane. Fractions 3 - 5 were combined to give 0.23 g (89% yield) of compound 10 as a white solid.

Chemical formula

[0267] Compound 10 (0.23 g, 0.45 mmol) was dissolved in DCM (3 mL), and trifluoroacetic acid (3 mL) was added. The solution was stirred overnight. After 18 hours, no SM was present by LC-MS. The reaction mixture was concentrated, and the residual TFA was removed by azeotropic distillation with toluene twice to afford 0.189 g (93%) of Compound 11 as a white solid.

Chemical formula

[0268] Compound 11 (0.189 g, 0.48 mmol) and COMU (0.215 g, 0.5 mmol) were dissolved in DCM (10 mL), and triethylamine (0.333 mL, 2.4 mmol) was added. The reaction mixture was stirred for about 5 minutes, then 6-amino-1-hexanol (0.059 g, 0.5 mmol) was added. After 1 hour, no starting material remained by LC-MS. The reaction mixture was concentrated, and water was added to the residue. The mixture was sonicated until all the materials were suspended in water, and the precipitate was collected by filtration and washed three times with water. The precipitate was dried in vacuo to afford 0.166 g (70% yield) of Compound 12 as a white solid.

Chemical formula

[0269] In a 100 mL round-bottom flask, compound 12 (0.166 g, 0.3 mmol) was dried by two consecutive evaporations of toluene. Diisopropylammonium tetrazolide (0.02 g, 0.12 mmol) and 4 Å molecular sieves were added to the flask. The flask was purged and refilled with nitrogen three times, and the solids were suspended in DCM (20 mL). The solids only partially dissolved. 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.14 g, 0.46 mmol) was added to the mixture, and the reaction was stirred for 18 hours. LC-MS indicated that no starting alcohol remained after 18 hours. The reaction was transferred to a separatory funnel and washed with saturated aqueous NaHCO3 (2 × 40 mL), water (40 mL), brine (40 mL), dried over magnesium sulfate, and concentrated to dryness. Hexane was added to the flask, and the residue was stirred in hexane for 1 hour to obtain a white precipitate. The white solid was collected by filtration, washed with hexane (2 × 20 mL), and dried under vacuum to give 0.116 g (51% yield) of LP-466p as a white waxy solid.

[0270] Synthesis of LP-493p (designated as LP-493p uridine) [Chemical formula]

[0271] To a solution of 1-bromohexadecane-16-ol (6.0 g, 18.7 mmol) in DCM (90 mL) was added triethylamine (2.9 mL, 20.5 mmol). The resulting solution was cooled to 0 °C in an ice-water bath. After cooling, acetyl chloride (1.46 mL, 20.5 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred at 0 °C for 1 h and then warmed to room temperature and stirred overnight. After about 18 h, the reaction mixture was washed with saturated NaHCO3 (20 mL), water, 1 M HCl (20 mL), water (2×20 mL), brine (20 mL), dried over sodium sulfate, and concentrated to give a white solid. The crude product was purified by silica gel chromatography (from 0:100 ethyl acetate:hexane to 20:80 ethyl acetate:hexane). The product was eluted with 10% ethyl acetate. Fractions 5 - 12 were concentrated to give 6.02 g (89% yield) of compound 29 as a white powder. [Chemical formula]

[0272] Compound 31 was prepared according to the literature procedure. Compound 31 (1.0 g, 2.1 mmol), compound 29 (1.53 g, 4.2 mmol), and tetrabutylammonium iodide (1.6 g, 0.42 mmol) were placed in a flask dried in an oven. The flask was evacuated and purged three times with nitrogen, and then anhydrous DMF (10 mL) was added to the flask. The solution was heated at 110 °C for 18 h. After 18 h, the reaction mixture was cooled to room temperature and the solvent was removed under vacuum. The residue was resuspended in DCM / MeOH and concentrated on silica gel for purification. The column was eluted from 3% MeOH / 97% DCM to 20% MeOH / 80% DCM. The fractions containing the 2' and 3' addition products were pooled and concentrated to give 0.236 g (21% yield) of compound 30 and the 3' addition product. [Chemical formula]

[0273] The compound 30 + 3' adduct (0.23 g, 0.44 mmol) was dried by successive evaporation of toluene and anhydrous pyridine using a rotary evaporator. DMAP (0.003 g, 0.022 mmol) and dimethoxytrityl chloride (0.165 g, 0.49 mmol) were added to the flask, and the flask was evacuated and purged with nitrogen three times. The solid was dissolved in pyridine (10 mL). The reaction mixture was stirred overnight at room temperature. All volatile substances were removed, and the remaining pyridine was removed by co-distillation with toluene. The residue was partitioned between DCM (20 mL) and aqueous NaHCO3 (20 mL). The organic phase was separated, the aqueous phase was extracted with DCM (20 mL), and the combined organic phases were dried (Na2SO4) and concentrated. The crude product was purified by silica gel chromatography. The silica was pretreated with a 50:50 mixture of hexane / ethyl acetate + 2% v / v triethylamine. The product was isolated by CombiFlash using a 40 g column (eluent: hexane - ethyl acetate + 1% triethylamine, 20 - 60%). The compound eluted with 60% ethyl acetate. The later fractions were contaminated with the 3' alkylated product. The fractions containing the pure 2' alkylated product were combined and concentrated to give 0.107 g (yield 27%) of compound 32 as a white solid.

Chemical formula

[0274] A 25 mL round-bottom flask was charged with compound 32 (0.150 g, 0.18 mmol), diisopropylammonium tetrazolide (0.043 g, 0.25 mmol), and 4 Å molecular sieves. The flask was evacuated three times with nitrogen. DCM (5 mL) was added, followed by dropwise addition of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (0.092 mL, 0.29 mmol). The reaction mixture was stirred overnight. The reaction mixture was quenched with approximately 2 mL of saturated NaHCO3, filtered into a separatory funnel, the layers were separated, and the NaHCO3 layer was extracted one more time with DCM (10 mL). The combined organic layers were dried over Na2SO4 and concentrated to a thick viscous liquid. The crude product was purified by silica gel chromatography (from 0:100 ethyl acetate:hexane to 100:0 ethyl acetate:hexane). The silica was pretreated with a 50:50 mixture of hexane / ethyl acetate + 2% v / v triethylamine. The product was eluted with 45% ethyl acetate. Fractions 15 - 35 contained the product with little contamination by the oxidized product. These were combined to give 0.088 g (47% yield) of compound 33 as a viscous colorless solid. Fractions 36 - 50 were combined to give 44 mg of a viscous colorless solid which contained a product with a higher amount of oxidized material.

[0275] (2C8C12) Phosphoramidite Synthesis [Chemical formula]

[0276] 2-Octyl-1-decanol (1.00 g, 3.35 mmol) and diisopropylammonium tetrazolide (0.2868 g, 1.68 mmol) were placed in a flask, and the flask was purged with nitrogen. DCM (50 mL) was added to the mixture, and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (2.66 mL, 8.37 mmol) was added dropwise. After the reaction was completed, 3 mL of triethylamine was added to the reaction mixture, and the reaction mixture was concentrated directly onto Celite for purification. The crude product was purified by silica gel chromatography (from 0:100 ethyl acetate:hexane + 2% triethylamine to 100:0 ethyl acetate:hexane + 2% triethylamine). The product was eluted with 100% hexane. The fractions containing the product were concentrated to give 1.268 g (76% yield) of a clear liquid.

[0277] (2C6C10) Phosphoramidite synthesis

Chemical formula

[0278] 2-Hexyl-1-decanol (1.00 g, 4.13 mmol) and diisopropylammonium tetrazolide (0.353 g, 2.06 mmol) were placed in a flask, and the flask was purged with nitrogen. DCM (50 mL) was added to the mixture, and 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (3.27 mL, 10.3 mmol) was added dropwise. After the reaction was completed, 3 mL of triethylamine was added to the reaction mixture, and the reaction mixture was concentrated directly onto Celite for purification. The crude product was purified by silica gel chromatography (from 0:100 ethyl acetate:hexane + 2% triethylamine to 100:0 ethyl acetate:hexane + 2% triethylamine). The product was eluted with 100% hexane. The fractions containing the product were concentrated to give 1.32 g (72% yield) of a clear liquid.

[0279] HO-C16 Phosphoramidite synthesis

Chemical formula

[0280] 1,16-Hexadecanediol, N,N-diisopropylethylamine (0.100 g) was dissolved in 2 mL of THF. 4,4'-Dimethoxytrityl chloride (2.2 g, 6.6 mmol) was slowly added as a solid. After 2 hours, the reaction mixture was concentrated on a rotary evaporator, and the product was purified by column chromatography (25% ethyl acetate / 75% hexane).

[0281] DMT-O-C 16 -OH (0.200 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.227 mL), and bisdiisopropylammonium tetrazolide (0.0611 g) were dissolved in anhydrous DCM at room temperature. The reaction vessel was capped and stirred overnight. The conversion rate was measured by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture, and the mixture was concentrated under vacuum until a white powder remained. The mixture was loaded onto a silica column (12 g) in a dry state using an ethyl acetate / hexane (1% triethylamine) solvent system to prevent hydrolysis from the silica gel. [1] 。The product was 31 characterized by 31P NMR, 1 1H NMR, and LC-MS.

[0282] Synthesis of C16 phosphoramidite

Chemical formula

[0283] Cetyl alcohol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.88 mL), and bisdiisopropylammonium tetrazolide (0.778 g) were dissolved in a DCM solution at room temperature. The reaction mixture was capped and stirred overnight. The conversion rate was measured by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture, and it was concentrated under vacuum until a white powder remained. The mixture was dry-packed onto a silica column (12 g) using an ethyl acetate / hexane (1% triethylamine) solvent system to prevent hydrolysis from the silica gel. The desired product was not retained on the column and eluted shortly after packing. Next, the isolated product was characterized by LC-MS, 1 1H NMR, and 31 31P NMR. Final yield: 856.5 mg (93.8%).

[0284] Synthesis of C22 Phosphoramidite

Chemical Structure

[0285] Docosanol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.1 mL), and bisdiisopropylammonium tetrazolide (0.577 g) were dissolved in a DCM solution at room temperature. The reaction mixture was capped and stirred overnight. The conversion rate was measured by LC-MS (0.25 M NH4HCO3:H2O buffer system). Celite® was added to the reaction mixture, and it was concentrated under vacuum until a white powder remained. The mixture was dry-packed onto a silica column (12 g) pretreated with 3 mL of triethylamine using an ethyl acetate / hexane (1% triethylamine) solvent system to prevent hydrolysis from the silica gel. Next, the isolated product was characterized by LC-MS, 1 1H NMR, and 31 31P NMR. Final yield: 2.1085 g (118.8%).

[0286] Example 3. Binding of Lipid PK / PD Modulator Precursor

[0287] Before or after annealing, and before or after the binding of one or more target ligands, one or more lipid PK / PD modulator precursors can be linked to the RNAi agents disclosed herein. The following describes the general binding process used to link lipid PK / PD modulator precursors to the constructs described in the examples shown herein.

[0288] A. Binding of Activated Ester PK / PD Modulators

[0289] The following procedure was used to bind PK / PD modulators having an activated ester moiety, such as TFP (tetrafluorophenoxy) or PNP (paranitrophenol), to RNAi agents having an amine-functionalized sense strand such as C6-NH2, NH2-C6, or (NH2-C6). The annealed RNAi agent dried by lyophilization was dissolved in DMSO and 10% water (v / v%) at 25 mg / mL. Next, 50 - 100 equivalents of TEA and 3 equivalents of the activated ester PK / PD modulator were added to the solution. The solution was reacted for 1 - 2 hours, during which it was monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on a Waters™ XBridge C18 column, Waters Corp.).

[0290] Next, 12 mL of acetonitrile and 0.4 mL of PBS were added to precipitate the product, and the solid was centrifuged to form a pellet. Next, the pellet was redissolved in 0.4 mL of 1×PBS and 12 mL of acetonitrile. The resulting pellet was dried under high vacuum for 1 hour.

[0291] B. Binding of Phosphoramidite PK / PD Modulators

[0292] PK / PD modulators having a phosphoramidite moiety can be attached to the resin using general oligonucleotide manufacturing conditions.

[0293] C. Hydrolysis of the PK / PD Modulator

[0294] Certain PK / PD modulators are hydrolyzed under the cleavage and deprotection conditions described in Example 1 above. For example, LP-429p, LP-456p, LP-462p, LP-463p, LP-464p, LP-466p, LP-493p, and HO-C16 phosphoramidite all contain moieties that are hydrolyzed under cleavage and deprotection conditions.

[0295] LP-465p binds to the oligonucleotide chain in a 0.5 - 1 M potassium carbonate solution in 1:1 methanol to water and is hydrolyzed after heating at 50 - 60 °C for about 4 hours.

[0296]

Table A-1

Table A-2

Table A-3

[0297] As used in Table A, the following notations are used to indicate modified nucleotides, target bases, and linking groups: A = adenosine-3′-phosphate C = cytidine-3′-phosphate G = guanosine-3′-phosphate U = uridine-3′-phosphate I = inosine-3′-phosphate a = 2′-O-methyladenosine-3′-phosphate as = 2′-O-methyladenosine-3′-phosphorothioate c = 2′-O-methylcytidine-3′-phosphate cs = 2′-O-methylcytidine-3′-phosphorothioate g = 2′-O-methylguanosine-3′-phosphate gs = 2′-O-methylguanosine-3′-phosphorothioate i = 2′-O-methylinosine-3′-phosphate is = 2′-O-methylinosine-3′-phosphorothioate t = 2′-O-methyl-5-methyluridine-3′-phosphate ts = 2′-O-methyl-5-methyluridine-3′-phosphorothioate u = 2′-O-methyluridine-3′-phosphate us = 2′-O-methyluridine-3′-phosphorothioate Af = 2′-fluoroadenosine-3′-phosphate Afs = 2′-fluoroadenosine-3′-phosphorothioate Cf = 2′-fluorocytidine-3′-phosphate Cfs = 2′-fluorocytidine-3′-phosphorothioate Gf = 2′-fluoroguanosine-3′-phosphate Gfs = 2′-fluoroguanosine-3′-phosphorothioate Tf = 2′-fluoro-5′-methyluridine-3′-phosphate Tfs = 2′-fluoro-5′-methyluridine-3′-phosphorothioate Uf = 2′-fluorouridine-3′-phosphate Ufs = 2′-fluorouridine-3′-phosphorothioate dT = 2′-deoxythymidine-3′-phosphate AUNA = 2′,3′-seco-adenosine-3′-phosphate AUNAs = 2′,3′-seco-adenosine-3′-phosphorothioate CUNA = 2′,3′-seco-cytidine-3′-phosphate CUNAs = 2′,3′-seco-cytidine-3′-phosphorothioate GUNA = 2′,3′-seco-guanosine-3′-phosphate GUNAs = 2′,3′-seco-guanosine-3′-phosphorothioate UUNA = 2′,3′-Seco-Uridine-3′-Phosphate UUNAs = 2′,3′-Seco-Uridine-3′-Phosphorothioate a_2N = Refer to Table 4 a_2Ns = Refer to Table 4 (invAb) = Inverted Abasic Deoxyribonucleotide-5′-Phosphate, Refer to Table 4 (invAb)s = Inverted Abasic Deoxyribonucleotide-5′-Phosphorothioate, Refer to Table 4 s = Phosphorothioate Linkage p = Terminal Phosphate (as Synthesized) vpdN = Vinylphosphonate Deoxyribonucleotide cPrpa = 5'-Cyclopropylphosphonate-2'-O-Methyladenosine-3'-Phosphate (Refer to Table 4) cPrpas = 5'-Cyclopropylphosphonate-2'-O-Methyladenosine-3'-Phosphorothioate (Refer to Table 4) cPrpu = 5'-Cyclopropylphosphonate-2'-O-Methyluridine-3'-Phosphate (Refer to Table 4) cPrpus = 5'-Cyclopropylphosphonate-2'-O-Methyluridine-3'-Phosphorothioate (Refer to Table 4) (Alk-SS-C6) = Refer to Table 4 (C6-SS-Alk) = Refer to Table 4 (C6-SS-C6) = Refer to Table 4 (6-SS-6) = Refer to Table 4 (C6-SS-Alk-Me) = Refer to Table 4 (NH2-C6) = Refer to Table 4 (NH-C6) = Refer to Table 4 (NH-C6)s = Refer to Table 4 -L6-C6- = Refer to Table 4 -L6-C6s- = Refer to Table 4 cC16 = Refer to Table 4 aC16 = Refer to Table 4 gC16 = Refer to Table 4 uC16 = Refer to Table 4 ALNA = Refer to Table 4

[0298] Example 4. In Vivo Administration of Lipid-Conjugated RNAi Agent in Mice

[0299] On the first day of the experiment, female C57bl / 6 mice were injected with either phosphate-buffered saline (PBS) or an RNAi agent formulated at 10 μg / μL in PBS. 10 μL of PBS or the RNAi agent solution was administered to 3 animals (n = 3) in each group. The animals were given intracerebroventricular injections according to the administration schedule in Table 5.

[0300] [Table 5]

[0301] On the 15th day of the experiment, the animals were sacrificed and the thoracic spinal cord, temporal lobe cortex, frontal lobe cortex, and cerebellum were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured using qPCR. The average SOD1 expression in each tissue of each animal was normalized relative to Group 1 (PBS). The results are shown in Tables 6a - 6d below.

[0302] [Table 6a]

[0303] [Table 6b]

[0304] [Table 6c]

[0305] [Table 6d]

[0306] Example 5. In Vivo Administration of Lipid-Conjugated RNAi Agent in Mice

[0307] On the first day of the test, female C57bl / 6 mice were injected with either phosphate-buffered saline (PBS) or an RNAi agent formulated at 10 μg / μL in PBS. 10 μL of PBS or the RNAi agent solution was administered to 3 animals (n = 3) in each group. The animals were intracerebroventricularly injected according to the administration schedule shown in Table 7.

[0308]

Table 7

[0309] On the 15th day of the test, the animals were sacrificed and the thoracic spinal cord, temporal lobe cortex and frontal lobe cortex, and cerebellum were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured using qPCR. The average SOD1 expression in each tissue of each animal was normalized compared to Group 1 (PBS). The results are shown in Tables 8a - 8d below.

[0310]

Table 8a

[0311]

Table 8b

[0312]

Table 8c

[0313]

Table 8d

[0314] Example 6 In Vivo Administration of Lipid-Conjugated RNAi Agent in Transgenic Mice

[0315] On the first day of the experiment, female TgSOD1G93A mice modified to express human SOD1 were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or an RNAi agent formulated at 3 μg / μL in aCSF. Three animals (n = 3) in each group were administered 10 μL of aCSF or the RNAi agent solution in aCSF. The animals were injected intracerebroventricularly according to the administration schedule shown in Table 9.

[0316]

Table 9

[0317] On the eighth day of the experiment, the animals were sacrificed and the thoracic spinal cord, cortex, cerebellum, and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured using qPCR. The average SOD1 expression in each tissue of each animal was normalized relative to Group 1 (PBS). The results are shown in Tables 10a - 10d below.

[0318]

Table 10a

[0319]

Table 10b

[0320]

Table 10c

[0321]

Table 10d

[0322] Example 7. In Vivo Administration of a Lipid - Linked RNAi Agent in Transgenic Mice

[0323] On the first day of the experiment, female TgSOD1G93A mice modified to express human SOD1 were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or an RNAi agent formulated at 3 μg / μL in aCSF. Three animals (n = 3) in each group were administered 10 μL of aCSF or the RNAi agent solution in aCSF. The animals were injected intracerebroventricularly according to the administration schedule shown in Table 11.

[0324]

Table 11

[0325] On the eighth day of the experiment, the animals were sacrificed and the thoracic spinal cord, cortex, cerebellum, and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured using qPCR. The average SOD1 expression in each tissue of each animal was normalized and compared to that of Group 1 (PBS). The results are shown in Tables 12a - 12d below.

[0326]

Table 12a

[0327]

Table 12b

[0328]

Table 12c

[0329]

Table 12d

[0330] Example 8. In Vivo Knockdown of SOD1 in Transgenic TgSOD1G93A Rats

[0331] On the first day of the experiment, TgSOD1G93A rats were injected with either 30 μL of artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 30 μL of a composite preparation at concentrations of 0.33, 1.0, 3.33, 10, and 30 mg / mL into each of groups 2 - 6 in aCSF according to Table 13 below.

Table 13

[0332] Four rats per group (n = 4) were administered. The rats were injected intrathecally on the first day. CSF was collected from each animal on day 85, and then the rats were euthanized. The left half of the brain and the thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right half of the brain of the thoracic spinal cord, cortex, cerebellum, and brainstem. The samples were analyzed by qPCR for SOD1 mRNA knockdown. The average of the results for each group is shown in Table 14 below:

Table 14

[0333] As shown in Table 14 above, a dose - dependent decrease in SOD1 mRNA expression was observed in transgenic rats treated with AD12261.

[0334] Example 9. In Vivo Knockdown of SOD1 in Cynomolgus Monkeys

[0335] On the first day of the experiment, cynomolgus monkeys were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or a composite preparation containing 45 mg of AD12261 in aCSF according to Table 15 below.

Table 15

[0336] In Group 1 (control), it was administered to 4 monkeys (n = 4), and in Groups 2, 3, and 4 (trigger treatment), it was administered to 5 monkeys (n = 5). The monkeys were injected intraspinally on Day 1. On Day 29 of the test, the animals in Groups 1 and 2 were euthanized, and brain and spinal cord tissues were collected from each animal. On Day 85 of the test, the animals in Group 3 were euthanized, and brain and spinal cord tissues were collected from each animal. On Day 168 of the test, the animals in Group 4 were euthanized, and brain and spinal cord tissues were collected from each animal. The samples were analyzed by qPCR for SOD1 mRNA knockdown. The average results of each group compared with Group 1 are shown in Table 16 below.

Table 16-1

Table 16-2

[0337] As shown in Table 16 above, a sustained (up to 168 days) decrease in SOD1 mRNA expression was observed in multiple tissues of non-human primates treated with AD12261.

[0338] Example 10. In Vivo Knockdown of SOD1 in Transgenic TgSOD1G93A Rats

[0339] On Day 1 of the test, male TgSOD1G93A rats (Sprague-Dawley) modified to express human SOD1 were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or an RNAi agent formulated at 10 mg / mL in aCSF. In each group, 4 animals (n = 4) were administered 30 μL of aCSF or the RNAi agent solution in aCSF. The animals were injected intraspinally (IT) according to the administration schedule in Table 17.

[0340]

Table 17

[0341] On the 8th day of the experiment, the animals were sacrificed, and the thoracic spinal cord, cortex (temporal), cerebellum, brainstem, and dorsal root ganglia (lumbar) were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured by qPCR using the PPIA control gene. The average SOD1 expression in each tissue of each animal was normalized compared to group 1 (aCSF). The results are shown in Table 18 below.

[0342]

Table 18

[0343] Example 11. In Vivo Knockdown of SOD1 in Transgenic TgSOD1G93A Rats

[0344] On the 1st day of the experiment, male TgSOD1G93A rats (Sprague-Dawley) modified to express human SOD1 were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or an RNAi agent formulated at 10 mg / mL in aCSF. Four animals (n = 4) in each group were administered 30 μL of aCSF or the RNAi agent solution in aCSF. The animals were injected intrathecally (IT) according to the dosing schedule in Table 19.

[0345]

Table 19

[0346] On the 8th day of the experiment, the animals were sacrificed, and the cortex (temporal), thoracic spinal cord, cerebellum, and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured by qPCR using the PPIA control gene. The average SOD1 expression in each tissue of each animal was normalized compared to group 1 (aCSF). The results are shown in Table 20 below.

[0347]

Table 20

[0348] Example 12. In Vivo Knockdown of SOD1 in Transgenic TgSOD1G93A Rats

[0349] On the first day of the test, male TgSOD1G93A rats (Sprague-Dawley) modified to express human SOD1 were injected with either artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or an RNAi agent formulated at 10 mg / mL in aCSF. Four animals (n = 4) in each group were administered 30 μL of aCSF or the RNAi agent solution in aCSF. The animals were injected intrathecally (IT) according to the administration schedule in Table 21.

[0350] [Table 21]

[0351] On the eighth day of the test, the animals were sacrificed and the cortex, thoracic spinal cord, cerebellum, brainstem, heart, midbrain, and hippocampus were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was measured by qPCR using the PPIA control gene. The average SOD1 expression in each tissue of each animal was normalized compared to Group 1 (aCSF). The results are shown in Table 22 below.

[0352] [Table 22]

[0353] Equivalents and Scope In the claims, articles such as "a", "an", and "the" may mean one or more unless the contrary is indicated or is not apparent from the context. A claim or description that includes "or" between one or more members of a group is considered satisfied if, unless the contrary is indicated or is not apparent from the context, one, more than one, or all of the members of the group are present in, used in, or relevant to a particular product or process. The present invention includes embodiments in which only one member of the group is present in, used in, or relevant to a particular product or process. The present invention includes embodiments in which two or more or all of the members of the group are present in, used in, or relevant to a particular product or process.

[0354] Furthermore, the present invention encompasses all modifications, combinations, and alterations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited claims are introduced into another claim. For example, a claim that depends on another claim can be modified to include one or more limitations found in other claims that depend on the same basic claim. When elements are presented as a list, for example, in Markush group format, each subgroup of the elements is also disclosed and any element can be removed from the group. Generally, when the present invention or an aspect of the present invention is referred to as including a particular element and / or feature, it is to be understood that a particular embodiment of the present invention or an aspect of the present invention consists of or consists essentially of such element and / or feature. For the sake of brevity, these embodiments are not specifically described verbatim herein. Also, note that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. When a range is indicated, the endpoints are also included. Further, unless otherwise specified or not apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can be assumed to be any specific value or subrange within the range described for different embodiments of the present invention, down to one tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0355] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are hereby incorporated by reference into this specification. In the event of any conflict between any of the incorporated references and this specification, this specification shall control. Further, specific embodiments of the present invention that are part of the prior art can be explicitly excluded from one or more claims. Such embodiments can be excluded even if not explicitly designated as such in this specification, as they are considered known to one of ordinary skill in the art. Specific embodiments of the present invention can be excluded from any claim for any reason, regardless of their relation to the existence of the prior art.

[0356] Other embodiments The present invention has been described in conjunction with its detailed description, but it should be understood that the foregoing description is for the purpose of explaining the scope of the present invention as defined by the scope of the appended claims and is not intended to limit the scope of the present invention. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. a) Oligonucleotides and, b) A compound comprising a lipid bonded to the 5' or 3' position of one terminal nucleotide of the oligonucleotide, Here, the oligonucleotide is a compound comprising at least 15 nucleotides complementary to the genes expressed in the CNS tissue.

2. The compound according to claim 1, wherein the oligonucleotide comprises a sense chain and an antisense chain, and the lipid is bonded to the 5' position of the sense chain.

3. The compound according to claim 1 or 2, wherein the lipid is saturated.

4. The compound according to claim 1 or 2, wherein the lipid is unsaturated.

5. The compound according to claim 1 or 2, wherein the lipid contains 10 to 30 or 15 to 20 carbon atoms.

6. The compound according to claim 5, wherein the lipid is selected from the group consisting of the following: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 During the ceremony, 【Chemistry 2】 The symbol indicates a connection point to the oligonucleotide, and the connection point includes a linker.

7. The compound according to claim 6, wherein the linker is (NH-C6)s: 【Transformation 3】

8. The compound according to claim 1 or 2, wherein the oligonucleotide is an RNAi agent.

9. The compound according to claim 2, wherein the sense strand further comprises an inverted debase at the 5' and / or 3' ends of the terminal nucleotide.

10. The compound according to claim 2, wherein the antisense chain includes a cyclopropylene (cPrp) modified nucleotide which is the 5' terminal nucleotide of the antisense chain.

11. Compounds selected from the following group: Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 In the formula, R includes oligonucleotides.

12. The compound according to claim 11, wherein the oligonucleotide is double-stranded and comprises a sense strand and an antisense strand, and the connection point to R is located on the 5' terminal nucleotide of the sense strand.

13. Compounds selected from the following group: Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 In the formula, R includes oligonucleotides.

14. The compound according to claim 13, wherein the oligonucleotide comprises a sense strand and an antisense strand, and the connection point to R is located on the 5' terminal nucleotide of the sense strand.

15. The compound according to claim 14, wherein the sense strand further comprises an inverted debase at the 5' and / or 3' ends of the terminal nucleotide, and the antisense strand comprises a cyclopropylene (cPrp) modified nucleotide which is the 5' terminal nucleotide of the antisense strand.

16. a) Oligonucleotides and, b) A compound comprising a hydroxylipid bonded to an internal nucleotide of the oligonucleotide, wherein the hydroxylipid contains a hydroxyl group, and Here, the oligonucleotide is a compound comprising at least 15 nucleotides complementary to the genes expressed in the CNS tissue.

17. A pharmaceutical composition for treating a disease or disorder of the central nervous system, comprising a compound according to any one of claims 1 to 16.

18. The pharmaceutical composition according to claim 17, wherein the disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and Lewy body dementia.

19. The compound comprises an oligonucleotide having a nucleotide sequence complementary to the gene expressed in CNS cells. The pharmaceutical composition according to claim 18, wherein the gene is selected from the group consisting of superoxide dismutase type 1 (SOD1), amyloid precursor protein (APP), ataxin 2 (ATXN2), ataxin 3 (ATXN3), sodium voltage-gated channel alpha subunit 9 (SCN9A), huntingtin (HTT), alpha-synuclein (SNCA), chromosome 9 open reading frame 72 (C9orf72), leucine-rich repeat kinase 2 (LRRK2), adrenergic receptor alpha 2A (ADRA2A), and androgen receptor (AR).

20. Compounds having the following structure: Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 or a pharmaceutically acceptable salt thereof.

21. A method for synthesizing a compound according to any one of claims 1 to 16, comprising reacting the compound according to claim 20 with an oligonucleotide-containing compound.

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