Lipid conjugates for delivery of therapeutic agents to CNS tissue

Abstract

Disclosed herein are compounds comprising lipid PK / PD modulators for delivering oligonucleotide-based agents (e.g., double-stranded RNAi agents) to certain cell types (e.g., central nervous system cells) in the body. When conjugated to oligonucleotide-based therapeutic or diagnostic agents, such as RNAi agents, the PK / PD modulators disclosed herein can enhance delivery of the compositions to specific targeted cells to promote inhibition of gene expression in those cells.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 495,505 filed on April 11, 2023 and U.S. Provisional Patent Application Serial No. 63 / 352,485 filed on June 15, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy is named 30707-WO_ST26_SeqListing.xml, created on June 13, 2023, and is 1519kb in size. Technical Field

[0005] The present disclosure relates to lipid conjugates for delivering oligonucleotide-based agents (eg, double-stranded RNAi agents) to certain central nervous system (CNS) cell types in vivo to inhibit genes expressed in those cells. Background Art

[0006] Oligonucleotide-based agents, such as antisense agents and double-stranded RNA interference (RNAi) agents, have shown great promise and are likely to revolutionize the medical field and provide patients with effective therapeutic treatment options. However, the effective delivery of oligonucleotide-based agents and particularly double-stranded therapeutic RNAi agents has long been a challenge in developing viable therapeutic pharmaceutical agents. This is especially true when attempting to achieve specific and selective delivery of oligonucleotide-based agents to extrahepatic (i.e., non-hepatocyte) cells.

[0007] Although various attempts have been made in the past few years to direct oligonucleotide-based agents to certain extrahepatic cell types, including central nervous system cells, adipocytes, cardiac myocytes, etc., such as the use of cholesterol conjugates (which are non-specific and have known disadvantages of being distributed to various undesirable tissues and organs) and lipid nanoparticles (LNPs) (which are often reported to have toxicity concerns), none has achieved suitable delivery to date. Therefore, there remains a need for a delivery vehicle to direct oligonucleotide-based agents, particularly RNAi agents, to non-hepatocyte cell types. Summary of the invention

[0008] Disclosed herein are compounds comprising a lipid conjugated (or linked) to an oligonucleotide-based agent for delivery to central nervous system tissue. Also disclosed herein are lipid PK / PD modulator precursors.

[0009] One aspect of the present invention provides a double-stranded oligonucleotide, wherein a lipid is conjugated to one of the terminal nucleotides of one of the chains. In certain embodiments, the lipid is conjugated to the 5' terminal nucleotide of one of the chains. In certain embodiments, the lipid is conjugated to the 3' terminal nucleotide of one of the chains. In certain embodiments, the lipid conjugated to the terminal nucleotide of one of the chains is saturated. In certain embodiments, the lipid is unsaturated. In certain embodiments, the lipid is a sterol. In certain embodiments, the lipid is a saturated lipid with 12 to 30 carbon atoms. In certain embodiments, the lipid is a straight-chain lipid with 16 carbon atoms. In certain embodiments, the lipid contains a hydroxyl moiety. In certain embodiments, the lipid is a cholesterol group.

[0010] In another aspect, the invention provides compounds comprising oligonucleotides, wherein hydroxy lipids are conjugated to internal nucleotides. In certain embodiments, the hydroxy lipids comprise aliphatic chains containing one or more hydroxyl (-OH) functional groups. In certain embodiments, the hydroxyl group is conjugated to the distal carbon of the aliphatic chain relative to the internal nucleotide (i.e., the carbon atom farthest from the internal nucleotide). In certain embodiments, the hydroxy lipids are conjugated to the 2' carbon of the internal nucleotide. In certain embodiments, the hydroxy lipids are composed of 12-24 carbon atoms. In certain embodiments, the hydroxy lipids are composed of 16 carbon atoms.

[0011] Unless otherwise defined, all technical and scientific terms used in this article have the same meanings as those of ordinary skill in the art are generally understood. Although methods and materials similar to or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in this article are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, the materials, methods and embodiments described are merely exemplary and are not intended to be restrictive.

[0012] Other objects, features, aspects and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings and from the claims.

[0013] Detailed Description

[0014] Lipid PK / PD Modulators

[0015] Compounds comprising PK / PD regulators conjugated to oligonucleotide-based agents are described herein to provide delivery of payloads (such as RNA interference (RNAi) agents) to cells in vivo. Without being limited to any particular theory, it is believed that the compounds described herein regulate the pharmacokinetic and / or pharmacodynamic properties of the corresponding delivery vehicles, thereby increasing RNAi-induced target gene knockdown in cells. Compounds described herein can facilitate delivery to certain cell types, including, but not limited to central nervous system cell types, including, but not limited to neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells.

[0016] The present invention provides lipid delivery platforms for oligonucleotides, methods of using the lipid delivery platforms, and methods of preparing the lipid delivery platforms.

[0017] As used herein and as understood by those skilled in the art, a polyethylene glycol (PEG) unit is represented by the formula -(CH 2 CH 2 It should be understood that in the chemical structures disclosed herein, the PEG unit can be depicted as -(CH 2 CH 2 O)-、-(OCH 2 CH 2 )-or-(CH 2 OCH 2 It is also to be understood that the numerals indicating the number of repeating PEG units may be placed on either side of the brackets depicting the PEG units.

[0018] Another aspect of the invention provides a method of preparing a compound comprising a RNAi agent and a lipid moiety.

[0019] In certain embodiments, the methods comprise conjugating an oligonucleotide-based agent comprising a first reactive moiety to a compound comprising a lipid and a second reactive moiety to form a compound comprising an RNAi agent and a lipid moiety.

[0020] In certain embodiments, the first reactive moiety is selected from hydroxyl and amine reactive groups. In certain embodiments, the first reactive moiety is an amine. In certain embodiments, the first reactive moiety is a hydroxyl group.

[0021] In certain embodiments, the second reactive moiety is selected from esters (including, but not limited to, activated esters such as tetrafluorophenoxy esters and p-nitrophenoxy esters), sulfones (including, but not limited to, sulfonyl halides), and phosphoramidites. In certain embodiments, the second reactive moiety is an ester. In certain embodiments, the second reactive moiety is a sulfone. In certain embodiments, the second reactive moiety is a phosphoramidite.

[0022] Formulas shown in Table 1 below and described herein 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, L The compounds P-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 pros” (hereinafter, referred to as “PK / PD modulator pros”). It will also be understood that a portion of the compounds may be referred to as "pharmacokinetic and / or pharmacodynamic modulators" (hereinafter, "PK / PD modulators").When used to represent the formula LP-128b, LP-132b, LP-183b, LP-183r-b shown in Tables 3 and 3a below, 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-462 b, 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), the term “PK / PD modulator” refers to the portion of the compound other than R (i.e., the oligonucleotide-based agent).

[0023] PK / PD regulators are connected to oligonucleotide-based agents, such as RNAi agents, to promote the delivery of RNAi agents to desired cells or tissues. PK / PD regulator precursors with reactive moieties (including, but not limited to activated ester groups and phosphoramidites) can be synthesized, which are easy to promote the connection with one or more linking groups on the RNAi agent. The chemical reaction synthesis of such PK / PD regulator precursors connected to RNAi agents is generally known in the art. The terms "PK / PD regulator" and "lipid PK / PD regulator" can be used interchangeably herein.

[0024] selected from 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, PK / PD modulator precursors of 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 can be used as starting materials for attachment to RNAi agents. The PK / PD modulator precursors can be covalently attached to RNAi agents using any method known in the art. For example, in certain embodiments, an activated ester PK / PD modulator precursor can be reacted with an amine-containing moiety on the 5' end of the sense strand.

[0025] Table 1. Lipid PK / PD modulator precursors

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] In certain embodiments, one or more PK / PD modulators can be conjugated with the RNAi agents described herein. In certain embodiments, one, two, three, four, five, six, seven or more PK / PD modulators can be conjugated with the RNAi agents described herein.

[0036] Using any known method in the art, a PK / PD regulator precursor can be conjugated to an RNAi agent. In certain embodiments, a PK / PD regulator precursor comprising an ester moiety can react with an RNAi agent comprising an amine to form a compound comprising a PK / PD regulator conjugated to an RNAi agent. In certain embodiments, the amine can be at the 5' or 3' end of the RNAi agent. In certain embodiments, the amine can be at the 5' end of the RNAi agent. In certain embodiments, the amine can be at the 3' end of the RNAi agent. In certain embodiments, a PK / PD regulator precursor comprising a sulfonyl moiety can react with an RNAi agent comprising an amine to form a compound comprising a PK / PD regulator conjugated to an RNAi agent. In certain embodiments, the amine can be at the 5' or 3' end of the RNAi agent. In certain embodiments, the amine can be at the 5' end of the RNAi agent. In certain embodiments, the moiety can be at the 3' end of the RNAi agent. In certain embodiments, a PK / PD modulator precursor comprising a phosphoramidite moiety can be reacted with a RNAi agent comprising a hydroxyl moiety to form a compound comprising a PK / PD modulator conjugated to an RNAi agent. In certain embodiments, the hydroxyl moiety can be at the 5' or 3' end of the RNAi agent. In certain embodiments, the hydroxyl moiety can be at the 5' end of the RNAi agent. In certain embodiments, the hydroxyl moiety can be at the 3' end of the RNAi agent.

[0037] In certain embodiments, the PK / PD modulator can be conjugated to the 5′ end of the sense or antisense strand, the 3′ end of the sense or antisense strand, or an internal nucleotide of the RNAi agent. In certain embodiments, the synthetic RNAi agent has a disulfide-containing moiety at the 3′ end of the sense strand, and the PK / PD modulator precursor can be conjugated to the 3′ end of the sense strand using any of the appropriate general synthetic schemes shown above.

[0038] In certain embodiments, lipid PK / PD modulators include compounds shown in Table 2.

[0039] Table 2. Lipid PK / PD modulators

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] in The point of attachment to the oligonucleotide is indicated.

[0048] In certain embodiments, the lipid PK / PD modulator is represented by a compound having the formula shown in Table 3.

[0049] Table 3. Lipid PK / PD modulators

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] wherein R comprises an oligonucleotide.

[0057] In certain embodiments, the lipid PK / PD modulator comprises an aliphatic linker between the lipid component and the oligonucleotide.Example PK / PD modulators are represented by compounds having the formula shown in Table 3a.

[0058] Table 3a. Lipid PK / PD modulators containing linkers

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] wherein R comprises an oligonucleotide. In certain embodiments, R is the point of attachment to the 5' terminal nucleotide of the oligonucleotide.

[0066] definition

[0067] As used herein, the terms "oligonucleotide" and "polynucleotide" refer to a polymer of linked nucleosides, each of which may independently be modified or unmodified.

[0068] As used herein, "RNAi agents" (also referred to as "RNAi triggers") refer to compositions containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting (e.g., degrading or inhibiting under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNAs in a sequence-specific manner. The RNAi agents used herein may act as follows: by an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or by alternative mechanisms or pathways. Although it is believed that, as the term is used herein, RNAi agents act primarily by RNA interference mechanisms, the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein comprise 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 targeted mRNA.The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0069] The term "lipid" as used herein refers to parts and molecules that are soluble in non-polar solvents. The term lipid includes amphiphilic molecules that include polar, water-soluble head groups and hydrophobic tails. The lipid can be natural or synthetic in 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. The term "saturated lipid" as used herein refers to a lipid that does not contain any degree of unsaturation. The term "unsaturated lipid" as used herein refers to a lipid that includes at least one (1) degree of unsaturation. The term "branched lipid" as used herein refers to a lipid that includes more than one straight chain, wherein each straight chain is covalently attached to at least one other straight chain. The term "straight chain lipid" as used herein refers to a lipid that does not have any branches.

[0070] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown" when referring to the expression of a given gene mean that when a cell, cell collection, tissue, organ, or subject is treated with an RNAi agent described herein, the expression of the gene is reduced as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from the mRNA in the cell, cell collection, tissue, organ, or subject in which the gene is transcribed, compared to a second cell, cell collection, tissue, organ, or subject not so treated.

[0071] The terms "sequence" and "nucleotide sequence" as used herein refer to a series or order of nucleobases or nucleotides described by consecutive letters using standard nomenclature.

[0072] As used herein, "base", "nucleotide base" or "nucleobase" is a heterocyclic pyrimidine or purine compound as a component of a nucleotide, and includes the primary purine bases adenine and guanine and the primary pyrimidine bases cytosine, thymine and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, bases with enlarged size and fluorinated bases (see, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including aminophosphite compounds comprising modified nucleobases) is known in the art.

[0073] As used herein and unless otherwise indicated, the term "complementary" when used to describe a first core base or nucleotide sequence (e.g., RNAi agent antisense strand or single-stranded antisense oligonucleotide) relative to a second core base or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA), refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) with an oligonucleotide or polynucleotide comprising a second nucleotide sequence and to 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 at least to the extent that the above hybridization requirements are met. Sequence identity or complementarity is unrelated to modification. For example, a and Af defined herein are complementary to U (or T) and identical to A for the purpose of determining identity or complementarity.

[0074] As used herein, "perfect complementarity" or "complete complementarity" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) bases in the contiguous sequence of the first oligonucleotide will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0075] As used herein, "partial complementarity" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% (but not all) of the bases in the contiguous sequence of the first oligonucleotide will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0076] As used herein, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% (but not all) of the bases in the contiguous sequence of the first oligonucleotide will hybridize to the same number of bases in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0077] As used herein, the terms "complementary," "fully complementary," "partially complementary," and "substantially complementary" are used with respect to nucleobase or nucleotide matches 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.

[0078] As used herein, the term "substantially identical" or "substantially identical" as applied to nucleic acid sequences refers to a nucleotide sequence (or a portion of a nucleotide sequence) having at least about 85% sequence identity or higher, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences in a comparison window. The percentage is calculated as follows: the number of positions at which the same type of nucleic acid base appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the window of comparison and the result is multiplied by 100 to produce the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences substantially identical to those disclosed herein.

[0079] As used herein, the terms "treatment," "treating," and the like refer to methods or procedures used to provide relief or reduction in the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treatment" and "treating" may include prophylactic treatment, management, preventative treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0080] When referring to RNAi agents, the phrase "introduced into a cell" as used herein refers to the functional delivery of the RNAi agent into the cell. The phrase "functional delivery" refers to the delivery of the RNAi agent to the cell in a manner such that the RNAi agent has the desired biological activity (e.g., sequence-specific inhibition of gene expression).

[0081] The term "isomer" as used herein refers to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of each other are termed "diastereomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers" or sometimes optical isomers. A carbon atom bonded to four different substituents is termed a "chiral center".

[0082] As used herein, unless specifically identified as having a specific conformation in a structure, for each structure in which an asymmetric center is present and thus produces enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including optically pure and racemic forms thereof. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[0083] The phrase "consisting of" as used in the claims herein excludes any elements, steps, or ingredients not specified in the claim. When used in the claims herein, the phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0084] Those of ordinary skill in the art will readily understand and appreciate 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 located. Thus, as used herein, the structures disclosed herein contemplate that certain functional groups (e.g., OH, SH, or NH) may be protonated or deprotonated. As those of ordinary skill in the art will readily appreciate, the disclosure herein is intended to encompass the disclosed compounds and compositions, regardless of their protonation state based on the environment (such as pH).

[0085] When referring to the connection between two compounds or molecules, the term "connection" or "conjugation" as used herein means that the two molecules are bound by covalent bonds or by non-covalent bonds (e.g., hydrogen bonds or ionic bonds). In certain embodiments, where the term "connection" or "conjugation" refers to the binding between two molecules by non-covalent bonds, the binding between the two different molecules has a specific affinity of less than 1 x 10 in a physiologically acceptable buffer (e.g., buffered saline). -4 M (for example, less than 1x10 - 5 M, less than 1x 10 -6 M or less than 1x 10 -7 M) K D Unless otherwise indicated, the terms "linked" and "conjugated" as used herein may refer to a connection between a first compound and a second compound with or without any intervening atoms or groups of atoms.

[0086] The linking group used herein is one or more atoms, which connects a molecule or a part of a molecule to a second molecule or the second part of a molecule. Similarly, as used in the art, the term support is sometimes used interchangeably with a linking group. A linking group may include any number of atoms or functional groups. In certain embodiments, a linking group may not promote any biological or drug response, and is only used to connect two bioactive molecules.

[0087] The term "alkyl" as used herein means a saturated aliphatic hydrocarbon group containing 1-12 (e.g., 1-8, 1-6, 1-4, or 1-3) carbon atoms. The alkyl group can be straight chain or branched. 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.

[0088] Unless otherwise specified, the symbols used in this document are The use of means that any one or more groups according to the scope of the invention described herein can be attached (or linked) thereto.

[0089] As used herein, the term "including" is used herein to refer to, and is used interchangeably with, the phrase "including, but not limited to". Unless the context clearly indicates otherwise, the term "or" is used herein to refer to, and is used interchangeably with, the term "and / or".

[0090] As used in the claims herein, the phrase "consisting of excludes any elements, steps, or ingredients not specified in the claim. When used in the claims herein, the phrase "consisting essentially of limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0091] Oligonucleotide-based agents, including RNAi agents

[0092] As used herein, an "oligonucleotide-based agent" is an agent containing about 10-50 (e.g., 10-48, 10-46, 10-44, 10-42, 10-40, 10-38, 10-36, 10-34, 10-32, 10-30, 10-28, 10-26, 10-24, 10-22, 10-20, 10-18, 10-16, 10-14, 10-12, 12-50, 12-48, 12-46, 12-44, 12-42, 12-40, 12-38, 12-36, 12-34, 12-32, 12-30, 12-28, 12-26, 12-24, 12-22, 12-20, 12-18, 12- 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, 1 6 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 3 2, 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, 2 6 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 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38,30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48 or 48 to 50) nucleotides or nucleotide base pairs. In certain embodiments, the oligonucleotide-based agent has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in a cell. In certain embodiments, the oligonucleotide-based agent is capable of inhibiting the expression of a basic gene after delivery to a cell expressing the gene, and is referred to herein as an "oligonucleotide-based agent that inhibits expression". Gene expression can be inhibited in vitro or in vivo. ,

[0093] "Oligonucleotide-based agents" include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules and dicer substrates. In certain embodiments, oligonucleotide-based agents are single-stranded oligonucleotides, such as antisense oligonucleotides. In certain embodiments, oligonucleotide-based agents are double-stranded oligonucleotides. In certain embodiments, oligonucleotide-based agents are double-stranded oligonucleotides as RNAi agents.

[0094] In certain embodiments, the oligonucleotide-based agent is a "RNAi agent", which, as defined herein, is a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The RNAi agent used herein can act as follows: by an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or by any alternative mechanism or pathway. Although it is believed that, as the term is used herein, RNAi agents act primarily through RNA interference mechanisms, the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein comprise 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 targeted mRNA. The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0095] Typically, the RNAi agent can be composed of at least a sense strand (also referred to as a passenger strand) comprising a first sequence and an antisense strand (also referred to as a guide strand) comprising a second sequence. The length of the sense strand and antisense strand of the RNAi agent can each be 16 to 49 nucleotides in length. In certain embodiments, the sense strand and antisense strand of the RNAi agent are independently 17 to 26 nucleotides in length. In certain embodiments, the sense strand and antisense strand are independently 19 to 26 nucleotides in length. In certain embodiments, the sense strand and antisense strand are independently 21 to 26 nucleotides in length. In certain embodiments, the sense strand and antisense strand are independently 21 to 24 nucleotides in length. The sense strand and antisense strand can be the same length or different lengths. The RNAi agent includes an antisense strand sequence that is at least partially complementary to a sequence in a target gene, and after delivery to a cell expressing the target, the RNAi agent can inhibit the expression of one or more target genes in vivo or in vitro.

[0096] General oligonucleotide-based agents and specific RNAi agents can be composed of modified nucleotides and / or one or more non-phosphodiester bonds. "Modified nucleotides" used herein are nucleotides other than ribonucleotides (2'-hydroxy nucleotides). In certain 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 used herein include, but are not limited to, deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3'-linked (inverted) nucleotides, nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids, 2',3'-open ring nucleotide mimetics (unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linked) nucleotides, 2'-F-arabino nucleotides, 5'-Me, 2'-fluoro nucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. 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, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides.

[0097] In addition, one or more nucleotides of an oligonucleotide-based agent (such as an RNAi agent) can be linked by a non-standard connection or backbone (i.e., a modified internucleoside connection or a modified backbone). The modified internucleoside connection can be a covalent internucleoside connection without a phosphate. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, borophosphates with normal 3'-5' linkages, 2'-5' linked analogs of borophosphates, or borophosphates with reversed polarity (wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0098] It is not necessary to uniformly modify all positions in a given compound. Instead, more than one modification may be incorporated into a single oligonucleotide-based agent or even into a single nucleotide thereof.

[0099] RNAi agent sense strand and antisense strand can be synthesized and / or modified by methods known in the art. Additional disclosures related to RNAi agents can be found in, for example, public documents about modifications, see, for example, Arrowhead Pharmaceuticals, Inc. International Patent Application No. PCT / US2017 / 045446 (WO2018027106), which is also incorporated herein by reference in its entirety.

[0100] Modified nucleotides

[0101] In certain embodiments, the RNAi agent contains one or more modified nucleotides. As used herein, "modified nucleotides" are nucleotides other than ribonucleotides (2'-hydroxy nucleotides). In certain 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. The modified nucleotides used herein may include, but are not limited to: deoxyribonucleotides, nucleotide mimetics, 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'-open ring nucleotide mimetics (unlocked nucleobase analogs, represented herein as N UNA or NUNA), locked nucleotides (denoted herein as N LNA or NLNA), 3'-O-methoxy (2' internucleoside linked) nucleotides (represented herein as 3'-OMen), 2'-F-arabinonucleotides (represented herein as NfANA or Nf ANA), 5'-Me, 2'-fluoro nucleotides (represented herein as 5Me-Nf), morpholino nucleotides, vinylphosphonate deoxyribonucleotides (represented herein as vpdN), vinylphosphonate-containing nucleotides and cyclopropylphosphonate-containing nucleotides (cPrpN). 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (also referred to herein as 2'-fluoro nucleotides and represented herein as Nf), 2'-deoxy nucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also referred to herein as 2'-MOE and represented herein as NM), 2'-amino nucleotides and 2'-alkyl nucleotides. It is not necessary to uniformly modify all positions of a given compound. Instead, more than one modification can be incorporated into a single target RNAi agent or even in a single nucleotide thereof. The target RNAi agent sense and antisense strands can be synthesized and / or modified by methods known in the art.Modification at one nucleotide is independent of modification at another nucleotide.

[0102] Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines and 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 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy 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-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0103] In certain embodiments, all or substantially all nucleotides of RNAi agents are modified nucleotides. As used herein, the RNAi agents in which substantially all nucleotides present are modified nucleotides are RNAi agents in which four or less (i.e., 0, 1, 2, 3 or 4) nucleotides are ribonucleotides (i.e., unmodified) in both sense strand and antisense strand. As used herein, the sense strand in which substantially all nucleotides present are modified nucleotides is the sense strand in which two or less (i.e., 0, 1 or 2) nucleotides are unmodified ribonucleotides in the sense strand. As used herein, the antisense sense strand in which substantially all nucleotides present are modified nucleotides is the antisense strand in which two or less (i.e., 0, 1 or 2) nucleotides are unmodified ribonucleotides in the antisense strand. In certain embodiments, one or more nucleotides of RNAi agents are unmodified ribonucleotides.

[0104] Modified internucleoside linkages

[0105] In certain embodiments, one or more nucleotides of an RNAi agent are connected by a non-standard connection or backbone (i.e., a modified internucleoside connection or a modified backbone). The modified internucleoside connection or backbone includes, but is not limited to: a thiophosphate group (represented herein as lowercase "s"), a chiral thiophosphate, a thiophosphate, a dithiophosphate, a phosphotriester, an aminoalkyl-phosphotriester, an alkylphosphonate (e.g., a methylphosphonate or a 3'-alkylenephosphonate), a chiral phosphonate, a phosphinate, an aminophosphoramidate (e.g., a 3'-aminophosphoramidate, an aminoalkylphosphoramidate or a thiocarbonylphosphoramidate), a thiocarbonylalkyl-phosphonate, a thiocarbonylalkylphosphotriester, a morpholino connection, a borophosphate with a normal 3'-5' connection, an analog of a 2'-5' connection of a borophosphate, or a borophosphate with an inverted polarity, wherein adjacent nucleoside units are connected 3'-5' to 5'-3' or 2'-5' to 5'-2'. In certain embodiments, the internucleoside of modification is connected or the main chain lacks phosphorus atom.The internucleoside of modification lacking phosphorus atom is connected including but not limited to short chain alkyl or cycloalkyl sugar connection, mixed heteroatoms and alkyl or cycloalkyl sugar connection or one or more short chain heteroatoms or heterocyclic sugar connection.In certain embodiments, the internucleoside main chain of modification includes but not limited to siloxane main chain, sulfide main chain, sulfoxide main chain, sulfone main chain, acetyl and thioacetyl main chain, methylene acetyl and thioacetyl main chain, main chain containing olefin, aminosulfonate main chain, methylene imino and methylene hydrazine main chain, sulfonate and sulfonamide main chain, amide main chain and other N, O, S and CH with mixing 2 The main chain of the component.

[0106] In certain embodiments, the sense strand of the RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, the antisense strand of the RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, or both the sense strand and the antisense strand can independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds. In certain embodiments, the sense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate bonds, the antisense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate bonds, or both the sense strand and the antisense strand can independently contain 1, 2, 3, or 4 phosphorothioate bonds.

[0107] In certain embodiments, the sense strand of RNAi agent contains at least 2 phosphorothioate nucleoside interlinks. In certain embodiments, the at least 2 phosphorothioate nucleoside interlinks are located between the nucleotides of 1-3 positions from the 3' end of the sense strand. In certain embodiments, a phosphorothioate nucleoside interlink is located at the 5' end of the sense strand, and another phosphorothioate bond is located at the 3' end of the sense strand. In certain embodiments, two phosphorothioate nucleoside interlinks are located at the 5' end of the sense strand, and another phosphorothioate bond is located at the 3' end of the sense strand. In certain embodiments, the sense strand does not include any phosphorothioate nucleoside interlinks between nucleotides, but contains 1,2 or 3 phosphorothioate bonds between the terminal nucleotides on the 5' and 3' ends, and optionally there is an inverted abasic residue end cap. In certain embodiments, the targeting ligand is connected to the sense strand via a phosphorothioate bond.

[0108] In certain embodiments, the RNAi agent antisense strand contains 4 phosphorothioate nucleoside interlinks. In certain embodiments, the 4 phosphorothioate nucleoside interlinks are located between nucleotides at positions 1-3 from the 5' end of the antisense strand, and between nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In certain embodiments, 3 phosphorothioate nucleoside interlinks are located between positions 1-4 from the 5' end of the antisense strand, and the fourth phosphorothioate nucleoside interlink is located between positions 20-21 from the 5' end of the antisense strand. In certain embodiments, the RNAi agent contains at least 3 or 4 phosphorothioate nucleoside interlinks in the antisense strand.

[0109] In certain embodiments, the RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In certain embodiments, 2'-modified nucleosides are combined with modified internucleoside linkages.

[0110] Linking groups and delivery agents

[0111] In certain embodiments, the oligonucleotide-based medicament (such as the RNAi medicament described herein) contains or is conjugated to one or more non-nucleotide groups, including, but not limited to, a linking group or a delivery agent. The non-nucleotide group can enhance the targeting, delivery or attachment of the RNAi medicament. The example of a linking group is provided in Table 4. The non-nucleotide group can be covalently linked to the 3' and / or 5' ends of the sense strand and / or antisense strand. In certain embodiments, the RNAi medicament contains a non-nucleotide group connected to the 3' and / or 5' ends of the sense strand. In certain embodiments, the non-nucleotide group is connected to the 5' end of the sense strand of the RNAi medicament. The non-nucleotide group can be directly or indirectly connected to the RNAi medicament via a joint / linking group. In certain embodiments, the non-nucleotide group is connected to the RNAi medicament via an unstable, cleavable or reversible bond or joint.

[0112] In certain embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached to improve the cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In certain embodiments, the non-nucleotide group enhances the endocytosis of the RNAi agent.

[0113] The RNAi agents described herein can be synthesized with reactive groups such as amino groups (also referred to herein as amines) at the 5'-end and / or 3'-end. The reactive groups can then be used to attach targeting moieties using typical methods in the art.

[0114] For example, in certain embodiments, the RNAi agent is synthesized with an NH 2 -C 6 The RNAi agents disclosed herein with a group. The terminal amino group can then react to form a conjugate, for example, with a specific group, which includes a compound with affinity for one or more integrins (i.e., and an integrin targeting ligand) or a PK enhancer. In certain embodiments, RNAi agents disclosed herein are synthesized with one or more alkyne groups at the 5'-end of the sense strand of the RNAi agent. The terminal alkyne group can then react to form a conjugate, for example, with a group including a targeting ligand.

[0115] In certain embodiments, an RNAi agent with a linking group is synthesized, which can then facilitate the covalent connection of the RNAi agent to a targeting ligand, a targeting group, a PK / PD modulator, or another type of delivery agent. The linking group can be connected to the 3' and / or 5' end of the sense strand or antisense strand of the RNAi agent. In certain embodiments, the linking group is connected to the sense strand of the RNAi agent. In certain embodiments, the linking group is conjugated to the 5' or 3' end of the sense strand of the RNAi agent. In certain embodiments, the linking group is conjugated to the 5' end of the sense strand of the RNAi agent. Examples of linking groups include, but are not limited to: C6-SS-Alk-Me, reactive groups such as primary amines and alkynes, alkyls, abasic residues / nucleotides, amino acids, tri-alkyne functionalized groups, ribitols, and / or PEG groups.

[0116] A linker or linking group is a connection between two atoms that connects a chemical group (such as an RNAi agent) or a target segment to another chemical group (such as a targeting ligand, a targeting group, a PK / PD modulator or a delivery agent) or a target segment via one or more covalent bonds. Unstable connections contain unstable bonds. The connection may optionally include a spacer that increases the distance between the two connected atoms. The spacer may further increase flexibility and / or length to the connection. The spacer includes, but is not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, and aralkyne; each of which may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the foregoing list is not intended to limit the scope of the specification.

[0117] In certain embodiments, without using additional joints, the targeting group is connected to the RNAi medicament. In certain embodiments, the targeting group is designed to have a joint that is easy to exist to promote connection with the RNAi medicament. In certain embodiments, when including two or more RNAi medicaments in the composition, the same joint can be used to connect two or more RNAi medicaments to their respective targeting groups. In certain embodiments, when including two or more RNAi medicaments in the composition, different joints are used to connect two or more RNAi medicaments to their respective targeting groups.

[0118] RNAi agents (whether modified or unmodified) can contain 3' and / or 5' targeting groups, linking groups, and / or can be conjugated to or include PK / PD modulators. Any RNAi agent sequence described herein that contains 3' or 5' targeting ligands, targeting groups, PK / PD modulators or linking groups may alternatively not contain 3' or 5' targeting ligands, targeting groups, linking groups or PK / PD modulators, or may contain different 3' or 5' targeting ligands, targeting groups, linking groups or PK / PD modulators, including, but not limited to, those shown in Tables 2 and 3. Any RNAi agent duplex listed in Table A, whether modified or unmodified, may further contain a targeting ligand, targeting group, linking group or PK / PD modulator, and the targeting group or linking group may be attached to the 3' or 5' end of the sense strand or antisense strand of the RNAi agent duplex.

[0119] In certain embodiments, the linking group can be synthetically conjugated to the 5' or 3' end of the sense strand of the RNAi agent described herein. In certain embodiments, the linking group is synthetically conjugated to the 5' end of the sense strand of the RNAi agent. In certain embodiments, the linking group conjugated to the RNAi agent can be a triyne linking group.

[0120] Examples of certain modified nucleotides, capping moieties, and linking groups are provided in Table 4.

[0121] Table 4: Structures representing various modified nucleotides, capping moieties, and linking groups

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Alternatively, other linking groups known in the art may be used.

[0129] In addition to or alternatively to connecting the RNAi agent to one or more targeting ligands, targeting groups and / or PK / PD modulators, in certain embodiments, a delivery agent can 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 may include, but is not limited to, a polymer (such as an amphiphilic polymer), a membrane active polymer, a peptide, melittin, melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine, or consisting thereof.

[0130] In certain embodiments, the RNAi medicament can be combined with lipid, nanoparticle, polymer, liposome, micelle, DPC or other delivery system available in the art. RNAi medicament can also be chemically conjugated to targeting group, lipid (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticle, polymer, liposome, micelle, DPC (see, for example, WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169 and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference) or other delivery system available in the art.

[0131] Pharmaceutical composition

[0132] In certain embodiments, the present disclosure provides a method comprising 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-305a, LP-306a, LP-307a, LP-308a, LP-309a, LP-310a, LP-311a, LP-312a, LP-313a, LP-314a, LP-315a, LP-316a, LP-317a, LP-318a, LP-319a, LP-319b 4a, 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, L P-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-287 b. 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, C 22b, 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,One or more 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), a pharmaceutical composition consisting of, or consisting essentially of, the same. ,

[0133] "Pharmaceutical composition" used herein comprises a pharmacologically effective amount of active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances intentionally included in drug delivery systems in addition to active pharmaceutical ingredients (API, therapeutic products). Excipients do not play a therapeutic effect or are not intended to play a therapeutic effect at the expected dose. Excipients may play the following roles: a) assist in the processing of drug delivery systems during manufacture, b) protect, support or enhance the stability, bioavailability or patient acceptability of API, c) assist in product identification, and / or d) enhance the overall safety and effectiveness of API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0134] Excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0135] The pharmaceutical compositions described herein may contain other additional components that are typically present in pharmaceutical compositions. In certain embodiments, the additional components are pharmaceutically active substances. Pharmaceutically active substances include, but are not limited to: antipruritic drugs, astringents, local anesthetics or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers and / or vaccines.

[0136] The pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavors, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They may also contain other agents with known therapeutic benefits.

[0137] The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be performed by any means known in the art, such as, but not limited to, topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by 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; subdermal (e.g., via implant device), intracranial, intraparenchymal, intrathecal and intraventricular administration. In certain embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, lozenges, hard or soft gelatin capsules, solutions, emulsions or suspensions. Rectal administration can also be performed, for example, using suppositories; topical or transdermal administration, for example, using ointments, creams, gels or solutions; or parenteral administration, for example, using injectable solutions.

[0138] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, In some embodiments, the composition of the present invention can be a phosphate buffered saline (PBS) or a phosphate buffered saline (PBS). It should be stable under manufacturing and storage conditions, and should be preserved for the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating agent such as lecithin by maintaining the required particle size (in the case of a dispersion) and by using a surfactant. In many cases, it will be preferred to include an isotonic agent in the composition, for example, sugar, a polyol such as mannitol, sorbitol and sodium chloride. By including an agent such as aluminum monostearate and gelatin that delays absorption in the composition, the extended absorption of the injectable composition can be achieved.

[0139] Sterile injectable solutions can be prepared as follows: the active compound is incorporated in the required amount in an appropriate solvent with a combination of one or more of the ingredients listed above as required, followed by filtration sterilization. Typically, dispersions are prepared as follows: the active compound is incorporated into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. For sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze drying, which produce powders of the active ingredient and any additional desired ingredients from their previously sterile filtered solutions.

[0140] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous formulation of any ligand described herein, which may be in microcrystalline form, such as in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present any ligand described herein for intra-articular and ophthalmic administration.

[0141] The active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Those skilled in the art will appreciate the methods for preparing such preparations. Liposomal 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.

[0142] The pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritic 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" means the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or preventive result.

[0143] Containing 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, LP-262c, LP-269c, LP-273c,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) A medicament of the invention is also an object of the invention, as is a method for producing such a medicament, which method comprises the step of: reacting one or more of 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-2 The compounds of LP-73c, 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 known therapeutic benefits are prepared in a pharmaceutically acceptable form.

[0144] The described 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 those containing 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-4 65b, LP-466b, LP-493b (2′ internal), (2C8C12)b, (2C6C10)b, HO-C16b, C16b, C22b, LP-1 28c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243 c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, LP-273c, LP- Pharmaceutical compositions of compounds of the invention may be packaged or included in a kit, container, bag or dispenser. 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-2 60a, 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-3 03a, 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-4 56a, 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,Compounds of 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 those containing 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, 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,Pharmaceutical compositions of compounds of LP-462c, LP-463c, LP-464c, LP-465c, LP-466c and LP-493c(2' internal) can be packaged in pre-filled syringes or vials.

[0145] Treatment methods and inhibition of expression

[0146] 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-29 6c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, LP Compounds of 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 condition that would benefit from the administration of such a compound. In certain embodiments, the formulas disclosed herein 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-277a, LP-278a, LP-279a, LP-280a, LP-281a, LP-282a, LP-283a, LP-284a, LP-285a, LP-286a, LP-287a, LP-288a, LP-289a, LP-300a, LP-310a, LP-311a, LP-312a, LP-313a, LP-314a, LP-315a, LP-316a, LP-317a, LP-318a, LP-319a, LP-320a, LP-321a, LP-324a, LP-325 -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-441 a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (inside 2′), (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-2 43b, 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, L P-441b, LP-456b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2′ internal), (2C8C12)b, (2C 6C10)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, L P-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-43 Compounds of LP-5c, 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 subjects (e.g., humans) who would benefit from reduction and / or inhibition of expression of target mRNA and / or protein levels, for example, subjects who have been diagnosed with or are suffering from symptoms associated with a central nervous system disease or lesion.

[0147] In certain embodiments, a therapeutically effective amount of 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-301a, LP-302a, LP-303a, LP-304a, LP-305a, LP-306a, LP-307a, LP-308a, LP-309a, LP-300b, LP-301c, LP-301d, LP-301d, LP-302d, LP-303a, LP-304a, LP-306b, LP-306c, LP-307a, LP-308a, LP-309b, LP-300c, LP-300d ... P-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-46 2a, 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-2 00b, 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-43 0b, 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, One or more compounds of 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). Treatment of a subject may include therapeutic and / or prophylactic treatment. A therapeutically effective amount 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-310 a. LP-383a, LP-395a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-44 1a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (2C8C12)a, (2C6C10)a, H O-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, L P-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-183 r-c, LP-200c, LP-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-25 9c, LP-260c, LP-262c, LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287 c, 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 subject can be a human, a patient or a human patient. The subject can be an adult, a teenager, a child or an infant. The pharmaceutical compositions described herein can be administered to humans or animals. ,

[0148] 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, 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 pathology associated with the target gene, or having a disease or pathology mediated at least in part by expression of the target gene. In certain embodiments, 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-283 a. 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, L P-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (2C8C12)a, (2C6C10)a, HO-C 16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-24 3b, 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- The compounds of 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 used to treat or manage clinical manifestations of a subject having a disease or condition that would benefit from or is at least partially mediated by a reduction in the mRNA of a target gene. A therapeutically effective amount 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-283 described herein is administered to a subject. a. 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-465 b, 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-286 c, 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) One or more of the compounds or compositions. In certain embodiments, the methods disclosed herein comprise administering to the subject to be treated a composition comprising 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, 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,Compounds of 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). In certain embodiments, a prophylactically effective amount of the described 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-286 a. 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, L P-465a, LP-466a, LP-493a (2′ internal), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132b, LP-183b, LP-183 r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-257b, LP-259b, LP-260b, LP-262b, LP-26 9b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289b, LP-290b, LP-293b, LP-296b, LP-300b, LP-303 b. 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), thereby treating the subject by preventing or inhibiting at least one symptom.

[0149] In certain embodiments, the present disclosure provides a method for treating a disease, disorder, condition or pathological condition mediated at least in part by target gene expression in a patient in need thereof, wherein the method comprises administering to the patient a compound 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-288a, LP-289a, LP-300a, LP-310a, LP-311a, LP-312a, LP-313a, LP-314a, LP-315a, LP-316a, LP-317a, LP-318a, LP-319a, LP-320a, LP-321a, LP-322a, LP-323a, LP-324a, LP-325 a. 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-C16 a, 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-3 10b,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,L P-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).

[0150] In certain embodiments, administration of a subject 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-291a, LP-293a, LP-294a, LP-295a, LP-296a, LP-297a, LP-298a, LP-300a, LP-301b, LP-302b, LP-303c, LP-304d, LP-305d, LP-306d, LP-307d, LP-308d, LP-309d, LP-310d, LP-311d, LP-312d, LP-313d, LP-314d, LP-315d P-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-44 0a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-132 b. 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-39 6b, 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-289 c. 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-4 The gene expression level and / or mRNA level of the target gene in a subject treated with a compound of LP-30c, 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 reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or greater than 99%. The gene expression level and / or mRNA level in the subject may be reduced in cells, cell collections and / or tissues of the subject.

[0151] In certain embodiments, a subject who has been administered 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 described herein is compared to a subject prior to administration of the compound, or compared to a subject who has not received the compound. , 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-4 40a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-128b, LP-13 2b, 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-3 96b, 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-2 89c, LP-290c, LP-293c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP-429c, The target protein level in a subject treated with a compound of 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 reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%. The protein level in the subject may be reduced in cells, cell collections, tissues, blood, and / or other fluids of the subject.

[0152] By any method known in the art, the reduction of target mRNA level and / or target protein level can be assessed. As used herein, the reduction or reduction of target mRNA level and / or protein level are collectively referred to as the reduction or reduction of target gene and / or protein level in this article, or suppress or reduce the expression of target gene.

[0153] In certain 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, LP-262c,LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-29 3c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, LP- The compounds of 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 prepare a pharmaceutical composition for treating a disease, lesion or symptom mediated at least in part by target gene expression. In certain embodiments, the disease, lesion or symptom mediated at least in part by target gene expression is a central nervous system disease or lesion.

[0154] In certain embodiments, the method of treating a subject is dependent on the subject's weight. In certain 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-291a, LP-293a, LP-294a, LP-295b, LP-296c, LP-297d, LP-298d, LP-299a, LP-300d, LP-301d, LP-302d, LP-303d, LP-304d, LP-305d, LP-306d, LP-307d, LP-308d, LP-309d, LP-310d, LP-311d, LP-312d, LP-313d, LP-314d, LP-315d, LP-316d, LP-317d, LP-318d, LP-319d, LP-320d, LP-321d, LP-323d, LP-324d, LP-325d, LP-326d, LP-327d, LP-328d 93a, 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-44 0a, LP-441a, LP-456a, LP-462a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (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-4 66b, 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, L Compounds of P-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). 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-270a, LP-271a, LP-272a, LP-273a, LP-274a, LP-275a, LP-276a, LP-277a, LP-278a, LP-279a, LP-280a, LP-281a, LP-282a, LP-283a, LP-284a, LP-285a, LP-286a, LP-287a, LP-288a, LP-289a, LP-290a, LP-300a, LP-310 LP-273a, LP-274a, LP-276a, LP-283a, LP-286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-30 0a, 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-200 b. 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,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).

[0155] In certain embodiments, 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-305a, LP-306a, LP-307a, LP-308a, LP-309a, LP-310a, LP-311a, LP-312a, LP-313a, LP-314a, LP-315a, LP-316a, LP-317a, LP-318a, LP-319a, LP-319b, LP-319c, LP-319d, LP-319e, LP-319f, LP-319f, LP-319g, LP-319h, LP-319h, LP-319i 4a, 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, L P-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-287 b. 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, C 22b, 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-40 9c, 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), which means that two doses are administered to the subject in a short (e.g., less than 24 hours) period of time. In certain embodiments, about half of the desired daily amount is administered in the initial administration, and the remaining about half of the desired daily amount is administered approximately 4 hours after the initial administration.

[0156] In certain 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-301a, LP-302a, LP-303a, LP-304a, LP-305a, LP-306a, LP-307a, LP-308a, LP-309a, LP-310a, LP-311a, LP-312a, LP-313a, LP-314a, LP-315a, LP-316a, LP-317a, LP-318a, LP-319a, LP-319b, LP-311c, LP-311d, LP-311d 3a, 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, L P-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-286 b. 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-C16 b. 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,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). 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-275a, LP-276a, LP-277a, LP-278a, LP-279a, LP-280a, LP-281a, LP-282a, LP-283a, LP-284a, LP-285a, LP-286a, LP-287a, LP-288a, LP-289a, LP-290a, LP-301a, LP-302a, LP-303a, LP-304a, LP-305a, LP-306a, LP-307b, LP-308a, LP-309a, LP-310a, LP-311a, LP-312b, LP-313a, LP-314a, LP-315b, LP-316a 6a, 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′ inside), (2C8C12)a, (2C6C10)a, HO-C16a, C16 a, 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-28 7b, 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-45 6b, LP-462b, LP-463b, LP-464b, LP-465b, LP-466b, LP-493b (2′ internal), (2C8C12)b, (2 C6C10)b, HO-C16b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, L P-232c, LP-233c, LP-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260 c, 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).

[0157] In certain 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, LP-262c,LP-269c, LP-273c, LP-274c, LP-276c, LP-283c, LP-286c, LP-287c, LP-289c, LP-290c, LP-2 93c, LP-296c, LP-300c, LP-303c, LP-304c, LP-310c, LP-383c, LP-395c, LP-396c, LP-409c, L Compounds of P-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 to treat diseases, lesions or symptoms mediated at least in part by target gene expression. In certain embodiments, the disease, lesion or symptom mediated at least in part by target gene expression is a central nervous system disease or lesion.

[0158] Another aspect of the invention provides a method for reducing target gene expression in vivo, the method comprising introducing into a cell a compound 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-294a, LP-295a, LP-296a, LP-297a, LP-298a, LP-299a, LP-300a, LP-301a, LP-302a, LP-303a, LP-304a, LP-305a, LP-306a, LP-307a, LP-308a 6a, 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-276 b. 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), wherein the compound comprises an RNAi agent that is at least substantially complementary to a target gene.

[0159] In certain embodiments, the cell is a central nervous system cell. In certain embodiments, the cell is in a subject. In certain embodiments, the subject has been diagnosed with a disease or lesion treated, prevented or improved by reducing the expression of a target gene. In certain embodiments, the disease or lesion is a central nervous system disease or lesion selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and Lewy body disease.

[0160] Another aspect of the invention provides any one of the lipid PK / PD regulators conjugated to an oligonucleotide-based agent described herein for use in treating, preventing or ameliorating a disease or lesion. In certain embodiments, the disease or lesion is a central nervous system disease or lesion selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and Lewy body disease.

[0161] Cells, tissues and non-human organisms

[0162] Contemplated are cells, tissues, and non-human organisms including those of the formulae described herein 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-4 63a, 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-4 31b, 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,At least one of 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). 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-284a, LP-286a, LP-287a, LP-288a, LP-289a, LP-300a, LP-311a, LP-312b, LP-313c, LP-314d, LP-315d, LP-316e, LP-317d, LP-318e, LP-319d, LP-320e, LP-321d, LP-323d, LP-324e, LP-325f, LP-326f, LP-327e -286a, LP-287a, LP-289a, LP-290a, LP-293a, LP-296a, LP-300a, LP-303a, LP-304a, LP-310a, LP-383a, LP-3 95a, LP-396a, LP-409a, LP-429a, LP-430a, LP-431a, LP-435a, LP-439a, LP-440a, LP-441a, LP-456a, LP-462 a, LP-463a, LP-464a, LP-465a, LP-466a, LP-493a (2′ inside), (2C8C12)a, (2C6C10)a, HO-C16a, C16a, C22a, LP-1 28b, LP-132b, LP-183b, LP-183r-b, LP-200b, LP-232b, LP-233b, LP-242b, LP-243b, LP-245b, LP-249b, LP-2 57b, LP-259b, LP-260b, LP-262b, LP-269b, LP-273b, LP-274b, LP-276b, LP-283b, LP-286b, LP-287b, LP-289 b. 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-C1 6b, C16b, C22b, LP-128c, LP-132c, LP-183c, LP-183r-c, LP-200c, LP-232c, LP-233c, L P-242c, LP-243c, LP-245c, LP-249c, LP-257c, LP-259c, LP-260c, LP-262c, LP-269c, L The invention relates to a method for delivering a compound of the invention comprising delivering the compound of the invention to a cell, tissue or non-human organism through the method of claim 1 , wherein the compound of the invention comprises a compound of the invention comprising: ... In some embodiments, the cell is a mammalian cell, including but not limited to a human cell. In some embodiments, the cell is a central nervous system cell.

[0163] The embodiments and items provided above are now illustrated with the following non-limiting examples. Example

[0164] The following examples are not limiting and are intended to illustrate certain embodiments disclosed herein.

[0165] Unless otherwise expressly stated, the numbers used to refer to compounds of a given embodiment are made with reference only to that particular embodiment and not to any other embodiment disclosed herein. For example, compound 1 in "Synthesis of LP-183 Phosphoramidite" in Example 2 is different from and does not represent compound 1 in "Synthesis of LP-232p" in Example 2. Similarly, it should be understood that specific compounds disclosed herein may be identified with different numbers in different embodiments. The compounds disclosed in the various tables throughout the detailed description (i.e., LPXXa, LPXXb, and LPXX-p, where XX is a number) are consistently referenced in the embodiments herein.

[0166] It should be understood that, unless expressly stated otherwise, the use of the term "EDC" in the examples herein refers to the commercially available EDC hydrochloride salt.

[0167] Example 1. Synthesis and composition of RNAi agents.

[0168] Described below are general protocols for the synthesis of certain RNAi agents and their conjugates, which are exemplified in the non-limiting Examples set forth herein.

[0169] Synthesis of RNAi Agents. RNAi agents can be synthesized using methods generally known in the art. With respect to the synthesis of RNAi agents exemplified in the Examples set forth herein, the sense and antisense strands of the RNAi agents were synthesized according to the solid phase phosphoramidite technique used in oligonucleotide synthesis. Depending on the scale, the sense and antisense strands of the RNAi agents were synthesized using (Bioautomation), (Bioautomation) or Oligopilot 100 (GE Healthcare). or Synthesis was performed on a solid support made from Prime Synthesis, Aston, PA, USA) or polystyrene (derived from Kinovate, Oceanside, CA, USA). All RNA and 2' modified RNA phosphoamidates were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA) or Hongene Biotech (Morrisville, NC, USA). Specifically, the following 2'-O-methylphosphoamidates used include the following: (5'-O-dimethoxytrityl-N 6-(Benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoamidate, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoamidoester, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoamidate and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoamidate. 2'-Deoxy-2'-fluoro-phosphoamidate and 2'-O-propargylphosphoamidate carry the same protecting groups as 2'-O-methylphosphoamidate. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoamidate was purchased from Glen Research (Virginia). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoamidate was purchased from ChemGenes. The following UNA phosphoamidates used included the following: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-sec-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoamidate, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-sec-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoamidate Esters, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-sec-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoamidate and 5'-(4,4'-dimethoxytrityl)-2',3'-sec-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoamidate. To introduce the phosphorothioate bond, a 100 mM solution of 3-phenyl 1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile or a 200 mM solution of xanthane hydride (TCI America, Portland, OR, USA) in pyridine was used.

[0170] TFA amino-linked phosphoamidate was also purchased commercially (ThermoFisher) to introduce the (NH2-C6) reactive group linker. TFA amino-linked phosphoamidate was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves 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. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me) and 60 seconds (2'F). Trialkyne-containing aminophosphites were synthesized to introduce various (TriAlk#) linkers. When used in conjunction with the RNAi agents presented in certain embodiments herein, the trialkyne-containing aminophosphites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidates were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves 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 min (RNA), 90 sec (2'O-Me) and 60 sec (2'F).

[0171] For some RNAi agents, a linker such as a C6-SS-C6 or 6-SS-6 group was introduced at the 3' terminal end of the sense strand. Pre-loaded resins were commercially obtained with various linkers. Alternatively, for some sense strands, a dT resin was used and various linkers were then added by standard phosphoamidate synthesis.

[0172] Cleavage and deprotection of support-bound oligomers. After completion of solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt.% aqueous methylamine and 28%-31% ammonium hydroxide solution (Aldrich) at 30° C. for 1.5 hours. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0173] Purification. Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13 μm column (available from Tosoh Biosciences) and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile, and buffer B was identical to buffer A except that 1.5 M sodium chloride was added. UV traces were recorded at 260 nm. Appropriate fractions were combined and then run on size exclusion HPLC using a GE Healthcare XK16 / 40 column filled with Sephadex G25 fine (available from Sigman Aldrich) using a running buffer of 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile or filtered water.

[0174] Annealing. The complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1× PBS (phosphate buffered saline, 1×, Corning, Cellgro) to form RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the absorbance of the solution on a UV-Vis spectrometer in 1× PBS. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was 0.037 mg / (mL·cm), or calculated from the extinction coefficient determined experimentally.

[0175] Example 2. Synthesis of lipid PK / PD modulator precursors

[0176] Synthesis of LP-183 Phosphoramidite

[0177]

[0178] To a solution of compound 2 (2.00 g) in DCM was added TEA (2.27 mL) at room temperature, followed by dropwise addition of compound 1 (4.931 g). The mixture was then stirred at room temperature for 2 h. The mixture was then filtered. The white solid was dried overnight. The product was a white solid, 4.267 g, 74% yield. LC-MS: [M+H] calculated value was 356.35, and the found value was 356.63.

[0179]

[0180] Compound 3 (0.61 g) was added to a mixture of compound 1 (2.54 g) in 120 mL DCM at room temperature, followed by dropwise addition of compound 2 (5.37 g). The mixture was then stirred overnight at room temperature. 5 mL TEA was added, followed by diatomaceous earth. After removing the solvent in a vacuum, the residue was loaded onto a 40 g column by a dry method. The product was purified using a gradient of hexanes (2% TEA) to 50% EtOAc (2% TEA) in hexanes (2% TEA). The product was a white waxy solid, 3.462 g, 87% yield. LC-MS: [M+H] calculated value was 556.46, and the measured value was 556.64.

[0181] Synthesis of LP-183r-p

[0182]

[0183] Compound 2 (299 mg) and EDC (498 mg) were added to a solution of compound 1 (312 mg) in 10 mL DCM at room temperature. The reaction mixture was stirred at room temperature for 1 h. After the solvent was removed in vacuo, the residue was dried and loaded onto a 12 g column. Hexanes to EtOAc were used as mobile phases. The product was a clear oil, 408 mg, 75% yield. LC-MS: [M+H] calculated value was 230.10, and the measured value was 230.34.

[0184]

[0185] Compound 2 (516 mg) and TEA (0.745 mL) were added to a solution of compound 1 (408 mg) in 20 mL DCM at room temperature. The reaction mixture was stirred overnight at room temperature. After removing the solvent in vacuo, the residue was recrystallized in MeOH. The product was a white solid, 555 mg, 88% yield. LC-MS: [M+H] calculated value was 356.35, and the measured value was 356.45.

[0186]

[0187] To a mixture of compound 1 (200 mg) in 10 mL of DCM was added compound 3 (33.2 mg) at room temperature, followed by dropwise addition of compound 2 (339 mg). The mixture was then stirred at room temperature overnight. 1 mL of TEA was added, followed by some After removing the solvent in vacuo, the residue was dry loaded onto a 4g column. A gradient of hexanes (2% TEA) to 50% EtOAc (2% TEA) in hexanes (2% TEA) was used as the mobile phase. The product was a white waxy solid, 95 mg, 30% yield. LC-MS: [M+H] calculated value was 556.46, found value was 556.82.

[0188] Synthesis of LP-232p

[0189]

[0190] Palmitoyl chloride (100 mg) was stirred in a solution of cis-4-(boc-amino)cyclohexylamine (0.0819 g) in 5 mL of DCM. After the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (hexanes to EtOAc). The product was 52 mg, 31%.

[0191]

[0192] 2 mL of dioxane: HCl (4 N) was added to 1 (0.0520 g) until the boc deprotection was complete. After removing the solvent in vacuo, 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 the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (DCM to 20% MeOH in DCM). The product was 45 mg, 65%.

[0193]

[0194] 2 mL of dioxane: HCl (4 N) was added to 1 (0.0449 g) until the OtBu deprotection was complete. After removing the solvent in vacuo, 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 the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (DCM to 20% MeOH in DCM). The product was 30 mg, 58%.

[0195] Synthesis of LP-233p

[0196]

[0197] Palmitic acid 1 (0.100 g) was stirred in a solution of 2 (0.0693 g), COMU (0.166 g), DIPEA (0.16 mL) in 5 mL of DCM. After the suspension was stirred overnight (heated at 40 °C), water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (hexanes to EtOAc). The product was 96 mg, 69%.

[0198]

[0199] 2 mL of dioxane: HCl (4 N) was added to 1 (0.0955 g) until the boc deprotection was complete. After removing the solvent in vacuo, the residue was stirred in a solution of 2 (0.0581 g), DIPEA (0.11 mL) and COMU (0.114 g) in 5 mL of DCM. After the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (DCM to 20% MeOH in DCM). The product was 68 mg, 54%.

[0200]

[0201] 2 mL of dioxane: HCl (4 N) was added to 1 (0.068 g) until the deprotection of otBu was complete. After removing the solvent in vacuo, 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 the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by column (DCM to 20% MeOH in DCM). The product was 22 mg, 28%.

[0202] Synthesis of LP-242p

[0203]

[0204] Palmitic acid (0.100 g) was stirred in 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 the suspension was stirred overnight (heated at 40 °C), water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0205]

[0206] 1 (0.017 g) was treated with HCl: dioxane and after 1 h, the crude reaction was dried in vacuo. A solution of 2 (0.0095 g), COMU (0.0186 g), DIPEA (0.0134 g) in 5 mL DCM was added. After the suspension was stirred, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0207]

[0208] 2 mL of dioxane: HCl (4 N) was added to 1 (0.121 g) until the deprotection of otBu was complete. After removing the solvent in vacuo, the crude 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 the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0209] Synthesis of LP-243p

[0210]

[0211] 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), DIPEA (0.161 mL) in 5 mL of DCM. After the suspension was stirred overnight (heated at 40 °C), water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0212]

[0213] 1 (0.0200 g) was treated with HCl: dioxane and after 1 h, the crude reaction was dried in vacuo. A solution of 2 (0.0119 g), COMU (0.0232 g), DIPEA (0.022 mL) in 5 mL DCM was added. After the suspension was stirred, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0214]

[0215] 2 mL of dioxane: HCl (4 N) was added to 1 (0.121 g) until the deprotection of otBu was complete. After removing the solvent in vacuo, the 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 the suspension was stirred overnight, water was added and the organic layer was extracted with DCM and purified by Na 2 SO 4 After filtration, the solvent was concentrated to dryness and the crude product was purified by flash chromatography.

[0216] Synthesis of LP-245p

[0217]

[0218] To a mixture of 1 (2.08 g) and 2 (1.98 g) in 50 mL of toluene was added TEA at room temperature. The reaction mixture was stirred at 90 ° C overnight. After cooling to room temperature, EtOAc and water were added for post-treatment. Purification was on a 40 g column. A gradient of hexanes to 30% EtOAc in hexanes was used for purification. The product was a light yellow oil, 1388 mg, 51%. LC-MS: [M+H] calculated value was 339.21, and the found value was 339.62.

[0219]

[0220] To a mixture of 1 (0.241 g) in MeOH / THF (4 mL / 4 mL) was added 1 N NaOH (6 mL) at room temperature. The reaction mixture was stirred at 60 °C for 1 h. After the organic solvent was removed in vacuo, 1 N HCl was added to adjust the mixture to pH ∼1. Then NaHCO 3The pH was adjusted to between 7 and 8. DCM was added for post-treatment. After the DCM was removed in vacuo, the residue was placed in high vacuum for 2 h. The residue was diluted with DCM, and then 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 1N HCl, NaHCO 3 and washed with brine. Purification was on a 12 g column. A gradient from hexanes to EtOAc was used for purification. The product was a brown oil, 285 mg, 74%. LC-MS: [M+H] calculated value was 540.34, found value was 541.07.

[0221]

[0222] To a mixture of 1 (0.0740 g) and Pd / C in EtOAc was charged H 2 (1 atmosphere). The reaction mixture was stirred at room temperature for 4 h. The mixture was stirred at room temperature for 2 h. Water was added for post-treatment. Purification was performed on a 12 g column. A gradient of DCM to 20% MeOH in DCM was used for purification. The product was a clear oil, 43 mg, 37%. LC-MS: [M+H] was calculated to be 836.71 and the found value was 837.68.

[0223]

[0224] A solution of 1 (0.0430 g) in 4N HCl / dioxane (3 mL) was stirred at room temperature overnight. After removing the solvent in a vacuum, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL DMF, 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 h. After removing the solvent in a vacuum, the residue was loaded on a 4 g post. The gradient of DCM to 20% MeOH in DCM was used for purification. The product was a light yellow oil, 34 mg, 37%. LC-MS: the calculated value of [M+H] was 928.64, and the measured value was 929.59.

[0225] Synthesis of LP-249p

[0226]

[0227] To a mixture of 1 (0.0600 g) and 2 (0.161 mL) in 4 mL DCM was added TEA (0.111 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 h. Water was added for post-treatment. Purification was on a 4 g column. A gradient from hexanes to EtOAc was used for purification. The product was a white solid, 74 mg, 60%. LC-MS: [M+H] calculated value was 465.41, and the found value was 465.91.

[0228]

[0229] To a solution of 1 (0.0740 g) in DCM was added TFA (50% in DCM) at room temperature. The reaction mixture was stirred at room temperature for 0.5 h. The solvent was removed in vacuo, and the residue was placed under high vacuum for 2 h. The residue was dissolved in DMF, and 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 h. The solvent was removed in vacuo. Purification was on a 12 g column. A gradient of DCM to 20% MeOH in DCM was used for purification. The product was a white solid, 56 mg, 58%. LC-MS: [M+H] was calculated to be 609.48, and the found value was 610.29.

[0230]

[0231] A solution of 1 (0.0560 g) in 4N HCl / dioxane (3 mL) was stirred at room temperature overnight. After removing the solvent in a vacuum, the residue was placed under high vacuum for 3 h. The residue was dissolved in 2 mL DMF, 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 h. After removing the solvent in a vacuum, the residue was loaded onto a 4 g post. The gradient of DCM to 20% MeOH in DCM was used for purification. The product was a pale solid, 16 mg, 25%. LC-MS: the calculated value of [M+H] was 701.42, and the measured value was 702.20.

[0232] Synthesis of LP-257p

[0233]

[0234] A solution of 1 (0.100 g) in 3 mL of DCM was added to 2 (0.331 mL) and TEA (0.304 mL) at room temperature. The reaction was stirred at room temperature for 1 h. EtOAc was added to dilute, and the mixture was then washed with 1N HCl, NaHCO 3The product was washed with brine. After removing the solvent in vacuo, the residue was loaded onto a 4 g column. A gradient from hexanes to EtOAc was used for purification. The product was a white solid, 134 mg, 58%. LC-MS: [M+H] Calculated value 422.36, found value 422.79.

[0235]

[0236] A solution of 1 (0.134 g) in 4N HCl / dioxane (8 mL) was stirred at room temperature overnight. After removing the solvent in vacuo, the residue was placed under high vacuum for 3 h. The product was a white solid, 118 mg, which was used in the next step without further purification. LC-MS: [M+H] calculated value was 366.30, found value was 366.62.

[0237]

[0238] A solution of 1 (0.0490 g) in 3 mL of DMF was added to COMU (0.086 g), DIPEA (0.047 mL) and 2 (0.045 g) at room temperature. The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc and then washed with 1N HCl, NaHCO 3 The product was washed with brine. After removing the solvent in vacuo, the residue was loaded onto a 4 g column. A gradient from hexanes to EtOAc was used for purification. The product was a white solid, 23 mg, 33%. LC-MS: [M+H] calculated value was 514.29, found value was 514.79.

[0239] Synthesis of LP-259p

[0240]

[0241] A solution of 1 (0.100 g) in 3 mL DCM was added to 2 (0.366 mL) and TEA (0.337 mL) at room temperature. The reactant was stirred at room temperature for 1 h. The reaction mixture was loaded onto a 12 g column. A gradient of hexanes to EtOAc was used for purification. The product was a white solid, 183 mg, 82%. LC-MS: [M+H] calculated value was 368.32, and the measured value was 368.60.

[0242]

[0243] 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 h. After cooling to room temperature, the MeOH / THF was removed in vacuo. The pH was adjusted to about 1 with 1N HCl. EtOAc and water were added for workup. After removing EtOAc in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL DMF, and COMU (0.136 g), DIPEA (0.085 mL) and 2 (0.053 g) were added at room temperature. The reaction was stirred at room temperature for 1 h. EtOAc was added to dilute the reaction mixture. The reaction mixture was washed with 1N HCl, NaHCO 3 The mixture was washed with brine. After removing EtOAc in vacuo, the residue was loaded onto a 12 g column. A gradient from hexanes to EtOAc was used for purification. The product was a white solid, 87 mg, 71%. LC-MS: [M+H] Calculated value 502.29, found value 502.72.

[0244] Synthesis of LP-260p

[0245]

[0246] A solution of 1 (0.100 g) in DDC was added to 2 (0.354 mL) and TEA (0.326 mL) at room temperature. The reactant was stirred at room temperature for 1 h. The reaction mixture was loaded onto a 12 g column. A gradient of hexanes to EtOAc was used for purification. The product was a white solid, 208 mg, 87%. LC-MS: [M+H] calculated value was 410.36, and the found value was 410.73.

[0247]

[0248] A solution of 1 (0.208 g) in 4N HCl / dioxane (8 mL) was stirred at room temperature overnight. After removing the solvent in vacuo, the residue was placed under high vacuum for 3 h. The product was a white solid, 179 mg, which was used in the next step without further purification. LC-MS: [M+H] calculated value was 354.30, found value was 354.65.

[0249]

[0250] A solution of 1 (0.0760 g) in 3 mL of DMF was added to COMU (0.120 g), DIPEA (0.072 mL) and 2 (0.0460 g) at room temperature. The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc and then washed with 1N HCl, NaHCO 3The product was washed with brine. After removing the solvent in vacuo, the residue was loaded onto a 12 g column. A gradient from hexanes to EtOAc was used for purification. The product was a white solid, 55 mg, 51%. LC-MS: [M+H] calculated value was 502.29, found value was 502.72.

[0251] Synthesis of LP-262p

[0252]

[0253] To a solution of 1 (0.0220 g) and 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 h. The reaction mixture was diluted with DCM. It was then washed with 1N HCl, saturated NaHCO 3 and washed with brine. Purification was performed on a 4 g column. Purification was performed using a gradient of DCM to 20% MeOH in DCM. The product was a clear solid, 77 mg, 65%. LC-MS: [M+2H]+H 2 O calculated value is 1294.76, found value is 1295.29; [M+3H]+H 2 The calculated value for O was 869.51, and the found value was 869.45; the calculated value for [M+4H] was 638.88, and the found value was 638.54.

[0254]

[0255] 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 in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL DMF, and then 2 (0.016 g) and TEA (0.013 mL) were added at room temperature. The reactants were stirred at room temperature for 1.5 h. After removing the solvent in vacuo, the residue was loaded onto a 4 g column. A gradient of DCM to 20% MeOH in DCM was used for purification. The product was a white solid, 61 mg, 78%. LC-MS: [M+2H]+H 2 The calculated value for O was 1302.84, and the found value was 1303.81; the calculated value for [M+4H] was 642.92, and the found value was 642.62.

[0256]

[0257] A solution of 1 (0.0610 g) in 4N HCl / dioxane (5 mL) was stirred at room temperature overnight. After removing the solvent in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL DMF, and then COMU (0.0152 g), DIPEA (0.009 mL) and 2 (0.0060 g) were added at room temperature. The reactant was stirred at room temperature for 1.5 h. After removing the solvent in vacuo, the residue was loaded onto a 4 g column. A gradient of DCM to 20% MeOH in DCM was used for purification. The product was a white solid, 13 mg, 21%. LC-MS: [M+2H]+H 2 O calculated value is 1348.80, found value is 1348.94; [M+3H]+H 2 The calculated value of O is 905.54 and the measured value is 905.09.

[0258] Synthesis of LP-269p

[0259]

[0260] To 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 TEA (0.418 mL, 3.000 mmol, 6.0 equiv) at ambient conditions. The reaction was stirred at room temperature for 3 hours, 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). The reaction was stirred at room temperature overnight. The reaction mixture was washed with 1 N HCl and then with brine. The mixture was then washed with Na 2 SO 4 Dry and concentrate. The residue is purified by centrifugation using silica gel as stationary phase with a gradient of EA to Hex 0-100%. Purification afforded 72 mg of product (19% yield).

[0261] Synthesis of LP-273p

[0262]

[0263] Compound 1 (0.200 g), NEt 3 To a solution of 2 (0.152 g) and COMU (0.261 g) in DCM was added 2. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was concentrated directly for isolation. The residue was loaded onto a 12-g column with a DCM liquid and a gradient of hexanes to 100% EtOAc was passed through Purification with the product eluting at 28% B. The product was concentrated in vacuo to afford a clear and light yellow oil. MS m / z: [M+H]+ Calcd. 477.23 m / z, Observed 477.52 m / z.

[0264] Synthesis of LP-274p

[0265]

[0266] To a solution of EPA 1 (60.5 mg, 0.200 mmol, 1 eq.) and 2 (36.5 mg, 0.220 mmol, 1.10 eq.) in 20 mL DCM was added COMU (94.2 mg, 0.220 mmol, 1.10 eq.) and then TEA (0.084 mL, 0.600 mmol, 3.0 eq.) at ambient conditions. The reaction 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 then washed with Na 2 SO 4 The reaction mixture was purified by centrifugation using a gradient of EA to Hex 0-50% using silica gel as stationary phase. Purification gave 69 mg of product (76% yield).

[0267] Synthesis of LP-283p

[0268]

[0269] Compound 1 (49 mg), NEt 3 To a solution of 1% 4-nitropropene (0.068 mL) and COMU (76.8 mg) in DMF was added compound 2 (29.8 mg). The reaction was stirred until complete conversion was observed by LC-MS. If conversion was not clearly observed by LC-MS, the reaction was stirred for 30 min until the bright yellow (before adding compound 2) turned into honey orange and all materials were observed to be dissolved. The reaction mixture was washed with water, extracted with DCM, and purified by Na 2 SO 4 Dry, filter, and concentrate in vacuo. The residue was loaded onto a 12-g column via a DCM liquid and passed through a gradient of hexanes to 100% EtOAc. Purification, where the product eluted at 31% B. The product was concentrated in vacuo to afford a white solid residue and purified by eluting in CDCl 3 1H NMR confirmed.

[0270] Synthesis of LP-286p

[0271]

[0272] To a solution of 1 (78.5 mg, 0.200 mmol, 1 eq.) and 2 (36.5 mg, 0.220 mmol, 1.10 eq.) in 20 mL of DCM was added COMU (94.2 mg, 0.220 mmol, 1.10 eq.) and then TEA (0.084 mL, 0.600 mmol, 3.0 eq.) at ambient conditions. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was washed with 1 N HCl and then brine. The mixture was washed with Na 2 SO 4 The reaction mixture was purified by centrifugation using a gradient of EA to Hex 0-50% using silica gel as stationary phase. Purification gave 69 mg of product (57% yield).

[0273] Synthesis of LP-287p

[0274]

[0275] To a solution of 1 (43.3 mg, 0.200 mmol, 1 eq) and 2 (36.5 mg, 0.220 mmol, 1.10 eq) in 20 mL DCM was added COMU (94.2 mg, 0.220 mmol, 1.10 eq) and then TEA (0.084 mL, 0.600 mmol, 3.0 eq) at ambient conditions. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was washed with 1 N HCl and then brine. The mixture was washed with Na 2 SO 4 The reaction mixture was purified by centrifugation using a gradient of EA to Hex 0-50% using silica gel as stationary phase. Purification afforded 52 mg of product (71% yield).

[0276] Synthesis of LP-290p

[0277]

[0278] Compound 1 (0.0540 g), NEt 3 To a solution of 2 (0.075 mL) and COMU (0.084 g) in DMF was added 2 (0.0327 g). The reaction was stirred for 30 min until the bright yellow (before the addition of 2) turned to honey orange and all materials were observed to be mostly dissolved. The reaction mixture was washed with water, extracted with DCM, and purified by Na 2 SO 4Dry, filter, and concentrate in vacuo. The residue was loaded onto a 12-g column via a DCM liquid and passed through a gradient of hexanes to 100% EtOAc. Purification, where the product eluted at 31% B. The product was concentrated in vacuo to afford a white solid residue and purified by eluting in CDCl 3 1H NMR confirmed in LC-MS: [M+H]+ calculated value is 428.14 m / z, observed value is 428.46 m / z.

[0279] Synthesis of LP-293p

[0280]

[0281] Compound 1 (73 mg), NEt 3 To a solution of 1,4-dihydro-1,4-dihydro-2-nitropropene (0.112 mL) and COMU (126 mg) in DMF was added compound 2 (48.9 mg). The reaction was stirred until complete conversion was observed by LC-MS. If conversion was not clearly observed by LC-MS, the reaction was stirred for 30 min until the bright yellow (before adding compound 2) turned into honey orange and all materials were observed to be mostly dissolved. The reaction mixture was then washed with water, extracted with DCM, and purified by Na 2 SO 4 Dry, filter, and concentrate in vacuo. The residue was loaded onto a 12-g column via a DCM liquid and passed through a gradient of hexanes to 100% EtOAc. Purification, where the product eluted at 30% B. The product was concentrated in vacuo to afford a white solid residue and purified by eluting in CDCl 3 1H NMR confirmed.

[0282] Synthesis of LP-296p

[0283]

[0284] Compound 1 (0.0344 g), NEt 3 To a solution of 2 (0.0117 g) and COMU (0.0182 g) in DCM was added 2 (0.0071 g). The reaction was stirred for 30 min until the bright yellow (before the addition of 2) turned to honey orange and all materials were observed to be mostly dissolved. The reaction mixture was concentrated directly for isolation. The residue was loaded onto a 4-g column with DCM to 20% MeOH / DCM (0% B to 20% B, to 40% B, to 50% B, then to 100% B) by DCM liquid. Purification, where the product eluted at 23% B. The product was concentrated under vacuum to afford a clear and colorless oil and purified by eluting in CDCl3 Confirmed by 1H NMR in. MS m / z: [M+H]+ calculated value is 1039.67m / z; observed value is 1040.36,671.78m / z.

[0285] Synthesis of LP-300p

[0286]

[0287] To a solution of 2 (5.29 g) in 100 mL toluene was added TEA (8.4 mL) at room temperature, followed by dropwise addition of 1 (5.20 g). The reaction mixture was stirred at 90 ° C for 16 h. After cooling to room temperature, EtOAc and water were added for post-treatment. Purification was performed on a 120 g column. A gradient of hexanes to 30% EtOAc in hexanes was used for purification. The product was a light yellow oil, 3658 mg, 54%. LC-MS: [M+H] calculated value was 339.21, and the found value was 339.17.

[0288]

[0289] A mixture of 1 (0.113 g) and 10% Pd / C (0.0036 g) in 10 mL of EtOAc was charged into H 2 (about 45psi). The reaction mixture was stirred at room temperature for 4h. After filtration, the solvent was removed in vacuo. The residue was then placed under high vacuum for 1h. The residue was dissolved in 10mL DCM, and TEA (0.279mL) and 2 (0.405mL) were added at room temperature. The reaction mixture was stirred at room temperature for 1h. Purification was performed on a 12g column. A gradient of hexanes to 50% EtOAc in hexanes was used for purification. The product was a white solid, 141mg, 66%. LC-MS: [M+H] calculated value was 635.57, and the found value was 635.95.

[0290]

[0291] A solution of 1 (0.141 g) in MeOH / THF (3 mL / 3 mL) was added to 1 N NaOH (3 mL) at room temperature. The mixture was stirred at room temperature for 2 h. After removing the organic solvent in vacuo, the residue was acidified to pH~1 with concentrated HCl. EtOAc was added to extract the product. After removing the solvent in vacuo, 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 EtOAc, then washed with 1 N HCl and brine. After removing the solvent in vacuo, the residue was loaded onto a 12 g column. A gradient of hexanes to 30% EtOAc in hexanes was used for purification. The product was a white solid, 80 mg, 47%. LC-MS: [M+H] calcd. 769.55, found 769.98.

[0292] Synthesis of LP-303p

[0293]

[0294] To a solution of vitamin D1 (185 mg, 0.500 mmol, 1 eq) and 2 (111 mg, 0.550 mmol, 1.10 eq) in 30 mL of DCM was added TEA (0.139 mL, 1.00 mmol, 2.0 eq) under ambient conditions. The reaction was stirred at room temperature for 8 hours. The reaction mixture was washed with 1N HCl and then with brine. The mixture was washed with Na 2 SO 4 Dry and concentrate. The residue is purified by centrifugation using silica gel as stationary phase with a gradient of EA to Hex 0-100%. Purification afforded 95 mg of product (35% yield).

[0295] Synthesis of LP-304p

[0296]

[0297] 1 (200 mg, 0.377 mmol, 1.0 eq) was hydrolyzed with a solution of LiOH (151 mg, 3.77 mmol, 10.0 eq) in MeOH / TFH / H2O (1:1:1, 90 mL). After removing all organic solvents, the aqueous phase was acidified to pH = 3 with 1N HCl. The reaction mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined and washed with Na 2 SO 4 Drying and concentration gave the crude acid.

[0298] To a solution of the above crude acid and tetrafluorophenol 4 (68.9 mg, 0.415 mmol, 1.10 equiv) in 30 mL DCM was added COMU (194 mg, 0.453 mmol, 1.20 equiv) and then TEA (0.158 mL, 1.13 mmol, 3.0 equiv) under ambient conditions. The reaction was stirred until complete conversion was observed by LC-MS. The reaction mixture was washed with 1N HCl and then with brine. The reaction mixture was washed with Na 2 SO 4 The reaction mixture was purified by centrifugation using a gradient of EA to Hex 0-100% using silica gel as the stationary phase. Purification afforded 170 mg of product (85% yield).

[0299] Synthesis of LP-310p

[0300]

[0301] DIPEA (0.057 mL), COMU (0.077 g) and 2 (0.0300 g) were added to a solution of 1 in DCM at room temperature. After stirring at room temperature for 2 h, the reaction was quenched 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. A gradient of hexanes to 50% hexanes in EtOAc was used for purification. The product was a white solid, 46 mg, 44%. LC-MS: [M+H] calculated value was 422.36, found value was 422.61.

[0302]

[0303] A solution of 1 (0.046 g) in 4N HCl / dioxane (2 mL) was stirred overnight at room temperature. After removing the solvent in a vacuum, the residue was placed under high vacuum for 3 h. The residue was then dissolved in DCM at room temperature, and COMU (0.0700 g), DIPEA (0.038 mL) and 2 (0.036 g) were then added at room temperature. After stirring at room temperature for 2 h, the solvent was removed in a vacuum. The residue was loaded onto a 4 g column. The gradient of hexanes to 50% hexanes in EtOAc was used for purification. The product was a white solid, 21 mg, 38%. LC-MS: The calculated value of [M+H] was 514.29, and the measured value was 514.61.

[0304] Synthesis of LP-383p

[0305]

[0306] Compound 2 (0.023 g) and EDC (0.039 g) were added to a solution of compound 1 (0.050 g) in 5 mL of DCM at room temperature. The mixture was stirred at room temperature for 1 h. After removing the solvent in vacuo, the residue was loaded onto a 4 g column by dry method. The product was purified using a solution of hexanes to 50% EtOAc in hexanes. The product was a white solid with a yield of 29 mg. LC-MS: [M+H+H 2 The calculated value is 388.27 and the measured value is 388.03.

[0307] Synthesis of LP-409p

[0308]

[0309] 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 product was obtained as a white precipitate, which was filtered and washed with acetone (20 mL). Use 1 H and 13 P NMR confirmed the structure of the compound.

[0310]

[0311] Compound 1 (1.9 g), 2 (0.846 g) and 3 (2.98 g) were dissolved in 100 mL 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 DCM, the product was dried and loaded onto a 24 g column. 0-50% (EA / Hex, 1% TEA was added) was used as the mobile phase to obtain the product as a white solid.

[0312] Synthesis of LP-429p

[0313]

[0314] 17-Hydroxyhexadecanoic acid (6) (3.53 g, 12.3 mmol) was added to a 500 mL RBF. The flask was purged with nitrogen and then DCM (150 mL) was added followed by acetic anhydride (18.6 mL, 197 mmol) and pyridine (30.8 mL, 382 mmol). The reaction was stirred overnight. The reaction mixture was concentrated and azeotroped with toluene 3 times to remove residual pyridine, acetic acid and acetic anhydride. The residue was then heated in 100 mL of 1:1 THF / NaHCO 3The aqueous solution mixture was stirred for 24 hours. About half of the THF was removed by rotary evaporator and the mixture was diluted with water and acidified with 3M HCl until pH was 1. The mixture became very foamy during the acidification. The product was collected by filtration and dried in a vacuum to produce 3.22g (80% yield) of compound 5 as a white solid. The product was not further purified.

[0315]

[0316] 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. Once cooled, N-methylmorpholine (1.4 mL, 12.7 mmol) and ethyl chloroformate (1.2 mL, 12.7 mmol) were added. The reactants were stirred at -15 ° C for 30 minutes. After 30 minutes, a solution of sodium azide (1.72 grams, 26.4 mmol) in water (6.6 mL) was added, and the reactants were stirred at -5 ° to 0 ° C in a water / salt / ice bath for 30 minutes. The reaction mixture was diluted with EtOAc (20 mL) and water (20 mL). The layers were separated and the water layer was extracted with EtOAc (2x 50 mL), and the combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate and concentrated to a white solid. Proton NMR showed that there was no remaining starting material based on the alpha proton of the carbonyl group. The solid was dissolved in toluene (55 mL) and heated to 65 ° C until gas generation ceased (about 30 minutes). The reactants were cooled to room temperature and 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 isocyanates were consumed after 2 hours, and another 2 equivalents of N-hydroxysuccinimide (2.43 g, 21.1 mmol) were added. The reactants were stirred overnight. After stirring overnight, proton NMR showed that no isocyanate remained. The reaction mixture was concentrated, and the resulting white powder was dissolved in EtOAc (100 mL) and poured into 300 mL of hexanes. The precipitate was collected by filtration. The proton NMR of the product showed residual N-hydroxysuccinimide. The product was dissolved in DCM and purified by silica gel chromatography 65:35 hexanes:EtOAc to 0:100 hexanes:EtOAc. The product began to elute at 50% EtOAc and was retained on the column. The fractions containing the product were combined to produce 2.25 g (48% yield) of compound 7 as a white solid.

[0317]

[0318] Compound 7 (1.00 g, 2.27 mmol) was added to 6-amino-1-hexanol (0.266 g, 2.27 mmol) and NEt 3 The mixture was stirred for 2 hours at 4 ℃ for 1 h at 4 ℃ for 1 h. The mixture was stirred for 2 hours at 4 ℃ for 3 hours ...

[0319]

[0320] In a 100 mL RBF, compound 8 (0.95 g, 2.14 mmol) was dried by three consecutive evaporations of toluene. Diisopropylammonium tetrazolium (0.146 g, 0.86 mmol) and 4 angstrom molecular sieves were added to the flask. The flask was purged and backfilled with nitrogen 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'-tetraisopropylphosphorodiamidite (0.98 g, 3.25 mmol) was added and the reaction was stirred for 18 hours. After 18 hours, LC-MS showed that no starting alcohol remained. The reaction was transferred to a separatory funnel and washed with saturated NaHCO 3 The mixture was stirred for 2 hours at room temperature for 1 hr at 4 000 rpm for 1 hr at 4 000 rpm. ...

[0321] Synthesis of LP-430p

[0322]

[0323] To a round-bottom flask containing a solution of hexadecyl isocyanate (1 eq.) in DCM (5 mL), a solution of 1,6-hexanediol (1 eq.) and TEA (2 eq.) in DCM (5 mL) was added. The mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by CombiFlash chromatography using 2% MeOH in DCM to produce compound 1 as an off-white solid in 20% yield. LC-MS [M+H] + 386.3634m / z, observed value is 386.3642m / z.

[0324] Compound 1 (1 eq.) was dried by evaporating toluene twice. Then, it was dissolved in anhydrous DCM (10 mL) and diisopropylammonium tetrazolium (1.4 eq.) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at N 2 The mixture was stirred at room temperature for 30 minutes under 4% N-butyl acetate. Then, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.6 equiv.) was added and stirring was continued at room temperature for 12 hours. Thereafter, 0.3 mL of TEA was added to quench the reaction and the mixture was loaded directly onto diatomaceous earth. CombiFlash chromatography using hexanes: ethyl acetate + 1% TEA (70:30) produced the pure product as a waxy off-white solid in 41.7% yield. LC-MS [M+H] + 586.4713m / z, observed value is 586.4720m / z.

[0325] Synthesis of LP-431p

[0326]

[0327] To a round-bottom flask containing a solution of 6-amino-1-hexanol (1.2 eq.) and TEA (2 eq.) in DCM (5 mL), a solution of hexadecyl chloroformate (1 eq.) in DCM (5 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure and purified by CombiFlash chromatography using 2% MeOH in DCM to produce compound 1 as an off-white solid in 20% yield. LC-MS [M+H] + 386.3634m / z, observed value is 386.3638m / z.

[0328] Compound 1 (1 eq.) was dried by evaporating toluene twice. Then, it was dissolved in anhydrous DCM (10 mL) and diisopropylammonium tetrazolium (1.4 eq.) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at N 2 Stir at room temperature for 30 minutes under 4% N-butylene glycol (2-cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite (1.6 equiv.) was then added and stirring continued at room temperature for 12 hours. Thereafter, 0.3 mL of TEA was added to quench the reaction and the mixture was loaded directly onto diatomaceous earth. CombiFlash chromatography using hexanes: ethyl acetate + 1% TEA (70:30) produced the pure product as a waxy off-white solid in 82.3% yield. LC-MS [M+H] + 586.4713m / z, observed value is 586.4705m / z.

[0329] Synthesis of LP-435p

[0330]

[0331] 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 was stirred overnight. 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 EtOAc: hexanes to 20:80 EtOAc: hexanes) and the product was eluted at 10% EtOAc. The fractions containing the product were concentrated to produce 0.975 g (43% yield) of compound 21 as a clear liquid.

[0332]

[0333] Compound 21 (0.975, 5.2 mmol) was dissolved in toluene (40 mL) and heated to 65 ° C for 1 hour. Gas generation was observed after reaching 65 ° C and stopped after about 30 min. 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). The toluene solution was added to the THF solution and a white precipitate was quickly formed. The reactants were stirred overnight. The reaction mixture was concentrated, and the crude product was recrystallized from isopropanol to produce 1.5558 g (77% yield) of compound 22 as a white solid.

[0334]

[0335] In a 100 mL RBF, compound 22 (1.55 g, 4.0 mmol) was dried by 2 consecutive evaporations of toluene. Diisopropylammonium tetrazolium (0.277 g, 1.6 mmol) and 4 angstrom molecular sieves were added to the flask. The flask was purged and backfilled with nitrogen 3 times and the solid was suspended in DCM (20 mL). The solid only partially dissolved. 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 showed that no starting alcohol remained. The reaction was transferred to a separatory funnel and washed with saturated NaHCO 3The mixture was stirred for 1 hour in a flask and stirred for 1 hour in a flask. The white solid was collected by filtration, washed with hexanes (2 x 20 mL), and dried under vacuum to produce 1.103 grams of white powder. Proton NMR showed that a large amount of water remained, and a large amount of material was insoluble in chloroform and DCM. The mixture was suspended in DCM, dried over magnesium sulfate, filtered through another magnesium sulfate pad, and concentrated to produce 0.46 g (19% yield) of compound LP-435p as an off-white powder.

[0336] Synthesis of LP-439p

[0337]

[0338] (3-aminobicyclo[1.1.1]pentane-1-yl)methanol (2) (0.20 g, 1.77 mmol) and hexadecylcarbamic acid 2,5-dioxopyrrolidine-1-yl ester (3) (0.67 g, 1.75 mmol) were dissolved in DCM (40 mL) and triethylamine (0.72 mL, 5.3 mmol) was subsequently added. The reactant was stirred overnight. After 18 hours, a precipitate was observed. The precipitate was collected by filtration and washed with DCM (2 x 10 mL). The precipitate was dried in a vacuum to produce 0.325 g (48% yield) of white solid. Proton NMR analysis was consistent with the product, and the crude material had an acceptable purity to proceed to the next step.

[0339]

[0340] Compound 1 (0.3 g, 0.79 mmol) was dried by evaporating toluene four times in succession, and then diisopropylammonium tetrazolium (0.054 g, 0.315 mmol) was added to the flask. The flask was purged and backfilled with nitrogen three times, the solid was suspended in DCM (20 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.39 mL, 1.214 mmol) was added, and the reaction was stirred for 18 hours. LC-MS analysis showed that no starting alcohol remained after 18 hours. The reaction was transferred to a separatory funnel and washed with saturated NaHCO 3 The mixture was stirred for 1 h at room temperature for 2 h. The mixture was stirred for 1 h at room temperature for 2 h. The mixture was stirred for 2 h. The mixture was stirred for 3 ...3 h. The mixture was stirred for 2 h.

[0341] Synthesis of LP-440p

[0342]

[0343] Anhydrous MeOH (8mL) is cooled to 0 ℃, potassium hydroxide (3 equivalents) is added, and the solution is stirred for 30min. Then a solution of 16-bromohexadecanoic acid (1 equivalent) in anhydrous MeOH (7mL) is added via a syringe. The reaction mixture is heated to reflux temperature and stirred overnight. After cooling to room temperature, MeOH is removed in a vacuum and the resulting crude mixture is reconstructed in 1N HCl (25mL) and ether (5mL). The crude product is extracted with ether (4x 30mL), the combined organic layer is washed with brine (30mL) and filtered through Na 2 SO 4 Dry, and then remove the solvent in vacuo. The product was then purified by column chromatography on silica gel using hexanes: ethyl acetate (85:15) to produce compound 1 as an oil in 86% yield. LC-MS [M+H] + 287.2586m / z, observed value is 287.2590.

[0344] To a solution of compound 1 (1 eq.) in DCM (50 mL) was added COMU (1.2 eq.) and DIPEA (2 eq.). The mixture was stirred at room temperature for 30 minutes. Then, 6-amino-1-hexanol (1.2 eq.) was added and the reaction mixture was stirred at room temperature for 12 hours. Then, the mixture was washed 3 times with 1M HCl (3 x 50 mL), washed once with brine (50 mL), and washed with Na 2 SO 4 The crude product was dried and concentrated under reduced pressure. ACN (100 mL) was added and carefully heated using a heat gun until all solids dissolved. The mixture was then left at room temperature, which resulted in the formation of white crystals. The precipitate was then collected by vacuum filtration and washed several times with ACN to remove the remaining pink. Compound 2 was obtained as a white solid in 74% yield. LC-MS [M+H] + 386.3634 m / z, observed value is 386.3626.

[0345] Compound 3 (1 eq.) was dried by evaporating toluene twice. Then, it was dissolved in anhydrous DCM (10 mL) and diisopropylammonium tetrazolium (0.4 eq.) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at N 2Stir at room temperature for 30 minutes under 4% N-butylene glycol (2-cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite (1.5 eq.) was then added and stirring continued at room temperature for 12 hours. Thereafter, 0.3 mL of TEA was added to quench the reaction and the mixture was loaded directly onto diatomaceous earth. CombiFlash chromatography using hexanes: ethyl acetate + 1% TEA (70:30) produced the pure product as a waxy off-white solid in 86% yield. LC-MS [M+H] + 586.4713m / z, observed value is 586.4705.

[0346] Synthesis of LP-441p

[0347]

[0348] To a round-bottom flask containing a solution of 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. The solution was then allowed to cool to room temperature and the solvent was removed in vacuo. The crude product was then dissolved in H 2 O (20 mL) and CH 3 The combined organic layers were washed once with brine (20 mL) and purified by Na 2 SO 4 Dry and concentrate under reduced pressure. The crude mixture 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] + 342.3736m / z, observed value is 342.3728.

[0349] In a round bottom flask containing a solution of compound 1 (1 eq) in MeOH (25 mL) was added ethyl trifluoroacetate (5 eq) and DIPEA (2 eq). The reaction mixture was stirred at 40 °C for 12 hours. Thereafter, the solvent was removed under reduced pressure and the crude was purified by CombiFlash chromatography using 4%-6% MeOH in DCM to produce compound 2 as an oil in 73% yield. LC-MS [M+H] + 438.3559 m / z, observed value 438.3551.

[0350] Compound 2 (1 eq.) was dried by evaporating toluene twice. Then, it was dissolved in anhydrous DCM (10 mL) and diisopropylammonium tetrazolium (0.4 eq.) was added, followed by activated molecular sieves (100 mg). The mixture was stirred at N 2Stir at room temperature for 30 minutes under 4% N-butylene glycol (2-cyanoethyl)-N,N,N',N'-tetraisopropylphosphorodiamidite (1.5 eq.) was then added and stirring continued at room temperature for 12 hours. Thereafter, 0.3 mL of TEA was added to quench the reaction and the mixture was loaded directly onto diatomaceous earth. CombiFlash chromatography using hexanes: ethyl acetate + 1% TEA (70:30) produced the pure product as a waxy off-white solid in 56% yield. LC-MS [M+H] + 638.4637m / z, observed value is 638.4629.

[0351] Synthesis of LP-456p

[0352]

[0353] 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 dry tetrahydrofuran (20 mL) at 0°C under nitrogen. The resulting solution was stirred at 0°C for 2 hours, then the cooling bath was removed and the mixture was stirred at room temperature overnight. Saturated aqueous sodium bicarbonate solution (50 mL) was added to quench the reaction. The mixture was then diluted with water (50 mL) and extracted 3 times with DCM (3 x 50 mL). The combined organic layers were washed with Na 2 SO 4 Dry and concentrate under reduced pressure. The crude 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] + 329.3056m / z, observed value is 329.5060.

[0354] A mixture of compound 1 (1 equivalent), silver carbonate (3 equivalents) and a catalytic amount of iodine in DCM (5 mL) was stirred with molecular sieves for 15 min. 2,3,4,6-tetra-O-acetyl-α-D-pyranose glucopyranose bromide (1.5 equivalents) in DCM (5 mL) was added to the mixture (also stirred with molecular sieves for 15 min). The resulting mixture was covered with aluminum foil and stirred at room temperature for 48 hours, then filtered through diatomaceous earth and washed with EtOAc. The filtrate was concentrated, and the crude product was purified by CombiFlash column chromatography using hexanes: ethyl acetate (80:20) to produce compound 2 as an oil in 33% yield. LC-MS: [M+H 2 O] + 676.4034 m / z, observed value 676.4041.

[0355] To a solution of compound 2 in DCM (5 mL) was added TFA (15 mL). The solution was stirred at room temperature for 2 h. Thereafter, the mixture was carefully poured into 100 mL of saturated NaHCO 3 Once neutralized, the aqueous phase was extracted 3 times with DCM (3 x 100 mL). The combined organic layers were washed with Na 2 SO 4 Drying and concentration under reduced pressure gave compound 3 as a white solid in 97% yield. LC-MS: [M+H] + 603.3381m / z, observed value 603.3388.

[0356] To a solution of compound 3 (1 eq.) in DCM (10 mL) was added COMU (1.2 eq.) and DIPEA (2 eq.). The mixture was stirred at room temperature for 30 minutes. Then, 6-amino-1-hexanol (1.2 eq.) was added and the reaction mixture was stirred at room temperature for 12 hours. Then, the mixture was washed 3 times with 1M HCl (3 x 10 mL), washed once with brine (10 mL), and washed with Na 2 SO 4 Dry and concentrate under reduced pressure. The crude was purified by CombiFlash chromatography using 0-100% hexanes:ethyl acetate over 40 min to give compound 4 as an oil in 83% yield. LC-MS [M+H] + 702.4429m / z, observed value is 702.4421.

[0357] Compound 4 (1 equivalent) was concentrated twice with toluene by rotary evaporator, and then anhydrous DCM (10 mL) was loaded into a reaction flask. The suspension was stirred at 900RPM with molecular sieves at ambient temperature under N2. 2-cyanoethyl N, N, N', N'-tetraisopropylphosphorodiamidite (1.5 equivalents) was added to the suspension, followed by diisopropylammonium tetrazolium (0.4 equivalents). After 12 hours, TEA (300uL) was added, and the reaction mixture was dried and loaded onto diatomaceous earth. The product was purified using hexanes: ethyl acetate + 1% TEA (60: 40) to produce LP-456p as an oil in 64% yield. LC-MS [M + H] + 902.5507m / z, observed value is 902.5517.

[0358] Synthesis of LP-462p

[0359]

[0360] To a round bottom flask containing 2099-117 (1 eq.) was added anhydrous THF (30 mL) and the solution was cooled to -20 °C. Ethyl chloroformate (1.2) and N-methylmorpholine (1.2 eq.) 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 eq.) in 1.5 mL of water was added to the reaction and the reaction was stirred at -7 °C for 90 minutes. The reaction was diluted with EtOAc. The aqueous layer was separated and extracted with EtOAc for another 2 times. The combined organic layers were washed with brine and purified by Na 2 SO 4 The mixture was stirred for 12 hours at room temperature for 2 hours.Then, the ... 2 O] + 717.4538 m / z, observed value 717.4530.

[0361] Compound 1 (1 equivalent) was rotary evaporated 2 times with toluene, and then anhydrous DCM (10 mL) was loaded into a reaction flask. The suspension was stirred with molecular sieves at 900RPM at ambient temperature under N2. 2-Cyanoethyl N, N, N', N'-tetraisopropylphosphorodiamidite (1.5 equivalents) was added to the suspension, followed by diisopropylammonium tetrazolium (0.4 equivalents). After 12 hours, TEA (300uL) was added, and the reaction mixture was dried and loaded onto diatomaceous earth. The product was purified using hexanes: ethyl acetate + 1% TEA (60:40) to produce LP-462p as an oil in 64% yield. LC-MS [M+H] + 916.5538m / z, observed value is 916.5543.

[0362] Synthesis of LP-463p

[0363]

[0364] 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) at room temperature, followed by pyridine (13.75 mL, 171 mmol). The mixture was stirred at room temperature overnight. After removing the solvent in vacuo, the residue was redissolved in DCM and dry loaded onto an 80 g column. Purification was performed using hexanes to 50% EtOAc in hexanes. Compound 24 was obtained as a white solid, 1.22 g, 62%. LC-MS: [M+H+H 2 The calculated value is 375.27 and the measured value is 374.80.

[0365]

[0366] Compound 24 (1.22 g, 3.4 mmol) was dissolved in ACN (40 mL) and saturated NaHCO 3 The suspension in aqueous solution (10 mL) was stirred at room temperature overnight. The pH was adjusted to 1 with 1N HCl. The precipitate was collected by suction filtration and washed with H 2 O and air-dried to yield 1.15 g (107% yield) of compound 25 as a white solid. Due to residual water, 1 H NMR determined greater than 100% yield. LC-MS: [M+H] calcd. 315.25, found 315.59.

[0367]

[0368] 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 hours. The reaction mixture was concentrated on silica gel and purified by column chromatography with 100% hexanes: 0% EtOAc to 0% hexanes: 100% EtOAc. The fractions containing the product were combined and the solvent was removed by a rotary evaporator to produce 1.66 g (94% yield) of compound 26 as a brown solid. LC-MS: [M+H] was calculated to be 480.37 and found to be 480.76.

[0369]

[0370] TFA (10 mL) was added to a solution of compound 26 in DCM (10 mL), and the reactant was stirred at room temperature for 1.5 hours. After removing the solvent in vacuo, the residue was dried under high vacuum for 2 hours. 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 hours. After removing part of the solvent in vacuo, the residue was recrystallized with ACN. The product was collected by suction filtration and dried in vacuo to produce 1.48 grams (82% yield) of compound 27 as an off-white solid. LC-MS: The calculated value of [M+H] was 523.41 and the measured value was 524.06.

[0371]

[0372] To a mixture of compound 27 (0.3 g, 0.57 mmol) in DCM (20 mL) was added diisopropylammonium tetrazolium (0.039 g, 0.23 mmol) at room temperature, followed by dropwise addition of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.277 g, 0.92 mmol). The mixture was then refluxed for 2 hours. After cooling to room temperature, the mixture was washed with saturated NaHCO 3 (aqueous solution) twice, followed by H 2 O washing. After removing almost all the solvent in vacuo, the residue was added to stirred hexanes and a white gel precipitate was formed. After filtration, the white solid was collected by suction filtration and washed twice with hexanes. The white solid was dried under high vacuum to produce 0.305 g (73% yield) of compound LP-463p as a white solid. LC-MS: [M+H] calculated value is 723.52, found value is 724.23.

[0373] Synthesis of LP-464p

[0374]

[0375] To a solution of 16-amino-hexadecanoic acid (1 eq.) in anhydrous MeOH (20 mL) was added ethyl trifluoroacetate (1.5 eq.) and TEA (1.1 eq.). The reaction was stirred at 50 °C for 12 h under a nitrogen atmosphere. The mixture was then concentrated under reduced pressure, diluted with EtOAc (30 mL) and washed with saturated KHSO 4 (15 mL) twice, washed once with brine (15 mL), and washed with Na 2 SO 4Drying and concentration under reduced pressure gave compound 1 as a white solid in 79% yield. LC-MS [M+H] + 368.2412 m / z, observed value 368.2419.

[0376] To a solution of compound 1 (1 eq.) in DCM (30 mL) was added COMU (1.2 eq.) and DIPEA (2 eq.). The mixture was stirred at room temperature for 30 minutes. Then, 6-amino-1-hexanol (1.2 eq.) was added and the reaction mixture was stirred at room temperature for 12 hours. Then, the mixture was washed 3 times with 1M HCl (3 x 15 mL), washed once with brine (15 mL), and washed with Na 2 SO 4 Dry and concentrate under reduced pressure. ACN (100 mL) was added to the crude product and carefully heated using a heat gun until all solids dissolved. The mixture was then left at room temperature, which resulted in the formation of white crystals. The precipitate was then collected by vacuum filtration and washed several times with ACN to remove residual pink. Compound 2 was obtained as a white solid in 82% yield. LC-MS [M+H] + 467.3461m / z, observed value is 467.3457.

[0377] Compound 2 (1 equivalent) was concentrated twice on a rotary evaporator with toluene, and then anhydrous DCM (10 mL) was loaded into a reaction flask. The suspension was stirred at 900RPM with molecular sieves at ambient temperature under N2. 2-Cyanoethyl N, N, N', N'-tetraisopropylphosphorodiamidite (1.5 equivalents) was added to the suspension, followed by diisopropylammonium tetrazolium (0.4 equivalents). After 12 hours, TEA (300uL) was added, and the reaction mixture was dried and loaded onto diatomaceous earth. The product was purified using hexanes: ethyl acetate + 1% TEA (60: 40) to produce LP-464p as a waxy solid in 77% yield. LC-MS [M + H] + 667.4539m / z, observed value is 667.4544.

[0378] Synthesis of LP-465p

[0379]

[0380] 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 reactant was stirred overnight. The reaction mixture was concentrated and the crude product was purified by silica gel chromatography (20:80 EtOAc: hexanes to 100:0 EtOAc: hexanes). The product was eluted at 10% EtOAc. It was found that fractions 1-4 contained the product, which was concentrated to produce 0.60 g (56% yield) of compound 17 as a white solid.

[0381]

[0382] Compound 17 (0.58 g, 1.7 mmol) was dissolved in toluene (20 mL) and heated to 65°C until no more gas evolution was observed (30 minutes). The solution was cooled to room temperature and then added to a solution of 6-amino-1-hexanol (0.2 g, 1.7 mmol) and pyridine (0.14, 1.7 mmol) in THF (20 mL). The reaction mixture was diluted with acetonitrile and the precipitate was collected by suction filtration, rinsed with acetonitrile, hexanes and dried in vacuo to produce 0.614 g (84% yield) of compound 19 as a white solid.

[0383]

[0384] In a 100 mL RBF, compound 19 (0.60 g, 1.4 mmol) was dried by 3 consecutive evaporations of toluene. Diisopropylammonium tetrazolium (0.096 g, 0.56 mmol) and 4 angstrom molecular sieves were added to the flask. The flask was purged and backfilled with nitrogen 3 times, and the solid was suspended in DCM (40 mL). The solid only partially dissolved. To the mixture, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.65 g, 2.2 mmol) was added and the reaction was stirred for 18 hours. After 18 hours, LC-MS showed that no starting alcohol remained. The reaction was transferred to a separatory funnel and washed with saturated NaHCO 3 The mixture was stirred for 2 h at 4 °C for 1 h. The mixture was stirred for 2 h at 4 °C for 3 ...

[0385] Synthesis of LP-466p

[0386]

[0387] Compound 7 (0.22g, 0.50mmol) and 3-aminobicyclo [1.1.1] pentane-1-carboxylic acid tert-butyl ester (0.0915g, 0.50mmol) were dissolved in DCM (10mL) and triethylamine (0.21mL, 1.5mmol) was added. After 18 hours, the remaining <2% of the starting NHS ester was determined by LC-MS. The reaction mixture was concentrated and directly loaded onto a silica gel column for purification. The product was purified by column chromatography with 0% EtOAc / 100% hexanes to 50% EtOAc 50% hexanes. Fractions 3-5 were merged to produce 0.23g (89% yield) of compound 10 as a white solid.

[0388]

[0389] 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, LC-MS showed that no starting material was present. The reaction mixture was concentrated and the residual TFA was removed by coevaporation with toluene 2 times to produce 0.189 mg (93%) of compound 11 as a white solid.

[0390]

[0391] 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 was stirred for about 5 minutes, and then 6-amino-1-hexanol (0.059 g, .5 mmol) was added. After 1 hour, LC-MS showed that there was no remaining starting material. 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 with water 3 times. The precipitate was dried in a vacuum to produce 0.166 g (70% yield) of compound 12 as a white solid.

[0392]

[0393] In a 100 mL RBF, compound 12 (0.166 g, 0.3 mmol) was dried by evaporating toluene twice in succession. Diisopropylammonium tetrazolium (0.02 g, 0.12 mmol) and 4 angstrom molecular sieves were added to the flask. The flask was purged and backfilled with nitrogen three times, and the solid was suspended in DCM (20 mL). The solid 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 showed that no starting alcohol remained after 18 hours. The reaction was transferred to a separatory funnel and washed with saturated NaHCO 3 The mixture was stirred for 1 h in 1% ethanol and then stirred for 2 h. The mixture was stirred for 3 h. The mixture was ...

[0394] Synthesis of LP-493p (shown as LP-493p uridine)

[0395]

[0396] 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 was stirred at 0°C for 1 hour, then allowed to warm to room temperature and stirred overnight. After about 18 hours, the reaction mixture was washed with saturated NaHCO 3 The mixture was stirred for 2 hours at 4 ℃ for 10 minutes. The mixture was stirred for 2 hours at 4 ℃ for 3 ...

[0397]

[0398] 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 dried flask. The flask was evacuated and purged with nitrogen three times, and then dry DMF (10 mL) was added to the flask. The solution was heated to 110°C for 18 hours. After 18 hours, the reaction was cooled to room temperature and the solvent was removed in vacuo. The residue was resuspended in DCM / MeOH and concentrated on silica gel for purification. The column was eluted with 3% MeOH / 97% DCM to 20% MeOH / 80% DCM. The fractions containing 2' and 3' addition products were combined and concentrated to produce 0.236 g (21% yield) of compound 30+3' addition product.

[0399]

[0400] Compound 30+3' addition product (0.23 g, 0.44 mmol) was dried by continuous 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 a flask and the flask was evacuated and purged with nitrogen three times. The solid was dissolved in pyridine (10 mL). The reactants were stirred at room temperature overnight. All volatiles were removed and the remaining pyridine was removed by co-distillation with toluene. The residue was dissolved in DCM (20 mL) and NaHCO 3 The organic phase was separated, the aqueous phase was extracted with DCM (20 mL), and the combined organic phases were dried (Na 2 SO 4 ) and concentrated. The crude product was purified by silica gel chromatography. The silica gel was pretreated with a 50:50 mixture of hexanes / EtOAc + 2% v / v triethylamine. The product was separated on a CombiFlash using a 40 g column, eluent: hexanes-ethyl acetate + 1% Et 3 N, 20-60% of the compound eluted at 60% EtOAc. The later fractions were contaminated with the 3' alkylated product. The fractions containing the pure 2' alkylated product were combined and concentrated to yield 0.107 g (27% yield) of compound 32 as a white solid.

[0401]

[0402] In a 25 mL RBF, compound 32 (0.150 g, 0.18 mmol), diisopropylammonium tetrazolium (0.043 g, 0.25 mmol) and 4 angstrom molecular sieves were placed, and the flask was evacuated and purged with nitrogen three times. DCM (5 mL) was added, followed by dropwise addition of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.092 mL, 0.29 mmol). The reactants were stirred overnight. The reaction mixture was washed with about 2 mL of saturated NaHCO 3 Quench, filter into a separatory funnel, separate the layers and add NaHCO 3 The layer was extracted with DCM (10 mL) once more. The combined organic layers were purified by Na 2 SO 4 The product was dried and concentrated into a thick viscous liquid. The crude product was purified by silica gel chromatography (0: 100 EtOAc: hexanes to 100: 0 EtOAc: hexanes). The silica gel was pretreated with a 50: 50 mixture of hexanes / EtOAc+2% v / v triethylamine. The product was eluted at 45% EtOAc. It was found that fractions 15-35 contained the product, which was contaminated by a small amount of oxidation products, and was merged to produce 0.088g (47% yield) of compound 33 as a sticky colorless solid. Fractions 36-50 were merged to produce 44mg of a sticky colorless solid, and contained the product with more oxidation substances.

[0403] Synthesis of (2C8C12)phosphoramidite

[0404]

[0405] 2-Octyl-1-decanol (1.00 g, 3.35 mmol) and diisopropylammonium tetrazolium (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'-tetraisopropylphosphorodiamidite (2.66 mL, 8.37 mmol) was added dropwise. After the reaction was completed, 3 mL of triethylamine was added to the reaction and the reactants were directly concentrated on diatomaceous earth for purification. The crude product was purified by silica gel chromatography (0:100 EtOAc: hexanes + 2% triethylamine to 100:0 EtOAc: hexanes + 2% triethylamine) and the product was eluted with 100% hexanes. The fraction containing the product was concentrated to 1.268 g (76% yield) of clear liquid.

[0406] Synthesis of (2C6C10)phosphoramidite

[0407]

[0408] 2-Hexyl-1-decanol (1.00 g, 4.13 mmol) and diisopropylammonium tetrazolium (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'-tetraisopropylphosphorodiamidite (3.27 mL, 10.3 mmol) was added dropwise. After the reaction was completed, 3 mL of triethylamine was added to the reaction and the reactants were directly concentrated on diatomaceous earth for purification. The crude product was purified by silica gel chromatography (0: 100 EtOAc: hexanes + 2% triethylamine to 100: 0 EtOAc: hexanes + 2% triethylamine), and the product was eluted with 100% hexanes. The fraction containing the product was concentrated to 1.32 g (72% yield) of clear liquid.

[0409] Synthesis of HO-C16 Phosphoramidite

[0410]

[0411] 1,16-Hexadecanediol, N,N-diisopropylethylamine (0.100 g) was dissolved in 2 mL THF. 4,4'-dimethoxytrityl chloride (2.2 g, 6.6 mmol) was slowly added as a solid. After 2 h, the reaction was concentrated by rotary evaporation and the product was purified by column chromatography (25% ethyl acetate / 75% hexanes).

[0412] DMT-OC 16 -OH (0.200 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.227 mL) and bis(diisopropylammonium tetrazolium) (0.0611 g) were dissolved in anhydrous DCM. The reaction was capped and stirred overnight. The results of LC-MS (0.25 M NH 4 HCO 3 :H 2 (0 buffer system) to determine the conversion rate. was added to the reaction mixture and it was concentrated under vacuum until a white powder remained. The mixture was dry loaded onto a silica gel column (12 g) using an EtOAc / hexanes (1% triethylamine) solvent system to prevent hydrolysis of the silica gel. [1] pass 31 PNMR, 1 The product was characterized by HNMR and LC-MS.

[0413] Synthesis of C16 Phosphoramidite

[0414]

[0415] Cetyl alcohol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.88 mL) and bis(diisopropylammonium tetrazolium) (0.778 g) were dissolved in a solution of DCM at room temperature. The reaction was capped and stirred overnight. The results were analyzed by LC-MS (0.25 M NH 4 HCO 3 :H 2 (0 buffer system) to determine the conversion rate. Add to the reaction mixture, and it is concentrated under vacuum until white powder remains. Use EtOAc / hexanes (1% triethylamine) solvent system to dry the mixture and load it onto a silica gel column (12 grams) to prevent silica gel hydrolysis. The expected product is not retained on the column and comes out soon after loading. Then by LC-MS, 1 HNMR and 31 The isolated product was characterized by PNMR. Final yield: 856.5 mg (93.8%).

[0416] Synthesis of C22 Phosphoramidite

[0417]

[0418] Docosanol (1.10 g), bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.1 mL) and bis(diisopropylammonium tetrazolium) (0.577 g) were dissolved in a solution of DCM at room temperature. The reaction was capped and stirred overnight. The results were analyzed by LC-MS (0.25 M NH 4 HCO 3 :H 2 (0 buffer system) to determine the conversion rate. Add to the reaction mixture and concentrate it under vacuum until white powder remains. The mixture is dry loaded onto a silica gel column (12 grams) pretreated with 3 mL of triethylamine using an EtOAc / hexane (1% triethylamine) solvent system to prevent hydrolysis of the silica gel. The column is then analyzed by LC-MS, 1 HNMR and 31 The isolated product was characterized by PNMR. Final yield: 2.1085 g (118.8%).

[0419] Example 3. Conjugation of lipid PK / PD modulator precursors

[0420] One or more lipid PK / PD modulator precursors can be linked to the RNAi agents disclosed herein before or after annealing and before or after conjugation to one or more targeting ligands. The general conjugation process for linking lipid PK / PD modulator precursors to the constructs described in the Examples depicted herein is described below.

[0421] A. Conjugation of Activated Ester PK / PD Modulators

[0422] The following procedure was used to conjugate a PK / PD modulator with an activated ester moiety such as TFP (tetrafluorophenoxy) or PNP (p-nitrophenol) to an RNAi agent with 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. 50-100 equivalents of TEA and 3 equivalents of the activated ester PK / PD modulator were then added to the solution. The solution was allowed to react for 1-2 hours while being analyzed by RP-HPLC-MS (mobile phase A 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile, Waters TM XBridge C18 column, Waters Corp.)

[0423] The product was then precipitated by adding 12 mL of acetonitrile and 0.4 mL of PBS, and the solid was centrifuged as a precipitate. The precipitate was then redissolved in 0.4 mL of 1XPBS and 12 mL of acetonitrile. The resulting precipitate was dried under high vacuum for 1 hour.

[0424] B. Conjugation of Phosphoramidite PK / PD Modulators

[0425] PK / PD modulators having a phosphoramidite moiety can be attached to the resin using typical oligonucleotide preparation conditions.

[0426] C. Hydrolysis of PK / PD Modulators

[0427] Certain PK / PD modulators are hydrolyzed under the cleavage and deprotection conditions described above in Example 1. For example, LP-429p, LP-456p, LP-462p, LP-463p, LP-464p, LP-466p, LP-493p, and HO-C16 phosphoramidite all include moieties that hydrolyze under the cleavage and deprotection conditions.

[0428] LP-465p was hydrolyzed after conjugation to the oligonucleotide strand in a solution of 0.5-1 M potassium carbonate in 1:1 methanol:water and heated to 50-60°C for approximately 4 hours.

[0429]

[0430]

[0431]

[0432]

[0433] As used in Table A, the following symbols are used to represent modified nucleotides, targeting groups, and linking groups:

[0434] A = adenosine-3'-phosphate

[0435] C = cytidine-3'-phosphate

[0436] G = guanosine-3'-phosphate

[0437] U = Uridine-3'-phosphate

[0438] I = inosine-3'-phosphate

[0439] a=2'-O-methyladenosine-3'-phosphate

[0440] as=2'-O-methyladenosine-3'-phosphorothioate

[0441] c = 2'-O-methylcytidine-3'-phosphate

[0442] cs = 2'-O-methylcytidine-3'-phosphorothioate

[0443] g = 2'-O-methylguanosine-3'-phosphate

[0444] gs = 2'-O-methylguanosine-3'-phosphorothioate

[0445] i = 2'-O-methylinosine-3'-phosphate

[0446] is=2'-O-methylinosine-3'-phosphorothioate

[0447] t=2'-O-methyl-5-methyluridine-3'-phosphate

[0448] ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate

[0449] u=2'-O-methyluridine-3'-phosphate

[0450] us = 2'-O-methyluridine-3'-phosphorothioate

[0451] Af = 2'-fluoroadenosine-3'-phosphate

[0452] Afs = 2'-fluoroadenosine-3'-phosphorothioate

[0453] Cf = 2'-fluorocytidine-3'-phosphate

[0454] Cfs = 2'-fluorocytidine-3'-phosphorothioate

[0455] Gf = 2'-fluoroguanosine-3'-phosphate

[0456] Gfs = 2'-fluoroguanosine-3'-phosphorothioate

[0457] Tf = 2'-fluoro-5'-methyluridine-3'-phosphate

[0458] Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate

[0459] Uf = 2'-fluorouridine-3'-phosphate

[0460] Ufs = 2'-fluorouridine-3'-phosphorothioate

[0461] dT = 2'-deoxythymidine-3'-phosphate

[0462] AUNA=2',3'-open-adenosine-3'-phosphate

[0463] AUNAs = 2',3'-open-adenosine-3'-phosphorothioate

[0464] CUNA = 2',3'-open-cytidine-3'-phosphate

[0465] CUNAs = 2',3'-open-cytidine-3'-phosphorothioate

[0466] GUNA=2',3'-open-guanosine-3'-phosphate

[0467] GUNAs = 2',3'-open-guanosine-3'-phosphorothioate

[0468] UUNA=2',3'-open-uridine-3'-phosphate

[0469] UUNAs=2',3'-open-uridine-3'-phosphorothioate

[0470] a_2N = see Table 4

[0471] a_2Ns = see Table 4

[0472] (invAb) = inverted abasic deoxyribonucleotide-5'-phosphate, see Table 4

[0473] (invAb)s = inverted abasic deoxyribonucleotide-5'-phosphorothioate, see Table 4

[0474] s = phosphorothioate bond

[0475] p = terminal phosphate (synthetic)

[0476] vpdN = vinylphosphonate deoxyribonucleotide

[0477] cPrpa = 5'-cyclopropylphosphonate-2'-O-methyladenosine-3'-phosphate (see Table 4)

[0478] cPrpas = 5'-cyclopropylphosphonate-2'-O-methyladenosine-3'-phosphorothioate (see Table 4)

[0479] cPrpu = 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphate (see Table 4)

[0480] cPrpus = 5'-cyclopropylphosphonate-2'-O-methyluridine-3'-phosphorothioate (see Table 4)

[0481] (Alk-SS-C6) = See Table 4

[0482] (C6-SS-Alk) = See Table 4

[0483] (C6-SS-C6) = See Table 4

[0484] (6-SS-6) = See Table 4

[0485] (C6-SS-Alk-Me) = See Table 4

[0486] (NH2-C6) = See Table 4

[0487] (NH-C6) = See Table 4

[0488] (NH-C6)s = See Table 4

[0489] -L6-C6- = See Table 4

[0490] -L6-C6s- = See Table 4

[0491] cC16 = see Table 4

[0492] aC16 = see Table 4

[0493] gC16 = See Table 4

[0494] uC16 = See Table 4

[0495] ALNA = See Table 4

[0496] Example 4. In vivo administration of lipid-linked RNAi agents in mice

[0497] On the first day of the study, female C57bl / 6 mice were injected with phosphate buffered saline (PBS) or RNAi agents formulated at 10 μg / μL in PBS. Three animals per group (n=3) were administered 10 μL of PBS or RNAi agent solution. Animals were injected intracerebroventricularly according to the dosing schedule in Table 5.

[0498] Table 5. Dosing regimen for mice of Example 4.

[0499]

[0500]

[0501] On the 15th day of the study, animals were sacrificed and thoracic spinal cord, temporal lobe and frontal cortex and cerebellum were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (PBS). The results are shown in Table 6a-6d below.

[0502] Table 6a. Mean relative SOD1 expression in the mice of Example 4 in the temporal cortex.

[0503] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.050 0.053 2 100ugAD08942 (without PK / PD regulator) 0.512 0.033 0.035 3 100ugAC001249(LP-183a) 0.406 0.095 0.124 4 100ugAC001374(LP-128a) 0.611 0.115 0.141 5 100ugAC001375(LP-200a) 0.525 0.095 0.117 6 100ugAC001380(LP-273a) 0.376 0.080 0.102 7 100ugAC001381(LP-274a) 0.418 0.101 0.133 8 100ugAC001382(LP-276a) 0.497 0.083 0.100 9 100ugAC001384(LP-293a) 0.254 0.127 0.255 10 100ugAC001389(LP-233a) 0.466 0.139 0.199 11 100ugAC001390(LP-242a) 0.322 0.086 0.118 12 100ugAC001391(LP-243a) 0.532 0.106 0.132 13 100ugAC001392(LP-249a) 0.459 0.096 0.121 14 100ugAC001393(LP-259a) 0.381 0.086 0.110 15 100ugAC001394(LP-260a) 0.293 0.075 0.101 16 100ugAC001377(LP-257a) 0.358 0.125 0.191

[0504] Table 6b. Mean relative SOD1 expression in the mice of Example 4 in the frontal cortex.

[0505]

[0506]

[0507] Table 6c. Average relative SOD1 expression in the mice of Example 4 in the thoracic spinal cord.

[0508] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.063 0.068 2 100ugAD08942 (without PK / PD regulator) 0.458 0.070 0.082 3 100ugAC001249(LP-183a) 0.150 0.011 0.012 4 100ugAC001374(LP-128a) 0.119 0.014 0.016 5 100ugAC001375(LP-200a) 0.145 0.038 0.051 6 100ugAC001380(LP-273a) 0.225 0.054 0.070 7 100ugAC001381(LP-274a) 0.229 0.024 0.027 8 100ugAC001382(LP-276a) 0.234 0.019 0.021 9 100ugAC001384(LP-293a) 0.234 0.030 0.035 10 100ugAC001389(LP-233a) 0.168 0.024 0.027 11 100ugAC001390(LP-242a) 0.301 0.100 0.149 12 100ugAC001391(LP-243a) 0.168 0.029 0.035 13 100ugAC001392(LP-249a) 0.168 0.018 0.020 14 100ugAC001393(LP-259a) ND* ND* ND* 15 100ugAC001394(LP-260a) 0.209 0.074 0.115 16 100ugAC001377(LP-257a) 0.148 0.018 0.021

[0509] *No data

[0510] Table 6d. Average relative SOD1 expression in the mice of Example 4 in the cerebellum.

[0511]

[0512]

[0513] Example 5. In vivo administration of lipid-linked RNAi agents in mice

[0514] On study day 1, female C57bl / 6 mice were injected with phosphate buffered saline (PBS) or RNAi agent formulated at 10 μg / μL in PBS. Three animals per group (n=3) were administered 10 μL of PBS or RNAi agent solution. Animals were injected intracerebroventricularly according to the dosing schedule in Table 7.

[0515] Table 7. Dosing regimen for mice of Example 5.

[0516] Group Dosed substance Dosing regimen 1 PBS Single injection on day 1 2 100ugAD08942 (without PK / PD regulator) Single injection on day 1 3 100ugAC001376(LP-245a) Single injection on day 1 4 100ugAC001378(LP-262a) Single injection on day 1 5 100ugAC001379(LP-269a) Single injection on day 1 6 100ugAC001395(LP-286a) Single injection on day 1 7 100ugAC001383(LP-287a) Single injection on day 1 8 100ugAC001396(LP-289a) Single injection on day 1 9 100ugAC001385(LP-296a) Single injection on day 1 10 100ugAD08908 (without PK / PD regulator) Single injection on day 1 11 100ugAC001385(LP-296a) Single injection on day 1 12 100ugAC001386(LP-300a) Single injection on day 1 13 100ugAC001388(LP-232a) Single injection on day 1 14 100ugAC001397(LP-290a) Single injection on day 1 15 100ugAC001442(LP-304a) Single injection on day 1 16 100ugAC001445(LP-310a) Single injection on day 1 17 100ugAC001446(LP-303a) Single injection on day 1 18 100ugAC001249(LP-183a) Single injection on day 1

[0517] On the 15th day of the study, animals were sacrificed and thoracic spinal cord, temporal lobe and frontal cortex and cerebellum were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (PBS). The results are shown in Table 8a-8d below.

[0518] Table 8a. Mean relative SOD1 expression in the mice of Example 5 in the temporal cortex.

[0519] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.066 0.070 2 100ugAD08942 (without PK / PD regulator) 0.846 0.017 0.017 3 100ugAC001376(LP-245a) 0.720 0.023 0.023 4 100ugAC001378(LP-262a) 0.682 0.126 0.154 5 100ugAC001379(LP-269a) 0.682 0.108 0.129 6 100ugAC001395(LP-286a) 0.999 0.085 0.093 7 100ugAC001383(LP-287a) 0.537 0.083 0.098 8 100ugAC001396(LP-289a) 0.554 0.108 0.135 9 100ugAC001385(LP-296a) 0.550 0.049 0.054 10 100ugAD08908 (without PK / PD regulator) 0.649 0.088 0.102 11 100ugAC001385(LP-296a) 0.588 0.052 0.057 12 100ugAC001386(LP-300a) 0.472 0.039 0.042 13 100ugAC001388(LP-232a) 0.318 0.048 0.057 14 100ugAC001397(LP-290a) 0.396 0.020 0.022 15 100ugAC001442(LP-304a) 0.416 0.011 0.011 16 100ugAC001445(LP-310a) 0.349 0.072 0.091 17 100ugAC001446(LP-303a) 0.384 0.042 0.047 18 100ugAC001249(LP-183a) 0.443 0.041 0.045

[0520] Table 8b. Mean relative SOD1 expression in the mice of Example 5 in the frontal cortex.

[0521] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.048 0.051 2 100ugAD08942 (without PK / PD regulator) 0.899 0.066 0.072 3 100ugAC001376(LP-245a) 0.761 0.036 0.038 4 100ugAC001378(LP-262a) 0.780 0.036 0.038 5 100ugAC001379(LP-269a) 0.855 0.049 0.052 6 100ugAC001395(LP-286a) 0.965 0.045 0.048 7 100ugAC001383(LP-287a) 0.682 0.034 0.036 8 100ugAC001396(LP-289a) 0.619 0.116 0.142 9 100ugAC001385(LP-296a) 0.735 0.070 0.077 10 100ugAD08908 (without PK / PD regulator) 0.700 0.028 0.029 11 100ugAC001385(LP-296a) 0.623 0.083 0.096 12 100ugAC001386(LP-300a) 0.660 0.094 0.110 13 100ugAC001388(LP-232a) 0.493 0.050 0.056 14 100ugAC001397(LP-290a) 0.518 0.084 0.100 15 100ugAC001442(LP-304a) 0.676 0.091 0.106 16 100ugAC001445(LP-310a) 0.520 0.120 0.156 17 100ugAC001446(LP-303a) 0.674 0.121 0.148 18 100ugAC001249(LP-183a) 0.795 0.082 0.092

[0522] Table 8c. Average relative SOD1 expression in the mice of Example 5 in the thoracic spinal cord.

[0523] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.067 0.072 2 100ugAD08942 (without PK / PD regulator) 0.409 0.036 0.040 3 100ugAC001376(LP-245a) 0.398 0.060 0.071 4 100ugAC001378(LP-262a) 0.282 0.068 0.090 5 100ugAC001379(LP-269a) 0.344 0.054 0.064 6 100ugAC001395(LP-286a) 0.646 0.075 0.085 7 100ugAC001383(LP-287a) 0.235 0.026 0.029 8 100ugAC001396(LP-289a) 0.331 0.064 0.080 9 100ugAC001385(LP-296a) 0.451 0.164 0.258 10 100ugAD08908 (without PK / PD regulator) 0.522 0.162 0.234 11 100ugAC001385(LP-296a) 0.537 0.065 0.074 12 100ugAC001386(LP-300a) 0.367 0.068 0.084 13 100ugAC001388(LP-232a) 0.252 0.059 0.078 14 100ugAC001397(LP-290a) 0.272 0.071 0.097 15 100ugAC001442(LP-304a) 0.194 0.022 0.024 16 100ugAC001445(LP-310a) 0.259 0.064 0.086 17 100ugAC001446(LP-303a) 0.144 0.030 0.038 18 100ugAC001249(LP-183a) 0.118 0.014 0.016

[0524] Table 8d. Average relative SOD1 expression in the mice of Example 5 in the cerebellum.

[0525] Group describe Relative SOD1 expression Error (high) Error (low) 1 PBS 1.000 0.062 0.066 2 100ugAD08942 (without PK / PD regulator) 0.623 0.028 0.029 3 100ugAC001376(LP-245a) 0.454 0.024 0.025 4 100ugAC001378(LP-262a) 0.438 0.040 0.044 5 100ugAC001379(LP-269a) 0.373 0.016 0.017 6 100ugAC001395(LP-286a) 0.767 0.138 0.168 7 100ugAC001383(LP-287a) 0.398 0.026 0.028 8 100ugAC001396(LP-289a) 0.493 0.019 0.020 9 100ugAC001385(LP-296a) 0.525 0.066 0.075 10 100ugAD08908 (without PK / PD regulator) 0.573 0.034 0.036 11 100ugAC001385(LP-296a) 0.512 0.049 0.054 12 100ugAC001386(LP-300a) 0.341 0.053 0.063 13 100ugAC001388(LP-232a) 0.270 0.014 0.015 14 100ugAC001397(LP-290a) 0.378 0.031 0.033 15 100ugAC001442(LP-304a) 0.414 0.032 0.035 16 100ugAC001445(LP-310a) 0.309 0.030 0.034 17 100ugAC001446(LP-303a) 0.441 0.016 0.017 18 100ugAC001249(LP-183a) 0.403 0.031 0.034

[0526] Example 6. In vivo administration of lipid-linked RNAi agents in transgenic mice

[0527] On the first day of the study, female TgSOD1G93A mice modified to express human SOD1 were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or RNAi agents formulated at 3 μg / μL in aCSF. Three animals per group (n=3) were administered 10 μL of aCSF or RNAi agent solution in aCSF. Animals were injected intracerebroventricularly according to the quantitative administration scheme of Table 9.

[0528] Table 9. Dosing regimen for mice of Example 6.

[0529] Group Dosed substance Dosing regimen 1 aCSF Single injection on day 1 2 30ugAD11556 (without PK / PD regulator) Single injection on day 1 3 30ugAC002478(LP183a) Single injection on day 1 4 30ugAD11758(C16a) Single injection on day 1 5 30ugAD11759(C22a) Single injection on day 1 6 30ugAD11821(HO-C16a) Single injection on day 1 7 30ugAD11841((2C8C12)a) Single injection on day 1 8 30ugAD11842((2C6C10)a) Single injection on day 1 9 30ugAD11276 (internal uC16) Single injection on day 1 10 30ugAD11278 (internal uC16) Single injection on day 1 11 30ugAD11731 (internal uC16) Single injection on day 1 12 30ugAD11732 (internal uC16) Single injection on day 1

[0530] On the 8th day of the study, animals were sacrificed and thoracic spinal cord, cortex, cerebellum and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (PBS). The results are shown in Tables 10a-10d below.

[0531] Table 10a. Mean relative SOD1 expression in the mice of Example 6 in the cortex

[0532] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.151 0.178 2 30ugAD11556 (without PK / PD regulator) 0.812 0.126 0.150 3 30ugAC002478(LP-183a) 0.591 0.109 0.133 4 30ugAD11758(C16a) 0.689 0.095 0.110 5 30ugAD11759(C22a) 0.809 0.130 0.155 6 30ugAD11821(HO-C16a) 0.632 0.105 0.125 7 30ugAD11841((2C8C12)a) 0.922 0.117 0.134 8 30ugAD11842((2C6C10)a) 0.724 0.129 0.157 9 30ugAD11276 (internal uC16) 0.661 0.150 0.195 10 30ugAD11278 (internal uC16) 0.617 0.093 0.109 11 30ugAD11731 (internal uC16) 0.648 0.117 0.143 12 30ugAD11732 (internal uC16) 0.698 0.167 0.219

[0533] Table 10b. Average relative SOD1 expression in the mice of Example 6 in the thoracic spinal cord.

[0534] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.134 0.155 2 30ugAD11556 (without PK / PD regulator) 0.544 0.086 0.101 3 30ugAC002478(LP-183a) 0.180 0.054 0.076 4 30ugAD11758(C16a) 0.165 0.018 0.020 5 30ugAD11759(C22a) 0.469 0.060 0.069 6 30ugAD11821(HO-C16a) 0.394 0.066 0.079 7 30ugAD11841((2C8C12)a) 0.281 0.055 0.069 8 30ugAD11842((2C6C10)a) 0.346 0.066 0.082 9 30ugAD11276 (internal uC16) 0.293 0.080 0.111 10 30ugAD11278 (internal uC16) 0.311 0.034 0.039 11 30ugAD11731 (internal uC16) 0.276 0.085 0.123 12 30ugAD11732 (internal uC16) 0.519 0.080 0.094

[0535] Table 10c. Average relative SOD1 expression in the mice of Example 6 in the cerebellum.

[0536] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.125 0.143 2 30ugAD11556 (without PK / PD regulator) 0.679 0.099 0.116 3 30ugAC002478(LP-183a) 0.473 0.069 0.080 4 30ugAD11758(C16a) 0.690 0.063 0.070 5 30ugAD11759(C22a) 0.532 0.173 0.256 6 30ugAD11821(HO-C16a) 0.474 0.100 0.127 7 30ugAD11841((2C8C12)a) 0.802 0.096 0.109 8 30ugAD11842((2C6C10)a) 0.473 0.062 0.071 9 30ugAD11276 (internal uC16) 0.359 0.058 0.069 10 30ugAD11278 (internal uC16) 0.559 0.075 0.087 11 30ugAD11731 (internal uC16) 0.506 0.122 0.162 12 30ugAD11732 (internal uC16) 0.524 0.045 0.049

[0537] Table 10d. Average relative SOD1 expression in the mice of Example 6 in the brainstem.

[0538]

[0539]

[0540] Example 7. In vivo administration of lipid-linked RNAi agents in transgenic mice

[0541] On the first day of the study, female TgSOD1G93A mice modified to express human SOD1 were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or RNAi agents formulated at 3 μg / μL in aCSF. Three animals per group (n=3) were administered 10 μL of aCSF or RNAi agent solution in aCSF. Animals were injected intracerebroventricularly according to the quantitative administration scheme of Table 11.

[0542] Table 11. Dosing regimen for mice of Example 7.

[0543] Group Dosed substance Dosing regimen 1 aCSF Single injection on day 1 2 30ugAD11556 (without PK / PD regulator) Single injection on day 1 3 30ugAC002478(LP-183a) Single injection on day 1 4 30ugAC002548(LP-283a) Single injection on day 1 5 30ugAC002549(LP-383a) Single injection on day 1 6 30ugAC002550(LP-396a) Single injection on day 1 7 30ugAD11739(LP-395c) Single injection on day 1 8 30ugAD11692(LP-183c) Single injection on day 1 9 30ugAD11728(LP-409c) Single injection on day 1

[0544] On the 8th day of the study, animals were sacrificed and thoracic spinal cord, cortex, cerebellum and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (PBS). The results are shown in Tables 12a-12d below.

[0545] Table 12a. Mean relative SOD1 expression in the mice of Example 7 in the cortex.

[0546] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.037 0.039 2 30ugAD11556 (without PK / PD regulator) 0.920 0.078 0.086 3 30ugAC002478(LP-183a) 0.763 0.088 0.100 4 30ugAC002548(LP-283a) 0.906 0.079 0.087 5 30ugAC002549(LP-383a) 0.840 0.050 0.053 6 30ugAC002550(LP-396a) 0.705 0.089 0.102 7 30ugAD11739(LP-395a) 0.815 0.117 0.136 8 30ugAD11692(LP-183a) 0.707 0.163 0.211 9 30ugAD11728(LP-409a) 0.472 0.124 0.168

[0547] Table 12b. Average relative SOD1 expression in the mice of Example 7 in the thoracic spinal cord.

[0548] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.051 0.054 2 30ugAD11556 (without PK / PD regulator) 0.770 0.155 0.194 3 30ugAC002478(LP-183a) 0.348 0.099 0.138 4 30ugAC002548(LP-283a) 0.765 0.186 0.246 5 30ugAC002549(LP-383a) 0.537 0.259 0.499 6 30ugAC002550(LP-396a) 0.616 0.035 0.037 7 30ugAD11739(LP-395a) 0.550 0.021 0.022 8 30ugAD11692(LP-183a) 0.368 0.059 0.070 9 30ugAD11728(LP-409a) 0.239 0.022 0.024

[0549] Table 12c. Average relative SOD1 expression in the mice of Example 7 in the cerebellum.

[0550] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.121 0.138 2 30ugAD11556 (without PK / PD regulator) 0.550 0.057 0.064 3 30ugAC002478(LP-183a) 0.380 0.103 0.141 4 30ugAC002548(LP-283a) 0.509 0.121 0.158 5 30ugAC002549(LP-383a) 0.576 0.183 0.269 6 30ugAC002550(LP-396a) 0.490 0.068 0.079 7 30ugAD11739(LP-395a) 0.606 0.188 0.272 8 30ugAD11692(LP-183a) 0.520 0.105 0.132 9 30ugAD11728(LP-409a) 0.442 0.055 0.062

[0551] Table 12d. Average relative SOD1 expression in the mice of Example 7 in the brainstem.

[0552] Group describe Relative SOD1 expression Error (high) Error (low) 1 aCSF 1.000 0.134 0.155 2 30ugAD11556 (without PK / PD regulator) 0.815 0.183 0.237 3 30ugAC002478(LP-183a) 0.333 0.049 0.057 4 30ugAC002548(LP-283a) 0.486 0.076 0.091 5 30ugAC002549(LP-383a) 0.518 0.179 0.274 6 30ugAC002550(LP-396a) 0.599 0.095 0.113 7 30ugAD11739(LP-395a) 0.463 0.149 0.219 8 30ugAD11692(LP-183a) 0.505 0.057 0.064 9 30ugAD11728(LP-409a) 0.529 0.182 0.279

[0553] Example 8. In vivo knockdown of SOD1 in transgenic TgSOD1 G93A rats

[0554] On study day 1, Tg SOD1 G93A rats were injected with 30 μL of artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or 30 μL of compound formulations in aCSF at concentrations of 0.33, 1.0, 3.33, 10 and 30 mg / mL (for Groups 2-6, respectively) according to Table 13 below:

[0555] Table 13: Quantitative administration groups of rats in Example 8.

[0556] Group ID Animals used Group 1 (aCSF) n＝4 Group 2 (10 μg AD12261) (LP-293a) n＝4 Group 3 (30 μg AD12261) (LP-293a) n＝4 Group 4 (100 μg AD12261) (LP-293a) n＝4 Group 5 (300 μg AD12261) (LP-293a) n＝4 Group 6 (900 μg AD12261) (LP-293a) n＝4

[0557] Four (n=4) rats per group were administered. Rats were injected intrathecally on day 1. On day 85, CSF was collected from each animal, and then the rats were euthanized and the left hemisphere and thoracic spinal cord were collected and stored in 10% NBF. Tissue samples were taken from the right hemisphere and thoracic spinal cord, cortex, cerebellum, and brainstem. The samples were analyzed for SOD1 mRNA knockdown by qPCR. The average results for each group are shown in Table 14 below:

[0558] Table 14. Relative expression of SOD1 mRNA in various tissues analyzed by qPCR for each quantitative administration group of Example 8.

[0559]

[0560] As shown in Table 14 above, a dose-dependent decrease in SOD1 mRNA expression was observed for transgenic rats treated with AD12261.

[0561] Example 9. In vivo knockdown of SOD1 in cynomolgus monkeys

[0562] On study day 1, cynomolgus monkeys were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or compound formulations containing 45 mg of AD12261 in aCSF according to Table 15 below:

[0563] Table 15: Dosage groups for non-human primates of Example 9.

[0564] Group ID Animals used Group 1 (aCSF) n＝4 Group 2 (45 mg AD12261) (LP-293a) - Day 29 n＝5 Group 3 (45 mg AD12261) (LP-293a) - Day 85 n＝5 Group 4 (45 mg AD12261) (LP-293a) - Day 168 n＝5

[0565] Four (n=4) monkeys in Group 1 (control group) were administered, and five (n=5) monkeys in Groups 2, 3, and 4 (trigger treatment) were administered. Monkeys were injected intrathecally on day 1. On study day 29, animals in Groups 1 and 2 were euthanized, and brain and spinal cord tissues were collected from each animal. On study day 85, animals in Group 3 were euthanized, and brain and spinal cord tissues were collected from each animal. On study day 168, animals in Group 4 were euthanized, and brain and spinal cord tissues were collected from each animal. Samples were analyzed for SOD1 mRNA knockdown by qPCR. Table 16 below shows the average results for each group relative to Group 1:

[0566] Table 16. Relative expression of SOD1 mRNA in various tissues of each quantitative administration group of Example 9 analyzed by qPCR.

[0567]

[0568]

[0569] As shown in Table 16 above, persistent (up to 168 days) reductions in SOD1 mRNA expression were observed in multiple tissues of non-human primates treated with AD12261.

[0570] Example 10. In vivo knockdown of SOD1 in transgenic Tg SOD1 G93A rats

[0571] On the first day of the study, male TgSOD1G93A rats (Sprague Dawley) modified to express human SOD1 were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or RNAi agents formulated at 10 mg / mL in aCSF. Four (n=4) animals per group were administered 30 μL of aCSF or RNAi agent solutions in aCSF. Animals were injected intrathecally (IT) according to the quantitative administration scheme of Table 17.

[0572] Table 17. Dosing regimen for mice of Example 10.

[0573] Group Dosed substance Dosing regimen 1 aCSF Single IT injection on day 1 2 300ugAD13302(10mg / mL)(LP-409a) Single IT injection on day 1 3 300ugAD13303(10mg / mL)(LP-430a) Single IT injection on day 1 4 300ugAD13304(10mg / mL)(LP-431a) Single IT injection on day 1 5 300ugAD13306(10mg / mL)(LP-435a) Single IT injection on day 1 6 300ugAD13308(10mg / mL)(LP-439a) Single IT injection on day 1 7 300ugAD13309(10mg / mL)(LP-429a) Single IT injection on day 1 8 300ugAD13310(10mg / mL)(LP-441a) Single IT injection on day 1

[0574] On the 8th day of the study, animals were sacrificed and thoracic spinal cord, cortex (temporal lobe), cerebellum, brain stem and dorsal root ganglion (lumbar region) were collected. PPIA was used to control genes and qPCR was used to determine the expression of superoxide dismutase 1 (SOD1) in each tissue. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (aCSF). The results are shown in Table 18 below.

[0575] Table 18. Mean relative SOD1 expression in rats of Example 10

[0576]

[0577]

[0578] Example 11. In vivo knockdown of SOD1 in transgenic Tg SOD1 G93A rats

[0579] On the first day of the study, male TgSOD1G93A rats (Sprague Dawley) modified to express human SOD1 were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or RNAi agents formulated at 10 mg / mL in aCSF. Four (n=4) animals per group were administered 30 μL of aCSF or RNAi agent solutions in aCSF. Animals were injected intrathecally (IT) according to the quantitative administration scheme of Table 19.

[0580] Table 19. Dosing regimen for mice of Example 11.

[0581]

[0582] On the 8th day of the study, the animals were sacrificed and the cortex (temporal lobe), thoracic spinal cord, cerebellum and brainstem were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR using PPIA control genes. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (aCSF). The results are shown in the following table 20.

[0583] Table 20. Mean relative SOD1 expression in rats of Example 11

[0584]

[0585] Example 12. In vivo knockdown of SOD1 in transgenic Tg SOD1 G93A rats

[0586] On the first day of the study, male TgSOD1G93A rats (Sprague Dawley) modified to express human SOD1 were injected with artificial cerebrospinal fluid (aCSF, obtained from a commercial supplier) or RNAi agents formulated at 10 mg / mL in aCSF. Four (n=4) animals per group were administered 30 μL of aCSF or RNAi agent solutions in aCSF. Animals were injected intrathecally (IT) according to the quantitative administration scheme of Table 21.

[0587] Table 21. Dosing regimen for mice of Example 12.

[0588] Group Dosed substance Dosing regimen 1 aCSF Single IT injection on day 1 2 300ugAC910361(10mg / mL)(internal uC16) Single IT injection on day 1 3 300ugAC910860(10mg / mL)(LP-293a) Single IT injection on day 1 4 300ugAC912620 (10mg / mL) (internal uLP-493a) Single IT injection on day 1

[0589] On the 8th day of the study, the animals were sacrificed and the cortex, thoracic spinal cord, cerebellum, brain stem, heart, midbrain and hippocampus were collected. The expression of superoxide dismutase 1 (SOD1) in each tissue was determined using qPCR using PPIA control genes. The average SOD1 expression of each animal in each tissue was normalized relative to the 1st group (aCSF). The results are shown in Table 22 below.

[0590] Table 22. Mean relative SOD1 expression in rats of Example 12

[0591]

[0592] Equivalent programs and scope

[0593] In the claims, articles such as "a", "an", and "the" may refer to one or more than one, unless indicated to the contrary or otherwise obvious from the context. Claims or descriptions including "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a specified product or process, unless indicated to the contrary or otherwise obvious from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or method. The invention includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise related to a given product or method.

[0594] In addition, the present invention encompasses all variations, combinations and arrangements, wherein one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim that is subordinate to another claim can be modified to include one or more limitations found in any other claim that is subordinate to the same basic claim. In the case where the elements are presented as a list, for example, in the Markush group format, each subgroup of the elements is also disclosed, and any element can be removed from the group. It should be understood that, in general, in the case where the present invention or aspects of the present invention are referred to as comprising specific elements and / or features, certain embodiments of the present invention or aspects of the present invention are composed of such elements and / or features, or are substantially composed of such elements and / or features. For simplicity, those embodiments are not specifically described in this article with such words. It should also be noted that the terms "comprising" and "containing" are intended to be open, and allow the inclusion of additional elements or steps. In the case of giving a range, endpoints are included. In addition, unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may be assumed in different embodiments of the present invention to be any specific value or sub-range within the stated range, to the tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0595] This application relates to various granted patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, the specification shall prevail. In addition, any specific embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if they are not explicitly set forth herein. Any specific embodiment of the present invention may be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[0596] Other Implementations

[0597] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative only and not to limit the scope of the invention, which is defined by the scope of the following claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A compound comprising: a) oligonucleotides; and b) a lipid conjugated to the 5' position or the 3' position of one of the terminal nucleotides of the oligonucleotide; The oligonucleotide comprises at least 15 nucleotides complementary to a gene expressed in CNS tissue.

2. The compound of claim 1, wherein the oligonucleotide is double-stranded.

3. The compound of claim 2, wherein the oligonucleotide comprises a sense strand and an antisense strand.

4. The compound of claim 3, wherein the lipid is conjugated to the sense strand.

5. The compound of claim 4, wherein the lipid is conjugated to the 5' position of the sense strand.

6. The compound of any one of claims 1-5, wherein the lipid is saturated.

7. The compound of any one of claims 1-5, wherein the lipid is unsaturated.

8. The compound of any one of claims 1-7, wherein the lipid comprises 10 to 30 carbon atoms.

9. The compound of any one of claims 1-8, wherein the lipid comprises 15 to 20 carbon atoms.

10. The compound of any one of claims 1-9, wherein the lipid is selected from: in The point of attachment to the oligonucleotide is indicated.

11. The compound of any one of claims 1-10, wherein the oligonucleotide is a RNAi agent.

12. The compound of any one of claims 1-11, wherein the antisense strand comprises cyclopropylene (cPrp) modified nucleotides.

13. The compound of claim 12, wherein the cyclopropylene-modified nucleotide is the 5'-terminal nucleotide of the antisense strand.

14. A compound selected from: wherein R comprises an oligonucleotide.

15. The compound of claim 14, wherein the oligonucleotide is double-stranded.

16. The compound of claim 15, wherein the oligonucleotide comprises a sense strand and an antisense strand.

17. The compound of claim 16, wherein the point of attachment to R is on the sense strand.

18. The compound of claim 17, wherein the point of attachment to R is at the 5' terminal nucleotide of the sense strand.

19. The compound of any one of claims 14-18, wherein the oligonucleotide is a RNAi agent.

20. A compound selected from: wherein R comprises an oligonucleotide.

21. The compound of claim 20, wherein the oligonucleotide is double-stranded.

22. The compound of claim 21, wherein the oligonucleotide comprises a sense strand and an antisense strand.

23. The compound of claim 22, wherein the point of attachment to R is on the sense strand.

24. The compound of claim 23, wherein the point of attachment to R is at the 5' terminal nucleotide of the sense strand.

25. The compound of any one of claims 20-24, wherein the oligonucleotide is a RNAi agent.

26. A compound comprising: a) oligonucleotides; and b) a hydroxy lipid conjugated to an internal nucleotide of the oligonucleotide; wherein the hydroxy lipid comprises a hydroxyl group, and The oligonucleotide comprises at least 15 nucleotides complementary to a gene expressed in CNS tissue.

27. The compound of claim 26, wherein the hydroxyl group is conjugated to a distal carbon of the hydroxylipid relative to the internal nucleotide.

28. The compound of claim 26 or claim 27, wherein the hydroxy lipid is conjugated to the 2' carbon of the internal nucleotide.

29. The compound of any one of claims 26-28, wherein the hydroxy lipid comprises 12 to 24 carbon atoms.

30. The compound of any one of claims 26-29, wherein the hydroxy lipid comprises 16 carbon atoms.

31. The compound of any one of claims 26-30, wherein the oligonucleotide is a RNAi agent.

32. The compound of claim 26, wherein the hydroxy lipid is in The point of attachment to the remainder of the oligonucleotide is indicated.

33. The compound of claim 32, wherein the oligonucleotide is a RNAi agent.

34. A method for delivering an oligonucleotide to a cell, the method comprising administering to a subject a compound according to any one of claims 1-33.

35. The method of claim 34, wherein the cell is part of the central nervous system (CNS).

36. A method of treating a disease or disorder of the central nervous system, the method comprising administering to a subject in need thereof a compound according to any one of claims 1-33.

37. The method of claim 36, 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 disease.

38. The method of claim 36 or 37, wherein the compound comprises an oligonucleotide comprising a nucleotide sequence complementary to a gene expressed in a CNS cell.

39. The method of claim 38, wherein the gene is selected from 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).

40. A compound having the following structure: or a pharmaceutically acceptable salt thereof.

41. A method of synthesizing a compound according to any one of claims 1 to 33, the method comprising reacting a compound according to claim 40 with a compound containing an oligonucleotide.

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