Lipid conjugates for delivery of therapeutic agents to adipose tissue

Abstract

Disclosed herein are compounds comprising lipid PK / PD modulators for in vivo delivery of oligonucleotide-based agents (e.g., double-stranded RNAi agents or antisense oligonucleotides) to certain tissues (e.g., adipose tissue) or cell types (e.g., adipocytes). The PK / PD modulators disclosed herein, when conjugated to oligonucleotide-based therapeutic or diagnostic agents, such as RNAi agents, can enhance in vivo delivery of the composition to adipocytes to facilitate inhibition of gene expression in those cells.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 478,795, filed January 6, 2023, and U.S. Provisional Patent Application No. 63 / 612,901, filed December 20, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted in XML format and is incorporated herein by reference in its entirety. The XML copy is named 30714-WO_SeqListing.xml, was created on January 5, 2024, and is 774kb in size.

[0003] The present disclosure relates to lipid conjugates for the in vivo delivery of oligonucleotide-based agents (e.g., double-stranded RNAi agents, e.g., small interfering RNA (siRNA) and antisense oligonucleotides) to adipose tissue and / or specific adipocyte cell types (e.g., adipocytes). [Background technology]

[0004] Oligonucleotide-based agents, such as double-stranded RNA interference (RNAi) agents and antisense oligonucleotides, show great promise and have the potential to revolutionize the field of medicine by providing patients with previously unavailable therapeutic treatment options.However, the effective in vivo delivery of oligonucleotide-based agents, especially double-stranded therapeutic RNAi agents, to desired cells and tissues of interest has long been a challenge in the development of viable therapeutic agents.Although consistent delivery to hepatocytes in the liver using carbohydrates such as N-acetyl-galactosamine has been well established in the field, significant challenges remain when trying to achieve specific and selective delivery of oligonucleotide-based agents to extrahepatic cells.

[0005] Over the past few years, various attempts have been made to target oligonucleotide-based agents to certain extrahepatic cell types, including cells of the central nervous system (CNS), adipocytes, cardiomyocytes, etc., using, for example, cholesterol conjugates (which have the disadvantage of being non-specific and therefore distributing to various undesirable tissues and organs) and lipid nanoparticles (LNPs) (which also have the disadvantage of being non-specific and are often reported to have toxicity concerns). However, to date, no suitable delivery has been achieved. Therefore, there remains a need for a delivery vehicle that can target oligonucleotide-based agents, especially RNAi agents, to non-hepatic cell types. As obesity has become a serious public health concern with its increasing incidence in adults and children, there is an ever-increasing need to selectively and effectively deliver therapeutic agents, such as oligonucleotide-based agents, to adipose tissue. Summary of the Invention

[0006] Disclosed herein are compounds (e.g., compounds of Formula (I)) comprising lipids conjugated (or linked) to oligonucleotide-based agents for delivery to adipose tissue or certain cell types (e.g., adipocytes). The lipids (also referred to herein as lipid PK / PD modulators) facilitate the delivery of the oligonucleotide-based agent payload to certain cell types (e.g., adipocytes) or adipose tissue. Also disclosed herein are lipid PK / PD modulator precursors (e.g., compounds of Formula (II)).

[0007] One aspect of the present disclosure provides a double-stranded oligonucleotide in which a lipid is conjugated to one of the terminal nucleotides of one of the strands. In some embodiments, the lipid is conjugated to the 5'-terminal nucleotide of one of the strands. In some embodiments, the lipid is conjugated to the 3'-terminal nucleotide of one of the strands. In some embodiments, the lipid is conjugated to both the 5'-terminal nucleotide and the 3'-terminal nucleotide of one of the strands. In some embodiments, the lipid is conjugated internally to one or more nucleotides of one or both of the strands (e.g., conjugated at the 2' position).

[0008] In another embodiment, the compound of formula (I) [ka]

[0009] and pharmaceutically acceptable salts thereof are disclosed herein, and R Z2 comprises an oligonucleotide containing from about 8 to about 50 independently modified or unmodified nucleotides, and L 1 and L 4 are each independently a lipid containing from about 10 to about 50 carbon atoms, and R Z1 , R Z3 , Y, Y 1 , L 2 , L 3 , t, and q are as defined herein. In some embodiments, a lipid (e.g., L 1 and / or L 4) is saturated. In some embodiments, the lipid is unsaturated. In some embodiments, the lipid is a sterol. In some embodiments, the lipid is a saturated lipid having 12-30 carbon atoms. In some embodiments, the lipid is a straight-chain lipid having 16 carbon atoms. In some embodiments, the lipid contains a hydroxyl moiety. In some embodiments, the lipid contains a carboxylic acid moiety.

[0010] Further provided herein is a method of treating a fat-related disorder (e.g., a metabolic disease such as obesity, type 2 diabetes, insulin resistance, metabolic syndrome, various lipodystrophies, lipedema, atherosclerotic vascular disease, a cardiometabolic disease or disorder, or other like disease or disorder) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compounds or compositions described herein.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

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

[0013] Lipid PK / PD modulators Described herein are compounds (e.g., compounds of Formula (I)) comprising a lipid PK / PD modulator conjugated to an oligonucleotide-based agent to provide in vivo cellular delivery of a payload, such as an RNA interference (RNAi) agent. Without being bound by any particular theory, it is believed that the compounds described herein modulate the pharmacokinetic and / or pharmacodynamic (PK / PD) properties of the RNAi agent, increasing delivery to adipose tissue and cells, thereby resulting in increased efficacy of the oligonucleotide-based therapeutic. The compounds described herein can facilitate delivery to certain cell types, including, but not limited to, adipocyte types, including white adipocytes and brown adipocytes.

[0014] Also, compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, as described herein, wherein: R Z1 and R Z3 each independently comprises a linking or capping residue; R Z2 and an oligonucleotide containing from about 8 to about 50 nucleotides, each of which may be independently modified or unmodified; Y is at least one L 1 , if present, L 2 To, or R Z1 is a bond or linker connecting Y 1 But there is at least one L 3 , if present, L 4 To, or R Z3 is a bond or linker connecting L 2 and L 3 is independently absent or a linker comprising 1 to 20 PEG units; q is 1, 2, or 3, valence permitting; t is 1, 2, or 3, valence permitting; L1 and L 4 are each independently a lipid or capping moiety containing from about 10 to about 50 carbon atoms.

[0015] The compounds of formula (I) contain a substituent R Z1 and R Z3 In one embodiment, R Z1 is an inverted abasic residue (e.g., R Z1 is (invAb)s as defined in Table 4). In one embodiment, R Z3 is an inverted abasic residue (e.g., R Z3 is (invAb) as defined in Table 4). In one embodiment, R Z1 is (invAb)s and R Z3 is (invAb). In one embodiment, R Z1 is R Z2 In one embodiment, R Z1 is a bond. In one embodiment, R Z3 is a bond.

[0016] The compounds of formula (I) contain a substituent R Z2 In one embodiment, R Z2 is a single-stranded oligonucleotide. In one embodiment, R Z2 is a double-stranded oligonucleotide. In one embodiment, R Z2 comprises an oligonucleotide, wherein the oligonucleotide comprises an antisense strand that is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in adipose tissue (e.g., human adipose tissue). Z2 The oligonucleotide comprises an oligonucleotide, wherein the oligonucleotide comprises an antisense strand that is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in an adipocyte (e.g., a human adipocyte). In some embodiments, the gene is expressed in a mature adipocyte. In some embodiments, the gene is expressed in a white adipocyte.

[0017] In certain embodiments, the compound of formula (I) contains a substituent Y. In certain embodiments, Y is a bond. In certain embodiments, Y is selected from the group consisting of at least one L 1 , if present, L 2 To, or L 1 R Z1 In one embodiment, Y is a linker that connects at least two L 1 Substituents, if any, are designated by L 2 or at least two L 1 Substituents are represented by R Z1 In some embodiments, Y is a divalent moiety that is connected to the formula: [ka] wherein Y a and Y c are each independently absent, —N(H)—, or —C(O)—; Y b is substituted or unsubstituted heteroalkylene, substituted or unsubstituted carbocyclene, substituted or unsubstituted heterocyclene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. a is —C(O)—. In one embodiment, Y a is —N(H)—. In one embodiment, Y c is —C(O)—. In one embodiment, Y c is —N(H)—. In one embodiment, Y b is a substituted or unsubstituted C1-C6 heteroalkylene. b is the expression [ka] In one embodiment, Y b is absent. b is a substituted or unsubstituted carbocyclene. b is the expression [ka] In one embodiment, Y b is substituted or unsubstituted heterocyclene. b is the expression [ka] In one embodiment, Y b is substituted or unsubstituted arylene. b is substituted or unsubstituted phenylene. b is the expression [ka] It is of the type.

[0018] In certain embodiments, Y is -N(H)-C(O)-, -C(O)-N(H)-, [ka] is selected from the group consisting of:

[0019] In one embodiment, Y is a group selected from two L 1 Group L 2 or two L 1 The group is R Z1 In some embodiments, Y is a trivalent moiety that is connected to the formula: [ka] It is of the type.

[0020] In some embodiments, Formula (I) is a group containing a substituent L 2 In one embodiment, L 2 is the expression [ka] wherein: L 2ais a bond or expression: [ka] wherein h is an integer from 1 to 12; L 2b is a bond or a chemical moiety formed by reacting a first reactive moiety with a second moiety; L 2c is a bond or a bidentate linking group.

[0021] In one embodiment, L 2a is a bond. 2a is the expression: [ka] wherein h is an integer from 1 to 12. In some embodiments, h is 2. In some embodiments, h is 3. In some embodiments, h is 5. In some embodiments, h is 9. In some embodiments, h is 10.

[0022] In one embodiment, L 2b is a bond. 2b is —C(O)—. In one embodiment, L 2b is the expression: [ka] It is of the type.

[0023] In one embodiment, L 2c is the expression: [ka] In one embodiment, L 2c is the expression: [ka] It is of the type.

[0024] The compounds of formula (I) contain t instances of substituents L 1 and q examples of substituents L 4 In one embodiment, L 1 and L 4 are each independently a lipid containing about 10 to about 50 carbon atoms.

[0025] In one embodiment, L 1 is a fatty acid, a fatty acid-derived group, a glycerolipid, a glycerolipid-derived group, a phospholipid, a phospholipid-derived group, a sphingolipid, a sphingolipid-derived group, a cholesterol ester, or a cholesterol ester-derived group. 1 At least one instance of L is independently a straight chain lipid. 1 At least one instance of L is independently a saturated lipid. 1 At least one example of L is independently an unsaturated lipid. 1 At least one instance of L is independently a branched lipid. 1 is a NEM (i.e., N-ethylmaleimide) capping moiety.

[0026] In one embodiment, L 1 At least one example of independently has the formula: [ka] wherein R L1a is H or CO2H, and r is an integer from 5 to 35. In one embodiment, R L1a is H. In one embodiment, R L1ais COH. In some embodiments, r is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, r is 9. In some embodiments, r is 11. In some embodiments, r is 12. In some embodiments, r is 13. In some embodiments, r is 14. In some embodiments, r is 15. In some embodiments, r is 17.

[0027] In one embodiment, L 1 At least one instance of independently has the formula [ka] wherein w is an integer of 2 to 25, and v is an integer of 2 to 25. 1 At least one example of independently has the formula: [ka] It is of the type.

[0028] In one embodiment, L 1 are independently straight-chain lipids containing 1 to 8 alkenylene moieties. 1 At least one example of independently has the formula: [ka] wherein R L1b is -CH3 or -CO2H, and R L1c is —CH— or —C(O)—, j is an integer of 0 to 20, k is an integer of 1 to 8, and o is an integer of 1 to 20. In one embodiment, R L1b is —H. In one embodiment, R L1b is —COH. In one embodiment, R L1c is -CH-. In one embodiment, R L1cis -C(O)-. In some embodiments, j is 0. In some embodiments, j is 1. In some embodiments, j is 4. In some embodiments, j is 7. In some embodiments, k is 1. In some embodiments, k is 2. In some embodiments, k is 3. In some embodiments, k is 4. In some embodiments, k is 5. In some embodiments, o is 2. In some embodiments, o is 3. In some embodiments, o is 6. In some embodiments, o is 10. In some embodiments, at least one L 1 are independently of the formula: [ka] It is of the type.

[0029] In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3.

[0030] In one embodiment, [ka] is selected from the group consisting of any of the PK / PD modulators shown in Table 2.

[0031] In one embodiment, L 4 is a fatty acid, a fatty acid-derived group, a glycerolipid, a glycerolipid-derived group, a phospholipid, a phospholipid-derived group, a sphingolipid, a sphingolipid-derived group, a cholesterol ester, or a cholesterol ester-derived group. 4 At least one instance of L is independently a straight chain lipid. 4 At least one instance of L is independently a saturated lipid. 4 At least one example of L is independently an unsaturated lipid. 4 At least one instance of L is independently a branched lipid.4 is a NEM (i.e., N-ethylmaleimide) capping moiety.

[0032] In one embodiment, L 4 At least one example of independently has the formula: [ka] wherein R L4a is H or CO2H, and d is an integer of 5 to 35. In one embodiment, R L4a is H. In one embodiment, R L4a is COH. In some embodiments, d is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, d is 9. In some embodiments, d is 11. In some embodiments, d is 12. In some embodiments, d is 13. In some embodiments, d is 14. In some embodiments, d is 15. In some embodiments, d is 17.

[0033] In one embodiment, L 4 At least one instance of independently has the formula [ka] wherein x is an integer of 2 to 25 and y is an integer of 2 to 25. 4 At least one example of independently has the formula: [ka] It is of the type.

[0034] In one embodiment, L 4 are independently straight-chain lipids containing 1 to 8 alkenylene moieties. 4 At least one example of independently has the formula: [ka] wherein R L4b is -CH3 or -CO2H, and R L4c is —CH— or —C(O)—, a is an integer of 0 to 20, b is an integer of 1 to 8, and c is an integer of 1 to 20. In one embodiment, R L4b is —H. In one embodiment, R L4b is —COH. In one embodiment, R L4c is -CH-. In one embodiment, R L4c is -C(O)-. In some embodiments, a is 0. In some embodiments, a is 1. In some embodiments, a is 4. In some embodiments, a is 7. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, c is 2. In some embodiments, c is 3. In some embodiments, c is 6. In some embodiments, c is 10. In some embodiments, at least one L 4 are independently of the formula: [ka] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[0035] In one embodiment, [ka] is selected from the group consisting of any of the PK / PD modulators shown in Table 2.

[0036] In some embodiments, the compound of formula (I) is a compound of formula (I) having a substituent Y 1 In one embodiment, Y 1 is a bond.1 must contain at least one L 4 , if present, L 3 To, or L 4 R Z3 In one embodiment, Y 1 must contain at least one L 4 , if present, L 3 Connect to L 4 R Z3 In one embodiment, Y 1 is the expression: [ka] wherein Y 1a and Y 1c are each independently absent, —N(H)—, or —C(O)—; Y 1b is substituted or unsubstituted heteroalkylene, substituted or unsubstituted carbocyclene, substituted or unsubstituted heterocyclene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. 1a is —C(O)—. In one embodiment, Y 1a is —N(H)—. In one embodiment, Y 1c is —C(O)—. In one embodiment, Y 1c is —N(H)—. In one embodiment, Y 1b is a substituted or unsubstituted C1-C6 heteroalkylene. 1b is the expression [ka] In one embodiment, Y 1b is a substituted or unsubstituted carbocyclene. 1b is the expression [ka] In one embodiment, Y 1bis substituted or unsubstituted heterocyclene. 1b is the expression [ka] In one embodiment, Y 1b is absent. 1b is substituted or unsubstituted arylene. 1b is substituted or unsubstituted phenylene. 1b is the expression [ka] It is of the type.

[0037] In one embodiment, Y 1 is -N(H)-C(O)-, -C(O)-N(H)-, [ka] is selected from the group consisting of:

[0038] In one embodiment, Y 1 There are two L 4 Group L 3 or two L 4 The group is R Z3 In some embodiments, Y is a trivalent moiety that is connected to 1 is the expression: [ka] It is of the type.

[0039] In some embodiments, Formula (I) is a group containing a substituent L 3 In one embodiment, L 3 is the expression [ka] wherein: L 3ais a bond or a bidentate linking group; L 3b is a bond or a chemical moiety formed by reacting a first reactive moiety with a second reactive moiety; L 3c is a bond or expression: [ka] wherein i is an integer from 1 to 12.

[0040] In one embodiment, L 3c is a bond. 3c is the expression: [ka] wherein h is an integer from 1 to 12. In some embodiments, h is 2. In some embodiments, h is 3. In some embodiments, h is 5. In some embodiments, h is 9. In some embodiments, h is 10.

[0041] In one embodiment, L 3b is a bond. 3b L 3b is a bond, -C(O)-, or the formula: [ka] It is of the type.

[0042] In one embodiment, L 3a is the expression: [ka] It is of the type.

[0043] The present disclosure further provides lipid delivery platforms for oligonucleotides, methods of using the lipid delivery platforms, and methods of manufacturing the lipid delivery platforms.

[0044] As used herein and understood by those of skill in the art, a polyethylene glycol (PEG) unit refers to a repeating unit of the formula -(CHCHO)-. It is understood that in the chemical structures disclosed herein, the PEG unit can be represented as -(CHCHO)-, -(OCHCH)-, or -(CHOCH)-. It is also understood that a number indicating the number of repeating PEG units can be placed on either side of the brackets representing the PEG unit.

[0045] Another aspect of the invention provides a process for producing a compound comprising an oligonucleotide (eg, a double-stranded or single-stranded oligonucleotide) and a lipid moiety.

[0046] In some embodiments, the method includes conjugating an oligonucleotide-based agent that includes a first reactive moiety to a compound that includes a lipid and a second reactive moiety to form a compound that includes both an RNAi agent and a lipid moiety.

[0047] In some embodiments, the first reactive moiety is selected from the group consisting of hydroxy and amine reactive groups. In some embodiments, the first reactive moiety is an amine. In some embodiments, the first reactive moiety is a hydroxy group. In some embodiments, the first reactive moiety is an alkyne. In some embodiments, the first reactive moiety is a disulfide.

[0048] In some embodiments, the second reactive moiety is selected from the group consisting of an ester (including but not limited to, activated esters such as succinimidyl esters, tetrafluorophenoxy esters, and para-nitrophenoxy esters), a sulfone (including but not limited to, methyl sulfone, sulfonyl halides), a maleimide, an azide, and a phosphoramidite. In some embodiments, the second reactive moiety is an ester. In some embodiments, the second reactive moiety is a sulfone. In some embodiments, the second reactive moiety is a phosphoramidite. In some embodiments, the second reactive moiety is a maleimide. In some embodiments, the second reactive moiety is an azide.

[0049] As shown in Table 1 below and as described herein, the compounds of formula (I), LP-4-p, LP-18-p, LP-128-p, LP-151-p, LP-183-p, LP-200-p, LP-208-p, LP-211-p, LP-232-p, LP-242-p, LP-243-p, LP-244-p, LP-245-p, LP-249-p, LP-274-p, LP-295-p, LP-310-p, LP-359-p, LP-361-p, LP-371-p, LP-374-p, LP-375-p, LP-377-p, LP-378-p, LP-379-p, LP-380-p, LP-40 3-p, LP-404-p, LP-412-p, LP-413-p, LP-416-p, LP-424-p, LP-425-p, LP-426-p, LP-427-p, LP-428-p, LP-432-p, LP-433-p, LP-444-p, LP-445-p, LP-446-p, LP-447-p, LP-453-p, LP-455-p, LP-457-p, LP-458-p, LP-459-p, LP-460-p, LP-461-p, LP-468-p, LP-469 phosphoramidite, LP-470 phosphoramidite, LP-473-p, LP-474-p, and CNR1 The SM2 phosphoramidite compounds may be referred to as "pharmacokinetic and / or pharmacodynamic modulator precursors" (hereinafter "PK / PD modulator precursors").

[0050] It will also be understood that some of the compounds may be referred to as "pharmacokinetic and / or pharmacodynamic modulators" (hereinafter "PK / PD modulators"). As shown in Table 3 below, formulas LP-4-b, LP-18-b, LP-128-b, LP-151-b, LP-183-b, LP-200-b, LP-208-b, LP-211-b, LP-232-b, LP-242-b, LP-243-b, LP-244-b, LP-245-b , LP-249-b, LP-274-b, LP-295-b, LP-310-b, LP-359-b, LP-361-b, LP-371-b, LP-374-b, LP-375-b, LP-377-b, LP-378-b, LP-379-b, LP-380-b, LP-403-b, L P-404-b, LP-412-b, LP-413-b, LP-416-a, LP-416-b, LP-424-b, LP-425-b, LP-426-b, LP-427-b, LP-428-b, LP-432-b, LP-433-b, LP-444-b, LP-445-b, LP- 446-b, LP-447-b, LP-453-b, LP-455-b, LP-457-b, LP-458-b, LP-459-b, LP-460-b, LP-461-b, LP-468-b, LP-469-b, LP-470-b, LP-473-b, LP-474-b, and CNR1 When used to refer to the portion of a compound of SM2-b, the term "PK / PD modulator" refers to the portion of the compound excluding R (ie, the oligonucleotide-based agent).

[0051] The lipid PK / PD modulator is linked to an oligonucleotide-based agent to facilitate delivery to desired adipocytes or tissues. The lipid PK / PD modulator precursor can be synthesized with a reactive moiety, including, but not limited to, an activated ester group and a phosphoramidite, which facilitates immediate linkage to one or more linking groups on an RNAi agent. Chemical synthesis methods for linking such PK / PD modulator precursors to oligonucleotides containing RNAi agents are generally known in the art. The terms "PK / PD modulator" and "lipid PK / PD modulator" can be used interchangeably herein.

[0052] In one embodiment, a compound of formula (II): [ka] or a salt thereof, provided herein, wherein: R g is a reactive moiety suitable for conjugation to an oligonucleotide-based agent; Y 2 But there is at least one L 5 , if present, L 6 To, or R g is a bond or linker connecting Each L 5 are independently lipids containing from about 10 to about 50 carbon atoms; L 6 is a linker comprising 1 to 20 PEG units; z is 1, 2, or 3, valence permitting.

[0053] The compound of formula (II) contains a substituent R g In one embodiment, R g is the expression: [ka] It is of the type.

[0054] In some embodiments, the compound of formula (II) comprises a substituent Y2 In one embodiment, Y 2 is a bond. 2 must contain at least one L 5 , if present, L 6 To, or L 5 R g In one embodiment, Y 2 If present, there is at least one L 5 L 6 Connect to L 5 R g In one embodiment, Y 2 is -N(H)-C(O)-, -C(O)-N(H)-, [ka] is selected from the group consisting of:

[0055] In one embodiment, Y 2 There are two L 5 Group L 6 or two L 5 The group is R g In some embodiments, Y is a trivalent moiety that is connected to 2 is the expression: [ka] It is of the type.

[0056] In some embodiments, the compound of formula (II) comprises a substituent L 6 In one embodiment, L 6 is a linker comprising 1 to 10 PEG units. 6 is the expression: [ka] In one embodiment, L 6 is the expression: [ka] In one embodiment, L 6 is the expression: [ka] In one embodiment, L 6 is the expression: [ka] In one embodiment, L 6 is the expression: [ka] It is of the type.

[0057] The compound of formula (II) contains a substituent L 5 In one embodiment, L 5 At least one instance of L is independently a saturated lipid. 5 At least one example of L is independently an unsaturated lipid. 5 At least one instance of L is independently a straight chain lipid. 5 At least one instance of L is independently a branched lipid. 5 At least one example of is independently a fatty acid, a fatty acid-derived group, a glycerolipid, a glycerolipid-derived group, a phospholipid, a phospholipid-derived group, a sphingolipid, a sphingolipid-derived group, a cholesterol ester, or a cholesterol ester-derived group.

[0058] In one embodiment, L 5 At least one example of independently has the formula: [ka] wherein R L5 is H or CO2H, and e is an integer from 5 to 35. In one embodiment, R L5 is H. In one embodiment, RL5 is COH. In some embodiments, e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, e is 9. In some embodiments, e is 11. In some embodiments, e is 12. In some embodiments, e is 13. In some embodiments, e is 14. In some embodiments, e is 15. In some embodiments, e is 17.

[0059] In one embodiment, L 5 At least one example of independently has the formula: [ka] In one embodiment, L 5 At least one example of independently has the formula: [ka] It is of the type.

[0060] In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3.

[0061] In certain embodiments, the compound of formula (II) can be any one of the compounds shown in Table 1 below.

[0062] PK / PD modulator precursors, such as compounds of formula (II), or LP-4-p, LP-18-p, LP-128-p, LP-151-p, LP-183-p, LP-200-p, LP-208-p, LP-211-p, LP-232-p, LP-242-p, LP-243-p, LP-244-p, LP-245-p, LP-249-p, LP-274-p, LP-295-p, LP-310-p, LP-359-p, LP-361-p, LP-371-p, LP-374-p, LP-375-p, LP-377-p, LP-378-p, LP-379-p, LP-380-p, LP-4 03-p, LP-404-p, LP-412-p, LP-413-p, LP-416-a, LP-416-b, LP-424-p, LP-425-p, LP-426-p, LP-427-p, LP-428-p, LP-432-p, LP-433-p, LP-444-p, LP-445-p, LP-446-p, LP-447-p, LP-453-p, LP-455-p, LP-457-p, LP-458-p, LP-459-p, LP-460-p, LP-461-p, LP-468-p, LP-469 phosphoramidite, LP-470 phosphoramidite, LP-473-p, LP-474-p, and CNR1 A compound selected from the group consisting of SM2 phosphoramidites can be used as a starting material for linking to an oligonucleotide-based agent, such as an RNAi agent or an antisense oligonucleotide. The PK / PD modulator precursor can be covalently attached to the oligonucleotide-based agent using any method known in the art. For example, in some embodiments, an activated ester PK / PD modulator precursor can be reacted with an amine-containing moiety on the 5' end of the sense strand. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] During the ceremony, [ka] indicates a solid support such as a resin.

[0063] In some embodiments, one or more lipids described herein can be conjugated to an oligonucleotide-based agent, such as an RNAi agent. In some embodiments, one, two, three, four, five, six, seven, or more lipids described herein can be conjugated to an oligonucleotide-based agent, such as an RNAi agent. In some embodiments, one lipid is conjugated to an oligonucleotide-based agent, such as an RNAi agent. In some embodiments, two lipids are conjugated to an oligonucleotide-based agent, such as an RNAi agent.

[0064] The lipid PK / PD modulator precursor can be conjugated to an oligonucleotide-based agent using any method known in the art. In some embodiments, a lipid PK / PD modulator precursor containing an ester moiety (e.g., an activated ester) can react with an amine-containing oligonucleotide-based agent to form a compound containing a PK / PD modulator conjugated to an oligonucleotide-based agent. An exemplary reaction scheme is shown below: [ka] In the formula, R ZZ contains an RNAi agent, R xcontains an ester moiety (eg, an activated ester such as a succinimidyl ester, a tetrafluorophenoxy ester, and a para-nitrophenoxy ester).

[0065] In some embodiments, the amine can be on the 5' or 3' end of the oligonucleotide-based agent. In some embodiments, the amine can be on the 5' end of the oligonucleotide-based agent. In some embodiments, the amine can be on the 3' end of the oligonucleotide-based agent. In some embodiments, the oligonucleotide-based agent is an RNAi agent, and the PK / PD modulator is conjugated to the sense (or passenger) strand of the RNAi agent.

[0066] In some embodiments, a PK / PD modulator precursor containing a maleimide moiety can be reacted with an RNAi agent containing a disulfide linkage to form a compound containing a PK / PD modulator conjugated to an RNAi agent. The disulfide can be reduced and added to the maleimide via a Michael addition reaction. An exemplary reaction scheme is shown below: [ka] In the formula, R ZZ comprises an RNAi agent, [ka] indicates a point of attachment to any suitable group known in the art. In some examples of the above reaction schemes, [ka] is hexyl (CH 13 ) and are bonded to alkyl groups such as

[0067] In some embodiments, the PK / PD modulator precursor may contain a sulfone moiety and may react with a disulfide. An exemplary reaction scheme is shown below: [ka] In the formula, R ZZ comprises an RNAi agent, [ka] indicates a point of attachment to any suitable group known in the art. In some examples of the above reaction schemes, [ka] is hexyl (CH 13 ) and are bonded to alkyl groups such as

[0068] In some embodiments, a PK / PD modulator precursor can include an azide moiety and can be reacted with an RNAi agent that includes an alkyne to form a compound that includes a PK / PD modulator conjugated to an RNAi agent according to the following general reaction scheme: [ka]

[0069] In the formula, R ZZcomprises an RNAi agent. In some embodiments, a lipid PK / PD modulator precursor comprising a sulfonyl moiety can be reacted with an oligonucleotide-based agent comprising an amine to form a compound comprising a lipid PK / PD modulator conjugated to an oligonucleotide-based agent. In some embodiments, the amine can be on the 5' or 3' end of the oligonucleotide-based agent. In some embodiments, the amine can be on the 5' end of the oligonucleotide-based agent. In some embodiments, the amine can be on the 3' end of the oligonucleotide-based agent. In some embodiments, a lipid PK / PD modulator precursor comprising a phosphoramidite moiety can be reacted with an oligonucleotide-based agent comprising a hydroxyl moiety to form a compound comprising a lipid PK / PD modulator conjugated to an oligonucleotide-based agent. In some embodiments, the oligonucleotide-based agent is an RNAi agent, and the hydroxyl moiety can be on the 5' or 3' end of the RNAi agent. In some embodiments, the hydroxyl moiety can be on the 5' end of the RNAi agent. In some embodiments, the hydroxyl moiety can be on the 3' end of the RNAi agent. In some embodiments, an azide-containing lipid PK / PD modulator precursor can be reacted with an alkyne-containing oligonucleotide-based agent to form a compound comprising a lipid PK / PD modulator conjugated to an oligonucleotide-based agent. In some embodiments, the alkyne can be on the 5' or 3' end of the oligonucleotide-based agent. In some embodiments, the alkyne can be on the 5' end of the oligonucleotide-based agent. In some embodiments, the alkyne can be on the 3' end of the oligonucleotide-based agent.

[0070] In some embodiments, the lipid 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 some embodiments, the lipid PK / PD modulator can be conjugated to the 2' position of a nucleotide of the sense or antisense strand. For example, a lipid PK / PD modulator can be conjugated to an oligonucleotide-based agent comprising a modified nucleotide containing an alkyne moiety (e.g., aAlk, aAlks, cAlk, cAlks, gAlk, gAlks, uAlk, uAlks, (see Table 4)) by contacting the alkyne with a PK / PD modulator precursor containing an azide moiety.

[0071] In some embodiments, the RNAi agent is synthesized with a disulfide-containing moiety at the 3' end of the sense strand, and the lipid 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.

[0072] In some embodiments, the lipid PK / PD modulator comprises a compound shown in Table 2.

[0073] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] During the ceremony, [ka] indicates the point of attachment to the oligonucleotide.

[0074] Each of the lipid PK / PD modulators described herein can be conjugated to an oligonucleotide at the 5'-terminus, the 3'-terminus, or both. Each of the lipid PK / PD modulators described herein can be combined with any one of the other lipid PK / PD modulators described herein. In some embodiments, the lipid PK / PD modulator can be directly conjugated to the 5'- or 3'-terminal nucleotide of the oligonucleotide, while in other embodiments, a linker can be used to conjugate the lipid PK / PD modulator to the terminal nucleotide (e.g., linker L6—see Table 4 for structural information). As shown in Table 2B, any combination of lipid PK / PD modulators can be used to synthesize an oligonucleotide-based agent.

[0075] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 [Table 3-27] [Table 3-28]

[0076] In some embodiments, the lipid PK / PD modulator may include a compound having a formula shown in Table 3.

[0077] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] wherein R comprises an oligonucleotide.

[0078] definition As used herein, the terms "oligonucleotide" and "polynucleoside" refer to a polymer of linked nucleosides, each of which may be independently modified or unmodified.

[0079] As used herein, the term "oligonucleotide-based agent" refers to a chemical composition composed of one or more oligonucleotides. "Oligonucleotide-based agent" includes, but is 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. General aspects of the manufacture, design, and synthesis of oligonucleotide-based agents are known in the art.

[0080] As used herein, "antisense oligonucleotide" means a single-stranded oligonucleotide molecule in which a portion of the nucleotide sequence is at least partially complementary to a messenger RNA (mRNA) and is thereby capable of hybridizing and thereby blocking translation of the mRNA into protein and inhibiting expression.

[0081] As used herein, an "RNAi agent" (also referred to as an "RNAi trigger") refers to a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can degrade or inhibit (e.g., under appropriate conditions) translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent may act via the RNA interference mechanism (i.e., induce RNA interference via interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway. Although RNAi agents, as that term is used herein, are believed to act primarily via the RNA interference mechanism, the RNAi agents of the present disclosure are not bound to or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are composed of a sense strand and an antisense strand and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of an RNAi agent described herein is at least partially complementary to a targeted mRNA. The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0082] As used herein, the term "lipid" refers to moieties and molecules that are soluble in nonpolar solvents. The term lipid includes amphipathic molecules containing a polar, water-soluble head group and a hydrophobic tail. Lipids can be of natural or synthetic origin. Non-limiting examples of lipids include fatty acids (e.g., saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids), glycerolipids (e.g., monoacylglycerols, diacylglycerols, and triacylglycerols), phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine), sphingolipids (e.g., sphingomyelin), and cholesterol esters. As used herein, the term "saturated lipid" refers to a lipid that does not contain any unsaturation. As used herein, the term "unsaturated lipid" refers to a lipid that contains at least one degree of unsaturation. As used herein, the term "branched lipid" refers to a lipid that contains two or more linear chains, each linear chain covalently linked to at least one other linear chain. As used herein, the term "straight chain lipid" refers to a lipid that does not contain any branching.

[0083] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown," when referring to the expression of a given gene, mean that expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with an oligonucleotide-based agent as described herein, compared to a second cell, group of cells, tissue, organ, or subject that has not been so treated.

[0084] As used herein, the terms "sequence" and "nucleotide sequence" mean the sequence or order of nucleic acid bases or nucleotides written as a sequence of letters using standard nomenclature.

[0085] As used herein, "base," "nucleotide base," or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, including the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified, including, but not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds containing modified nucleobases) is known in the art.

[0086] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleobase or nucleotide sequence (e.g., an RNAi agent sense strand or a target mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., an RNAi agent antisense strand or a single-stranded antisense oligonucleotide), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a duplex or double-helix structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specific standard conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for purposes of determining identity or complementarity.

[0087] As used herein, "fully complementary" or "sufficiently complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0088] As used herein, "partially complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, at least 70% (but not all) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0089] As used herein, "substantially complementary" means that in a hybridized pair of nucleic acid or nucleotide sequence molecules, at least 85% (but not all) of the bases in a contiguous sequence of a first oligonucleotide hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence can include all or a portion of the first or second nucleotide sequence.

[0090] As used herein, the terms "complementary," "fully complementary," "partially complementary," and "substantially complementary" are used in reference to matching nucleobases or nucleotides between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of a target mRNA.

[0091] As used herein, the term "substantially identical" or "substantial identity" as applied to a nucleic acid sequence means that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or more sequence identity, 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 over a comparison window. The percentage is calculated by determining the number of positions where the same type of nucleobase is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences substantially identical to those disclosed herein.

[0092] As used herein, the terms "treat," "treatment," and the like refer to methods or steps taken to provide relief or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" can include preventative treatment, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0093] As used herein, the phrase "introducing into a cell," when referring to an oligonucleotide-based agent, means functionally delivering the oligonucleotide-based agent to a cell. The phrase "functional delivery" means delivering the oligonucleotide-based agent to a cell in a manner that allows the oligonucleotide-based agent to have its expected biological activity, e.g., sequence-specific inhibition of gene expression.

[0094] As used herein, the term "isomers" refers to compounds that have identical molecular formulae but differ in the nature or sequence 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 called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereomers," and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0095] As used herein, for each structure in which asymmetric centers exist and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, unless the structure is specifically identified as having a particular configuration, 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.

[0096] As used in the claims herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. As used in the claims herein, the phrase "consisting essentially of" limits the claim to specified substances or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0097] Those skilled 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 placed. Thus, as used herein, the structures disclosed herein contemplate that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of their state of protonation based on the environment (e.g., pH), as will be readily understood by those skilled in the art.

[0098] As used herein, the term "linked" or "conjugated," when referring to a connection between two compounds or molecules, means that the two molecules are linked by a covalent bond or associated through a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples, when the term "linked" or "conjugated" refers to an association between two molecules through a non-covalent bond, the association between the two different molecules is such that the two molecules are at a concentration of 1×10 in a physiologically acceptable buffer (e.g., buffered saline). -4 Less than M (e.g., 1 × 10 -5 Less than M, 1 x 10 -6 Less than M or 1 x 10 -7 K (less than M) D Unless otherwise stated, 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.

[0099] As used herein, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another second molecule or a second portion of a molecule. Similarly, when used in the art, the term scaffold may be used interchangeably with linking group. A linking group may contain any number of atoms or functional groups. In some embodiments, a linking group may simply serve to link two biologically active molecules, without facilitating any biological or pharmaceutical response.

[0100] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group containing 1 to 12 (e.g., 1 to 8, 1 to 6, 1 to 4, or 1 to 3) carbon atoms. Alkyl groups can be straight-chained 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.

[0101] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds). In an alkenyl group, a C=C double bond of unspecified stereochemistry (e.g., -CH=CHCH3) can be either an (E)- or a (Z)-double bond.

[0102] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (eg, 1, 2, 3, or 4 triple bonds).

[0103] The term "carbocyclyl" or "carbocyclic" refers to a ring system having 3 to 14 ring carbon atoms, such as "C 3~14 "Carbocyclyl" refers to a non-aromatic cyclic hydrocarbon group having zero heteroatoms. Carbocyclyl groups can be monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing fused, bridged, or spiro ring systems), saturated, or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, and the point of attachment is on the carbocyclyl ring; in such cases, the number of carbons continues to designate the number of carbons in the carbocyclic ring system.

[0104] The term "heterocyclyl" or "heterocyclic" refers to a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, valence permitting. Heterocyclyl groups may be either monocyclic (a "monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems, e.g., bicyclic systems), saturated, or contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, with the point of attachment being on either the carbocyclyl ring or the heterocyclyl ring, or in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, with the point of attachment being on the heterocyclyl ring, in which case the number of ring members continues to designate the number of ring members in the heterocyclyl ring system.

[0105] The term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-14 aryl"). In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C10 aryl", e.g., naphthyl, such as 1-naphthyl, 2-naphthyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, and the radical or point of attachment is on the aryl ring; in such cases, the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system.

[0106] The term "heteroaryl" refers to a radical of a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1 to 4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be at a carbon or nitrogen atom, valence permitting. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring; in such cases, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, and the point of attachment is on either the aryl or heteroaryl ring; in such cases, the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on the ring containing a heteroatom (e.g., 2-indolyl) or on the ring without a heteroatom (e.g., 5-indolyl).

[0107] Unless otherwise stated, symbols used herein [ka] The use of means that any group according to the scope of the invention described herein can be linked (or connected) to it.

[0108] symbol [ka] is used twice in a structure, the structure is divalent and is understood to be connected to two other groups. In some embodiments, where chemically feasible, the divalent structure may be oriented so that the structure can be attached in either direction, for example, [ka] Such structures include: [ka] It can be read as:

[0109] The addition of the suffix "-ene" to a group indicates that the group is a divalent moiety, for example, alkylene is a divalent moiety of alkyl, alkenylene is a divalent moiety of alkenyl, alkynylene is a divalent moiety of alkynyl, heteroalkylene is a divalent moiety of heteroalkyl, carbocyclylene is a divalent moiety of carbocyclyl, heterocyclylene is a divalent moiety of heterocyclyl, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0110] As used herein, the term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." Unless context clearly indicates otherwise, the term "or" is used herein to mean, and is used interchangeably with, the term "and / or."

[0111] As used in the claims herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. As used in the claims herein, the phrase "consisting essentially of" limits the claim to certain substances or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0112] Oligonucleotide-based agents, including RNAi agents As used herein, an "oligonucleotide-based agent" refers to an oligonucleotide-based agent having a length of about 8-50 (e.g., 8-48, 8-46, 8-44, 8-42, 8-40, 8-38, 8-36, 8-34, 8-32, 8-30, 8-28, 8-26, 8-24, 8-22, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 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, 12-14, 14-50 , 14-48, 14-46, 14-44, 14-42, 14-40, 14-38, 14-36, 14-34, 14-32, 14-30, 14-28, 14-26, 14-24, 14-22, 14-20, 14-18, 14-16, 16-50, 16-48, 16-46, 16-4 4, 16-42, 16-40, 16-38, 16-36, 16-34, 16-32, 16-30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 18-50, 18-48, 18-46, 18-44, 18-42, 18-40, 18-38, 18- 36, 18-34, 18-32, 18-30, 18-28, 18-26, 18-24, 18-22, 18-20, 20-50, 20-48, 20-46, 20-44, 20-42, 20-40, 20-38, 20-36, 20-34, 20-32, 20-30, 20-28, 20 ~26, 20~24, 20~22, 22~50, 22~48, 22~46, 22~44, 22~42, 22~40, 22~38, 22~36, 22~34, 22~32, 22~30, 22~28, 22~26, 22~24, 24~50, 24~48, 24~46, 24~44, 2 4~42, 24~40, 24~38, 24~36, 24~34, 24~32, 24~30, 24~28, 24~26, 26~50, 26~48, 26~46, 26~44, 26~42, 26~40, 26~38, 26~36, 26~34, 26~32, 26~30, 26~28,28~50, 28~48, 28~46, 28~44, 28~42, 28~40, 28~38, 28~36, 28~34, 28~32, 28~30, 30~50, 30~48, 30~46, 30~44, 30~42, 30~40, 30~38, 30~36, 30~34, 30~32, 32~50, 32~48, 32~46, 32~44, 32~42, 32~40, 32~38, 32~36, 32~34, 34~50, 34~48, 34~46, 34~44, 34~42, 34~40, 34~38, 3 In some embodiments, the oligonucleotide-based agent comprises at least one nucleotide sequence containing 4-36, 36-50, 36-48, 36-46, 36-44, 36-42, 36-40, 36-38, 38-50, 38-48, 38-46, 38-44, 38-42, 38-40, 40-50, 40-48, 40-46, 40-44, 40-42, 42-50, 42-48, 42-46, 42-44, 44-50, 44-48, 44-46, 46-50, 46-48, or 48-50 nucleotides or nucleotide base pairs. In some 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 some embodiments, the oligonucleotide-based agent can inhibit expression of the underlying gene (e.g., ALK7, Adipoq) when delivered to cells that express the gene, and is referred to herein as an "expression-inhibiting oligonucleotide-based agent." Gene expression can be inhibited in vitro or in vivo.

[0113] In some embodiments, the oligonucleotide-based agent is a single-stranded oligonucleotide, such as an antisense oligonucleotide. In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide, such as an RNAi agent, such as an siRNA. In some embodiments, the oligonucleotide-based agent is a double-stranded oligonucleotide that is an RNAi agent.

[0114] In some embodiments, the oligonucleotide-based agent is an RNAi agent. Typically, an RNAi agent can be composed of at least a sense strand (also called a passenger strand) comprising a first sequence and an antisense strand (also called a guide strand) comprising a second sequence. The sense strand of the RNAi agent can be 15-49 nucleotides in length, and the antisense strand can be 17-49 nucleotides in length. In some embodiments, the sense strand and antisense strand of the RNAi agent are independently 17-26 nucleotides in length. In some embodiments, the sense strand and antisense strand are independently 19-26 nucleotides in length. In some embodiments, the sense strand and antisense strand are independently 21-26 nucleotides in length. In some embodiments, the sense strand and antisense strand are independently 21-24 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length and the antisense strand is 19 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides long, and the antisense strand is 23 nucleotides long. The sense strand and the antisense strand can be the same length or different lengths. The RNAi agent comprises an antisense strand sequence that is at least partially complementary to a sequence in a target gene (e.g., ALK7, Adipoq), and upon delivery to a cell that expresses the target, the RNAi agent can inhibit the expression of one or more target genes in vivo or in vitro.

[0115] Oligonucleotide-based agents generally may be specifically composed of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides may include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted nucleotides), nucleotides containing unnatural bases, bridged nucleotides, peptide nucleic acids, 2',3'-seconucleotide mimics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, 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 five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-fluoro nucleotides (alternatively referred to herein and in the art as 2'-deoxy-2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides.

[0116] Moreover, one or more nucleotides of an oligonucleotide-based agent, such as an RNAi agent, can be linked by a non-standard bond or backbone (i.e., a modified internucleoside linkage or a modified backbone). The modified internucleoside linkage can be a non-phosphate-containing covalent internucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidates, aminoalkyl phosphoramidates, or thionophosphoramidates), thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with reverse polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0117] It is not necessary for all positions in a given compound to be uniformly modified; conversely, more than one modification may be incorporated into a single oligonucleotide-based agent, or even into a single nucleotide thereof.

[0118] RNAi agent sense and antisense strands can be synthesized and / or modified by methods known in the art. Further disclosure related to RNAi agents can be found, for example, in the disclosure of modifications, for example, in International Patent Application No. PCT / US2017 / 045446 (WO2018 / 027106) to Arrowhead Pharmaceuticals, Inc., which is also incorporated herein by reference in its entirety.

[0119] Modified Nucleotides In some embodiments, an oligonucleotide-based agent contains one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include deoxyribonucleotides, nucleotide mimics, abasic nucleotides (referred to herein as Ab), 2'-modified nucleotides, 3'-to-3' linked (inverted) nucleotides (referred to herein as invdN, invN, invn), modified nucleobase-containing nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seconucleotide mimics (unlocked nucleobase analogs, referred to herein as N UNA or NUNA), locked nucleotides (referred to herein as N LNA or NLNA), 3'-O-methoxy (2' internucleoside linkage) nucleotides (referred to herein as 3'-Omen), 2'-F-arabinonucleotides (referred to herein as NfANA or Nf ANAnucleotides (represented herein as 5Me-Nf), morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides (cPrpN). 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lowercase "n" in nucleotide sequences), 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-methoxylethyl) nucleotides (also referred to herein as 2'-MOE and represented herein as NM), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary that all positions in a given compound be uniformly modified. Conversely, two or more modifications can be incorporated into a single oligonucleotide-based agent, or even into a single nucleotide thereof. Oligonucleotide-based agents can be synthesized and / or modified by methods known in the art. The modification in one nucleotide is independent of the modification in another nucleotide.

[0120] Modified nucleobases include 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-thiothymine, 2-thio Included are synthetic and natural nucleobases such as cytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0121] In some embodiments, all or substantially all of the nucleotides of an oligonucleotide-based agent, such as an RNAi agent, are modified nucleotides. As used herein, an oligonucleotide-based agent in which substantially all of the nucleotides present are modified nucleotides is an agent in which there are two or fewer (i.e., 0, 1, or 2) nucleotides present that are ribonucleotides (i.e., unmodified) in each oligonucleotide strand. As used herein, when an oligonucleotide-based agent is an RNAi agent such as an siRNA or double-stranded RNA, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent in which (i) the sense strand has two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand, and (ii) the antisense strand has two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides. In some embodiments, one or more nucleotides of an oligonucleotide-based agent are unmodified ribonucleotides.

[0122] Modified internucleoside linkages In some embodiments, one or more nucleotides of an oligonucleotide-based agent are linked by a non-standard linkage or backbone (ie, a modified internucleoside linkage or a modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with reverse polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short-chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH moieties.

[0123] In some embodiments, the oligonucleotide-based agent contains one or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all of the nucleotides of the oligonucleotide-based agent are phosphorothioate and / or phosphorodithioate linkages. In some embodiments, only 1, 2, 3, 4, or 5 nucleotides at each end of the oligonucleotide-based agent are phosphorothioate or phosphorodithioate linkages.

[0124] In some embodiments, the oligonucleotide-based agent is an RNAi agent, and in some embodiments, the sense strand of the RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of the RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate or phosphorodithioate linkages, or both the sense and antisense strands can independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate or phosphorodithioate linkages. In some embodiments, the sense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of the RNAi agent can contain 1, 2, 3, or 4 phosphorothioate or phosphorodithioate linkages, or both the sense and antisense strands can independently contain 1, 2, 3, or 4 phosphorothioate or phosphorodithioate linkages. In some embodiments, the RNAi agent sense strand contains at least two phosphorothioate or phosphorodithioate internucleoside linkages. In some embodiments, at least two phosphorothioate or phosphorodithioate internucleoside linkages are between nucleotides 1 to 3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5' end of the sense strand and another phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, two phosphorothioate internucleoside linkages are located at the 5' end of the sense strand and another phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate internucleoside linkages between nucleotides, but does contain one, two, or three phosphorothioate linkages between the terminal nucleotides at both the 5' and 3' ends and the optional inverted abasic residue end cap. In some embodiments, the targeting ligand or PK / KD modulator is linked to the sense strand via a phosphorothioate linkage.In some embodiments, the RNAi agent antisense strand contains four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are between nucleotides 1-3 from the 5' end of the antisense strand and between nucleotides 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end of the antisense strand. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5' end of the antisense strand, and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5' end of the antisense strand. In some embodiments, the RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand. In some embodiments, the RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, 2'-modified nucleosides are combined with modified internucleoside linkages.

[0125] Capping residues or moieties In some embodiments, the sense strand of an oligonucleotide or RNAi agent as described herein may contain one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of a nucleotide sequence of an RNAi agent disclosed herein. Capping residues can, in some cases, provide an RNAi agent with certain beneficial properties, such as protection against exonuclease degradation. In some embodiments, an inverted abasic residue (invAb) (also referred to in the art as an "inverted abasic site") is added as a capping residue (see Table 4). (See, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art and include, for example, inverted abasic residues and carbon chains, such as terminal C3H7 (propyl), C6H 13 (hexyl), or C 12 H 25 (dodecyl) group. In some embodiments, the capping residue is present at either the 5'-end, the 3'-end, or both the 5'- and 3'-ends of the sense strand. In some embodiments, the 5'-end and / or the 3'-end of the sense strand may contain two or more inverted abasic deoxyribose moieties as capping residues.

[0126] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3'-end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the PK / KD modulator and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, inclusion of one or more inverted abasic residues or inverted abasic sites at or near one or more ends of the sense strand of the RNAi agent allows for enhanced activity or other desired properties of the RNAi agent.

[0127] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues can be inserted between the PK / KD modulator and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues can be linked via a phosphate, phosphorothioate (e.g., designated herein as (invAb) (see Table 4)), or other internucleoside linkage. In some embodiments, including one or more inverted abasic residues at or near one or more ends of the sense strand of the RNAi agent can enhance the activity or other desired properties of the RNAi agent. In some embodiments, the inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence or the 3' end of the antisense strand sequence can include an inverted abasic residue. The chemical structure of an inverted abasic deoxyribose residue is shown in Table 4.

[0128] Linking Groups and Other Delivery Moieties As described herein, oligonucleotide-based agents, such as RNAi agents, contain or are conjugated to one or more non-nucleotide groups, including, but not limited to, lipid PK / KD modulators, linking groups, or another type of targeting or delivery moiety. The non-nucleotide group can enhance targeting, delivery, or binding of the oligonucleotide-based agent. Examples of linking groups are shown in Table 4. The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, the RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the RNAi agent sense strand. The non-nucleotide group can be linked to the RNAi agent directly or indirectly via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0129] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached, improving tissue-specific distribution and cell-specific uptake of the conjugate, hi some embodiments, the non-nucleotide group enhances endocytosis of the RNAi agent.

[0130] The RNAi agents described herein can be synthesized with reactive groups, such as amino groups (also referred to herein as amines), at the 5' and / or 3' ends, which can then be used to attach targeting moieties using methods typical in the art.

[0131] For example, in some embodiments, RNAi agents disclosed herein are synthesized with an NH2-C6 group at the 5'-end of the sense strand of the RNAi agent. The terminal amino group can then be reacted to form a conjugate with, for example, a group comprising a compound having affinity for one or more integrins (i.e., integrin-targeting ligands) or a PK promoter. In some 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 be reacted to form a conjugate with, for example, a group comprising a targeting ligand.

[0132] In some embodiments, RNAi agents are synthesized presenting a linking group, which can then facilitate covalent attachment of the RNAi agent to a targeting ligand, targeting group, PK / PD modulator, or another type of delivery agent. The linking group can be attached to the 3' and / or 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is attached to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' end of the RNAi agent sense strand. Examples of linking groups include, but are not limited to, C6-SS-Alk-Me, reactive groups such as primary amines and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and / or PEG groups.

[0133] A linker or linking group is a connection between two atoms that connects one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting ligand, targeting group, PK / PD modulator, or delivery agent) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. The linkage can optionally include a spacer that increases the distance between the two linked atoms. The spacer can further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, 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 preceding list is not intended to limit the scope of the description.

[0134] In some embodiments, targeting group is linked to RNAi agent without using additional linker.In some embodiments, targeting group is designed to have linker that exists to facilitate linking to RNAi agent.In some embodiments, when two or more RNAi agents are contained in the composition, two or more RNAi agents can be linked to their respective targeting groups using the same linker.In some embodiments, when two or more RNAi agents are contained in the composition, two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0135] RNAi agents, whether modified or unmodified, can contain 3' and / or 5' targeting groups, linking groups, and / or can be conjugated to or contain PK / PD modulators. Any of the RNAi agent sequences otherwise described herein that contain a 3' or 5' targeting ligand, targeting group, PK / PD modulator, or linking group may alternatively not contain a 3' or 5' targeting ligand, targeting group, linking group, or PK / PD modulator, or may contain a different 3' or 5' targeting ligand, targeting group, linking group, or PK / PD modulator, including, but not limited to, those depicted in Tables 2 and 3. Any of the RNAi agent duplexes listed in Table A, whether modified or unmodified, can further comprise a targeting ligand, targeting group, linking group, or PK / PD modulator, and the targeting group or linking group can be attached to the 3' or 5' end of either the sense or antisense strand of the RNAi agent duplex.

[0136] In some embodiments, a linking group can be synthetically conjugated to the 5' or 3' end of the sense strand of an RNAi agent described herein. In some embodiments, a linking group is synthetically conjugated to the 5' end of the sense strand of an RNAi agent. In some embodiments, the linking group conjugated to an RNAi agent can be a trialkyne linking group.

[0137] The following notations are used herein to denote modified nucleotides, targeting groups, or linking groups: A = adenosine-3'-phosphate C = cytidine-3'-phosphate G = guanosine-3'-phosphate U = uridine-3'-phosphate I = inosine-3'-phosphate a = 2'-O-methyladenosine-3'-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3'-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i = 2'-O-methylinosine-3'-phosphate is = 2'-O-methylinosine-3'-phosphorothioate t = 2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us = 2'-O-methyluridine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate dT = 2'-deoxythymidine-3'-phosphate AUNA = 2',3'-seco-adenosine-3'-phosphate AUNAs = 2',3'-seco-adenosine-3'-phosphorothioate CUNA = 2',3'-seco-cytidine-3'-phosphate CUNAs = 2',3'-seco-cytidine-3'-phosphorothioate GUNA = 2',3'-seco-guanosine-3'-phosphate GUNAs = 2',3'-seco-guanosine-3'-phosphorothioate UUNA = 2',3'-seco-uridine-3'-phosphate UUNAs = 2',3'-seco-uridine-3'-phosphorothioates s = phosphorothioate linkage p = terminal phosphate (as synthesized)

[0138] The structures of certain modified nucleotides, capping residues, and linking groups used herein are provided in Table 4.

[0139] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0140] Alternatively, other linking groups known in the art may be used.

[0141] In addition to, or instead of, linking an RNAi agent to one or more targeting ligands, targeting groups, and / or PK / PD modulators, in some embodiments, the RNAi agent may be delivered to a cell or tissue using a delivery agent. A delivery agent is a compound that can improve 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, a melittin peptide, a melittin-like peptide (mLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane-active polyamine.

[0142] In some embodiments, RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art.RNAi agents can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference), or other delivery systems available in the art.

[0143] Pharmaceutical Compositions In some embodiments, the present disclosure relates to Formula (I), LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1Pharmaceutical compositions comprising, consisting of, or consisting essentially of one or more compounds of SM2-a, and / or CNR1 SM2-b are provided.

[0144] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and, optionally, one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance other than the active pharmaceutical ingredient (API, therapeutic product) that is intentionally included in a drug delivery system. The excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dose. An excipient may a) aid in processing of the drug delivery system during manufacturing; b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) act to enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0145] 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, bulking agents, fillers, flavorings, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0146] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active substance. Pharmaceutically active substances include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0147] Pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for varying osmotic pressure, buffers, coating agents, or antioxidants. Pharmaceutical compositions may also contain other agents with known therapeutic benefits.

[0148] Pharmaceutical compositions can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be by any method generally known in the art, including, but not limited to, topical (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including nebulizers, intratracheal, or intranasal), epidermal, transdermal, oral, or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous (e.g., via an implantation device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection. Pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions, or suspensions. It can also be administered rectally, for example using suppositories; topically or transdermally, for example using ointments, creams, gels, or solutions; or parenterally, for example using injection solutions.

[0149] 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 dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF, Parsippany, NJ), or phosphate-buffered saline. The carrier should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0150] Sterile injection solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the ingredients listed above as necessary, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other ingredients listed above as necessary.For the preparation of sterile powder for sterile injection solution, the preparation method includes vacuum drying and freeze-drying, and obtains powder of active ingredient and any other desired ingredients from the solution that has been previously sterilized and filtered.

[0151] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any of the ligands described herein, which may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present any of the ligands described herein for both intra-articular and ocular administration.

[0152] 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.The method for preparing such formulations will be clear to those skilled in the art.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.

[0153] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, a "pharmacologically effective amount," a "therapeutically effective amount," or simply an "effective amount" refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or preventative result.

[0154] Formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a,A pharmaceutical product containing the compound of CNR1 SM2-b is also an object of the present invention, and the manufacturing process of such a pharmaceutical product is also an object of the present invention. The process includes the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a,The present invention includes providing one or more compounds of LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b, and optionally one or more other substances with known therapeutic benefits, in a pharmaceutically acceptable form.

[0155] The compounds of formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-band the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a disclosed in this specificationA pharmaceutical composition comprising a compound of CNR1 SM2-b can be packaged in or contained in a kit, container, pack, or dispenser. The formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b,A compound of LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b, and a compound of formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-bPharmaceutical compositions containing the compounds LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b may be packaged in pre-filled syringes or vials.

[0156] Methods of treating and inhibiting expression The formulas (I), LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a disclosed in this specificationor CNR1 SM2-b can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from the administration of such a compound. In some embodiments, compounds of formula (I), LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b , LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-36 1-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP -379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b , LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-43 3-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP -455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b,The LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b compounds can be used to treat subjects (e.g., humans) who would benefit from a reduction and / or inhibition in the expression of target mRNA and / or protein levels, for example, subjects diagnosed with or suffering from symptoms associated with a lipid disease or disorder.

[0157] The present disclosure also provides a method of modulating (i.e., inhibiting or increasing) gene activity (e.g., abnormal activity, e.g., increased or decreased activity) in adipose tissue of a subject, biological sample, or cell (e.g., adipocyte), comprising administering an effective amount of one or more compounds of Formula (I) to the subject, biological sample, or cell. The present disclosure also provides a method of treating a wide range of diseases, such as diseases associated with abnormal activity (e.g., increased or decreased activity) of genes expressed in adipose tissue or adipocytes, in a subject in need thereof, comprising administering a therapeutically effective amount of one or more compounds of Formula (I) to the subject.

[0158] In some embodiments, one or more therapeutically effective dosages (I) are disclosed in this specification. -b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP-244-a、LP-24 4-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-310-a、LP-310-b、LP-359-a、LP-359-b、LP-361-a、LP-3 61-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、LP-375-a、LP-375-b、LP-377a、LP-377-b、LP-378-a、LP-378-b、LP-379-a、LP-379-b、LP-380-a、LP-378-a 380-b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413a、LP-413-b、LP-416-a、LP-416-b、LP-424-a、LP-424-b、LP-425-a、LP -425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432a、LP-432-b、LP-433-a、LP-433-b、LP-444-a、LP-444-b、LP-445-a、L P-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455a、LP-455-b、LP-457-a、LP-457-b、LP-458-a、LP-458-b、LP-459a、 LP-459-b、LP-460-a、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP-469a、LP-469-b、LP-470-a、LP-470-b、LP-473a、LP-473-b、LP-474a、LP-474-b、CNR1 SM2a、or CNR1 SM2-b's compound is administered. Targeted treatment includes treatment and / or preventive treatment. Targeted treatment includes 1 or more types of LP-4-a, LP-4-b, LP-18-a, LP-128-a, LP-128-b, LP-128-b 151-a、LP-151-b、LP-183-a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b 、LP-243-a、LP-243-b、LP-244-a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-310-a、LP-310-a 310-b、LP-359-a、LP-359-b、LP-361-a、LP-361-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、LP-375-a、LP-375-b、LP-377-a、LP-377-b、LP-378-a 、LP-378-b、LP-379-a、LP-379-b、LP-380-a、LP-380-b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-413-a 416-a、LP-416-b、LP-424-a、LP-424-b、LP-425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432a、LP-432-b 、LP-433-a、LP-433-b、LP-444-a、LP-444-b、LP-445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455-a、LP-447-a 55-b、LP-457-a、LP-457-b、LP-458-a、LP-458-b、LP-459-a、LP-459-b、LP-460a、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP-469-a、The subject may be a human, a patient, or a human patient. The subject may be an adult, an adolescent, a child, or an infant. The administration of the pharmaceutical compositions described herein may be to a human or an animal.

[0159] The formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a described in this specificationAlternatively, the CNR1 SM2-b compound can be used to treat at least one symptom in a subject having a disease or disorder associated with a target gene (e.g., ALK7, Adipoq) or having a disease or disorder mediated at least in part by expression of a target gene (e.g., ALK7, Adipoq). In some embodiments, the formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200 -a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-2 44-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP -359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412 -b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-4 27-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP -446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a,The LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b compounds are used to treat or manage the clinical symptoms of a subject having a disease or disorder that would benefit from, or that would be mediated at least in part by, a reduction in the mRNA of a target gene. The subject may administer a therapeutically effective amount of one or more of the compounds of the formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b ... -a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-3 71-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379- b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP -416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-42 8-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a,A compound or composition of LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b is administered. In some embodiments, the methods disclosed herein are of the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, described herein.LP-444-a、LP-444-b、LP-445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、L P-453-a、LP-453-b、LP-455-a、LP-455-b、LP-457-a、LP-457-b、LP-458-a、LP-458-b、LP -459-a、LP-459-b、LP-460-a、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP- 469-a、LP-469-b、LP-470-a、LP-470-b、LP-473-a、LP-473-b、LP-474-a、LP-474-b、CNR1 SM2-a, or CNR1 SM2-b's compound contains a composition, which is to be administered to the patient. 8-b、LP-128-a、LP-128-b、LP-151-a、LP-151-b、LP-183-a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP -208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP-244- a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-3 10-a、LP-310-b、LP-359-a、LP-359-b、LP-361-a、LP-361-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、 LP-375-a、LP-375-b、LP-377-a、LP-377-b、LP-378-a、LP-378-b、LP-379-a、LP-379-b、LP-380a、LP-380 -b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-416-a、LP- 416-b、LP-424-a、LP-424-b、LP-425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445- a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-4, and administering any one or more of the described compounds, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b, thereby treating the subject by preventing or inhibiting at least one symptom.

[0160] In certain embodiments, the present disclosure provides a method of treating a disease, disorder, condition, or pathological state mediated at least in part by target gene expression in a patient in need thereof, the method comprising administering to a patient a compound of formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-232-b, LP-232-c, LP-232-d, LP-232-e, LP-232-f, LP-232-g, LP-232-h, LP-232-i, LP-232-j, LP-232-m ... a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP-244-a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295 -a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-37 7-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP- 413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP -432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, L P-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b,The method includes administering to a patient any of the compounds LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b.

[0161] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 described hereinIn a subject administered a compound SM2-a or CNR1 SM2-b, the gene expression level and / or mRNA level of the target gene is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or more than 99% compared to the subject before administration of the compound or a subject not administered the compound. The gene expression level and / or mRNA level in a subject can be reduced in a cell, cell group, and / or tissue of the subject.

[0162] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 in this specificationIn a subject administered a compound SM2-a or CNR1 SM2-b, target protein levels are reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to the subject before administration of the compound or to a subject not administered the compound. Protein levels in a subject can be reduced in cells, cell populations, tissues, blood, and / or other bodily fluids of the subject.

[0163] Reduction of target mRNA levels and / or target protein levels can be assessed by any method known in the art. As used herein, reduction or decrease of target mRNA levels and / or protein levels is collectively referred to herein as reduction or decrease of target gene and / or protein levels, or inhibition or reduction of target gene expression.

[0164] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 described hereinThe SM2-a or CNR1 SM2-b compound can be used to prepare a pharmaceutical composition for use in treating a disease, disorder, or condition mediated at least in part by target gene expression. In some embodiments, the disease, disorder, or condition mediated at least in part by target gene expression is a lipid-related disease or disorder.

[0165] In some embodiments, it depends on the weight of the object. a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP-244 -a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-310-a、LP-310-b、LP-359-a、LP-359-b、LP-36 1-a、LP-361-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、LP-375-a、LP-375-b、LP-377-a、LP-377-b、LP-378-a、LP-378-b、LP-379-a、LP-379-b、LP-379-a 80-a、LP-380-b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-416-a、LP-416-b、LP-424-a、LP-424-b、LP-413-a 425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432-a、LP-432-b、LP-433-a、LP-433-b、LP-444-a、LP-444-b、LP -445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455a、LP-455-b、LP-457-a、LP-457-b、LP-458a、LP-458-b、L P-459-a、LP-459-b、LP-460-a、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP-469-a、LP-469-b、LP-470-a、LP-470-b、LP-473a、LP-473-b、The compounds LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b can be administered at a dose of about 0.05 mg / kg to about 40.0 mg / kg of the subject's body weight. In other embodiments, the compounds of formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a,The compounds LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b may be administered at a dose of about 5 mg / kg to about 20 mg / kg of the subject's body weight.

[0166] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1The SM2-b compound may be administered in split doses, meaning that two doses are administered to a subject a short time (e.g., less than 24 hours) apart. In some embodiments, about half of the desired daily dose is administered in a first dose, and about half of the remaining desired daily dose is administered about 4 hours after the first dose.

[0167] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, described herein...Or CNR1 The compound of SM2-b can be administered every day (ie, every day). In some embodiments, the specifications described in this specification are LP-4-a、LP-4-b、LP-18-a、LP-18-b、LP-128-a、LP-128-b、LP-151-a、LP-151-b、LP-1 83-a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP -244-a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295a、LP-295-b、LP-310-a、LP-310-b、LP-359a、LP-359-b、 LP-361-a、LP-361-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、LP-375-a、LP-375-b、LP-377a、LP-377-b、LP-378-a、LP-378-b、LP-379a、LP-379-a b、LP-380-a、LP-380-b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-416-a、LP-416-b、LP-424-a、LP-42 4-b、LP-425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432-a、LP-432-b、LP-433-a、LP-433-b、LP-444-a、LP-428-a 444-b、LP-445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455-a、LP-455-b、LP-457-a、LP-457-b、LP-458-a、L P-458-b、LP-459-a、LP-459-b、LP-460-a、LP-460-b、LP-461-a、LP-461-b、LP-468a、LP-468-b、LP-469-a、LP-469-b、LP-470-a、LP-470-b、LP-473a、The compounds LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b can be administered once a week (i.e., weekly). In other embodiments, the compounds of formula LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a,The LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b compound may be administered biweekly (once every other week).

[0168] In some embodiments, the formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 described hereinSM2-a or CNR1 SM2-b compounds or compositions containing such compounds can be used to treat diseases, disorders, or conditions mediated at least in part by target gene expression. In some embodiments, the disease, disorder, or condition mediated at least in part by target gene expression is a lipid-related disease or disorder.

[0169] Another aspect of the present invention is a method of reducing target gene expression in the system. a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP-242-b、LP-243-a、LP-243-b、LP-244 -a、LP-244-b、LP-245-a、LP-245-b、LP-249-a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-310-a、LP-310-b、LP-359-a、LP-359-b、LP-36 1-a、LP-361-b、LP-371-a、LP-371-b、LP-374-a、LP-374-b、LP-375-a、LP-375-b、LP-377-a、LP-377-b、LP-378-a、LP-378-b、LP-379-a、LP-379-b、LP-379-a 80-a、LP-380-b、LP-403-a、LP-403-b、LP-404-a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-416-a、LP-416-b、LP-424-a、LP-424-b、LP-413-a 425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432-a、LP-432-b、LP-433-a、LP-433-b、LP-444-a、LP-444-b、LP -445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455a、LP-455-b、LP-457-a、LP-457-b、LP-458a、LP-458-b、L P-459-a、LP-459-b、LP-460-a、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP-469-a、LP-469-b、LP-470-a、LP-470-b、LP-473a、LP-473-b、The present invention provides a method comprising introducing a compound selected from the group consisting of LP-474-a, LP-474-b, CNR1 SM2-a, and CNR1 SM2-b, wherein the compound comprises an oligonucleotide-based agent at least substantially complementary to a target gene. In some embodiments, the cell is an adipocyte. In some embodiments, the cell is present in a subject. In some embodiments, the subject has been diagnosed with a disease or disorder that can be treated, prevented, or ameliorated by reducing expression of the target gene.

[0170] Another aspect of the present invention provides a use of any one of the lipid PK / PD modulators conjugated to an oligonucleotide-based agent described herein for the treatment, prevention, or amelioration of a disease or disorder. In some embodiments, the disease or disorder is a fat-related disease (such as lipodystrophy or lipedema) or disorder selected from the group consisting of obesity, type 2 diabetes, insulin resistance, metabolic syndrome, various lipodystrophies, atherosclerotic vascular diseases, cardiometabolic diseases or disorders, or other similar diseases or disorders.

[0171] Cells, tissues, and non-human organisms The formulas LP-4-a, LP-4-b, LP-18-a, LP-18-b, LP-128-a, LP-128-b, LP-151-a, LP-151-b, LP-183-a, LP-183-b, LP-200-a, LP-200-b, LP-208-a, LP-208-b, LP-211-a, LP-211-b, LP-232-a, LP-232-b, LP-242-a, LP-242-b, LP-243-a, LP-243-b, LP-244-a, LP-244-b, LP-245-a, LP-245-b, LP-249-a, LP-249-b, LP-274-a, LP-274-b, LP-295-a, LP-295-b, LP-310-a, LP-310-b, LP-359-a, LP-359-b, LP-361-a, LP-361-b, LP-371-a, LP-371-b, LP-374-a, LP-374-b, LP-375-a, LP-375-b, LP-377-a, LP-377-b, LP-378-a, LP-378-b, LP-379-a, LP-379-b, LP-380-a, LP-380-b, LP-403-a, LP-403-b, LP-404-a, LP-404-b, LP-412-a, LP-412-b, LP-413-a, LP-413-b, LP-416-a, LP-416-b, LP-424-a, LP-424-b, LP-425-a, LP-425-b, LP-426-a, LP-426-b, LP-427-a, LP-427-b, LP-428-a, LP-428-b, LP-432-a, LP-432-b, LP-433-a, LP-433-b, LP-444-a, LP-444-b, LP-445-a, LP-445-b, LP-446-a, LP-446-b, LP-447-a, LP-447-b, LP-453-a, LP-453-b, LP-455-a, LP-455-b, LP-457-a, LP-457-b, LP-458-a, LP-458-b, LP-459-a, LP-459-b, LP-460-a, LP-460-b, LP-461-a, LP-461-b, LP-468-a, LP-468-b, LP-469-a, LP-469-b, LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a described in this specificationOr CNR1 Among the compounds of SM2-b, at least 1 cell, tissue, and non-human organism are intended. 28-b、LP-151-a、LP-151-b、LP-183-a、LP-183-b、LP-200-a、LP-200-b、LP-208-a、LP-208-b、LP-211-a、LP-211-b、LP-232-a、LP-232-b、LP-242-a、LP -242-b、LP-243-a、LP-243-b、LP-244-a、LP-244-b、LP-245-a、LP-245-b、LP-249a、LP-249-b、LP-274-a、LP-274-b、LP-295-a、LP-295-b、LP-310a、 LP-310-b、LP-359-a、LP-359-b、LP-361-a、LP-361-b、LP-371-a、LP-371-b、LP-374a、LP-374-b、LP-375-a、LP-375-b、LP-377-a、LP-377-b、LP-378- a、LP-378-b、LP-379-a、LP-379-b、LP-380-a、LP-380-b、LP-403-a、LP-403-b、LP-404a、LP-404-b、LP-412-a、LP-412-b、LP-413-a、LP-413-b、LP-41 6-a、LP-416-b、LP-424-a、LP-424-b、LP-425-a、LP-425-b、LP-426-a、LP-426-b、LP-427-a、LP-427-b、LP-428-a、LP-428-b、LP-432-a、LP-432-b、LP-428-a 433-a、LP-433-b、LP-444-a、LP-444-b、LP-445-a、LP-445-b、LP-446-a、LP-446-b、LP-447-a、LP-447-b、LP-453-a、LP-453-b、LP-455-a、LP-455-b、L P-457-a、LP-457-b、LP-458-a、LP-458-b、LP-459-a、LP-459-b、LP-460、LP-460-b、LP-461-a、LP-461-b、LP-468-a、LP-468-b、LP-469-a、LP-469-b、The compound LP-470-a, LP-470-b, LP-473-a, LP-473-b, LP-474-a, LP-474-b, CNR1 SM2-a, or CNR1 SM2-b is delivered to a cell, tissue, or non-human organism. In some embodiments, the cell is a mammalian cell, including but not limited to a human cell. In some embodiments, the cell is an adipocyte.

[0172] The embodiments and provisions provided above will now be illustrated with the following non-limiting examples. [Example]

[0173] The following examples are intended to illustrate, but not to limit, certain embodiments disclosed herein.

[0174] Unless otherwise specified, numbers used to refer to compounds in a given Example are made with reference only to that particular Example and not to any other Examples disclosed herein. For example, compound 1 in Example 2, "Synthesis of LP-4-p Phosphoramidite," is different from and does not refer to compound 1 in Example 2, "Synthesis of LP-18-p." Similarly, it will be understood that specific compounds disclosed herein may be identified by different numbers in different Examples. Compounds disclosed in various tables throughout the detailed description (i.e., LPXXa, LPXXb, and LPXX-p, where XX is a number) are consistently referred to throughout the Examples herein.

[0175] Unless otherwise specified, it will be understood that the term "EDC" as used in the examples herein refers to commercially available N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0176] Example 1. Synthesis of RNAi agents and compositions. The following describes general procedures for the synthesis of oligonucleotide-based agents, such as RNAi agents and antisense oligonucleotides, and conjugates thereof, as exemplified in the non-limiting examples described herein.

[0177] Synthesis of Oligonucleotide-Based Agents. Oligonucleotide-based agents can generally be synthesized using methods known in the art. For the synthesis of the RNAi agents exemplified in the Examples described 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, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or Oligopilot 100 (GE Healthcare) was used. Synthesis was carried out on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA) or polystyrene (obtained from Kinovate, Oceanside, CA, USA). All RNAs and 2'-modified RNA phosphoramidites were obtained from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or other companies. Hungry The 2'-O-methylphosphoramidites used were purchased from Biotech (Morrisville, NC, USA). Specifically, the following 2'-O-methylphosphoramidites were used: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-N 2The protecting groups used for the 2'-O-methylphosphoramidites were 5'-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-Deoxy-2'-fluoro-phosphoramidite and 2'-O-propargylphosphoramidite had the same protecting groups as the 2'-O-methylphosphoramidite. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes. The following UNA phosphoramidites were used: 5'-(4,4'-dimethoxytrityl)-N-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5' The phosphorothioate linkages were introduced using a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, available from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile or a 200 mM solution of hydrogenated xanthan gum (TCI America, Portland, OR, USA) in pyridine.Antisense oligonucleotides can be manufactured using the same general methods used in connection with the RNAi agent conjugates described in the Examples herein.

[0178] TFA AminoLink phosphoramidite was also purchased commercially (ThermoFisher). Linker L6 was purchased from BroadPharm as propargyl-PEG 5-NHS (catalog number BP-20907) and coupled to the NH2-C6 group from AminoLink phosphoramidite using standard coupling conditions to form -L6-C6-. Linker Alk-cyHex was similarly purchased commercially as a propargyl-containing phosphoramidite compound from Lumiprobe (alkyne phosphoramidite, 5'-terminus) to form linker-Alk-cyHex-. In each case, phosphorothioate linkages were introduced as specified using the conditions described herein. Cyclopropylphosphonate phosphoramidite was synthesized according to WO 2017 / 214112 (see also Altenhofer et.al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).

[0179] For some RNAi agents disclosed herein, a linker such as a C6-SS-C6 or 6-SS-6 group was introduced at the 3' end of the sense strand. Pre-loaded resins with the respective linkers were commercially available. Alternatively, for some sense strands, dT resin was used, and then the respective linkers were added via standard phosphoramidite synthesis.

[0180] 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 weight (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).

[0181] Purification. Crude oligomers were purified by anion-exchange HPLC using a TSKgel® SuperQ-5PW 13 μm column (commercially available from Tosoh Biosciences) and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% ​​acetonitrile, and buffer B was the same as buffer A supplemented with 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled and then subjected to size-exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex® G-25 fine (commercially available from Sigma-Aldrich) with a running buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile or filtered water. Alternatively, pooled fractions were desalted and exchanged into the appropriate buffer or solvent system via tangential flow filtration.

[0182] Annealing. For the RNAi agents disclosed in the Examples herein, complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) to form the RNAi agent. Some RNAi agents were lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the solution absorbance in 1x PBS with a UV-Vis spectrometer. The solution absorbance at 260 nm was then multiplied by a conversion factor and a dilution factor to determine the duplex concentration. The conversion factor used was 0.037 mg / (mL / cm) or calculated from the experimentally determined extinction coefficient.

[0183] Example 2. Synthesis of lipid PK / PD modulator precursors Synthesis of LP-4-p [ka]

[0184] Decanoic acid (182 mg, 1.06 mmol) was stirred in DMF (5 mL). N-boc-ethylenediamine (0.185 mL, 1.16 mmol) was added, followed by TBTU (409 mg, 1.27 mmol) and DIPEA (0.555 mL, 3.18 mmol). The suspension was stirred for 16 h, then the reaction was diluted with HO and extracted with EtOAc. The organic layer was dried over NaSO, filtered, and concentrated to dryness. The crude product was purified by flash chromatography (EtOAc / hexanes) to give 1 (232 mg). To intermediate 1 (232 mg, 0.738 mmol) was added TFA:DCM (1:1), and the reaction was stirred until complete by LCMS. The reaction was concentrated, and the residue was dissolved in DCM (5 mL), and maleimide-PEG2-NHS ester (300 mg, 0.848 mmol) and DIPEA (0.369 mL, 2.12 mmol) were added. The reaction was stirred overnight, then diluted with HO, extracted with EtOAc, dried over NaSO, filtered, and concentrated. The crude product was purified by flash chromatography (EtOAc / hexanes) to give LP-4-p. LC / MS (ESI+) m / z calculated 524.32 (M), found 524.65 (M+H+).

[0185] Synthesis of LP-18-p [ka]

[0186] Arachidonic acid (188 mg, 0.619 mmol) was dissolved in DMF (5 mL), then azido-PEG3-amine (148 mg, 0.682 mmol), TBTU (238 mg, 0.743 mmol), and DIPEA (0.324 mL, 1.85 mmol) were added, and the suspension was stirred overnight. The reaction was diluted with HO and extracted with DCM containing 20% ​​trifluoroethanol. The extract was dried over NaSO, filtered, and concentrated to dryness to give the crude product, which was purified by flash chromatography to give LP-18-p. LC / MS (ESI+) m / z calculated 502.35 (M), found 504.71 (M+H+).

[0187] Synthesis of LP-151-p [ka]

[0188] Intermediate 2: To a solution of eicosapentaenoic acid (100 mg, 0.330 mmol) in DMF, N-boc-ethylenediamine (53 mg, 0.330 mmol), TBTU (106 mg, 0.330 mmol), and DIPEA (0.23 mL, 1.32 mmol) were added. The reaction mixture was stirred until complete conversion was observed by LCMS. The reaction was extracted with EtOAc and washed with saturated NaHCO3, HO, and saturated NH4Cl. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (0–20% MeOH / DCM) using silica gel as the stationary phase to give 1 (147 mg) as a white oily residue in 65% yield. LC / MS calculated [M+H] 445.34 m / z, observed 445.56. To compound 1 (96 mg, 0.216 mmol) was added 4 M HCl in dioxane (10 equiv.) and the reaction was stirred until complete conversion was observed by LCMS. The reaction was azeotroped with PhMe / MeOH and concentrated to give 2 as a white solid. LC / MS calculated [M+H] 345.28 m / z, observed 345.59.

[0189] A solution of 2 (82.5 mg, 0.216 mmol) and triethylamine (0.151 mL, 0.109 mmol) in dry DCM was stirred under N. Maleimide-amide-PEG-NHS ester (92.1 mg, 0.216 mmol) was added slowly, and the reaction was stirred until complete conversion was observed by LCMS. The reaction was washed with saturated NaHCO and NH. Cl, dried over Na. SO, filtered, and concentrated. The crude residue was purified by flash chromatography on a 12 g silica gel column (0–8% MeOH / DCM over 20 min, product eluted at approximately 7% MeOH) to give LP-151-p (141 mg) in 46% yield. LC / MS calculated [M+H] 655.40 m / z, observed 656.06.

[0190] Synthesis of LP-208-p [ka]

[0191] To a solution of amine-PEG 2-acid (40 mg, 0.226 mmol) in DMF at room temperature, triethylamine (0.157 mL, 1.12 mmol) was added, followed by palmitic acid NHS ester (90 mg, 0.248 mmol). The mixture was stirred at room temperature for 15 min, and then COMU (97 mg, 0.225 mmol) and 2,3,5,6-tetrafluorophenol (37 mg, 0.225 mmol) were added, and the mixture was stirred at room temperature for 30 min. The reaction was diluted with EtOAc, washed successively with brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was purified on a 4 g silica gel column (0-50% EtOAc / hexanes) to give LP-208-p (61 mg) as a white wax in 48% yield. LC-MS: calculated [M+H] 564.33, found 564.83.

[0192] Synthesis of LP-211-p [ka]

[0193] Compound 1 (Asta Tech® #64704, 0.75 g) was dissolved in 16 mL of DMF, followed by the addition of TBTU (0.71 g) and DIPEA (0.89 mL). The mixture was stirred for 10 min, followed by the addition of compound 2 (0.68 g) in DMF. The reaction was allowed to proceed for 1 h, after which the mixture was diluted with 120 mL of EtOAc, washed with 5% citric acid, water (3 × 20 mL), NaCl (1 × 15 mL), dried over NaSO, filtered, and concentrated on a rotary evaporator and high vacuum. The product was purified on a silica gel column using MeOH / DCM (0–5%) over 25 min. Yield: 927 mg. LC-MS: calculated [M+H] 658.92, found 659.90. [ka]

[0194] Compound 1 (0.92 g) was dissolved in a mixture of 1:1 DCM:TFA (6 mL). The reaction was stirred for 1 hour. The compound was concentrated in vacuo. Yield: 851 mg. LC-MS: calculated [M+H] 602.81, found 603.84.

[0195] Synthesis of LP-232-p [ka]

[0196] Palmitoyl chloride (100 mg) was stirred in a 5 mL DCM solution of cis-4-(boc-amino)cyclohexylamine (0.0819 g). After stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography (hexane: EtOAc). Yield 52 mg, 31%. [ka]

[0197] To 1 (0.0520 g) was added 2 mL of dioxane:HCl (4 N) until the boc deprotection was complete. After removing the solvent in vacuo, a solution of 2 (0.0316 g), DIPEA (0.0445 g), and COMU (0.0620 g) in 5 mL of DCM was added to the crude residue. After stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by column chromatography (MeOH / DCM 0-20%). Yield 45 mg, 65%. [ka]

[0198] To 1 (0.0449 g) was added 2 mL of dioxane:HCl (4 N) until 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 stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography (MeOH / DCM 0-20%). Yield 30 mg, 58%.

[0199] Synthesis of LP-242-p [ka]

[0200] tert-Butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (300 mg, 1.17 mmol) was dissolved in DCM and triethylamine (0.82 mL, 5.89 mmol) was added. Palmitic acid NHS ester (416 mg, 0.46 mL, 1.17 mmol) was then added to the solution, and the reaction was stirred for 60 min. LC-MS indicated the reaction was complete. The reaction was quenched with 2 M HCl (20 mL), and the layers were separated. The organic layer was washed with 1 M HCl (2 × 20 mL), HO (3 × 20 mL), saturated NaCl (20 mL), dried over NaSO, and concentrated to give 1 (484 mg) as a white solid in 83% yield, which was purified by silica gel chromatography (MeOH / DCM 0–4%). Intermediate 1 (484 mg, 0.982 mmol) was dissolved in DCM and cooled to 0 °C, and TFA (2 mL, 26 mmol) was added. After the addition of TFA, the reaction was warmed to room temperature and stirred for 60 min. LC-MS showed the reaction was complete. The reaction was quenched with saturated aqueous NaHCO until the solution remained basic. The mixture was then extracted with DCM (3 × 15 mL), washed with brine (2 × 30 mL), dried over NaSO, and concentrated to give 2 (385 mg) as an off-white solid, which was used without further purification. Acid-PEG2-tert-butyl ester (252 mg, 0.962 mmol) was dissolved in DCM, followed by the addition of triethylamine (3.4 mL, 24.5 mmol) and COMU (412 mg, 0.962 mmol). After stirring the reaction for 3 minutes, intermediate 2 (385 mg, 0.982 mmol) was added and the reaction was stirred for 30 minutes. LC-MS confirmed the reaction was complete. The reaction was quenched with 2M HCl (20 mL) and the layers were separated. The organic layer was washed with HO (3 x 20 mL), saturated NaCl (20 mL), dried over NaSO, and concentrated to give a crude white solid, which was purified by silica gel chromatography (MeOH / DCM 0-10%) to give 3 (585 mg) as a white solid in 95% yield. LC / MS (ESI + ) m / z calculated 636.96, found 637.46 (M+H + ).

[0201] Intermediate 3 (585 mg, 0.918 mmol) was dissolved in 4 M HCl in dioxane (15 mL) and the reaction was stirred until no starting material was observed by LC-MS. Nitrogen was bubbled through the reaction to remove most of the HCl, then the solvent was removed under reduced pressure and the solid was dried in vacuo to give 4 (538 mg) as a white solid, which was used without purification. Intermediate 4 (538 mg, 0.926 mmol) and COMU (436 mg, 1.01 mmol) were dissolved in DCM (10 mL) and triethylamine (1.3 mL, 9.26 mmol) was added. The reaction was stirred for 3 minutes, then 2,3,5,6-tetrafluorophenol (169 mg, 1.01 mmol) was added. After 20 minutes, LC-MS indicated the reaction was complete. The reaction mixture was concentrated, loaded directly onto a silica gel column, and purified by silica gel chromatography MeOH / DCM (0-20%) to give LP-242-p (372 mg) as an off-white solid in 50% yield. LC / MS (ESI + ) m / z calculated 728.91, observed 729.63 (M+H + ).

[0202] Synthesis of LP-243-p [ka]

[0203] A suspension of palmitic acid (0.100 g), tBu-3,9-diazaspiro[5,5]undecane-3-carboxylate HCl (0.0732 g), COMU (0.166 g), and DIPEA (0.161 mL) in 5 mL of DCM was stirred at 40 °C (oil bath temperature) overnight. Water was added, and the organics were extracted with DCM and dried over Na SO . After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography (MeOH / DCM 0-5%). [ka]

[0204] 1 (0.0200 g) was treated with 4 M HCl:dioxane and stirred for 1 h. The reaction was evaporated in vacuo. To the crude product was added a solution of 2 (0.0119 g), COMU (0.0232 g), and DIPEA (0.022 mL). After stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography. [ka]

[0205] To 1 (0.121 g), 2 mL of dioxane:HCl (4 N) was added until otBu deprotection was complete. After removing the solvent in vacuo, the crude product 1 was stirred in a solution of tetrafluorophenol (0.0363 g), DIPEA (0.104 mL), and COMU (0.112 g) in 5 mL of DCM. After stirring the suspension overnight, water was added, and the organics were extracted with DCM and dried over Na2SO4. After filtration, the solvent was concentrated to dryness, and the crude product was purified by flash chromatography.

[0206] Synthesis of LP-244-p [ka]

[0207] To a mixture of 1 (0.100 g) and 2 (0.251 mL) in DCM was added TEA (0.174 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with water, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude material, which was purified by flash chromatography (EtOAc / hexanes). The product was a white solid (147 mg, 74%). LC-MS: calculated [M+H] 480.42, found 480.85. [ka]

[0208] To a solution of 1 (0.147 g) in DCM, TFA (1 mL) was added 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 then placed under high vacuum for 2 h. The residue was dissolved in 3 mL of DMF, and then 2 (0.0800 g), DIPEA (0.0160 mL), and COMU (0.197 g) were added at room temperature. The mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo. Purification was performed on a 12 g column. Purification was performed using a gradient from DCM to 20% MeOH in DCM. The product was a clear oil (173 mg, 91%). LC-MS: calculated [M+H] 624.50, found 625.33. [ka]

[0209] A solution of 1 (0.173 g) in 4 N HCl / dioxane (6 mL) was stirred overnight at room temperature. After the solvent was removed in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL of DMF, and then DIPEA (0.145 mL), COMU (0.356 g), and 2 (0.0920 g) were added. The mixture was stirred at room temperature for 2 h. After the solvent was removed in vacuo, the residue was loaded onto a 12 g column. Purification was performed using a gradient from DCM to 20% MeOH in DCM. The product was a pale yellow solid (68 mg, 37%). LC-MS: calculated [M+H] 716.43, found 717.23.

[0210] Synthesis of LP-245-p [ka]

[0211] 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, the reaction was diluted with EtOAc, washed with HO (×2), brine, dried over NaSO, filtered, and concentrated under reduced pressure. Purification was carried out on a 40 g column (EtOAc / hexane, 0-30%). The product was a pale yellow oil (yield 1388 mg, 51%). LC-MS: calculated [M+H] 339.21, found 339.62. [ka]

[0212] To a mixture of 1 (0.241 g) in MeOH / THF (4 mL / 4 mL) was added 1N 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, 1N HCl was added to adjust the mixture to pH 1. NaHCO3 was added to adjust the pH to 7-8. The product was extracted with DCM, dried over Na2SO4, filtered, concentrated, and placed under vacuum. The residue was dissolved in 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, NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification was carried out on a 12 g column (EtOAc / hexane). The product was a brown oil (285 mg, 74%). LC-MS: calculated [M+H] 540.34, found 541.07. [ka]

[0213] A mixture of 1 (0.0740 g) and Pd / C in EtOAc was degassed with N2 and then charged with H2 (1 atm) at room temperature. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was filtered through Celite®. After EtOAc was removed in vacuo, the residue was further dried under high vacuum for 1 h. The residue was dissolved in 3 mL of DCM, and 2 (0.166 mL) and TEA (0.115 mL) were added at room temperature. The mixture was stirred at room temperature for 2 h. The reaction was washed with H2O, brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude material, which was purified on a 12 g silica gel column (MeOH / DCM, 0-20%). The product was a clear oil (43 mg, 37%). LC-MS: calculated [M+H] 836.71, found 837.68. [ka]

[0214] A solution of 1 (0.0430 g) in 4 N HCl / dioxane (3 mL) was stirred overnight at room temperature. After the solvent was removed in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 3 mL of DMF, and then DIPEA (0.027 g), COMU (0.0660 g), and 2 (0.017 g) were added. The mixture was stirred at room temperature for 2 h. After the solvent was removed in vacuo, the residue was loaded directly onto a 4 g column and purified by flash chromatography (MeOH / DCM, 0-20%). The product was a pale yellow oil (34 mg, 37%). LC-MS: calculated [M+H] 928.64, found 929.59.

[0215] Synthesis of LP-249p [ka]

[0216] To a mixture of 1 (0.0600 g) and 2 (0.161 mL) in 4 mL of DCM was added TEA (0.111 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was washed with HO, dried over NaSO, filtered, and concentrated under reduced pressure to give the crude material, which was purified on a 4 g silica gel column (EtOAc / hexane). The product was a white solid (74 mg, 60%). LC-MS: calculated [M+H] 465.41, found 465.91. [ka]

[0217] 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 then placed under high vacuum for 2 h. The residue was dissolved in DMF, and then 2 (0.0420 g), DIPEA (0.084 mL), and COMU (0.102 g) were added at room temperature. The mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo, and the crude reaction was dry-loaded onto a 12 g column and purified by flash chromatography (MeOH / DCM, 0-20%). The product was a white solid (56 mg, 58%). LC-MS: calculated [M+H] 609.48, found 610.29. [ka]

[0218] A solution of 1 (0.0560 g) in 4 N HCl / dioxane (3 mL) was stirred overnight at room temperature. After the solvent was removed in vacuo, the residue was placed under high vacuum for 3 h. The residue was dissolved in 2 mL of DMF, and then DIPEA (0.048 mL), COMU (0.118 g), and 2 (0.031 g) were added. The mixture was stirred at room temperature for 2 h. After the solvent was removed in vacuo, the residue was dry-loaded onto a 4 g column and purified by flash chromatography (MeOH / DCM, 0-20%). The product was an off-white solid (16 mg, 25%). LC-MS: calculated [M+H] 701.42, found 702.20.

[0219] Synthesis of LP-274-p [ka]

[0220] To a solution of EPA1 (60.5 mg, 0.200 mmol, 1 equiv.) and 2 (36.5 mg, 0.220 mmol, 1.10 equiv.) in 20 mL of DCM, COMU (94.2 mg, 0.220 mmol, 1.10 equiv.) was added, followed by TEA (0.084 mL, 0.600 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 1 N HCl, brine, dried over Na2SO4, filtered, and concentrated. The reaction mixture was purified by CombiFlash® using silica gel as the stationary phase with a gradient of 0-50% EtOAc / hexanes. 69 mg of product was obtained (76% yield).

[0221] Synthesis of LP-295-p [ka]

[0222] To a 40 mL vial equipped with a stir bar under N2, α-linolenic acid (100 mg, 0.359 mmol), 2,3,5,6-tetrafluorophenol (72 mg, 0.431 mmol), DCM (4 mL), and triethylamine (0.15 mL, 1.08 mmol) were added. COMU (185 mg, 0.431 mmol) was added, and the reaction was wrapped in foil and stirred at room temperature for 2 hours. TLC (kMnO4 staining) confirmed the reaction was complete. The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO3 (x3), brine, dried over MgSO4, filtered, and concentrated under reduced pressure (protecting the product from light) to give LP-295-p (152 mg) as a crude red oil, which was used without further purification. LC / MS (ESI) + ) m / z calculated value 426.22 (M), observed value 427.48 (M+H + ). 1H NMR(400MHz,CDCl3)δ =7.02-6.94(m,1H),5.44-5.28(m,6H),2.80(app.t.,4H),2.66(t,2H),2.10-2.02(m,4H),1.77(q,2H),1.48-1.31(br.m.,8H),0.972(t,3H).

[0223] Synthesis of LP-310-p [ka]

[0224] To a solution of 1 in DCM was added DIPEA (0.057 mL), COMU (0.077 g), and 2 (0.0300 g) 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, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was loaded onto a 4 g column and purified by flash chromatography (EtOAc / hexane, 0-50%). The product was a white solid (46 mg, 44%). LC-MS: calculated [M+H] 422.36, found 422.61. [ka]

[0225] A solution of 1 (0.046 g) in 4 N HCl / dioxane (2 mL) was stirred overnight at room temperature. After the solvent was removed in vacuo, the residue was placed under high vacuum for 3 h. The residue was then dissolved in DCM at room temperature, followed by the addition of COMU (0.0700 g), DIPEA (0.038 mL), and 2 (0.036 g) at room temperature. After stirring at room temperature for 2 h, the solvent was removed in vacuo. The residue was loaded onto a 4 g silica column and purified by flash chromatography (EtOAc / hexane, 0-50%). The product was a white solid (21 mg, 38%). LC-MS: calculated [M+H] 514.29, found 514.61.

[0226] Synthesis of LP-359-p [ka]

[0227] Compound 1 (Asta Tech® #64704, 250 mg) was dissolved in 6 mL of DMF. TBTU (238 mg) and DIPEA (0.48 mL) were then added. The mixture was stirred for 10 minutes, followed by the addition of compound 2 (129 mg) in DMF. After stirring for 1 hour, the reaction mixture was diluted with 75 mL of EtOAc, washed with 3% citric acid (3 × 8 mL), HO (2 × 8 mL), NaCl (1 × 8 mL), dried over NaSO, filtered, and concentrated on a rotary evaporator and high vacuum. The product was purified by dry-loading in 5 mL of silica onto a 12G RediSep Gold Rf column (MeOH / DCM, 0–5% over 30 minutes) (yield 287 mg). LC-MS: calculated [M+H] 526.76, found 527.39. [ka]

[0228] Compound 1 (287 mg) was dissolved in 10 mL of MeOH. Then, 104 mg of Pd / C was added. The vessel was purged with N2, and H2 was introduced via a balloon. The reaction was stirred overnight. The mixture was filtered through Celite®, and the pad was washed with ethanol. The product was concentrated on a rotary evaporator and high vacuum. Yield 254 mg. LC-MS: calculated [M+H] 500.77, found 501.46. [ka]

[0229] Compound 1 (20 mg) was dissolved in 1.2 mL of THF. TEA (0.033 mL) and compound 2 (22 mg) were then added. The reaction was stirred at room temperature for 1 h. The mixture was concentrated in vacuo and then dry-loaded onto a silica gel column with Celite and purified by flash chromatography using dry MeOH / DCM 0-10% over 25 min. Yield: 19 mg. LC-MS: calculated [M+H] 750.99, found 751.85. [ka]

[0230] Compound 1 (19 mg) was dissolved in 1:1 DCM:TFA (1.0 mL). The reaction was stirred at room temperature for 1 hour. A very small amount of toluene was added, and the reaction was concentrated in a water bath at 28°C, followed by rotary evaporation and high vacuum concentration. Yield: 16 mg. LC-MS: calculated [M+H] 694.89, found 695.99.

[0231] Synthesis of LP-361-p [ka]

[0232] To a 40 mL vial equipped with a stir bar under N2, N-(2-aminospiro[3.3]hept-6-yl)carbamic acid tert-butyl ester (350 mg, 1.55 mmol), DCM (10 mL), and triethylamine (0.65 mL, 4.65 mmol) were added. The reaction was stirred and cooled in an ice bath for 10 min, then palmitoyl chloride (0.51 mL, 1.7 mmol) was added dropwise over 30 s. A white precipitate formed immediately, and the reaction was stirred on ice for 10 min. The ice bath was removed, and the reaction was stirred overnight while warming to room temperature. TLC (ninhydrin) confirmed the reaction was complete. The reaction was dry-loaded onto a 24 g silica gel column with silica and purified by flash chromatography (EtOAc / hexanes, 0–70% over 35 min, ELS detection). After this period, the column was flushed with 20% MeOH / DCM to facilitate elution of the product. Fractions were pooled and concentrated under reduced pressure to give 1 (602 mg) as a white solid in 84% yield. LC / MS (ESI + ) m / z calculated value 464.40 (M), observed value 465.64 (M+H + ).

[0233] To a 40 mL vial equipped with a stir bar was added Intermediate 1 (165 mg, 0.355 mmol) and 4 M HCl in dioxane (30 mL, 128.8 mmol). The vial was tightly capped and stirred for 2 h. The reaction immediately turned orange, and then a white precipitate formed within 10 min. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure, azeotroped with PhMe (5 mL), and dried overnight to give 2 (142 mg) as a white solid HCl salt in quantitative yield. Intermediate 2 (142 mg, 0.354 mmol) was suspended in DCM (10 mL), and triethylamine (0.15 mL, 1.06 mmol) and a stir bar were added. NHS-PEG2-NHBoc (145 mg) was finally added, and the reaction was stirred under N2 for 2 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with saturated NaHCO3 (x2), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (8 mL), dry-loaded onto a 12 g silica column, and purified by flash chromatography (MeOH / DCM 0-5%, ELS detection) to give Intermediate 3 (172 mg) as a white solid in 78% yield. LC / MS (ESI + ) m / z calculated 623.49 (M), observed 624.78 (M+H + ).

[0234] Intermediate 3 (170 mg, 0.272 mmol) was stirred in 4 M HCl in dioxane (7 mL, 27.2 mmol) in a sealed RB flask for 2 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure, azeotroped with PhMe (5 mL), and dried overnight to give 4 (154 mg) as a white solid HCl salt in quantitative yield. LC / MS (ESI + ) m / z calculated value 523.43 (M), observed value 524.60 (M+H + ).

[0235] To an oven-dried 40 mL flask equipped with a stir bar under N2, intermediate 4 (75 mg, 0.135 mmol), sieve-dried THF (3 mL), and triethylamine (0.12 mL, 0.815 mmol) were added. 2,3,4,5,6-Pentafluorophenyl 4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl]benzoate sulfone (71 mg, 0.0163 mmol) was added, and the reaction was stirred for 2 h. The reaction slowly became very heterogeneous. A small aliquot was taken, diluted with MeCN, and analyzed by LCMS to confirm completion of the reaction. The reaction was dry-loaded onto a 12 g silica gel column with Celite and purified by flash chromatography (MeOH / DCM 0–8% over 45 min, ELS detection) to give LP-361-p (52 mg) as a white solid in 52% yield. LC / MS (ESI) + ) m / z calculated value 773.44 (M), observed value 775.11 (M+H + ).

[0236] Synthesis of LP-371-p [ka]

[0237] Compound 1 (palmitic acid, 2.50 g) was dissolved in 60 mL of DMF. TBTU (3.44 g) and DIPEA (6.9 mL) were then added. The reaction was stirred for 10 minutes, and then compound 2 (2.66 g in DMF) was added. The reaction was complete in 1 hour. The mixture was diluted with 300 mL of EtOAc and washed with 3% citric acid (3 × 60 mL), HO (2 × 60 mL), and NaCl (1 × 60 mL), then dried over NaSO. The product was filtered and concentrated on a rotary evaporator and high vacuum. The product was purified using column chromatography (80G RediSep Gold Rf column loaded in DCM (15 mL) with 1 drop of MeOH, mobile phase MeOH / DCM, 0–5% over 30 minutes). Yield 4.094 g, LC-MS: calculated [M+H] 486.74, found 488.11. [ka]

[0238] Compound 1 (4.094 g) was dissolved in 4 M HCl in dioxane (28 mL) at 0° C. for 10 min. The reaction was warmed to room temperature and stirred for 2 h. The product was concentrated by rotary evaporation and high vacuum. Yield 3.485 g. LC-MS: calculated [M+H] 386.57, found 388.02. [ka]

[0239] Compound 1 (2.3 g) was dissolved in 80 mL of THF. TEA (4.975 mL) and compound 2 (3.10 g) were then added. The reaction was stirred at room temperature for 1 h. The reaction was dry-packed with Celite 545, and the mixture was concentrated in a 28 °C water bath and placed under high vacuum to dry completely. The product was purified by flash chromatography (MeOH / DCM, 0-6% over 40 min) (yield 2.905 g). LC-MS: calculated [M+H] 636.85, found 637.95.

[0240] Synthesis of LP-374-p [ka]

[0241] Compound 1 (oleic acid, 200 mg) was dissolved in 6.5 mL of DMF. TBTU (250 mg) and DIPEA (0.501 mL) were then added to the reaction mixture. The reaction was stirred for 10 minutes, and then compound 2 (193 mg in DMF) was added. The reaction was complete in 1 hour. The reaction was diluted with 75 mL of EtOAc, washed with 3% citric acid (3 × 8 mL), HO (2 × 8 mL), NaCl (1 × 8 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The product was purified by column chromatography (12G column loaded in 1 mL of DCM, MeOH / DCM, 0–3% over 30 minutes) (yield 254 mg). LC-MS: calculated [M+H] 512.76, found 513.62. [ka]

[0242] Compound 1 (150 mg) was dissolved in 4 M HCl in dioxane at 0 °C. The mixture was warmed to room temperature and the reaction was stirred for 90 min. The product was concentrated on a rotary evaporator and further dried under high vacuum. Yield 110 mg, LC-MS: calculated [M+H] 412.66, found 413.35. [ka]

[0243] Compound 1 (110 mg) was dissolved in 4.5 mL of THF. Compound 2 (145 mg) and TEA (0.223 mL) were added. The reaction was stirred at room temperature for 1 h. The mixture was concentrated in a 28 °C water bath, dissolved in sieve-dried DCM, loaded onto a column, and purified by flash chromatography (dry MeOH / DCM, 0-8% over 30 min). Yield 92 mg. LC-MS: calculated [M+H] 662.86, found 664.12.

[0244] Synthesis of LP-375-p [ka]

[0245] Compound 1 (linoleic acid, 200 mg) was dissolved in 6.5 mL of DMF. TBTU (252 mg) and DIPEA (0.505 mL) were then added, and the mixture was stirred for 10 min. Compound 2 (195 mg) was then added, and the reaction was stirred for 1 h. The mixture was then diluted with 75 mL of EtOAc, washed with 3% citric acid (3 × 8 mL), HO (2 × 8 mL), NaCl (1 × 8 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The product was loaded onto a 12G column in 1 mL of DCM and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield 266 mg. LC-MS: calculated [M+H] 510.76, found 511.90. [ka]

[0246] Compound 1 (166 mg) was dissolved in 2.5 mL of 4 M HCl in dioxane. The mixture was stirred for 1 hour. The product was concentrated on a rotary evaporator and placed under high vacuum. Yield 125 mg, LC-MS: calculated [M+H] 410.64, found 411.18. [ka]

[0247] Compound 1 (125 mg) was dissolved in 4.5 mL of THF. Compound 2 (165 mg) and TEA (0.255 mL) were then added. The mixture was stirred for 1 h. The compound was concentrated in a 28 °C water bath, and sieve-dried DCM was loaded onto a column and purified by flash chromatography (dry MeOH / DCM, 0-8% over 25 min). Yield 110 mg. LC-MS: calculated [M+H] 660.87, found 662.05.

[0248] Synthesis of LP-377-p [ka]

[0249] Compound 1 (palmitic acid, 175 mg) was dissolved in 6.5 mL of DMF. TBTU (252 mg) and DIPEA (0.483 mL) were then added. The mixture was stirred for 10 min. Compound 2 (183 mg in DMF) was then added. The reaction was stirred at room temperature for 1 h. The mixture was then diluted with 75 mL of EtOAc and washed with 3% citric acid (3 × 8 mL), HO (2 × 8 mL), and NaCl (1 × 8 mL). It was then dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was wet-loaded in 2 mL of DCM onto a 12G column and purified by flash chromatography (MeOH / DCM, 0–3% over 30 min). Yield 277 mg. LC-MS: calculated [M+H] 471.72, found 472.57. [ka]

[0250] Compound 1 (277 mg) was dissolved in 6 mL of 1:1 DCM:TFA. The reaction was stirred at room temperature for 1 hour. The product was concentrated on a rotary evaporator and placed under high vacuum. Yield 249 mg. LC-MS: calculated [M+H] 415.62, found 416.22. [ka]

[0251] Compound 1 (244 mg) was dissolved in 10 mL of DCM. EDC·HCl (141 mg), NHS (135 mg), and DMAP (14 mg) were then added sequentially. The reaction was stirred overnight at room temperature. The reaction mixture was then diluted with 70 mL of DCM and washed with citric acid (pH 3, 3 × 8 mL), followed by NaCl (1 × 8 mL, 1 drop of 10% citric acid solution), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was wet-loaded in 2.5 mL of DCM onto a 12G column and purified by flash chromatography (MeOH / DCM, 0–3% over 30 min). Yield: 63 mg. LC-MS: calculated [M+H] 512.69, found 513.53.

[0252] Synthesis of LP-378-p [ka]

[0253] Compound 1 (myristic acid, 2.50 g) was dissolved in 50 mL of DMF. TBTU (3.87 g) and DIPEA (7.7 mL) were then added, and the mixture was stirred for 10 min. Compound 2 (2.99 g in DMF) was then added, and the reaction was stirred for 1 h. The reaction mixture was diluted with 300 mL of EtOAc, washed with 3% citric acid (3 × 60 mL), HO (2 × 60 mL), and NaCl (1 × 60 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was loaded onto an 80G RediSep Gold Rf column in 15 mL of DCM and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield 4.172 g. LC-MS: calculated [M+H] 458.68, found 459.83. [ka]

[0254] Compound 1 (4.172 g) was dissolved in 28 mL of 4 M HCl in dioxane at 0 °C, and the mixture was stirred for 10 min. The reaction was warmed to room temperature and stirred for 2 h. The product was concentrated on a rotary evaporator and placed under high vacuum. Yield 3.436 g, LC-MS: calculated [M+H] 358.57, found 359.76. [ka]

[0255] Compound 1 (2.5 g) was dissolved in 80 mL of THF. Then, TEA (5.83 mL) and compound 2 (3.48 g) were added. The reaction was stirred for 1 h. Celite® 545 was added, and the mixture was concentrated in a 28°C water bath and placed under high vacuum. The compound was dry-loaded onto a 120 G column and purified by flash chromatography (MeOH / DCM, 0-7% over 40 min) (yield 2.33 g). LC-MS: calculated [M+H] 608.80, found 610.12.

[0256] Synthesis of LP-379-p [ka]

[0257] Compound 1 (Asta Tech® #89929, 3.00 g) was dissolved in 50 mL of DMF. TBTU (3.09 g) and DIPEA (6.2 mL) were then added, and the mixture was stirred for 10 minutes. Compound 2 (1.68 g in DMF) was then added. The mixture was stirred for 1 hour. The reaction mixture was then diluted with 300 mL of EtOAc, washed with 3% citric acid (3 × 60 mL), HO (2 × 60 mL), and NaCl (1 × 60 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was loaded onto an 80 g column in 14 mL of DCM with 2 drops of MeOH and purified by flash chromatography (MeOH / DCM, 0–4% over 40 minutes). Yield: 4.033 g. LC-MS: calculated [M+H] 498.71, found 499.85. [ka]

[0258] Compound 1 (4.033 g) was dissolved in 50 mL of 1:1 DCM:TFA. The mixture was stirred for 1 hour. The product was concentrated on a rotary evaporator and placed under high vacuum. The product was then dissolved in ACN and concentrated, then dissolved in DCM and concentrated again. Yield 3.839 g, LC-MS: calculated [M+H] 442.60, found 443.81.

[0259] Synthesis of LP-380-p [ka]

[0260] Compound 1 (lauric acid NHS ester, 150 mg) was dissolved in 6 mL of DMF. DIPEA (0.357 mL) and compound 2 (138 mg) were then added, and the reaction was stirred for 1 h. The mixture was diluted with 75 mL of EtOAc and washed with 3% citric acid in water (3 × 8 mL), HO (2 × 8 mL), and NaCl (1 × 8 mL), then dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was loaded onto a 12G column in 2 mL of DCM and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield 177 mg. LC-MS: calculated [M+H] 430.63, found 431.43. [ka]

[0261] Compound 1 (177 mg) was dissolved in 4 mL of 4 M HCl in dioxane at 0° C. and stirred for 10 min. The mixture was warmed to room temperature and stirred for 1 h. The product was concentrated on a rotary evaporator and placed under high vacuum, then dissolved and concentrated twice with DCM / toluene and placed under high vacuum. Yield 150 mg. LC-MS: calculated [M+H] 330.51, found 331.16. [ka]

[0262] Compound 1 (150 mg) was dissolved in 6 mL of THF, followed by the addition of TEA (0.380 mL) and compound 2 (246 mg). The reaction was stirred at room temperature for 1 h. Celite® 545 was then added, and the mixture was concentrated in a 28°C water bath. The crude product was dry-loaded onto a 12G column and purified by flash chromatography (MeOH / DCM, 0-4% over 25 min). Yield 148 mg. LC-MS: calculated [M+H] 580.74, found 581.94.

[0263] Synthesis of LP-403-p [ka]

[0264] To a 250 mL RB flask equipped with a stir bar under N2 was added erucic acid (5 g, 14.7 mmol) and dry DCM (125 mL). The reaction was cooled in an ice bath for 10 min, and then mCPBA (77%, 4.3 g, 19.1 mmol) was added portionwise over 5 min. The reaction was stirred on ice for 10 min, then the ice bath was removed and the reaction was stirred for 18 h while warming to room temperature. NMR confirmed the reaction was complete. The reaction was dry-loaded directly onto a 220 g silica column with silica and purified by flash chromatography (MeOH / DCM, 0–5%) to give the intermediate epoxide (4.4 g) as a white solid in 83% yield. To a 200 mL RB flask equipped with a stir bar was added the intermediate epoxide (2.5 g, 7.04 mL), dioxane (36 mL), and HO (12 mL). H2SO4 (3 M solution in H2O, 24 mL, 70.2 mmol) was added and the reaction was stirred at 100 °C (oil bath temperature) for 18 h. HPLC with ELSD confirmed the reaction was complete. The reaction was diluted with H2O (50 mL) and extracted with EtOAc (150 mL x 3). The pooled organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give 1 (2.5 g) as a white solid in 95% yield. LC / MS (ESI - ) m / z calculated value 372.32 (M), observed value 371.48- (MH + ).

[0265] To an oven-dried 500 mL RB flask equipped with a stir bar under N2 was added Intermediate 1 (5.4 g, 14.5 mmol) and sieve-dried MeOH (200 mL). The resulting suspension was cooled in an ice bath for 10 min, then thionyl chloride (1.1 mL, 15.2 mmol) was added dropwise over 1 min. The reaction was stirred on ice for 10 min, then the ice bath was removed and the reaction was stirred for 2 h while warming to room temperature. HPLC with ELSD confirmed the reaction was complete. The reaction was concentrated to a small volume (25 mL), diluted with DCM, washed with saturated NaHCO3 (x2), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give 2 (5.4 g) as an oil in 97% yield. 1 H NMR(400MHz, CDCl3)δ=3.65(s,3H),3.37-3.41(m,2H),2.29(t,2H),1.99(br.s. ,2H),1.66-1.54(m,3H),1.54-1.37(m,5H),1.37-1.20(overlapping m,26H),0.87(t,3H).

[0266] To a 500 mL RB flask equipped with a stir bar was added intermediate 2 (5.4 g, 13.9 mmol), THF (150 mL), and HO (50 mL). NaIO (4.4 g, 20.9 mmol) was added and the reaction was stirred for 18 h. HPLC with ELSD confirmed the reaction was complete. The reaction was concentrated under reduced pressure to remove THF, further diluted with HO (50 mL), and extracted with DCM (100 mL x 2). The pooled organic layers were washed with brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (20 mL), loaded onto a 120 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–10% over 60 min, ELS detection) to give 3 (1.6 g) as a colorless oil in 50% yield. 1 HNMR (400MHz, CD2Cl2) δ=9.73(t,1H),3.63(s,3H),2.40(dt,2H),2.29(t,2H),1.65-1.57(m,2H),1.36-1.26(m,16H).

[0267] To an RB flask already containing 3 (1.6 g) under N2, a stir bar, piperidine (0.85 mL, 8.57 mmol), and sieve-dried MeCN (60 mL) were added. 1,1-Dimethylethyl 2-(phenylsulfinyl)acetate (1.74 g, 7.25 mmol) was added, and the reaction was stirred for 18 h. HPLC with ELSD confirmed the reaction was complete. The reaction was concentrated under reduced pressure, dissolved in DCM (20 mL), loaded onto a 120 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–30% over 60 min) to give 4 (1.8 g) as a colorless oil in 77% yield. 1 HNMR(400MHz,CDCl3)δ 6.83(dd,1H),5.94(dd,1H),4.29-4.24(m,1H),3.66(s,3H),2.29(t,2H),1.65-1.52(m,4H),1.48(s,9H),1.32-1.23(br.s.,14H).

[0268] To a 100 mL RB flask equipped with a stir bar under N2, intermediate 4 (800 mg, 2.24 mmol), DCM (210 mL), and imidazole (381 mg, 5.6 mmol) were added. The resulting solution was cooled in an ice bath for 10 min, and then TBDMS-Cl (405 mg, 2.69 mmol) was added. The reaction was stirred on ice for 10 min, then the ice bath was removed and the reaction was stirred for 16 h while warming to room temperature. HPLC with ELSD confirmed the reaction was complete. The reaction was diluted with DCM, washed with HO (×2), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (4 mL), loaded onto a 40 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–10% over 30 min, ELS detection) to give the silyl-protected intermediate (447 mg) as a colorless oil in 43% yield. A small amount of this intermediate (105 mg, 0.22 mmol) was added to a 40 mL vial, and THF (1.5 mL), HO (0.5 mL), and LiOH (1 M solution in HO, 0.512 mL, 0.512 mmol) were added sequentially. The reaction was stirred for 12 h. Completion of the reaction was confirmed by HPLC using an ELSD. THF was removed under reduced pressure, and the reaction was acidified with 10% citric acid (1 mL) and extracted with EtOAc (×2). The pooled extracts were washed with brine, dried over MgSO, filtered, and concentrated under reduced pressure to give 5 (82 mg) as a colorless oil in 82% yield. To a 40 mL vial equipped with a stir bar under N2 was added 5 (335 mg, 0.733 mmol), azido-PEG5-amine (270 mg, 0.88 mmol), DIPEA (0.38 mL, 2.2 mmol), and sieve-dried DCM (12 mL). HBTU (334 mg, 0.88 mmol) was added, and the reaction was stirred at room temperature for 3 h. Completion of the reaction was confirmed using both LCMS and HPLC with ELSD.The reaction was diluted with DCM, washed with saturated NaHCO3 (x4), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (5 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography MeOH / DCM (0-5% over 30 min, ELS detection, product eluted at 2%) to give 6 (401 mg) as a colorless oil in 73% yield by LC / MS (ESI). + ) m / z calculated value 744.51 (M), observed value 745.86 (M+H + ).

[0269] To a 40 mL vial equipped with a stir bar under N2, 6 (95 mg, 0.134 mmol), DCM (1 mL), and TFA (1.25 mL, 11.1 mmol) were added. The reaction was stirred at room temperature for 4.5 h. Completion of the reaction was confirmed by both LCMS and HPLC with ELSD. The reaction was concentrated under reduced pressure, dissolved in DCM (5 mL), concentrated again, and then dried on a vacuum pump for 1 h to give the acid intermediate (73 mg), which was used without purification. The 40 mL vial containing the acid intermediate (73 mg, 0.127 mmol) was placed under N2 and dissolved in sieve-dried DCM (1.5 mL). The reaction was cooled in an ice bath for 10 min, and then Dess-Martin periodinane (57 mg, 0.133 mmol) was added. The reaction was stirred on ice for 30 min, then the ice bath was removed and the reaction was stirred while warming to room temperature for 3 h. Completion of the reaction was confirmed by LCMS. The reaction mixture was suspended in 10% MeOH / DCM, dry-packed onto a 12 g silica gel column, and purified by flash chromatography (MeOH / DCM 0-10%, product elution at approximately 5%) to give LP-403-p (11.3 mg) as a white solid in 16% yield. LC / MS (ESI) + ) m / z calculated value 572.34 (M), observed value 573.56 (M+H + ).

[0270] Synthesis of LP-404-p [ka]

[0271] To an oven-dried 100 mL round-bottom flask equipped with a stir bar under N2 was added Fmoc-Ser-OtBu (3 g, 7.8 mmol), sieve-dried DCM (25 mL), DIPEA (3.4 mL, 19.5 mmol), and activated molecular sieves. The resulting solution was cooled in an ice bath for 10 min, and then 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (2.3 mL, 10.1 mmol) was added dropwise over 1 min. The reaction was gently stirred on ice for 15 min, then the ice bath was removed and the reaction was gently stirred for 2 h while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was quenched with MeOH (10 mL), filtered (to remove sieves), and concentrated under reduced pressure to give a crude yellow oil, which was dissolved in DCM (20 mL), loaded onto a 120 g silica gel column, and purified by flash chromatography (1% EtN in EtOAc / hexanes, 10-80% gradient over 40 min). The pooled product fractions were concentrated and azeotroped with PhMe (10 mL) to give 1 (3.29 g) as a colorless oil in 73% yield. LC / MS (ESI) - ) m / z calculated value 583.28 (M), observed value 584.91 (M+H + ).

[0272] A round-bottom flask containing Intermediate 1 (3.29 g, 5.63 mmol) was placed under N2 and dissolved in sieve-dried MeCN (10 mL). 2-Hydroxy-N,N,N-trimethylethanaminium 4-methylbenzenesulfonate (1.7 g, 6.2 mmol) was added as a solution in sieve-dried MeCN (10 mL). ETT (0.75 M in MeCN, 7.24 mL, 5.63 mmol) was added slowly, and the reaction was stirred under N2 for 2 h. LCMS confirmed the reaction was complete. The reaction was cooled in an ice bath for 10 min, and then mCPBA (77%, 2.47 g, 11.26 mmol) was added in portions. The reaction was stirred on ice for 15 min, then the ice bath was removed and the reaction was stirred for 1 h while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure at 28 °C and dried on a vacuum pump for 2 h to give a white oily solid. The solid was dissolved in DCM (20 mL, sonication required) and then loaded onto an 80 g silica gel column and purified by flash chromatography (MeOH / DCM, 0-20% over 40 min) to give 2 (1.66 g) as a colorless oil / tetrazolium salt in 40% yield. LC / MS (ESI) + ) m / z calculated 602.26 (M + ), actual value 602.82 (M + ).

[0273] A round-bottom flask containing intermediate 2 (1.66 g, 2.14 mmol) was placed under N2 and dissolved in sieve-dried DCM (20 mL). Triisopropylsilane (1.3 mL, 6.41 mmol) and TFA (20 mL, 261 mmol) were added sequentially, and the reaction was stirred for 2.5 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure, dissolved in DCM (15 mL), concentrated again, and then azeotroped with PhMe (15 mL) to give the intermediate acid (2.48 g), which was used without further purification. A round-bottom flask containing the crude intermediate acid (2.2 g) was placed under N2 and dissolved in sieve-dried DMF (25 mL). The reaction was cooled in an ice bath for 10 minutes, then DIPEA (0.625 mL, 3.60 mmol) and HBTU (1.36 g, 3.60 mmol) were added, and the reaction was stirred on ice for 10 minutes. BocNH-PEG2-amine (893 mg, 3.60 mmol) was added slowly as a solution in DMF, and the reaction was stirred on ice for 10 minutes. The ice bath was then removed, and the reaction was stirred for 7 hours while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure, then DMF (15 mL) was added, and the reaction was again concentrated under reduced pressure. The crude reaction was dissolved in DCM (30 mL), and half of the reaction was loaded onto a 24 g silica gel column and purified by flash chromatography (MeOH / DCM, 0–20% over 1 hour). The second half of the reaction was purified in the same manner to give Intermediate 3 (790 mg) as a colorless oil and TFA salt in 49% yield. LC / MS (ESI + ) m / z calculated 776.36 (M + ), actual value 777.09 (M + ).

[0274] To a 40 mL vial equipped with a stir bar was added Intermediate 3 (125 mg, 0.163 mmol), followed by methylamine (40% in HO, 3.75 mL, 33.8 mmol), and the reaction was stirred for 2 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and then azeotroped with PhMe (2 mL × 2) to give the crude product, which was dissolved in sieve-dried DCM / DMF (1:1, 3 mL). DIPEA (0.14 mL, 0.815 mmol), palmitic acid (73 mg, 0.285 mmol), and HBTU (109 mg, 0.285 mmol) were added sequentially, and the reaction was stirred under N for 16 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure, re-evaporated with PhMe (10 mL), and dried on a vacuum pump for 3 h. The crude material was dry-packed onto a glass silica gel column with silica and purified by manual flash column chromatography (MeOH / CHCl3 0-10% for 20 min, then switched to 15:9:1 CHCl3 / MeOH / HO for 30 min) to give 4 (42 mg) as a yellowish oil in 35% yield. LC / MS (ESI) + ) m / z calculated value 738.49 (M), observed value 740.19 (M + +H).

[0275] To a 40 mL vial was added a stir bar, Intermediate 4 (42 mg, 0.057 mmol), and 4 M HCl in dioxane (2 mL, 8 mmol). The reaction vial was tightly capped and stirred for 2 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and then dried on a vacuum pump for 3 h. The crude intermediate was dissolved in dry DMF (1 mL), then DIPEA (30 μL, 0.171 mmol) and maleimide-C5-NHS ester (23 mg, 0.074 mmol) were added sequentially, and the reaction was stirred for 4.5 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure, reconstituted with PhMe (3 mL), and dried on a vacuum pump for 3 h. The crude material was dry-packed onto a glass silica gel column with silica and purified by manual flash column chromatography (MeOH / CHCl3 0-30% for 25 min, then switched to 55:41:4 MeOH / CHCl3 / HO for 30 min) to give intermediate LP-404-p (22 mg) as a colorless oil in 50% yield. LC / MS (ESI) + ) m / z calculated value 831.51 (M), observed value 833.27 (M+H + ).

[0276] Synthesis of LP-412-p [ka]

[0277] To a 40 mL vial equipped with a stir bar, under N2, was added arachidonic acid (100 mg, 0.3218 mmol) and sieve-dried DCM (3 mL). DIPEA (0.17 mL, 0.99 mmol), 2,3,5,6-tetrafluorophenol (66 mg, 0.394 mmol), and COMU (169 mg, 0.394 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 3 h. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO (×3, or until the aqueous extract was colorless), brine, dried over MgSO4, filtered, and concentrated under reduced pressure (protecting the product from light) to give LP-412-p (156 mg) as a brown oil, which was used without further purification. LC / MS (ESI) + ) m / z calculated value 452.23 (M), observed value 454.04 (M+H + ).

[0278] Synthesis of LP-413-p [ka]

[0279] Compound 1 (Asta Tech® #W15452, 1.00 g) was dissolved in 22 mL of DMF. TBTU (1.212 g) and DIPEA (2.33 mL) were then added. The mixture was stirred for 10 minutes, and then compound 2 (1.05 g in DMF) was added. The reaction was covered with foil and stirred for 1 hour. The mixture was then diluted with EtOAc (225 mL), washed with 3% citric acid in water (3 × 30 mL), HO (2 × 30 mL), and NaCl (1 × 30 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was loaded onto a 40 G column with DCM (4 mL) and purified by flash chromatography (MeOH / DCM, 0–4% over 30 minutes). Yield 1.158 g, LC-MS: calculated [M+H] 563.82, found 564.83. [ka]

[0280] Compound 1 (1.158 g) was dissolved in 10.5 mL of 4 M HCl in dioxane at 0 °C. The mixture was stirred in the dark for 10 min, then warmed to room temperature, wrapped in foil, and stirred for 4 h. The product was concentrated on a rotary evaporator, then dissolved and concentrated twice using DCM / toluene, and then placed under high vacuum. Yield 1.0135 g. LC-MS: calculated [M+H] 507.71, found 508.93. [ka]

[0281] Compound 1 (1.0135 g) was dissolved in 16 mL of DCM. Tetrafluorophenol (Sigma® #196789, 0.497 g) was then added. After stirring for 10 min, EDC·-HCl (Sigma® #E7750, 0.574 g) was added. The mixture was stirred for 2 h. The reaction was concentrated in vacuo, and the crude product was dry-loaded onto a 40 G column with silica and purified by flash chromatography (EtOAc / hexane, 0–70% over 30 min) (yield 655 mg). LC-MS: calculated [M+H] 655.77, found 656.90.

[0282] Synthesis of LP-424-p [ka]

[0283] To a 40 mL vial equipped with a stir bar, α-linolenic acid (200 mg, 0.718 mmol) and sieve-dried DCM (4 mL) were added under N2. Amine-PEG-tert-butyl ester (239 mg, 0.862 mmol), DIPEA (0.37 mL, 2.15 mmol), and HBTU (327 mg, 0.862 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 5 h. Completion of the reaction was confirmed by TLC (β-cresol green and KMnO4 staining). The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x3), dried over MgSO, filtered, and concentrated under reduced pressure (protecting the product from light) to give the crude material, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0-75% over 50 min) to give Intermediate 1 (293 mg) as a colorless oil in 76% yield. LC / MS (ESI) + ) m / z calculated value 537.40 (M), observed value 538.61 (M+H + ).

[0284] To a 40 mL vial equipped with a stir bar was added Intermediate 1 (155 mg, 0.288 mmol) and 4 M HCl in dioxane (3.5 mL, 14.4 mmol). The vial was tightly capped, wrapped in foil, and the reaction was stirred for 4 h. Completion of the reaction was confirmed by TLc (KMnO staining) and LCMS. The reaction was concentrated under reduced pressure (shielding the product from light) and dried overnight on a vacuum pump (shielding the product from light) to give Intermediate 2 (145 mg) as an oil, which was placed under N2 and dissolved in sieve-dried DCM (3 mL). Triethylamine (0.12 mL, 0.864 mmol), 2,3,5,6-tetrafluorophenol (58 mg, 0.346 mmol), and COMU (148 mg, 0.346 mmol) were added sequentially, and the reaction was stirred for 3 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure (protecting the product from light) to give the crude material, which was dissolved in DCM (4 mL), loaded onto a 12 g column, and purified by flash chromatography (0-100% EtOAc / hexanes, product eluted with 60% EtOAc) to give LP-424-p (54 mg) as an orange oil in 30% yield. LC / MS (ESI) + ) m / z calculated 629.33 (M), observed 630.53 (M+H + ).

[0285] Synthesis of LP-425-p [ka]

[0286] To a 40 mL vial equipped with a stir bar, under N2, was added linoleic acid (100 mg, 0.356 mmol) and sieve-dried DCM (3 mL). Triethylamine (0.15 mL, 1.06 mmol), 2,3,5,6-tetrafluorophenol (65 mg, 0.392 mmol), and COMU (167 mg, 0.392 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 3 h. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO (×3, or until the aqueous extract was colorless), brine, dried over MgSO4, filtered, and concentrated under reduced pressure (protecting the product from light) to give LP-425-p (163 mg), which was used without further purification. LC / MS (ESI) + ) m / z calculated 428.51, observed 429.55 (M+H + ).

[0287] Synthesis of LP-426-p [ka]

[0288] To a 40 mL vial equipped with a stir bar, under N2, was added linoleic acid (100 mg, 0.356 mmol) and sieve-dried DCM (3 mL). DIPEA (0.14 mL, 1.06 mmol), amine-PEG3-tert-butyl ester (118 mg, 0.427 mmol), and HBTU (162 mg, 0.427 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 4 h. Completion of the reaction was confirmed by TLc (KMnO4 staining). The reaction was diluted with DCM, washed with HO (×3), saturated NaHCO (×3), brine, dried over MgSO, filtered, and concentrated under reduced pressure (protecting the product from light) to give the crude product, which was dissolved in DCM (2.5 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–75%, product eluted with 50% EtOAc) to give 1 (131 mg) as a colorless oil in 68% yield. A 40 mL vial already containing 1 (131 mg, 0.242 mmol) was wrapped in foil and stirred in 4 M HCl in dioxane (3.5 mL, 12.1 mmol) for 3.5 h. The reaction was complete as confirmed by TLC and LCMS. The reaction was concentrated under reduced pressure (protecting the product from light) and dried on a vacuum pump for 2 h (protecting the product from light) to give 2 (117 mg, 0.242 mmol) as a colorless oil, which was placed under N2 and dissolved in sieve-dried DCM (3 mL). Triethylamine (0.1 mL, 0.726 mmol), 2,3,5,6-tetrafluorophenol (44 mg, 0.266 mmol), and COMU (114 mg, 0.266 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 1.5 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure (protecting the product from light) to give the crude material, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0-100%) to give LP-426-p (49 mg) as a colorless oil in 32% yield. LC / MS (ESI) + ) m / z calculated value 631.35 (M), observed value 633.21 (M+H +).

[0289] Synthesis of LP-427-p [ka]

[0290] To a 40 mL vial equipped with a stir bar was added oleic acid NHS ester (100 mg, 0.284 mmol) and sieve-dried DCM (2.5 mL) under N2. Triethylamine (0.12 mL, 0.852 mmol) and amino-PEG 3-tert-butyl ester (87 mg, 0.312 mmol) were added sequentially, and the reaction was stirred for 3 h. Completion of the reaction was confirmed by TLc (KMnO4 staining). The reaction was diluted with DCM, washed with saturated NaHCO3 (x2), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (2.5 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (0–75% EtOAc / hexanes over 40 min, product eluted with 50% EtOAc) to give 1 (101 mg) as a colorless oil in 70% yield. A 40 mL vial already containing 1 (101 mg, 0.186 mmol) was stirred in 4 M HCl in dioxane (2 mL, 7.5 mmol) for 2.5 h. Completion of the reaction was confirmed by TLC and LCMS. The reaction was concentrated under reduced pressure and dried overnight on a vacuum pump to give 2 (90 mg, 0.185 mmol) as an oil, which was placed under N2 and dissolved in sieve-dried DCM (3 mL). Triethylamine (0.08 mL, 0.555 mmol), 2,3,5,6-tetrafluorophenol (34 mg, 0.203 mmol), and COMU (87 mg, 0.203 mmol) were added sequentially, and the reaction was stirred for 3 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (2.5 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0-75%, product eluted with 50% EtOAc) to give LP-427-p (55 mg) as a colorless oil in 47% yield. LC / MS (ESI) + ) m / z calculated value 633.37 (M), observed value 635.28 (M+H + ).

[0291] Synthesis of LP-428-p [ka]

[0292] To a round-bottom flask was added 6-hydroxyhexanoic acid (2.82 g, 21.3 mmol), DCM (28.3 mL), triethylamine (2.9 mL, 21.3 mmol), and TBTU (6.29 g, 19.5 mmol). The reaction was stirred for 1 minute, then 2-amino-boc-proline-OMe HCl (5 g, 17.8 mmol) was added and the reaction was stirred until complete by HPLC (ELSD). The reaction was quenched with HO (30 mL). The layers were separated and the organic layer was washed with 1 M HCl (30 mL) and saturated NaHCO (30 mL). A large emulsion formed, which was then centrifuged. The layers were separated, and the organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give a crude yellow oil, which was loaded onto a 220 g silica gel column and purified by flash chromatography (MeOH / DCM, 0-10%) to give 1 (3.32 g) as a colorless oil in 52% yield. LC / MS calculated 358.21 (M), found 359.32 (M+H). + ). To a round-bottom flask was added 1 (1.27 g, 3.56 mmol) and THF (7.5 mL). Aqueous LiOH (5 mL, 7.49 mmol) was added and the reaction was stirred for 1 h. The reaction was confirmed to be complete by HPLC with ELSD. To the reaction was added 2 M HCl (21.6 mL, 43.1 mmol) and the reaction was stirred at 40 °C for 3 h. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 12 h to give 2 (1.4 g, HCl salt) as a thick, viscous, colorless oil, which was used without purification.

[0293] To a round-bottom flask was added myristic acid (8 g, 35.1 mmol), THF (40 mL), TSTU (11 g, 38.5 mmol), and triethylamine (5.8 mL, 42 mmol). The reaction was diluted with MeCN (40 mL) to give a solution, which was stirred at room temperature for 17.5 h. The reaction was poured into HO (500 mL), and the crude precipitated product was vacuum filtered, collected, suspended in MeCN, and concentrated under reduced pressure to give crude 3 (11.2 g), which was used without further purification. To a 100 mL round-bottom flask was added amino-PEG2-acid (2.4 g, 13.9 mmol), DCM (44 mL), and triethylamine (4.3 mL, 31.3 mmol). To the resulting suspension was added crude 3 (4.4 g, 13.6 mmol) over 4 min. The reaction was stirred for 19 h and slowly became homogeneous. The reaction completion was confirmed by HPLC with ELSD. The reaction was quenched with piperidine (0.188 mL), stirred for 30 min, and then diluted with HO (45 mL) and concentrated HCl (3 mL). The layers were separated, and the organic layer was washed with 0.5 M HCl (45 mL x 2), HO (40 mL x 2), dried over NaSO, filtered, and concentrated under reduced pressure to give 4 (4.9 g) as a white solid in 94% yield. LC / MS calculated m / z 387.30 (M), found 388.38 (M+H). + ) and 386.36(MH + ). A portion of crude 4 (3 g, 7.88 mmol) was added to a round-bottom flask and suspended in MeCN (25 mL) and triethylamine (1.3 mL, 9.5 mmol). TSTU (2.6 g, 8.7 mmol) and THF (15 mL) were added, and the resulting yellow solution was stirred for 19 h. The reaction was partially concentrated under reduced pressure and then poured into HO (250 mL). A white precipitate formed, which was filtered by vacuum filtration, suspended in MeCN, concentrated under reduced pressure, and then dried overnight to give 5 (3.6 g) as a white solid. 1HNMR(400MHz,CDCl3)δ =6.02(t,1H),3.85(t,2H),3.66-3.60(m,2H),3.57-3.52(m,2H),3.47-3.41(m,2H),2.8 9(t,2H),2.83(s,4H),2.16(t,2H),1.66-1.58(m,2H),1.34-1.22(m,22H),0.877(t,3H).

[0294] To a round-bottom flask was added 2 (1 g, 3.56 mmol), DMF (20 mL), and triethylamine (2.4 mL, 17.2 mmol). Intermediate 5 (1.3 g, 2.74 mmol) was added, and the suspension was stirred vigorously for 16 h. HPLC and LCMS showed a mixture of 6 and a by-product; both the acid and amine of 2 had reacted with 5. The reaction was diluted with HO (20 mL), and then 1 M NaOH (5 mL) and solid LiOH (98 mg) were added (pH 12), and the reaction was stirred for 2.5 h. The reaction was acidified with 2 M HCl (40 mL), diluted with HO (110 mL), and extracted with DCM (60 mL × 3). The pooled organic layers were washed with 1 M HCl (75 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give crude 6 (1.76 g), which was used without purification. To a round-bottom flask was added crude 6 (1.68 g, 2.74 mmol), DCM (17 mL), pyridine (17 mL), and DMT-Cl (1.02 g, 3.01 mmol). The orange solution was stirred for 17 h. LCMS confirmed the reaction was complete. The reaction was quenched with MeOH (5 mL), concentrated under reduced pressure, suspended in EtOAc (75 mL), and washed with HO (75 mL × 2). The resulting emulsion was dissolved by the addition of brine (10 mL). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to give crude 7 (3.19 g), which was dissolved in DCM (8 mL), loaded onto an 80 g silica gel column, and purified by flash chromatography (MeOH / DCM with 1% EtN, 0–10%) to give 7 (1.83 g) as the triethylammonium salt in 66% yield. LC / MS (ESI - ) m / z calculated value 915.56 (M), observed value 914.58 (MH + ).

[0295] To a round-bottom flask was added 7 (1.84 g, 1.80 mmol) and MeCN (26 mL). Triethylamine (0.630 mL, 4.52 mmol) and TBTU (580 mg, 1.80 mmol) were added, and the reaction was stirred for 2 min and then added to a reaction vessel containing NittoPhase HL native resin (0.572 mmol / g hydroxy loading). The reaction was shaken for 16 h, drained, and washed with MeCN (100 mL). To the resin was added NMI / MeCN (1:4, 12 mL), collidine / MeCN (3:2, 6 mL), AcO / MeCN (2:3, 6 mL), and DMAP (92 mg). The capping reaction was shaken for 3 h, and the resin was drained, washed with MeCN (150 mL), MeOH (100 mL), transferred to a plastic bottle, and dried in vacuo for 16 h. The loading of the resin was determined to be 0.371 mmol / g.

[0296] Synthesis of LP-432-p [ka]

[0297] Compound 1 (gamma-linolenic acid, 150 mg) was dissolved in 5 mL of DCM. Tetrafluorophenol (Sigma #196789, 134 mg) was then added, and the mixture was stirred for 10 min. EDC·-HCl (155 mg) was then added, and the mixture was stirred for 2 h. The mixture was concentrated, and the crude product was suspended in DCM (5 mL), dry-loaded onto a 12 G column with silica, and purified by flash chromatography (EtOAc / hexane, 0–30% over 35 min). Yield: 197 mg. LC-MS: calculated [M+H] 426.50, found 427.66.

[0298] Synthesis of LP-433-p [ka]

[0299] Compound 1 (Sigma® #L2378, 250 mg) was dissolved in DMF, followed by the addition of TBTU (331 mg) and DIPEA (0.636 mL). The mixture was stirred for 10 min, followed by the addition of compound 2 (286 mg in DMF). The mixture was stirred at room temperature in the dark for 1 h. The mixture was then diluted with EtOAc (70 mL) and washed with 3% citric acid in water (3 × 8 mL), HO (2 × 8 mL), and NaCl (1 × 8 mL), then dried over NaSO, filtered, and concentrated on a rotary evaporator. The crude product was dissolved in 3 mL of DCM, loaded onto a 24 G column, and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield: 420 mg. LC-MS: calculated [M+H] 537.78, found 538.70. [ka]

[0300] Compound 1 (400 mg) was dissolved in 6 mL of 4 M HCl in dioxane at 0° C. The mixture was stirred in the dark for 10 minutes, then warmed to room temperature and stirred for 4 hours. The product was concentrated on a rotary evaporator and placed under high vacuum. The product was dissolved in DCM / toluene, concentrated twice, and then placed under high vacuum overnight. Yield 358 mg. LC-MS: calculated [M+H] 481.67, found 482.37. [ka]

[0301] Compound 1 (358 mg) was dissolved in 7.5 mL of DCM. Tetrafluorophenol (185 mg) was then added. The mixture was stirred for 10 min, and then EDC·-HCl (214 mg) was added. The mixture was stirred for 2 h. The crude reaction was then concentrated in vacuo, suspended in DCM, dry-loaded onto a 12 G column with silica, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 35 min) (yield 373 mg). LC-MS: calculated [M+H] 629.73, found 630.71.

[0302] Synthesis of LP-444-p [ka]

[0303] To a 100 mL round-bottom flask equipped with a stir bar under N2 was added tert-butyl (3-aminophenyl)carbamate (500 mg, 2.4 mmol), sieve-dried DCM (25 mL), and triethylamine (1 mL, 7.2 mmol). The solution was cooled in an ice bath for 10 min, and then palmitoyl chloride (0.8 mL, 2.6 mmol) was added dropwise over 1 min. The reaction was stirred on ice for 15 min, then the ice bath was removed and the reaction was stirred for 18 h while warming to room temperature. Completion of the reaction was confirmed by TLC (ninhydrin stain). The reaction was quenched with 7N NH3 in MeOH (0.5 mL) and stirred for an additional 10 min. The reaction was vacuum filtered, concentrated under reduced pressure, and then dried on a vacuum pump for 2 h to give 1 (1.07 g), which was used without purification. A round-bottom flask containing 1 (1.07 g, 2.39 mmol) was placed under N and cooled in an ice bath for 10 min. Dry DCM (15 mL) and TFA (10 mL) were added sequentially, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 2 h while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure to give crude 2, which was dissolved in DCM (50 mL) and concentrated again. The reaction was dissolved in DCM, washed with NaCO (10% aqueous solution, ×3 washes), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give 2 (853 mg, free base) as a gray solid, which was carried forward without further purification. To a 40 mL vial equipped with a stir bar under N was added 2 (273 mg, 0.784 mmol), sieve-dried DCM (15 mL), DIPEA (0.54 mL, 3.13 mmol), and acid-PEG-2-tert-butyl ester (239 mg, 0.862 mmol). HBTU (327 mg, 0.862 mmol) and DMAP (10 mol%, 10 mg) were added, and the reaction was stirred for 4 h. LCMS indicated the reaction was approximately 90% complete.The reaction was diluted with DCM, washed with HO (×2), saturated NaHCO (×4), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (5 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (MeOH / DCM 0–5%) to give 3 (309 mg) as a tan solid in 67% yield. LC / MS (ESI). + ) m / z calculated value 590.43 (M), observed value 591.83 (M+H + ).

[0304] To a 40 mL vial equipped with a stir bar was added 3 (309 mg, 0.522 mmol) and 4 M HCl in dioxane (6.5 mL, 26.1 mmol). The reaction was tightly capped and stirred for 2.5 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give 4, which was carried forward without purification. Crude 4 (280 mg, 0.523 mmol) was dissolved in sieve-dried DCM (10 mL) in a 40 mL vial equipped with a stir bar under N2. 2,3,5,6-Tetrafluorophenol (96 mg, 0.575 mmol) and triethylamine (0.22 mL, 1.56 mmol) were added, and the resulting solution was cooled in an ice bath for 15 min. COMU (246 mg, 0.575 mmol) was added slowly and the reaction was stirred on ice for 10 minutes. The ice bath was then removed and the reaction was stirred for 3 hours. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (×4), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (3.5 mL), loaded onto a 12 g silica column, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 40 minutes, product eluted with 45% EtOAc) to give LP-444-p (178 mg) as a white solid in 50% yield. LC / MS (ESI + ) m / z calculated 682.36 (M), observed 683.99 (M+H + ).

[0305] Synthesis of LP-445-p

change

[0306] To a 100 mL round-bottom flask equipped with a stir bar under N2 was added tert-butyl (2-aminophenyl)carbamate (500 mg, 2.4 mmol), sieve-dried DCM (25 mL), and triethylamine (1 mL, 7.2 mmol). The solution was cooled in an ice bath for 10 min, and then palmitoyl chloride (0.8 mL, 2.6 mmol) was added dropwise over 1 min. The reaction was stirred on ice for 15 min, then the ice bath was removed and the reaction was stirred for 1 h while warming to room temperature. Completion of the reaction was confirmed by TLC (ninhydrin stain). The reaction was quenched with 7N NH3 in MeOH (0.5 mL) and stirred for an additional 20 min. The reaction was concentrated under reduced pressure and then dried on a vacuum pump for 2 h to give 1 (1.07 g), which was used without purification. A round-bottom flask containing 1 (1.07 g, 2.39 mmol) was placed under N and cooled in an ice bath for 10 min. Dry DCM (15 mL) and TFA (10 mL) were added sequentially, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 1.5 h while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure to give crude 2, which was suspended in DCM (50 mL), concentrated again, and dried on a vacuum pump for 16 h. The crude material was suspended in DCM, washed with NaCO (10% aqueous solution, ×4 washes), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give 2 (807 mg, free base) as a gray solid, which was carried forward without further purification. To a 40 mL vial equipped with a stir bar under N was added 2 (187 mg, 0.539 mmol), sieve-dried DCM (12 mL), DIPEA (0.375 mL, 2.16 mmol), acid-PEG2-tert-butyl ester (165 mg, 0.593 mmol), and DMAP (10 mol%, 6.5 mg, 0.054 mmol). The reaction was cooled in an ice bath for 10 min, then HBTU (226 mg, 0.593 mmol) was added slowly, and the reaction was stirred on ice for 10 min. The ice bath was removed, and the reaction was stirred for 18 h. LCMS confirmed the reaction was complete.The reaction was diluted with DCM, washed with HO (×2), saturated NaHCO (×3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (5 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (MeOH / DCM 0–8% over 60 min) to give 3 (232 mg, oily solid) as a mixture of 3 and a tetramethylurea byproduct. LC / MS (ESI). + ) m / z calculated value 590.43 (M), observed value 591.83 (M+H + ).

[0307] To a 40 mL vial equipped with a stir bar was added 3 (232 mg, 0.392 mmol) and 4 M HCl in dioxane (4.5 mL, 19.6 mmol). The reaction was tightly capped and stirred for 3 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give 4, which was carried forward without purification. Crude 4 (210 mg, 0.393 mmol) was dissolved in sieve-dried DCM (10 mL) in a 40 mL vial equipped with a stir bar under N2. 2,3,5,6-Tetrafluorophenol (72 mg, 0.432 mmol) and triethylamine (0.16 mL, 1.18 mmol) were added, and the resulting solution was cooled in an ice bath for 15 min. COMU (185 mg, 0.432 mmol) was added slowly, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 3 h. The reaction was confirmed to be complete by LCMS. The reaction was diluted with DCM, washed with HO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (4 mL), loaded onto a 12 g silica column, and purified by flash chromatography (EtOAc / hexanes, 0-75% over 50 min, product eluted with 35% EtOAc) to give LP-445-p (157 mg) as a white solid in 59% yield. LC / MS (ESI + ) m / z calculated value 682.36 (M), observed value 684.08 (M+H + ).

[0308] Synthesis of LP-446-p

change

[0309] To a 100 mL round-bottom flask equipped with a stir bar under N2, tert-butyl (4-aminophenyl)carbamate (250 mg, 1.2 mmol), sieve-dried DCM (12 mL), and triethylamine (0.5 mL, 3.6 mmol) were added. The solution was cooled in an ice bath for 10 min, and then palmitoyl chloride (0.4 mL, 1.3 mmol) was added dropwise over 1 min. The reaction was stirred on ice for 15 min, then the ice bath was removed, and the reaction was stirred for 16 h while warming to room temperature. The reaction remained heterogeneous. Completion of the reaction was confirmed by TLC (a sample aliquot was diluted with MeOH to make it more homogeneous before spotting onto the plate, and ninhydrin staining was used for visualization). MeOH (25 mL) was added to quench the reaction, and the reaction was stirred for 1 h. The reaction was concentrated under reduced pressure and then dried on a vacuum pump for 2 h to give 1 (535 mg), which was used without purification. A round-bottom flask containing 1 (535 mg, 1.2 mmol) was placed under N and cooled in an ice bath for 10 min. Dry DCM (5 mL) and TFA (5 mL) were added sequentially, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 2 h while warming to room temperature. The reaction started out heterogeneous and slowly became homogeneous. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure to give crude 2, which was dissolved in DCM (50 mL) and concentrated again, then suspended in PhMe (15 mL), concentrated, and dried on a vacuum pump for 16 h. The reaction was dissolved in DCM, washed with NaCO (10% aqueous solution, ×3 washes), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give 2 (395 mg, free base) as a gray solid, which was carried forward without further purification. To a 40 mL vial equipped with a stir bar under N2 was added 2 (250 mg, 0.721 mmol), sieve-dried DCM (15 mL), DIPEA (0.5 mL, 2.88 mmol), acid-PEG2-tert-butyl ester (220 mg, 0.793 mmol), and DMAP (10 mol%, 9 mg, 0.0721 mmol). The resulting suspension was cooled in an ice bath for 10 min, then HBTU (301 mg, 0.793 mmol) was added slowly, and the reaction was stirred on ice for 10 min. The ice bath was removed, and the reaction was stirred for 18 h.The reaction became homogeneous after 1 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM and washed with HO (×2), saturated NaHCO (×5, a slight emulsion was observed), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (7 mL, sonication required), loaded onto a 24 g silica gel column, and purified by flash chromatography (MeOH / DCM 0–3% over 40 min) to give 3 (163 mg) as a white solid in 38% yield. LC / MS (ESI). + ) m / z calculated value 590.43 (M), observed value 591.83 (M+H + ).

[0310] To a 40 mL vial equipped with a stir bar was added 3 (163 mg, 0.275 mmol) and 4 M HCl in dioxane (4 mL, 13.8 mmol). The reaction was tightly capped and stirred for 3 h. The reaction remained a heterogeneous suspension. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give 4, which was carried forward without purification. Crude 4 (147 mg, 0.274 mmol) was dissolved in sieve-dried DCM (10 mL) in a 40 mL vial equipped with a stir bar under N2. To the suspension were added 2,3,5,6-tetrafluorophenol (50 mg, 0.302 mmol), triethylamine (0.11 mL, 0.822 mmol), and COMU (129 mg, 0.302 mmol) sequentially, and the reaction was stirred for 2 h. The reaction remained heterogeneous. The reaction was confirmed to be complete by LCMS. The reaction was diluted with DCM, washed with HO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (20 mL), dry-loaded onto a 12 g silica gel column with silica, and purified by flash chromatography (EtOAc / hexanes, 0-75%, product eluted with 55% EtOAc) to give LP-446-p (39 mg) as a white solid in 20% yield. LC / MS (ESI + ) m / z calculated value 682.36 (M), observed value 684.08 (M+H + ).

[0311] Synthesis of LP-447-p [ka]

[0312] To a 40 mL vial equipped with a stir bar under N2, stearic acid (100 mg, 0.352 mmol) and sieve-dried DCM (3 mL) were added. DIPEA (0.18 mL, 1.06 mmol), amine-PEG 3-tert-butyl ester (117 mg, 0.421 mmol), and HBTU (162 mg, 0.427 mmol) were added sequentially, and the reaction was stirred for 3 h. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO, saturated NaHCO3 (x3), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (0–75% EtOAc / hexanes, product eluted with 50% EtOAc) to give 1 (140 mg) as a white solid in 74% yield. A 40 mL vial already containing 1 (140 mg, 0.257 mmol) was stirred in 4 M HCl in dioxane (3 mL) for 2.5 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give 2 (125 mg, 0.256 mmol) as a white solid, which was placed under N2 and dissolved in sieve-dried DCM (3 mL). Triethylamine (0.1 mL, 0.768 mmol), 2,3,5,6-tetrafluorophenol (47 mg, 0.281 mmol), and COMU (120 mg, 0.281 mmol) were added sequentially, and the reaction was stirred for 2.5 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (x3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (2.5 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0-75% over 40 min, product eluted at 50% EtOAc) to give LP-447-p (90 mg) as a colorless oil in 56% yield. LC / MS (ESI) + ) m / z calculated value 635.38 (M), observed value 636.56 (M+H + ).

[0313] Synthesis of LP-453-p [ka]

[0314] Compound 1 (palmitic acid, 400 mg) was dissolved in 9 mL of DMF. TBTU (551 mg) and DIPEA (1.104 mL) were then added, and the mixture was stirred for 10 min. Compound 2 (368 mg in DMF) was then added, and the mixture was stirred for 1 h. The mixture was then diluted with 125 mL of EtOAc, washed with 3% aqueous citric acid (3 × 10 mL), HO (2 × 10 mL), and NaCl (1 × 10 mL), dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was loaded onto a 24 G column in 3 mL of DCM and purified by flash chromatography (MeOH / DCM, 0–5% over 30 min). Yield: 555 mg. LC-MS: calculated [M+H] 452.72, found 453.85. [ka]

[0315] Compound 1 (555 mg) was dissolved in 8 mL of 4 M HCl in dioxane. The mixture was stirred for 2 hours. The product was concentrated on a rotary evaporator and placed under high vacuum, then redissolved in DCM / toluene, concentrated twice, and placed under high vacuum overnight. Yield 432 mg. LC-MS: calculated [M+H] 352.61, found 353.49. [ka]

[0316] Compound 1 (432 mg) was dissolved in 10 mL of DMF. TBTU (452 ​​mg) and DIPEA (0.867 mL) were then added, and the mixture was stirred for 10 min. Compound 2 (370 mg in DMF) was then added, and the mixture was stirred for 1 h. The mixture was diluted with 150 mL of EtOAc and washed with 3% citric acid in water (3 × 13 mL), HO (2 × 13 mL), NaHCO (2 × 13 mL), and NaCl (1 × 13 mL), then dried over NaSO, filtered, and concentrated on a rotary evaporator. The crude product was dissolved in 3 mL of DCM, loaded onto a 24G column, and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield: 390 mg. LC-MS: calculated [M+H] 596.89, found 597.86. [ka]

[0317] Compound 1 (400 mg) was dissolved in 6 mL of 4 M HCl in dioxane. The mixture was stirred for 2 hours. The product was then concentrated on a rotary evaporator and placed under high vacuum. The product was redissolved in DCM / toluene, concentrated twice, and placed under high vacuum overnight. Yield 390 mg. LC-MS: calculated [M+H] 540.79, found 541.79. [ka]

[0318] Compound 1 (362 mg) was dissolved in 7 mL of DCM. Tetrafluorophenol (167 mg) was then added, and the mixture was stirred for 10 min. EDC·HCl (193 mg) was then added. The mixture was stirred for 2 h. The crude reaction was concentrated, suspended in DCM, dry-loaded onto a 12 G column with silica, and purified by flash chromatography (EtOAc / hexane, 0–100% over 40 min) (yield 127 mg). LC-MS: calculated [M+H] 688.85, found 689.93.

[0319] Synthesis of LP-455-p [ka]

[0320] Compound 2 (61 mg) was dissolved in 2 mL of DMF, followed by the addition of TBTU (64 mg) and DIPEA (0.122 mL), and the mixture was stirred for 10 min. Compound 1 (50 mg in DMF) was then added. The flask was covered with foil, and the mixture was stirred for 1 h. The mixture was then diluted with EtOAc (25 mL) and washed with 3% aqueous citric acid (3 × 3 mL), HO (2 × 3 mL), sodium bicarbonate (1 × 3 mL), and NaCl (1 × 3 mL), then dried over NaSO, filtered, and concentrated on a rotary evaporator. The crude product was taken up in 1 mL of DCM, loaded onto a 4G column, and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min) (yield 64 mg). LC-MS: calculated [M+H] 577.85, found 578.87. [ka]

[0321] Compound 1 (64 mg) was dissolved in 2 mL of 4 M HCl in dioxane. The mixture was stirred in the dark for 2 hours. The product was concentrated on a rotary evaporator and placed under high vacuum, then dissolved and concentrated twice with DCM / toluene and placed under high vacuum overnight. Yield 59 mg. LC-MS: calculated [M+H] 521.74, found 522.71. [ka]

[0322] Compound 1 (57 mg) was dissolved in 3 mL of DCM. Tetrafluorophenol (27 mg) was then added, and the mixture was stirred for 10 min. EDC·HCl (31 mg) was then added, and the mixture was stirred for 2 h. The reaction mixture was concentrated, dissolved in 4 mL of DCM, dry-loaded onto a 4G column with silica, and purified by flash chromatography (EtOAc / hexane, 0–100% over 35 min) (yield 59 mg). Yield LC-MS: calculated [M+H] 669.80, found 670.71.

[0323] Synthesis of LP-457-p [ka]

[0324] Arachidonic acid (142 mg, 0.466 mmol) and sieve-dried DCM (10 mL) were added to a 40 mL vial equipped with a stir bar under N2. Triethylamine (0.32 mL, 2.33 mmol), amine-PEG5-tert-butyl ester (187 mg, 0.513 mmol), and COMU (219 mg, 0.513 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 2 h. Completion of the reaction was confirmed by TLc (KMnO4 staining). The reaction was diluted with DCM, washed with HO (×3), saturated NaHCO (×2), brine, dried over MgSO, filtered, and concentrated under reduced pressure (protecting the product from light) to give the crude product, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 20 min, product eluted with 80% EtOAc) to give 1 (172 mg) as a white solid in 57% yield. The 40 mL vial already containing 1 (172 mg, 0.264 mmol) was wrapped in foil and stirred in 4 M HCl in dioxane (5 mL) for 3 h. The reaction was complete by LCMS. The reaction was concentrated under reduced pressure (protecting the sample from light) and dried on a vacuum pump for 16 hours to give 2 (157 mg, 0.264 mmol) as a white solid, which was placed under N2 and dissolved in sieve-dried DCM (10 mL). Triethylamine (0.18 mL, 1.32 mmol) and 2,3,5,6-tetrafluorophenol (48 mg, 0.289 mmol) were added, and the reaction was cooled in an ice bath for 10 minutes. COMU (124 mg, 0.289 mmol) was added, and the reaction was stirred on ice for 15 minutes. The ice bath was then removed, the reaction was wrapped in foil, and stirred for 2 hours while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (×3, a large emulsion formed which became clear after 20 min), brine, dried over MgSO, filtered, and concentrated under reduced pressure (protect the product from light) to give the crude material, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 30 min, product eluted with 80% EtOAc) to give LP-457-p (56 mg) as a yellow oil in 29% yield.LC / MS (ESI). + ) m / z calculated value 743.40 (M), observed value 745.20 (M+H + ).

[0325] Synthesis of LP-458-p [ka]

[0326] Arachidonic acid (150 mg, 0.492 mmol) and sieve-dried DCM (10 mL) were added to a 40 mL vial equipped with a stir bar under N2. Triethylamine (0.34 mL, 2.46 mmol), amine-PEG10-tert-butyl ester (317 mg, 0.541 mmol), and COMU (232 mg, 0.541 mmol) were added sequentially, and the reaction was wrapped in foil and stirred for 5 h. Completion of the reaction was confirmed by TLC (b-cresol green and KMnO4 staining). The reaction was concentrated to 5 mL (shielded from light), loaded onto a 24 g silica gel column, and purified by flash chromatography (0–4% MeOH / DCM over 40 min, product eluted with 2% MeOH) to give 1 (254 mg) as a colorless oil in 60% yield. A 40 mL vial already containing 1 (254 mg, 0.291 mmol) was wrapped in foil and stirred in 4 M HCl in dioxane (7 mL) for 3 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure (protecting the product from light) and dried on a vacuum pump for 16 h to give 2 (237 mg, 0.29 mmol) as a white solid, which was placed under N2 and dissolved in sieve-dried DCM (8 mL). Triethylamine (0.16 mL, 1.16 mmol), 2,3,5,6-tetrafluorophenol (58 mg, 0.348 mmol), and COMU (150 mg, 0.348 mmol) were added, and the reaction was wrapped in foil and stirred for 2 h. The reaction was not complete after 2 hours, so additional 2,3,5,6-tetrafluorophenol (0.5 equiv.) and COMU (0.5 equiv.) were added, and the reaction was stirred for an additional hour. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure (protecting the product from light) to give the crude material, which was dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (MeOH / DCM, 0-4% over 50 minutes, product eluted with 3% MeOH) to give LP-458-p (63 mg) as an oil in 23% yield. LC / MS (ESI + ) m / z calculated value 963.53 (M), observed value 965.34 (M+H + ).

[0327] Synthesis of LP-459-p [ka]

[0328] To a 40 mL vial equipped with a stir bar under N2, stearic acid (300 mg, 1.05 mmol), sieve-dried DCM (15 mL), DIPEA (0.73 mL, 4.2 mmol), and HBTU (441 mg, 1.16 mmol) were added. The reaction was stirred for 2 min, then N-(2-aminospiro[3.3]hept-6-yl)carbamic acid tert-butyl ester (262 mg, 1.16 mmol) was added, and the reaction was stirred for 1.5 h. TLC (b-cresol green stain) confirmed the reaction was complete. The reaction was diluted with DCM (50 mL) and washed with saturated NaHCO3 (x4) and brine. The organic phase remained heterogeneous, while the aqueous phase was homogeneous during workup. After washing, the organic layer was diluted with MeOH until homogeneous, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM / MeOH (5:1), dry-loaded with silica gel onto a 24 g silica gel column, and purified by flash chromatography (MeOH / DCM, 0-15% over 60 min, ELS detection, product eluted with 2.5% MeOH) to give 1 (227 mg) as a white solid in 44% yield. LC / MS (ESI) + ) m / z calculated value 492.43 (M), observed value 493.90 (M+H + ).

[0329] To a 40 mL vial containing 1 (227 mg, 0.461 mmol) was added a stir bar and 4 M HCl in dioxane (5 mL). The vial was tightly capped and stirred for 2 h. The reaction remained heterogeneous. An aliquot was taken, diluted with MeOH / DCM (1:1), and analyzed by LCMS to confirm completion. The reaction was concentrated under reduced pressure and dried overnight to give 2 (197 mg) as a white solid HCl salt in quantitative yield. Intermediate 2 (197 mg, 0.459 mmol) was suspended in sieve-dried DCM (15 mL), and triethylamine (0.32 mL, 2.3 mmol) and acid-PEG 2-tert-butyl ester (140 mg, 0.504 mmol) were added. The reaction was cooled in an ice bath for 10 min, then COMU (216 mg, 0.504 mmol) was added, and the reaction was stirred on ice for 10 min. The ice bath was then removed and the reaction was stirred for 3 hours while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (×4), saturated NaHCO (×3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (5 mL, sonication required), loaded onto a 24 g silica column, and purified by flash chromatography (MeOH / DCM 0–7% over 60 minutes, ELS detection, product eluted with 3.75% MeOH) to give 3 (195 mg) as a white solid in 67% yield. LC / MS (ESI + ) m / z calculated 636.51 (M), observed 638.18 (M+H + ).

[0330] Intermediate 3 (195 mg, 0.306 mmol) was stirred in 4 M HCl in dioxane (3.5 mL) in a sealed vial for 2 hours. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and dried overnight to give 4 (178 mg) as a white solid in quantitative yield. The vial containing 4 (178 mg, 0.306 mmol) was placed under N2, a stir bar was added, and triethylamine (0.21 mL, 1.53 mmol) and 2,3,5,6-tetrafluorophenol (56 mg, 0.337 mmol) were added. The reaction was cooled in an ice bath for 10 minutes, then COMU (144 mg, 0.337 mmol) was added, and the reaction was stirred on ice for 10 minutes. The ice bath was then removed, and the reaction was stirred for 3 hours while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was diluted with DCM, washed with HO (x3), dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (3.5 mL), loaded onto a 12 g silica column, and purified by flash chromatography (EtOAc / hexanes, 0-100%, 30 min, ELS detection, product eluted with 100% EtOAc) to give LP-459-p (82 mg) as a white solid in 37% yield. LC / MS (ESI) + ) m / z calculated value 728.44 (M), observed value 730.17 (M+H + ).

[0331] Synthesis of LP-460-p [ka]

[0332] To a 40 mL vial equipped with a stir bar, under N2, were added arachidic acid (300 mg, 0.959 mmol), sieve-dried DCM (15 mL), triethylamine (0.53 mL, 3.83 mmol), and N-(2-aminospiro[3.3]hept-6-yl)carbamic acid tert-butyl ester (238 mg, 1.05 mmol). COMU (450 mg, 1.05 mmol) was added in one portion, and the reaction was stirred for 3 h. TLC (b-cresol green staining) confirmed the reaction was complete. The reaction was diluted with 10% MeOH / DCM and washed with HO (x3) and saturated NaHCO3 (x4). A large emulsion developed during the washes, which required 20 min to dissolve per wash. The pooled organic layer was diluted with MeOH, but never became homogeneous. The reaction was not dried over MgSO4, but rather directly concentrated under reduced pressure, azeotroped with PhMe (10 mL), and then dried on a vacuum pump for 14 h. The crude material was suspended in DCM / MeOH (5:1), dry-loaded onto a 40 g silica gel column with silica, and purified by flash chromatography (MeOH / DCM 0–15% over 60 min, ELS detection; product eluted at approximately 3% MeOH) to give 1 (250 mg) as a white solid in 50% yield. LC / MS (ESI) + ) m / z calculated value 520.46 (M), observed value 521.90 (M+H + ).

[0333] To a 40 mL vial containing 1 (250 mg, 0.479 mmol) was added a stir bar and 4 M HCl in dioxane (5 mL). The vial was tightly capped and stirred for 2 h. The reaction remained heterogeneous. An aliquot was taken, diluted with MeOH / DCM (1:1), and analyzed by LCMS to confirm completion. The reaction was concentrated under reduced pressure and dried overnight to give 2 (219 mg) as a white solid HCl salt in quantitative yield. Intermediate 2 (219 mg, 0.479 mmol) was suspended in sieve-dried DCM (10 mL) and triethylamine (0.33 mL, 2.4 mmol) and acid-PEG 2-tert-butyl ester (146 mg, 0.526 mmol) were added. The reaction was cooled in an ice bath for 10 min, then COMU (225 mg, 0.526 mmol) was added and the reaction was stirred on ice for 10 min. The ice bath was then removed and the reaction was stirred for 2 hours while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x2), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (10 mL, sonication required), loaded onto a 24 g silica column, and purified by flash chromatography (MeOH / DCM 0-10%, ELS detection) to give Intermediate 3 (238 mg) as a white solid in 75% yield. LC / MS (ESI + ) m / z calculated 664.54 (M), observed 666.26 (M+H + ).

[0334] Intermediate 3 (238 mg, 0.358 mmol) was stirred in a sealed vial in 4 M HCl in dioxane (7 mL) for 2.5 h. The reaction started homogeneously but became heterogeneous after 30 min. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and dried overnight to give 4 (218 mg) as a white solid in quantitative yield. The vial containing 4 (218 mg, 0.358 mmol) was placed under N2, a stir bar was added, and triethylamine (0.25 mL, 1.79 mmol) and 2,3,5,6-tetrafluorophenol (65 mg, 0.394 mmol) were added. The reaction was cooled in an ice bath for 10 min, then COMU (169 mg, 0.394 mmol) was added, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 2 h while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was diluted with DCM, washed with HO (x3), dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (4 mL), loaded onto a 12 g silica column, and purified by flash chromatography (MeOH / DCM 0-3% over 35 min, ELS detection, product eluted with 3% MeOH) to give LP-460-p (81 mg) as a white solid in 30% yield. LC / MS (ESI) + ) m / z calculated value 756.47 (M), observed value 758.16 (M+H + ).

[0335] Synthesis of LP-461-p [ka]

[0336] To a 40 mL vial equipped with a stir bar under N2, 16-(tert-butoxy)-16-oxohexadecanoic acid (300 mg, 0.875 mmol), amine-PEG3-methyl ester HCl salt (219 mg, 0.963 mmol), sieve-dried DCM (10 mL), and triethylamine (0.61 mL, 4.38 mmol) were added. The stirred solution was cooled in an ice bath for 10 min, then COMU (412 mg, 0.963 mmol) was added, and the reaction was stirred on ice for 10 min. The ice bath was then removed, and the reaction was stirred for 2 h while warming to room temperature. Completion of the reaction was confirmed by LCMS and TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO (x3), saturated NaHCO (x2), dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (5 mL), loaded onto a 24 g silica column, and purified by flash chromatography (0-100% EtOAc / hexanes over 50 min, ELS detection, product eluted at 60% EtOAc) to give 1 (309 mg) as an oily, colorless solid in 69% yield. LC / MS (ESI) + ) m / z calculated value 515.38 (M), observed value 516.86 (M+H + ).

[0337] To a 40 mL vial equipped with a stir bar was added 1 (100 mg, 0.194 mmol), MeOH (0.97 mL), and 0.5 M NaOH (0.97 mL, 0.485 mmol). The vial was tightly capped and stirred for 2.5 h. The reaction started out heterogeneous and slowly became homogeneous. Completion of the reaction was confirmed by LCMS. The reaction was diluted with HO (7 mL), acidified with 1 M HCl until the pH was 3, and extracted with DCM (15 mL × 3). The pooled organic extracts were washed with brine, dried over MgSO, filtered, and concentrated under reduced pressure in a 40 mL vial to give 2 (75 mg) as an oily solid, which was placed under N and dissolved in sieve-dried DCM (5 mL). To this solution was added a stir bar, triethylamine (80 μL, 0.596 mmol), and 2,3,5,6-tetrafluorophenol (30 mg, 0.179 mmol). COMU (77 mg, 0.179 mmol) was added, and the reaction was stirred for 3 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure, dried on a vacuum pump for 3 h, dissolved in DCM (3 mL), loaded onto a 12 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 40 min, ELS detection; product eluted with 45% EtOAc) to give 3 (40 mg) as a colorless oil in 41% yield. To an oven-dried 40 mL vial equipped with a stir bar under N2 was added 3 (35 mg, 0.0538 mmol), sieve-dried dioxane (0.5 mL), and 4 M HCl in dioxane (1.35 mL, 5.38 mmol). The reaction was tightly capped and stirred for 5 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated at 28 °C and dried on a vacuum pump for 16 h to give LP-461-p (30 mg). LC / MS (ESI + ) m / z calculated value 593.30 (M), observed value 594.98 (M+H + ).

[0338] Synthesis of LP-468-p [ka]

[0339] To a 40 mL vial equipped with a stir bar under N2, arachidic acid (300 mg, 0.959 mmol), sieve-dried DCM (15 mL), and DIPEA (0.66 mL, 3.83 mmol) were added. HBTU (399 mg, 1.05 mmol) was added, and the reaction was stirred for 10 minutes. Amine-PEG3-tert-butyl ester (292 mg, 1.05 mmol) was added as a DCM solution (5 mL), and the reaction was stirred for 3.5 hours. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with saturated NaHCO3 (x4), dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (6 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (0–75% EtOAc / hexanes over 50 min, ELS detection; product eluted with 50% EtOAc) to give 1 (452 ​​mg) as a white oily foam in 83% yield. A 40 mL vial already containing 1 (450 mg, 0.786 mmol) was stirred in 4 M HCl in dioxane (6 mL) for 2.5 h (the vial was tightly capped). LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give 2 (405 mg). Intermediate 2 (405 mg, 0.78 mmol) was dissolved in sieve-dried DCM (15 mL), a stir bar was added, and the column was placed under N2. Triethylamine (0.43 mL, 3.12 mmol), 2,3,5,6-tetrafluorophenol (156 mg, 0.94 mmol), and COMU (402 mg, 0.94 mmol) were added sequentially, and the reaction was stirred for 2.5 h. After this period, the reaction was not complete (LCMS), so additional 2,3,5,6-tetrafluorophenol (0.6 equiv.) was added, and the reaction was stirred for an additional 1.5 h. The reaction was confirmed to be complete by LCMS. The reaction was diluted with DCM, washed with HO (×3), dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (6 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–100% over 45 min, ELS detection).The cleanest fractions were pooled and concentrated under reduced pressure to give LP-468-p (187 mg) as a colorless oil in 36% yield. LC / MS (ESI). + ) m / z calculated value 663.41 (M), observed value 665.09 (M+H + ).

[0340] Synthesis of Lp-469 phosphoramidite [ka]

[0341] Compound 1 (Asta Tech® #W15452, 500 mg) was dissolved in 11 mL of DMF. TBTU (606 mg) and DIPEA (1.162 mL) were then added, and the mixture was stirred in the dark for 10 min. Compound 2 (365 mg in DMF) was then added, and the mixture was stirred for 1 h. The mixture was diluted with EtOAc (140 mL), washed with 3% aqueous citric acid (3 × 15 mL), HO (2 × 15 mL), and NaCl (1 × 15 mL), then dried over NaSO, filtered, concentrated on a rotary evaporator, and placed under high vacuum. The crude product was dissolved in 4 mL of DCM, loaded onto a 24 G column, and purified by flash chromatography (MeOH / DCM, 0–4% over 30 min). Yield: 615 mg. LC-MS: calculated [M+H] 479.70, found 480.85. [ka]

[0342] Compound 1 (613 mg) was added to a 100 mL round-bottom flask containing sieves and purged with N2. Then, 12 mL of DCM was added, followed by DIPEA (0.678 mL). The mixture was cooled to 0 °C. Compound 2 (0.356 mL) was added dropwise via syringe, and the mixture was stirred on ice for 10 min. The ice bath was then removed, and the reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was filtered through Celite® and concentrated. The crude product was dry-loaded onto a 24 G column with Celite and purified by flash chromatography (EtOAc / hexanes with 1% triethylamine, 0-75% over 35 min). Yield: 311 mg. 1H NMR(400MHz,CD2Cl2)δ 6.09(t,1H),5.45-5.32(m,8H),3.85--3.8(m,3H),3.70(m,1H),3.60(d,12H),3.52(t,2H),3.40(t,2H),2.90--2.80 (dd,6H),2.65(t,2H),2.18(t,2H),2.16-2.04(m,4H),1.70(m,2H),1.40--1.25(m,6H),1.20(dd,12H),0.80(t,3H). 31P NMR (400MHz, CD2Cl2) δ 148.458 (s, 1P).

[0343] Synthesis of LP-470 phosphoramidite [ka]

[0344] Compound 1 (Asta Tech® #W15452, 250 mg) was dissolved in 10 mL of DMF. TBTU (303 mg) and DIPEA (0.581 mL) were then added. The mixture was stirred in the dark for 10 minutes. Compound 2 (473 mg in DMF) was then added, and the reaction was stirred in the dark for 1 hour. The reaction was concentrated on a rotary evaporator and placed under high vacuum. The crude material was dissolved in 4 mL of DCM, loaded onto a 24G column, and purified by flash chromatography (MeOH / DCM, 0-5% over 30 minutes). Yield 462 mg. LC-MS: calculated [M+H] 788.07, found 789.30. [ka]

[0345] Compound 1 (462 mg) was added to a 100 mL round-bottom flask containing sieves and purged with N2. Then, 7 mL of DCM was added, followed by DIPEA (0.311 mL), and the mixture was cooled to 0 °C. Compound 2 (0.163 mL) was then added dropwise via syringe. The reaction mixture was stirred on ice for 10 min, then the ice bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred overnight. The crude reaction was filtered through Celite® and concentrated. The crude product was dry-loaded onto a 12G column with Celite and purified by flash chromatography (EtOAc / hexanes, 0-100% over 30 min, hold at 100% EtOAc for 15 min). Yield: 166mg 1H NMR(400MHz,CD2Cl2)δ 6.09(t,1H),5.45-5.32(m,8H),3.85--3.8(m,3H),3.70(m,1H),3.60(d,40H),3.52(t,2H),3.40(t,2H),2.90--2.80 (dd,6H),2.65(t,2H),2.18(t,2H),2.16-2.04(m,4H),1.70(m,2H),1.40--1.25(m,6H),1.20(dd,12H),0.80(t,3H). 31P NMR (400MHz, CD2Cl2) δ 148.458 (s, 1P).

[0346] Synthesis of LP-473-p [ka]

[0347] To a 40 mL vial equipped with a stir bar under N2, myristic acid (500 mg, 2.19 mmol), sieve-dried DCM (15 mL), and DIPEA (1.5 mL, 8.76 mmol) were added. HBTU (915 mg, 2.41 mmol) was added, and the reaction was stirred for 10 minutes. Amine-PEG2-tert-butyl ester (560 mg, 2.41 mmol) was added as a DCM solution (5 mL), and the reaction was stirred for 2.5 hours. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO (×2), saturated NaHCO (×3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (10 mL), loaded onto a 40 g silica gel column, and purified by flash chromatography (MeOH / DCM 0–5%, ELS detection) to give 1 (1.17 g, oily foam) as a mixture of 1 and the tetramethylurea byproduct. To an oven-dried 100 mL round-bottom flask was added 1 (1.17 g), a stir bar, and 4 M HCl in dioxane (16.4 mL). The reaction was sealed with a septum and stirred for 3 h. LCMS confirmed the reaction was complete. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give crude 2 (846 mg, theoretical maximum). In a 100 mL round-bottom flask, 2 (846 mg, 2.19 mmol, theoretical maximum) was dissolved in sieve-dried DCM (20 mL), a stir bar was added, and the flask was placed under N2. Triethylamine (1.2 mL, 8.76 mmol) and 2,3,5,6-tetrafluorophenol (437 mg, 2.62 mmol) were added, and the reaction was cooled in an ice bath for 10 min. COMU (1.12 g, 2.62 mmol) was added in portions, and the reaction was stirred on ice for 30 min. The ice bath was removed, and the reaction was stirred for 2 h while warming to room temperature. LCMS confirmed the reaction was complete. The reaction was diluted with DCM, washed with HO (×3, 20 mL), dried over MgSO, filtered, and concentrated under reduced pressure to give the crude material, which was dissolved in DCM (10 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0–100%, 40 min, ELS detection) to give LP-473-p (558 mg) as a yellow solid. LC / MS (ESI+ ) m / z calculated value 535.29 (M), observed value 536.93 (M+H + ).

[0348] Synthesis of LP-474-p [ka]

[0349] To a 40 mL vial equipped with a stir bar under N2, myristic acid (562 mg, 2.46 mmol), sieve-dried DCM (15 mL), and DIPEA (1.7 mL, 9.84 mmol) were added. HBTU (1.12 g, 2.95 mmol) was added and the reaction was stirred for 5 minutes. Amine-PEG2-NHBoc (675 mg, 2.95 mmol) was added as a solution in DCM (5 mL) and the reaction was stirred for 3 hours. Completion of the reaction was confirmed by TLC (b-cresol green staining). The reaction was diluted with DCM, washed with HO (×2), saturated NaHCO (×3), brine, dried over MgSO, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (6 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (0–85% EtOAc / hexanes over 45 min, ELS detection) to give 1 (916 mg) as a white solid in 82% yield. LC / MS (ESI) + ) m / z calculated value 458.37 (M), observed value 459.79 (M+H + ).

[0350] To a round-bottom flask already containing 1 (916 mg, 1.99 mmol) was added a stir bar and 4 M HCl in dioxane (12.5 mL). The reaction was sealed with a septum and stirred for 2.5 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 16 h to give crude 2 (789 mg, theoretical maximum), which was dissolved in sieve-dried DCM (20 mL), a stir bar was added, and the reaction was placed under N2. DIPEA (1 mL, 5.97 mmol) was added and the reaction was stirred for 10 min. 4-tert-Butoxycarbonyl)benzoic acid (529 mg, 2.38 mmol) and HBTU (904 mg, 2.38 mmol) were added sequentially and the reaction was stirred for 4 h. Completion of the reaction was confirmed by LCMS. The reaction was diluted with DCM, washed with saturated NaHCO3 (x4), brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give the crude product, which was dissolved in DCM (7 mL), loaded onto a 24 g silica gel column, and purified by flash chromatography (0-100% EtOAc / hexanes over 45 min, ELS detection, product eluted at 70% EtOAc) to give 3 (1 g) as a white solid in 89% yield. LC / MS (ESI) + ) m / z calculated value 562.40 (M), observed value 564.02 (M+H + ).

[0351] To a round-bottom flask already containing 3 (1 g, 1.77 mmol), a stir bar and 4 M HCl in dioxane (17 mL) were added. The reaction was sealed with a septum and stirred for 4 h. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 4 h to give crude 4 (900 mg, theoretical maximum), which was dissolved in sieve-dried DCM (20 mL), a stir bar was added, and the mixture was placed under N2. Triethylamine (0.98 mL, 7.08 mmol) and 2,3,5,6-tetrafluorophenol (440 mg, 2.65 mmol) were added, and the reaction was cooled in an ice bath for 10 min. COMU (1.13 g, 2.65 mmol) was added in portions, and the reaction was stirred on ice for 10 min. The ice bath was removed, and the reaction was stirred for 2.5 h while warming to room temperature. Completion of the reaction was confirmed by LCMS. The reaction was concentrated under reduced pressure and dried on a vacuum pump for 2 hours. The crude material was dissolved in DCM (8 mL), loaded onto a 40 g silica gel column, and purified by flash chromatography (EtOAc / hexanes, 0-100% over 50 min, ELS detection) to give LP-474-p (1.3 g, a mixture of product and urea by-product) as an orange solid, which was not further purified. LC / MS (ESI) + ) m / z calculated value 654.33 (M), observed value 656.00 (M+H + ).

[0352] Synthesis of CNR1 SM2-p [ka]

[0353] Compound 1 (Asta Tech® #W15452, 915 mg) was dissolved in 20 mL of DMF. TBTU (1.1 g) and DIPEA (2.1 mL) were then added, and the mixture was stirred for 10 minutes. Compound 2 (Asta Tech #F11105, 677 mg) was then added, and the reaction was covered with foil and stirred for 1 hour. The reaction was then diluted with 200 mL of EtOAc and washed with 3% aqueous citric acid (3 × 25 mL), HO (2 × 25 mL), and NaCl (1 × 25 mL), then dried over NaSO, filtered, and concentrated on a rotary evaporator. The crude product was dissolved in 5 mL of DCM, loaded onto a 40 G column, and purified by flash chromatography (MeOH / DCM, 0–3% over 30 minutes). Yield: 977 mg. LC-MS: calculated [M+H] 447.66, found 448.87. [ka]

[0354] Compound 1 (977 mg) was dissolved in 12 mL of 4 M HCl in dioxane at 0° C. and stirred for 10 minutes. The flask was then covered with foil, and the reaction mixture was allowed to warm to room temperature and stirred for 5 hours. The product was concentrated on a rotary evaporator and placed under high vacuum, then twice dissolved in DCM / toluene, concentrated, and placed under high vacuum overnight. Yield 879 mg. LC-MS: calculated [M+H] 391.55, found 392.83. [ka]

[0355] Compound 1 (854 mg) was dissolved in 20 mL of DCM. NHS (Sigma® #130672, 251 mg) was then added, and the mixture was stirred for 10 minutes. EDC·HCl (Sigma® #E7750, 418 mg) was then added, and the reaction mixture was stirred in the dark for 4 hours. The reaction mixture was diluted with 90 mL of DCM and washed with 3% aqueous citric acid (2 × 10 mL), HO (1 × 10 mL), and then NaCl (1 × 10 mL), then dried over NaSO, filtered, and concentrated (yield 970 mg). The product was used without further purification. LC-MS: calculated [M+H] 488.63, found 489.79.

[0356] Example 3. Conjugation of lipid PK / PD modulator precursors One or more lipid PK / PD modulator precursors can be linked to the oligonucleotide-based agent either before or after annealing one or more targeting ligands, and either before or after conjugation. The following describes the general conjugation process used to link lipid PK / PD modulator precursors to the RNAi agent constructs described in the examples presented herein.

[0357] A. Conjugation of Activated Ester PK / PD Modulators

[0358] Using the following procedure, PK / PD modulators bearing activated ester moieties, such as TFP (tetrafluorophenoxy) or PNP (para-nitrophenol), were conjugated to RNAi agents bearing amine-functionalized sense strands, such as C6-NH2, NH2-C6, or (NH2-C6). The annealed RNAi agents, dried by lyophilization, were dissolved in DMSO and 10% water (v / v%) at 25 mg / mL. Then, 50–100 equivalents of TEA and 3 equivalents of activated ester PK / PD modulator were added to the solution. The solution was allowed to react for 1–2 hours, during which time it was monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on a Waters™ XBridge C18 column, Waters Corp.).

[0359] The product was then precipitated by adding 12 mL of acetonitrile and 0.4 mL of PBS, and the solid was centrifuged to pellet. The pellet was then redissolved in 0.4 mL of 1×PBS and 12 mL of acetonitrile. The resulting pellet was dried under high vacuum for 1 hour.

[0360] B. Conjugation of Maleimide-Containing Lipid PK / PD Modulator Precursors

[0361] The following describes a general process used to link maleimide-containing lipid PK / PD modulator precursors to (C6-SS-C6) or (6-SS-6) functionalized sense strands of RNAi agents by dithiothreitol reduction of the disulfide followed by thiol-Michael addition of the respective maleimide-containing lipid PK / PD modulator precursors. In a vial, the functionalized sense strand was dissolved in sterile water at 50 mg / mL. Then, 20 equivalents each of 0.1 M Hepes pH 8.5 buffer and dithiothreitol were added. The mixture was allowed to react for 1 hour, and then the conjugate was precipitated in acetonitrile and PBS, and the solid was centrifuged to pellet.

[0362] The pellet was placed in a 70 / 30 mixture of DMSO / water at a solid concentration of 30 mg / mL. Then, 1.5 equivalents of maleimide-containing lipid PK / PD modulator precursor was added. The mixture was allowed to react for 30 minutes. The product was purified by AEX-HPLC (mobile phase A: 25 mM TRIS pH = 7.2, 1 mM EDTA, 50% acetonitrile; mobile phase B: 25 mM TRIS pH = 7.2, 1 mM EDTA, 500 mM NaBr, 50% acetonitrile; solid phase TSKgel-30, 1.5 cm x 10 cm). The solvent was removed by rotary evaporation and desalted on a 3K spin column using 2 x 10 mL exchanges with sterile water. The solid product was dried using lyophilization and stored for later use.

[0363] C. Conjugation of Sulfone-Containing Lipid PK / PD Modulator Precursors

[0364] The functionalized sense strand was dissolved in sterile water at 50 mg / mL in a vial. 20 equivalents each of 0.1 M Hepes pH 8.5 buffer and dithiothreitol were then added. The mixture was allowed to react for 1 hour, after which the conjugate was precipitated in acetonitrile and PBS, and the solid was centrifuged to pellet.

[0365] The pellet was placed in a 70 / 30 mixture of DMSO / water at a solid concentration of 30 mg / mL. Then, 1.5 equivalents of sulfone-containing lipid PK / PD modulator precursor was added. The vial was purged with N2 and heated to 40 °C with stirring. The mixture was allowed to react for 1 hour. The product was purified by AEX-HPLC (mobile phase A: 25 mM TRIS pH = 7.2, 1 mM EDTA, 50% acetonitrile; mobile phase B: 25 mM TRIS pH = 7.2, 1 mM EDTA, 500 mM NaBr, 50% acetonitrile; solid phase TSKgel-30, 1.5 cm x 10 cm). The solvent was removed by rotary evaporation and desalted on a 3K spin column using 2 x 10 mL exchanges with sterile water. The solid product was dried using lyophilization and stored for later use.

[0366] D. Conjugation of Azide-Containing Lipid PK / PD Modulator Precursors

[0367] One molar equivalent of Cu(I)-loaded TG-TBTA resin was weighed into a glass vial. The vial was purged with N2 for 15 minutes. The functionalized sense strand was then dissolved in a separate vial in sterile water at a concentration of 100 mg / mL. Two equivalents of an azide-containing lipid PK / PD modulator precursor (50 mg / mL in DMF) were then added to the vial. TEA, DMF, and water were then added until the final reaction conditions were 33 mM TEA, 60% DMF, and 20 mg / mL of conjugate product. The solution was then transferred via syringe to the vial containing the resin. The N2 purge was removed, the vial was sealed, and the mixture was transferred to a 40°C stir plate. The mixture was allowed to react for 16 hours. The resin was filtered off using a 0.45 μm filter.

[0368] The product was purified using AEX purification (Mobile Phase A: 25 mM TRIS pH=7.2, 1 mM EDTA, 50% acetonitrile; Mobile Phase B: 25 mM TRIS pH=7.2, 1 mM EDTA, 500 mM NaBr, 50% acetonitrile; Solid Phase TSKgel-30, 1.5 cm x 10 cm). The acetonitrile was removed using a rotary evaporator and desalted on a 3K spin column using 2 x 10 mL exchanges with sterile water. The solid product was dried using lyophilization and stored for later use.

[0369] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] (Ac = acetyl); (NH-C6)s, (invAb), (C6-S), L6, NEM, DBCO-C6s, TDA Pep 1, WAT-homing Pep, see Table 4 for chemical structure information.

[0370] Example 4. In vivo administration of lipid-conjugated RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 5.

[0371] [Table 7]

[0372] The lipid-conjugated RNAi agent was designed to contain an antisense nucleotide sequence complementary to the Adipoq gene transcript, which expresses the protein hormone adiponectin primarily in adipose tissue. Therefore, the lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, the animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Cat. MRP300) and normalized to pre-dosing and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The mean Adipoq expression for each animal in each tissue was normalized to pre-dose and control Group 1 (saline). The results are shown in Tables 6A and 6B below.

[0373] [Table 8]

[0374] Groups 2 to 10 showed a reduction in serum adiponectin at all time points. In particular, animals in Group 3, which received an RNAi agent containing the 5'-terminal LP-413-a and 3'-terminal LP-378-a moieties, showed a greater than 90% reduction in serum adiponectin on days 15 and 22.

[0375] [Table 9]

[0376] Groups 2-10 showed a reduction in Adipoq in both harvested tissues on day 22. In particular, Group 3 showed an approximately 80% reduction in iWAT and pgWAT on day 22.

[0377] Example 5. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.25 mg / kg, 0.5 mg / kg, or 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 7.

[0378] [Table 10]

[0379] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin levels were analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 8A and 8B below.

[0380] [Table 11]

[0381] Groups 2-13 showed a reduction in serum adiponectin at all time points measured. In particular, animals in Group 10, which received an RNAi agent containing the 5'-terminal LP-446a and 3'-terminal LP-378a moieties, showed a greater than 90% reduction in serum adiponectin on days 15 and 22.

[0382] [Table 12]

[0383] Groups 2-13 showed a reduction in Adipoq in both tissues. In particular, Group 10 showed a more than 80% reduction in adiponectin in both iWAT and pgWAT on day 22.

[0384] Example 6. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.25 mg / kg, 0.5 mg / kg, or 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 9.

[0385] [Table 13]

[0386] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, the animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 10A and 10B below.

[0387] [Table 14]

[0388] Groups 2 to 13 showed reduced serum adiponectin levels. In particular, animals in Group 13, which received an RNAi agent containing the 5'-terminal LP-208a and 3'-terminal LP-378a moieties, showed greater than 90% adiponectin knockdown on days 15 and 22.

[0389] [Table 15]

[0390] Groups 2 to 13 showed a reduction in Adipoq in both tissues. In particular, Group 13 showed approximately 70% knockdown of adiponectin in both iWAT and pgWAT on day 22.

[0391] Example 7. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 0.5 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 11.

[0392] [Table 16]

[0393] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 12A and 12B below.

[0394] [Table 17]

[0395] Groups 2-10 showed reduced serum adiponectin at all time points. In particular, animals in Group 3, which received an RNAi agent containing the 5'-terminal LP-455a and 3'-terminal LP-378a moieties, showed greater than 80% adiponectin knockdown at days 15 and 22.

[0396] [Table 18]

[0397] Groups 2 to 10 showed a reduction in Adipoq in both tissues. In particular, Group 3 showed approximately 70% knockdown of adiponectin in both pgWAT and iWAT on day 22.

[0398] Example 8. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.5 mg / kg or 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 13.

[0399] [Table 19]

[0400] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, the animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 14A and 14B below.

[0401] [Table 20]

[0402] Groups 2 to 11 showed reduced serum adiponectin at all time points measured. In particular, animals in groups 3 and 9, which received RNAi agents containing the 5'-terminal LP-413a and 3'-terminal LP-378a moieties and the 5'-terminal LP-426a and 3'-terminal LP-378a moieties, respectively, showed approximately 90% knockdown of adiponectin on days 15 and 22.

[0403] [Table 21]

[0404] Groups 2-11 showed a reduction in Adipoq in both tissues. In particular, both Groups 3 and 9 showed a greater than 80% adiponectin knockdown in iWAT and a greater than 60% adiponectin knockdown in pgWAT.

[0405] Example 9. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 0.5 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 15.

[0406] [Table 22]

[0407] Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 16A and 16B below.

[0408] [Table 23]

[0409] Groups 2-7 and 9-12 showed decreased adiponectin on day 8. Groups 2-7, 9, 10, and 12 showed decreased adiponectin on day 15. Groups 2-7 and 9-12 showed decreased adiponectin on day 22.

[0410] [Table 24]

[0411] Groups 2-7 and 9-12 showed a reduction in Adipoq in both tissues. In particular, groups 5 and 6 showed approximately 62% and 59% knockdown of adiponectin in iWAT and approximately 67% knockdown of adiponectin in pgWAT, respectively.

[0412] Example 10. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.75 mg / kg, 1.5 mg / kg, or 3 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 17.

[0413] [Table 25]

[0414] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 18A and 18B below.

[0415] [Table 26]

[0416] Groups 2-13 showed a reduction in serum adiponectin at all time points measured, with all groups showing approximately 85% or greater knockdown on days 15 and 22.

[0417] [Table 27]

[0418] Groups 2-13 showed a reduction in Adipoq in both tissues. Notably, animals in groups 7 and 10, which received RNAi agents (at 3 mg / kg) containing the 5'-terminal LP-18a and 3'-terminal LP-378a moieties, and the 5'-terminal LP-413a and 3'-terminal LP-378a moieties, respectively, showed greater than 90% adiponectin knockdown in both iWAT and pgWAT.

[0419] Example 11. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 19.

[0420] [Table 28]

[0421] The lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, the animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 20A and 20B below.

[0422] [Table 29]

[0423] Groups 2-11 showed a reduction in serum adiponectin at all time points measured. Group 2, which contained mice administered an RNAi agent containing a NEM cap instead of a lipid, showed only a small reduction in serum adiponectin compared to the larger reductions in the other groups whose RNAi agents contained at least one lipid at the 5'- or 3'-terminal nucleotide. Notably, animals in Group 7, which were administered an RNAi agent containing a 5'-terminal LP-379a and a 3'-terminal LP-378a moiety, showed greater than 85% knockdown of serum adiponectin at days 15 and 22.

[0424] [Table 30]

[0425] Groups 3, 5-7, and 9-11 showed reduced Adipoq in iWAT. Groups 3-11 showed reduced Adipoq in pgWAT. Group 2, which contained mice administered an RNAi agent containing a NEM cap instead of lipid, showed a small increase in Adipoq expression in tissues compared to a larger reduction in the other groups whose RNAi agents contained at least one lipid at the 5' or 3' terminal nucleotide.

[0426] Example 12. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five (n=5) (for groups 1-4 and groups 6-11) or four (n=4) (for group 5) animals were administered 250 μL / 25 g body weight of saline or RNAi agent solution (1 mg / kg) in each group. Animals were injected subcutaneously (SQ) with the dosing regimen in Table 21.

[0427] [Table 31]

[0428] Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 22A and 22B below.

[0429] [Table 32]

[0430] Groups 2-11 showed reduced serum adiponectin at all time points measured. In particular, animals in Group 10, which received an RNAi agent containing the 5'-terminal LP-128a and 3'-terminal LP-378a moieties, showed greater than 85% knockdown of serum adiponectin on days 15 and 22.

[0431] [Table 33]

[0432] Groups 2 to 11 showed a reduction in Adipoq in both tissues. In particular, Group 10 showed approximately 68% knockdown of adiponectin in iWAT and approximately 80% knockdown of adiponectin in pgWAT.

[0433] Example 13. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.75 mg / kg, 1.5 mg / kg, or 3 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 23.

[0434] [Table 34]

[0435] Serum was collected on day 1 before dosing. Serum was collected on days 8 and 15 after dosing. On day 15 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 24A and 24B below.

[0436] [Table 35]

[0437] Groups 2-10 showed reduced serum adiponectin at both time points measured. Notably, animals in Group 8, which received an RNAi agent containing the 5'-terminal CNR1 SM2-1 and 3'-terminal LP-378a moieties, showed greater than 90% knockdown of adiponectin at day 15 at a dose of only 0.75 mg / kg.

[0438] [Table 36]

[0439] Groups 2-10 showed a reduction in Adipoq in both tissues. Notably, Group 8 showed approximately 70% knockdown of adiponectin in both iWAT and pgWAT on day 15 at a dose of only 0.75 mg / kg.

[0440] Example 14. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 1.5 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 25.

[0441] [Table 37]

[0442] Serum was collected on day 1 before dosing. Serum was collected on days 8 and 15 after dosing. On day 15 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 26A and 26B below.

[0443] [Table 38]

[0444] Groups 2-9 showed reduced serum adiponectin at both time points. Notably, Group 4 animals administered an RNAi agent containing the 5'-terminal 379-a and 3'-terminal LP-371a moieties showed greater than 90% knockdown of serum adiponectin at day 15.

[0445] [Table 39]

[0446] Groups 2-9 showed a reduction in Adipoq in both tissues, with Group 4 in particular showing a greater than 75% knockdown in both iWAT and pgWAT at day 15.

[0447] Example 15. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 1.5 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 27.

[0448] [Table 40]

[0449] Serum was collected on day 1 before dosing. Serum was collected on days 8 and 15 after dosing. On day 15 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 28A and 28B below.

[0450] [Table 41]

[0451] Groups 2-7 showed reduced serum adiponectin at both time points. Notably, animals in Group 5, which received an RNAi agent containing the 5'-terminal 379-a and 3'-terminal LP-378a moieties, showed greater than 90% knockdown of serum adiponectin at day 15.

[0452] [Table 42]

[0453] Groups 2-7 showed a reduction in Adipoq in both tissues, with Group 5 in particular showing a greater than 70% knockdown in iWAT and pgWAT on day 15.

[0454] Example 16. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (at 2 mg / kg). Animals were injected subcutaneously (SQ) or intravenously (IV) via the left posterior tibial vein (LPTV) with the dosing regimens in Table 29.

[0455] [Table 43]

[0456] Serum was collected on day 1 before dosing. Serum was collected on days 8 and 15 after dosing. On day 15 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 30A and 30B below.

[0457] [Table 44]

[0458] Groups 2-12 showed reduced serum adiponectin at both time points. Notably, animals in groups 4 (IV) and 9 (SQ), which received RNAi agents containing the 5'-terminal 379-LP-371a moiety and the 3'-terminal LP-233a moiety, showed approximately 95% knockdown of serum adiponectin at day 15.

[0459] [Table 45]

[0460] Groups 2-12 showed a reduction in Adipoq in both tissues, with groups 4 and 9 in particular showing impressive knockdown in both tissues.

[0461] Example 17. In vivo administration of lipid-linked RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.75 mg / kg, 1.5 mg / kg, or 3 mg / kg). Animals were injected intravenously (IV) via the left posterior tibial vein (LPTV) with the dosing regimen in Table 31.

[0462] [Table 46]

[0463] Serum was collected on day 1 before dosing. Serum was collected on days 8 and 15 after dosing. On day 15 after dosing, animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Mouse adiponectin in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, catalog MRP300) and normalized to pre-dose and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR with mouse mArl1 as a control. The average Adipoq expression for each animal in each tissue was normalized to pre-dose and control group 1 (saline). The results are shown in Tables 32A and 32B below.

[0464] [Table 47]

[0465] Groups 2-10 showed reduced serum adiponectin at both time points. Notably, animals in Group 8, which received an RNAi agent containing the 5'-terminal CNR1 SM2-1 and 3'-terminal LP-378 moieties, showed greater than 90% knockdown of serum adiponectin at day 15 at a dose of only 0.75 mg / kg.

[0466] [Table 48]

[0467] Groups 2 to 10 showed a reduction in Adipoq in both tissues, with Group 8 in particular showing approximately 70% knockdown of adiponectin in both iWAT and pgWAT.

[0468] Example 18. In vivo administration of ALK7 RNAi agents in mice. ALK7 RNAi agents were evaluated in vivo in mice using oligonucleotides containing the PK / PD modulators LP-371-a and LP-379-a. On day 1, five (n=5) female C57bl / 6 mouse test animals received a single subcutaneous (SQ) injection of 250 μl per 25 g of body weight containing either 1.0 mg / kg (mpk), 3.0 mg / kg (mpk) of ALK7 RNAi agent, or saline. Dosing was according to Table 33 below. [Table 49]

[0469] ALK7 RNAi agents AC004391, AC004390, AC004392, and AC005181 target and initiate RNAi and the RNA-induced silencing complex (RISC) of mouse ALK7. ALK7 RNAi agents AC005824 and AC005823 target and initiate RNAi and the RNA-induced silencing complex (RISC) of human ALK7.

[0470] Five mice (n=5) were dosed in each group. Mice were injected subcutaneously (SQ) on day 1. On day 15, mice were euthanized and approximately 50 mg of adipose tissue (inguinal white adipose tissue iWAT, perigonadal white adipose tissue pgWAT) was collected for analysis. Samples were analyzed for mALK7 mRNA knockdown by qPCR using mARL1 as an endogenous control reference gene and normalized to Group 1 (saline). The average results for each group are shown in Table 34 below. [Table 50]

[0471] As shown in Table 34, Groups 2-13 demonstrated a reduction in ALK7 in both iWAT and pgWAT compared to Group 1, which did not receive an ALK7 RNAi agent. More specifically, a 3.0 mg / kg dose of the ALK7 RNAi agent AC005824 achieved approximately 70% ALK7 inhibition (0.294) in iWAT at Day 15. A 3.0 mg / kg dose of the ALK7 RNAi agent AC005181 achieved approximately 81% ALK7 inhibition (0.187) in pgWAT at Day 15. A dose response was observed in Groups 4 and 5, 6 and 7, 8 and 9, 10 and 11, and 12 and 13 in iWAT, and also in Groups 2 and 3, 4 and 5, 8 and 9, 10 and 11, and 12 and 13 in pgWAT.

[0472] Example 19. In vivo administration of ALK7 RNAi agents in cynomolgus monkeys. ALK7 RNAi agents, including the PK / PD modulators LP-371-a and LP-379-a, are currently being tested in cynomolgus monkeys for the inhibition of ALK7. On days 1 and 29, three male cynomolgus monkey test animals (n=3) per test group are administered ALK7 RNAi agents (3.0 mg / kg) formulated in saline via subcutaneous (SQ) injection using a syringe and needle in the mid-scapular region at a dose volume of 0.3 mL / kg. Fat biopsies are taken from all test animals on days -7 (pre-dose), 15, 29, 57, and 85. The dosing regimen is as shown in Table 35 below. [Table 51]

[0473] All animals are fasted for at least 12 hours but no longer than 24 hours for scheduled blood collection and biopsy procedures. The blood collection site is the femoral vein. The saphenous vein (not used for dose administration) may be used as an alternative collection site.

[0474] Fat biopsies are taken as a sedated procedure. Sedation is achieved using ketamine HCl (10 mg / kg) or Telazol (5–8 mg / kg), administered as an intramuscular (IM) injection and supplemented with ketamine (5 mg / kg) as needed.

[0475] An approximately 3-5 cm skin incision is made, after which adipose tissue (optimally 50-150 mg) is harvested. The skin is then closed in a conventional manner using sutures (or alternative) that maintain sterile technique.

[0476] Each biopsy site is separated by at least 1-2 cm. The biopsy is divided into two fragments (one fragment approximately 25-75 mg, the second fragment approximately 25-75 mg) for each collection time point. Analgesics may be administered at the veterinarian's discretion.

[0477] Blood is collected in tubes without anticoagulant (serum separator tubes) and allowed to clot at ambient temperature before centrifugation to obtain serum.

[0478] Blood will be collected on days -7, 1, 15, 29, 57, and 85 prior to liver biopsy sampling or dose administration (if applicable), and from any animals found moribund or sacrificed at unscheduled intervals.

[0479] Individual doses of ALK7 RNAi agents are calculated based on body weight recorded on each day of dosing.

[0480] Fat biopsies and serum collected from test animals are used for analysis of ALK7 expression and further biological parameters. Serum ALK7 mRNA expression levels are quantified via qPCR using cARL1 as an endogenous control reference gene and normalized to day -7.

[0481] This study is currently ongoing and the currently available data is presented in Table 36 below. [Table 52]

[0482] Both Groups 1 and 2, AC006188 and AC006189, demonstrated inhibition of ALK7 in cynomolgus monkeys.

[0483] Example 20. In vivo administration of lipid-conjugated RNAi agents in mice. On study day 1, female C57bl / 6 mice were injected with either saline or lipid-conjugated RNAi agents formulated in saline. Five animals (n=5) in each group received 250 μL / 25 g body weight of saline or RNAi agent solution (0.5 mg / kg or 1 mg / kg). Animals were injected subcutaneously (SQ) with the dosing regimen in Table 37.

[0484] [Table 53]

[0485] The lipid-conjugated RNAi agent was designed to contain an antisense nucleotide sequence complementary to the Adipoq gene transcript, which expresses the protein hormone adiponectin primarily in adipose tissue. Therefore, the lipid-conjugated RNAi agent was designed to inhibit Adipoq gene expression. Serum was collected on day 1 before dosing. Serum was collected on days 8, 15, and 22 after dosing. On day 22 after dosing, the animals were sacrificed and adipose tissue was collected, including inguinal white adipose tissue (iWAT) and perigonadal white adipose tissue (pgWAT). Serum mouse adiponectin was analyzed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Cat. MRP300) and normalized to pre-dosing and control group 1 (saline). Mouse Adipoq expression in each tissue was determined using qPCR, with mouse mArl1 as a control. The mean Adipoq expression for each animal in each tissue was normalized to pre-dose and control Group 1 (saline). The results are shown in Tables 6A and 6B below.

[0486] [Table 54]

[0487] Groups 4-9 showed reduced serum adiponectin at all time points. Groups 2 and 3, which did not contain lipid moiety conjugates, showed poor knockdown. In particular, Groups 4 and 5, which were treated with RNAi agents containing the 5'-terminal LP-379-a and 3'-terminal LP-371-a moieties, showed impressive reductions in serum adiponectin compared to dose levels on days 15 and 22.

[0488] [Table 55] In particular, groups 4 and 5 showed impressive adiponectin reduction in iWAT and pgWAT on day 22.

[0489] Equivalents and Scope In the claims, articles such as "a," "an," and "the" may mean one or more unless indicated otherwise or clear from the context. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which two or more or all group members are present in, employed in, or relevant to a given product or process.

[0490] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends from the same base claim. When elements are presented as lists (e.g., in Markush group format), each subgroup of elements is also disclosed, and any element can be removed from the group. In general, when the invention or aspects of the invention are referred to as comprising particular elements and / or features, it is to be understood that certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements and / or features. For the sake of brevity, these embodiments have not been specifically described verbatim herein. Also, note that the terms "comprising" and "containing" are intended to be open, permitting the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value or subrange within the ranges described in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0491] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Furthermore, any particular embodiment of the present invention that falls within the prior art may be expressly excluded from any one or more claims. Because such embodiments are deemed known to those of skill in the art, they may be excluded even if the exclusion is not explicitly set forth herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0492] Other embodiments While the invention has been described in conjunction with the detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A compound of formula (I) 【Chemistry 1】 During the ceremony, R Ｚ１ and R Ｚ３ are each independently a linking or capping residue; R Ｚ２ comprises an oligonucleotide containing from about 8 to about 50 nucleotides, each of which may independently be modified or unmodified; Y is at least one L １ and, if present, L ２ to, or R Ｚ１ is a bond or linker connecting Y １ However, at least one L ３ and, if present, L ４ to, or R Ｚ３ is a bond or linker connecting L ２ and L ３ are each independently a linker; q is 1, 2, or 3, valence permitting; t is 1, 2, or 3, valence permitting; L １ and L ４ is each independently a lipid containing from about 10 to about 50 carbon atoms, or a pharmaceutically acceptable salt thereof.

2. R Ｚ１ But, R Ｚ２ 2. The compound or pharmaceutically acceptable salt of claim 1, wherein:

3. 3. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein Y is a bond.

4. Y is at least one L １ and, if present, L ２ To, or L １ R Ｚ１ 4. The compound or pharmaceutically acceptable salt according to claim 1, wherein

5. Y is at least one L １ and, if present, L ２ or connect to L １ R Ｚ１ 5. The compound or pharmaceutically acceptable salt of any one of claims 1 to 4, wherein:

6. Y is a group of the formula: 【Chemistry 2】 wherein: Y ａ and Y ｃ are each independently absent, —N(H)—, or —C(O)—; Y ｂ or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 5, wherein R is absent or is substituted or unsubstituted heteroalkylene, substituted or unsubstituted carbocyclene, substituted or unsubstituted heterocyclene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

7. Y ａ but, 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, which is -C(O)-.

8. Y ａ The compound or pharmaceutically acceptable salt of claim 6, wherein is -N(H)-.

9. Y ｃ The compound or pharmaceutically acceptable salt thereof according to any one of claims 6 to 8, wherein is -(CO)-.

10. Y ｃ The compound or pharmaceutically acceptable salt thereof according to any one of claims 6 to 8, wherein is -N(H)-.

11. Y ｂ is substituted or unsubstituted C １ ~C ６ The compound or pharmaceutically acceptable salt of any one of claims 6 to 10, which is heteroalkylene.

12. Y ｂ But, the formula 【Transformation 3】 12. The compound or pharmaceutically acceptable salt thereof according to any one of claims 6 to 11, wherein

13. Y ｂ The compound or pharmaceutically acceptable salt according to any one of claims 6 to 10, wherein is a substituted or unsubstituted carbocyclene.

14. Y ｂ But, the formula 【Chemistry 4】 14. The compound or pharmaceutically acceptable salt according to any one of claims 6 to 10, or 13, wherein

15. Y ｂ The compound or pharmaceutically acceptable salt according to any one of claims 6 to 10, wherein is a substituted or unsubstituted heterocyclene.

16. Y ｂ But, the formula 【Transformation 5】 16. The compound or pharmaceutically acceptable salt of any one of claims 6 to 10, or 15, wherein

17. Y ｂ The compound or pharmaceutically acceptable salt thereof according to any one of claims 6 to 10, wherein is substituted or unsubstituted arylene.

18. Y ｂ 18. The compound or pharmaceutically acceptable salt thereof according to any one of claims 6 to 10 or 17, wherein is substituted or unsubstituted phenylene.

19. Y ｂ But, the formula 【Transformation 6】 19. The compound or pharmaceutically acceptable salt according to any one of claims 6 to 10, or 17 to 18, wherein

20. Y is -N(H)-C(O)-, -C(O)-N(H)-, 【Transformation 7】 20. The compound or pharmaceutically acceptable salt of any one of claims 1 to 19, selected from the group consisting of:

21. Y is two L １ The group is L ２ or two L １ The group is R Ｚ１ 3. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein:

22. Y is a group of the formula: 【Transformation 8】 22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, which is:

23. L ２ But, the formula 【Chemistry 9】 wherein: L ２ａ is a bond or the formula: 【Chemistry 10】 wherein h is an integer from 1 to 12; L ２ｂ is a bond or a chemical moiety formed by reacting a first reactive moiety with a second reactive moiety; L ２ｃ 23. The compound or pharmaceutically acceptable salt of any one of claims 1 to 22, wherein is a bond or a bidentate linking group.

24. L ２ｂ is a bond, —C(O)—, or formula: 【Chemistry 11】 24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, which is:

25. L ２ｃ But the formula: 【Chemistry 12】 25. The compound or pharmaceutically acceptable salt of claim 23 or 24, wherein:

26. 26. The compound or pharmaceutically acceptable salt according to any one of claims 23 to 25, wherein h is 2, 3, 5, 9, or 10.

27. L １ 27. The compound or pharmaceutically acceptable salt of any one of claims 1 to 26, wherein at least one instance of is independently a straight-chain lipid.

28. L １ 28. The compound or pharmaceutically acceptable salt of any one of claims 1 to 27, wherein at least one instance of is independently a saturated lipid.

29. L １ At least one instance of independently has the formula: 【Chemistry 13】 wherein: R Ｌ１ａ is H or CO ２ H, 29. The compound or pharmaceutically acceptable salt according to any one of claims 1 to 28, wherein r is an integer from 5 to 35.

30. R Ｌ１ａ 30. The compound of claim 29, or a pharmaceutically acceptable salt thereof, wherein:

31. R Ｌ１ａ But CO ２ 31. The compound of claim 29 or 30, or a pharmaceutically acceptable salt thereof, wherein:

32. 32. The compound or pharmaceutically acceptable salt of any one of claims 29 to 31, wherein r is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

33. 33. The compound or pharmaceutically acceptable salt of any one of claims 29 to 32, wherein r is 9, 11, 12, 13, 14, 15, or 17.

34. 30. The compound or pharmaceutically acceptable salt of claim 29, wherein r is 13.

35. R Ｌ１ａ or a pharmaceutically acceptable salt thereof, of claim 34, wherein

36. 30. The compound or pharmaceutically acceptable salt of claim 29, wherein r is 12.

37. R Ｌ１ａ But CO ２ 37. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein:

38. L １ 38. The compound or pharmaceutically acceptable salt of any one of claims 1 to 37, wherein at least one instance of is independently a branched lipid.

39. L １ At least one instance of independently has the formula 【Chemistry 14】 wherein: w is an integer from 2 to 25; 39. The compound or pharmaceutically acceptable salt of any one of claims 1 to 38, wherein v is an integer from 2 to 25.

40. L １ At least one instance of independently has the formula: 【Chemistry 15】 40. The compound or pharmaceutically acceptable salt of any one of claims 1 to 39, wherein:

41. L １ 41. The compound or pharmaceutically acceptable salt of any one of claims 1 to 40, wherein at least one instance of is independently an unsaturated lipid.

42. L １ 42. The compound or pharmaceutically acceptable salt of any one of claims 1 to 41, wherein at least one instance of is independently a straight chain lipid comprising 1 to 8 alkenylene moieties.

43. L １ At least one instance of independently has the formula: 【Chemistry 16】 wherein: R Ｌ１ｂ But -CH ３ , or -CO ２ H, R Ｌ１ｃ But -CH ２ - or -C(O)-, j is an integer from 0 to 20; k is an integer from 1 to 8; 43. The compound or pharmaceutically acceptable salt according to any one of claims 1 to 42, wherein o is an integer from 1 to 20.

44. R Ｌ１ｂ or a pharmaceutically acceptable salt thereof, of claim 43, wherein:

45. R Ｌ１ｂ But -CO ２ 44. The compound of claim 43, or a pharmaceutically acceptable salt thereof, wherein:

46. R Ｌ１ｃ But -CH ２ 46. ​​The compound or pharmaceutically acceptable salt thereof according to any one of claims 43 to 45, wherein

47. R Ｌ１ｃ The compound or pharmaceutically acceptable salt according to any one of claims 43 to 45, wherein is -C(O)-.

48. 48. The compound or pharmaceutically acceptable salt of any one of claims 43 to 47, wherein j is 0, 1, 4, or 7.

49. 49. The compound or pharmaceutically acceptable salt of any one of claims 43 to 48, wherein k is 1, 2, 3, 4, or 5.

50. 50. The compound or pharmaceutically acceptable salt of any one of claims 43 to 49, wherein o is 2, 3, 6, or 10.

51. L １ At least one instance of independently has the formula: 【Chemistry 17】 51. The compound or pharmaceutically acceptable salt of any one of claims 1 to 50, wherein:

52. Y １ 52. The compound or pharmaceutically acceptable salt of any one of claims 1 to 51, wherein is a bond.

53. Y １ However, at least one L ４ and, if present, L ３ To, or L ４ R Ｚ３ 53. The compound or pharmaceutically acceptable salt of any one of claims 1 to 52, which is a linker connecting

54. Y １ However, at least one L ４ and, if present, L ３ or connect to L ４ R Ｚ３ 54. The compound or pharmaceutically acceptable salt of any one of claims 1 to 53, wherein:

55. Y １ But the formula: [Chemistry 18] wherein: Y １ａ and Y １ｃ are each independently absent, —N(H)—, or —C(O)—; Y １ｂ or a pharmaceutically acceptable salt thereof.

55. The compound of any one of claims 1 to 54, wherein:

56. Y １ａ or a pharmaceutically acceptable salt thereof, according to claim 55, wherein: is -C(O)-.

57. Y １ａ or a pharmaceutically acceptable salt thereof, of claim 55, wherein is -N(H)-.

58. Y １ｃ 58. The compound or pharmaceutically acceptable salt according to any one of claims 55 to 57, wherein is -(CO)-.

59. Y １ｃ 58. The compound or pharmaceutically acceptable salt of any one of claims 55 to 57, wherein is -N(H)-.

60. Y １ｂ is substituted or unsubstituted C １ ~C ６ 60. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, which is heteroalkylene.

61. Y １ｂ But, the formula 【Chemistry 19】 61. The compound or pharmaceutically acceptable salt of any one of claims 55 to 60, wherein:

62. Y １ｂ 60. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, wherein is a substituted or unsubstituted carbocyclene.

63. Y １ｂ But, the formula 【Chemistry 20】 63. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, or 62, wherein

64. Y １ｂ 60. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, wherein is a substituted or unsubstituted heterocyclene.

65. Y １ｂ But, the formula 【Chemistry 21】 65. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, or 64, wherein

66. Y １ｂ 60. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, wherein is substituted or unsubstituted arylene.

67. Y １ｂ 67. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, or 66, wherein is substituted or unsubstituted phenylene.

68. Y １ｂ But, the formula 【Chemistry 22】 68. The compound or pharmaceutically acceptable salt of any one of claims 55 to 59, or 66 to 67, wherein

69. Y １ が、-N(H)-C(O)-、-C(O)-N(H)-、 【Chemistry 23】 69. The compound of any one of claims 1 to 68, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

70. Y １ But two L ４ The group is L ３ or two L ４ The group is R Ｚ３ 52. The compound or pharmaceutically acceptable salt of any one of claims 1 to 51, wherein:

71. Y １ But the formula: 【Chemistry 24】 71. The compound of claim 70, or a pharmaceutically acceptable salt thereof, which is:

72. L ３ But, the formula 【Chemistry 25】 wherein: L ３ａ is a bond or a bidentate linking group; L ３ｂ is a bond or a chemical moiety formed by reacting a first reactive moiety with a second reactive moiety; L ３ｃ is a bond or the formula: 【Chemistry 26】 72. The compound of any one of claims 1 to 71, or a pharmaceutically acceptable salt thereof, wherein i is an integer from 1 to 12.

73. L ３ｂ is a bond, —C(O)—, or a group of the formula: 【Chemistry 27】 73. The compound of claim 72, or a pharmaceutically acceptable salt thereof, which is:

74. L ３ａ But the formula: 【Chemistry 28】 74. The compound or pharmaceutically acceptable salt of claim 72 or 73, wherein:

75. L ４ 75. The compound or pharmaceutically acceptable salt of any one of claims 1 to 74, wherein at least one instance of is independently a saturated lipid.

76. L ４ 76. The compound or pharmaceutically acceptable salt of any one of claims 1 to 75, wherein at least one instance of is independently a straight-chain lipid.

77. L ４ At least one instance of independently has the formula: 【Chemistry 29】 wherein: R Ｌ４ａ is H or CO ２ H, 77. The compound or pharmaceutically acceptable salt of any one of claims 1 to 76, wherein d is an integer from 5 to 35.

78. R Ｌ４ａ or a pharmaceutically acceptable salt thereof.

79. R Ｌ４ａ But CO ２ 79. The compound of claim 77 or 78, or a pharmaceutically acceptable salt thereof, wherein:

80. 80. The compound or pharmaceutically acceptable salt of any one of claims 77 to 79, wherein d is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

81. 81. The compound or pharmaceutically acceptable salt of any one of claims 77 to 80, wherein d is 9, 11, 12, 13, 14, 15, or 17.

82. 78. The compound or pharmaceutically acceptable salt of claim 77, wherein d is 13.

83. R Ｌ４ａ or a pharmaceutically acceptable salt thereof.

84. 78. The compound or pharmaceutically acceptable salt of claim 77, wherein d is 12.

85. R Ｌ４ａ But CO ２ 85. The compound of claim 84, or a pharmaceutically acceptable salt thereof, wherein:

86. L ４ 86. The compound or pharmaceutically acceptable salt of any one of claims 1 to 85, wherein at least one instance of is independently a branched lipid.

87. L ４ At least one instance of independently has the formula 【Transformation 30】 wherein: x is an integer from 2 to 25; 87. The compound or pharmaceutically acceptable salt of any one of claims 1 to 86, wherein y is an integer from 2 to 25.

88. L ４ At least one instance of independently has the formula: 【Chemistry 31】 88. The compound or pharmaceutically acceptable salt of any one of claims 1 to 87, wherein:

89. L ４ 89. The compound or pharmaceutically acceptable salt of any one of claims 1 to 88, wherein at least one instance of is independently an unsaturated lipid.

90. L ４ or a pharmaceutically acceptable salt thereof, wherein at least one instance of is independently a straight chain lipid comprising 1 to 8 alkenylene moieties.

91. L ４ At least one instance of independently has the formula 【Chemistry 32】 wherein: R Ｌ４ｂ But -CH ３ , or -CO ２ H, R Ｌ４ｃ But -CH ２ - or -C(O)-, a is an integer from 0 to 20, b is an integer from 1 to 8; 91. The compound or pharmaceutically acceptable salt of any one of claims 1 to 90, wherein c is an integer from 1 to 20.

92. R Ｌ４ｂ or a pharmaceutically acceptable salt thereof.

93. R Ｌ４ｂ But -CO ２ 92. The compound of claim 91, or a pharmaceutically acceptable salt thereof, wherein:

94. R Ｌ４ｃ But -CH ２ 94. The compound or pharmaceutically acceptable salt according to any one of claims 91 to 93, wherein:

95. R Ｌ４ｃ 94. The compound or pharmaceutically acceptable salt according to any one of claims 91 to 93, wherein is -C(O)-.

96. 96. The compound or pharmaceutically acceptable salt of any one of claims 91 to 95, wherein a is 0, 1, 4, or 7.

97. 97. The compound or pharmaceutically acceptable salt of any one of claims 91 to 96, wherein b is 1, 2, 3, 4, or 5.

98. 98. The compound or pharmaceutically acceptable salt of any one of claims 91 to 97, wherein c is 2, 3, 6, or 10.

99. L ４ At least one instance of independently has the formula: 【Transformation 33】 99. The compound or pharmaceutically acceptable salt of any one of claims 1 to 98, wherein [Request Item 100] [Chemistry 34] or a pharmaceutically acceptable salt thereof, wherein: 【Request Item 101】 【Chemistry 35】 is selected from the group consisting of any of the PK / PD modulators shown in Table 2, or a pharmaceutically acceptable salt thereof.

102. R Ｚ１ The compound or pharmaceutically acceptable salt of any one of claims 1 to 101, wherein is an inverted abasic residue.

103. R Ｚ１ But the formula: 【Transformation 36】 103. The compound or pharmaceutically acceptable salt of any one of claims 1 to 102, wherein

104. R Ｚ３ The compound or pharmaceutically acceptable salt of any one of claims 1 to 103, wherein is an inverted abasic residue.

105. R Ｚ３ But the formula: 【Chemistry 37】 105. The compound or pharmaceutically acceptable salt of any one of claims 1 to 104, wherein:

106. a) an oligonucleotide-based agent comprising an oligonucleotide, and b) at least one lipid conjugated to the oligonucleotide-based agent, wherein the at least one lipid is conjugated to the 5'-end or the 3'-end of the oligonucleotide; A compound wherein the oligonucleotide-based agent comprises at least 15 nucleotides complementary to a gene expressed in adipose tissue.

107. 107. The compound of claim 106, wherein the oligonucleotide-based agent is double-stranded.

108. 108. The compound of claim 106 or 107, wherein the oligonucleotide-based agent comprises a sense strand and an antisense strand.

109. 109. The compound of claim 108, wherein the at least one lipid is conjugated to the sense strand.

110. 110. The compound of claim 109, wherein the at least one lipid is conjugated to the 5' position of the sense strand.

111. 111. The compound of any one of claims 106 to 110, wherein the at least one lipid is saturated.

112. 111. The compound of any one of claims 106 to 110, wherein the at least one lipid is unsaturated.

113. 113. The compound of any one of claims 106 to 112, wherein the at least one lipid comprises from 10 to 30 carbon atoms.

114. 114. The compound of any one of claims 106 to 113, wherein the at least one lipid comprises 15 to 20 carbon atoms.

115. The at least one lipid is Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 wherein: 【Transformation 38】 115. The compound of any one of claims 106 to 114, wherein: denotes a point of attachment to the oligonucleotide.

116. 116. The compound of any one of claims 106-115, wherein the oligonucleotide-based agent is conjugated to at least two lipids.

117. 117. The compound of any one of claims 106-116, wherein the oligonucleotide-based agent is conjugated to at least three lipids.

118. 117. The compound of claim 116, wherein the at least two lipids are conjugated to the 5' end of the oligonucleotide and the 3' end of the oligonucleotide.

119. 119. The compound of any one of claims 106-118, wherein the oligonucleotide-based agent is an RNAi agent and the antisense strand comprises cyclopropyl-phosphonate modified nucleotides (cPrp) or vinyl-phosphonate modified nucleotides (vp).

120. 120. The compound of claim 119, wherein the cyclopropyl-phosphonate or vinyl-phosphonate modified nucleotide is the 5' terminal nucleotide of the antisense strand. 【Request Item 121】 【Table 2-1】 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 wherein R comprises an oligonucleotide.

122. 122. The compound of claim 121, wherein the oligonucleotide is double-stranded.

123. 123. The compound of claim 122, wherein the oligonucleotide comprises a sense strand and an antisense strand.

124. 124. The compound of claim 123, wherein the point of attachment to R is on the sense strand.

125. 125. The compound of claim 124, wherein the point of attachment to R is on the 5'-terminal nucleotide of the sense strand.

126. 1. An RNAi agent comprising a sense strand and an antisense strand, 126. An RNAi agent, wherein the sense strand is a compound of any one of claims 1 to 125, and the antisense strand is at least 70%, 85%, or 90% complementary to the sense strand.

127. The RNAi agent of claim 126, wherein the antisense strand is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in adipose tissue.

128. 128. The RNAi agent of claim 126 or 127, wherein the antisense strand is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in human adipose tissue.

129. The RNAi agent of any one of claims 126 to 128, wherein the antisense strand is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in an adipocyte.

130. The RNAi agent of any one of claims 126 to 129, wherein the antisense strand is at least 70%, 80%, or 90% complementary to the mRNA of a gene expressed in human adipocytes.

131. The RNAi agent of claim 129 or 130, wherein the gene is expressed in white adipocytes.

132. The RNAi agent of any one of claims 129 to 131, wherein the gene is expressed in mature adipocytes.

133. 132. A pharmaceutical composition comprising a compound of any one of claims 1 to 125 or a pharmaceutically acceptable salt thereof, or an RNAi agent of any one of claims 126 to 132, and a pharmaceutically acceptable excipient.

134. 132. A method for delivering an oligonucleotide to adipose tissue, the method comprising administering to a subject a compound of any one of claims 1-125, an RNAi agent of any one of claims 123-132, or a pharmaceutical composition of claim 133.

135. 132. A method for delivering an oligonucleotide to a cell, the method comprising administering to a subject a compound of any one of claims 1-125, an RNAi agent of any one of claims 126-132, or a pharmaceutical composition of claim 133.

136. 136. The method of claim 135, wherein the cells are adipocytes.

137. 133。 A method of modulating the activity of a gene in a subject, a biological sample, or a cell, said method comprising introducing into said subject, biological sample, or cell an effective amount of a compound of any one of claims 1 to 125, an RNAi agent of any one of claims 126 to 132, or a pharmaceutical composition of claim 133, wherein said gene is a gene expressed in adipose tissue or adipocytes.

138. 138. The method of claim 137, wherein said expression of said gene is inhibited.

139. 139. The method of claim 137 or 138, wherein said expression is inhibited by at least 50%, 60%, 70%, 80%, or 90% compared to expression before administration.

140. 140. The method of any one of claims 137 to 139, wherein the gene is overexpressed in the subject, biological sample, or cell.

141. The method of any one of claims 137 to 140, wherein the gene is expressed in white adipocytes.

142. 142. The method of any one of claims 137 to 141, wherein the gene is expressed in mature adipocytes.

143. Compound of formula (II): 【Chemistry 39】 or a pharmaceutically acceptable salt thereof, wherein: R ｇ is a reactive moiety suitable for conjugation to an oligonucleotide-based agent; Y ２ However, at least one L ５ and, if present, L ６ to, or R ｇ is a bond or linker connecting Each L ５ are independently lipids containing from about 10 to about 50 carbon atoms; L ６ is a linker comprising 1 to 20 PEG units; A compound or a pharmaceutically acceptable salt thereof, wherein z is 1, 2, or 3, as valence permits.

144. Y ２ 144. The compound or pharmaceutically acceptable salt of claim 143, wherein: is a bond.

145. Y ２ However, at least one L ５ and, if present, L ６ To, or L ５ R ｇ 144. The compound or pharmaceutically acceptable salt of claim 143, wherein:

146. Y ２ However, at least one L ５ and, if present, L ６ To, or L ５ R ｇ 146. The compound or pharmaceutically acceptable salt of any one of claims 143 to 145, wherein:

147. Y ２ が、-N(H)-C(O)-、-C(O)-N(H)-、 【Chemistry 40】 147. The compound of claim 145 or 146, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

148. Y ２ But two L ５ The group is L ６ or two L ５ The group is R ｇ 144. The compound or pharmaceutically acceptable salt of claim 143, wherein:

149. Y ２ But the formula: 【Chemistry 41】 149. The compound of claim 148, or a pharmaceutically acceptable salt thereof, which is:

150. L ６ 150. The compound or pharmaceutically acceptable salt of any one of claims 143 to 149, wherein is a linker comprising 1 to 10 PEG units.

151. L ６ But the formula: 【Chemistry 42】 151. The compound or pharmaceutically acceptable salt of any one of claims 143 to 150,

152. L ６ But the formula: 【Chemistry 43】 152. The compound or pharmaceutically acceptable salt of any one of claims 143 to 151,

153. 153. The compound or pharmaceutically acceptable salt of any one of claims 143 to 152, wherein z is 1 or 2, valence permitting.

154. R ｇ But the formula: 【Chemistry 44】 154. The compound or pharmaceutically acceptable salt of any one of claims 143 to 153,

155. L ５ or a pharmaceutically acceptable salt thereof, wherein at least one instance of is independently a saturated lipid.

156. L ５ or a pharmaceutically acceptable salt thereof, wherein at least one instance of is independently an unsaturated lipid.

157. L ５ or a pharmaceutically acceptable salt thereof, wherein at least one instance of is independently a straight chain lipid.

158. L ５ At least one instance of independently has the formula: 【Chemistry 45】 wherein: R Ｌ５ is H or CO ２ H, 158. The compound or pharmaceutically acceptable salt of any one of claims 143 to 157, wherein e is an integer from 5 to 35.

159. R Ｌ５ or a pharmaceutically acceptable salt thereof.

160. R Ｌ５ But CO ２ 160. The compound of claim 158 or 159, or a pharmaceutically acceptable salt thereof, wherein:

161. 161. The compound or pharmaceutically acceptable salt of any one of claims 158 to 160, wherein e is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

162. 162. The compound or pharmaceutically acceptable salt of any one of claims 158 to 161, wherein e is 9, 11, 12, 13, 14, 15, or 17.

163. 159. The compound or pharmaceutically acceptable salt of claim 158, wherein e is 13.

164. R Ｌ５ or a pharmaceutically acceptable salt thereof.

165. 159. The compound or pharmaceutically acceptable salt of claim 158, wherein e is 12.

166. R Ｌ５ But CO ２ 166. The compound of claim 165, or a pharmaceutically acceptable salt thereof, wherein:

167. L ５ or a pharmaceutically acceptable salt thereof, wherein at least one instance of is independently a branched lipid.

168. L ５ At least one instance of independently has the formula: 【Chemistry 46】 168. The compound or pharmaceutically acceptable salt of any one of claims 143 to 167,

169. L ５ At least one instance of independently has the formula: 【Chemistry 47】 169. The compound or pharmaceutically acceptable salt of any one of claims 143 to 168,

170. The following formula: Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 wherein: 【Chemistry 48】 indicates a solid support such as a resin, or a pharmaceutically acceptable salt thereof.

171. A method of synthesizing a lipid-oligonucleotide conjugate, comprising contacting an oligonucleotide with any one of compounds 143-170.

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