Novel RNA therapeutics and their uses

Abstract

The present invention relates to novel therapeutic compounds known as RNAi agents that decrease the expression of the ANGPTL8 receptor (expressed by the ANGPTL8 gene), thereby decreasing mRNA expression and protein expression. Such RNAi agents are useful in the treatment of diseases such as dyslipidemia, cardiovascular disorders, and cardiometabolic disorders, which involve the regulation of ANGPTL8 expression and function.

Description

[Technical Field]

[0001] The present disclosure relates to novel RNAi agents designed to reduce the expression of ANGPTL8 in the liver, wherein the RNAi comprises a delivery moiety optionally conjugated to an oligonucleotide via a linker. The RNAi agents are useful for treating diseases involving the regulation of ANGPTL8 expression. [Background technology]

[0002] Angiopoietin-like protein 8 (ANGPTL8) is primarily expressed in the liver and adipose tissue and plays an important role in triglyceride metabolism. ANGPTL8, together with ANGPTL3 and ANGPTL4, is thought to regulate triglyceride levels by inhibiting the enzymatic activity of lipoprotein lipase (LPL). When active, it hydrolyzes triglycerides10, reducing circulating plasma triglycerides. Increased ANGPTL8 levels are observed in or are associated with cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), renal dysfunction, hypertension, nonalcoholic fatty liver disease (NSHIP) such as nonalcoholic steatohepatitis (NASH), and obesity.

[0003] ANGPTL8 siRNA and ASO have been described, such as those disclosed in WO 2020 / 104649(A2), but none have provided advances for patient treatment. The use of ANGPTL8 RNAi agents herein to reduce ANGPTL8 expression can be used, for example, to treat patients in need of treatment for cardiometabolic disorders and related disorders such as dyslipidemia. Summary of the Invention

[0004]

[0013] In one aspect, provided herein is an RNAi agent for reducing ANGPTL8 gene expression, the RNAi agent comprising a delivery moiety of Formula I conjugated to R, wherein R is double stranded RNA (dsRNA) comprising an antisense strand and a sense strand;

[0005] [ka] R is conjugated to connection point E of Formula I, optionally via a linker, wherein the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages. In some embodiments, Formula I is conjugated to the sense strand, optionally via a linker. In some embodiments, Formula I is conjugated to the 3'-terminal nucleotide of the sense strand, optionally via a linker.

[0006] In one embodiment, provided herein is an RNAi agent for reducing ANGPTL8 gene expression, the RNAi agent comprising a delivery moiety of Formula I conjugated to R, wherein R is double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand;

[0007] [ka] R is conjugated to connection point E of Formula I, optionally via a linker, and the sense strand and the antisense strand form a duplex region, and the antisense strand comprises any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a sequence having 90% sequence identity thereto, or an antisense strand sequence set forth in Table 3A, Table 3B, and Table 4, or a sequence having 90% sequence identity thereto, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages. In some embodiments, Formula I is conjugated to the sense strand, optionally via a linker. In some embodiments, Formula I is conjugated to the 3'-terminal nucleotide of the sense strand, optionally via a linker.

[0008] In some embodiments, the antisense strand is 15 to 50 nucleotides in length. In some embodiments, the sense strand is 15 to 50 nucleotides in length. In some embodiments, the antisense strand is 18 to 23 nucleotides in length. In some embodiments, the sense strand is 18 to 21 nucleotides in length. In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

[0009] In some embodiments, the sense strand or the antisense strand comprises a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein. In some embodiments, the sense strand and the antisense strand comprise a sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein.

[0010] In some embodiments, R is conjugated to Formula I via a linker. In further embodiments, the linker comprises a linker of Formula II having connection points A and B, or the linker comprises Formula III having connection points C and D,

[0011] [ka] a. Formula I is conjugated at connection point E to connection point A of Formula II, and Formula II is conjugated to a phosphate group at connection point B, which is further conjugated to R; or b. Formula I is conjugated at connection point E to connection point C of Formula III, and Formula III is conjugated to a phosphate group at connection point D, which is further conjugated to R.

[0012] In another aspect, provided herein is a pharmaceutical composition comprising an ANGPTL8 RNAi agent described herein and one or more pharmaceutically acceptable excipients.

[0013] In another aspect, provided herein are methods of treating a patient in need of treatment for cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), abnormal renal function, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), or obesity, the methods comprising administering to the patient an ANGPTL8 RNAi agent or pharmaceutical composition thereof described herein.

[0014] In another aspect, the present invention provides an ANGPTL8 RNAi agent for use in therapy.The present invention also provides the use of an ANGPTL8 RNAi agent in the manufacture of a medicament for treating cardiovascular disease, diabetes, dyslipidemia (including high triglyceride levels), renal dysfunction, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), or obesity. DETAILED DESCRIPTION OF THE INVENTION

[0015] Such siRNAs may exhibit one or more of the following: improved knockdown in the liver, improved tissue exposure, improved exposure in liver hepatocytes, improved durability response, improved pharmacokinetic profile, reduced off-target effects, and / or improved toxicity profile, compared to other liver-targeting siRNAs, such as ANGPTL8 siRNAs containing different delivery ligands, different sequences, or different modified sequences, or compared to treatment with a vehicle control. Other embodiments of the ANGPTL8 RNAi agents herein may include one or more of reduced side effects, improved toxicity profile, improved safety profile, improved tolerability or compliance, and / or improved liver function tests, compared to statins or other standard therapies. Still other siRNAs herein may have other advantages, including, for example, improved and / or simplified synthesis, a synthetic process with fewer degradation products, or a combination thereof, in combination with any of the preceding advantages or as independent advantages.

[0016] The RNAi agents herein include a sense strand and an antisense strand, each of which is an oligonucleotide. In some embodiments, the RNAi agents described herein also include a delivery moiety. As used herein, "nucleotide" refers to an organic compound having a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) and a phosphate group. A "nucleotide" can function as a monomer unit of a nucleic acid polymer, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0017] As used herein, "oligonucleotide" refers to a short nucleic acid compound (e.g., less than about 100 nucleotides in length). An oligonucleotide may be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a duplex region. As a non-limiting set of examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (DsiRNA), or an antisense oligonucleotide (ASO).

[0018] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at the 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions of the nucleobase, sugar, or phosphate group.

[0019] As used herein, a "modified internucleotide linkage" refers to an internucleotide linkage that has one or more chemical modifications compared to a reference internucleotide linkage having a phosphodiester bond. The modified internucleotide linkage may be a non-naturally occurring linkage.

[0020] As used herein, "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. The modified nucleotide may be a non-naturally occurring nucleotide. The modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, the modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0021] The term "percent sequence identity" with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleic acid bases in a candidate sequence that are identical to the nucleotides, nucleosides, or nucleic acid bases in the reference nucleic acid sequence, after optimally aligning the sequences and, if necessary, introducing gaps or overhangs to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.1), including BLAST, BLAST-2, ALIGN, Clustal W2.0, Clustal X2.0, or Megalign (DNASTAR) software. In one embodiment herein, sequence identity is calculated using Clustal W2.0 or Clustal X2.0. In another embodiment, sequence identity is calculated using Clustal W2.0. In another embodiment, sequence identity is calculated using Clustal X2.0. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, and the fragment of nucleic acid sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.The percentage can be calculated by determining the number of positions where the same nucleotide, nucleoside, or nucleobase occurs in both sequences to determine 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 determine the percentage sequence identity. The output is the percent identity of the subject sequence to the query sequence. In some embodiments, the percent sequence identity is the percentage of nucleotide residues that are identical between the two strands using a PID3 calculation, which is the number of identical nucleotide residues divided by the total number of nucleotides in the shorter of the two sequences, multiplied by 100. See, e.g., Raghava, G., Barton, G.J. Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0022] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate. The 5'-phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates, such as 5'-methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide can have a phosphate analog at the 4'-carbon position of the sugar of the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of an oxymethyl group is attached to the sugar moiety (e.g., at the 4'-carbon) or an analog thereof. See, e.g., WO 2018 / 045317. Other modifications of the 5' end of oligonucleotides have also been developed (see, e.g., WO 2011 / 133871, U.S. Pat. No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0023] As used herein, "region of complementarity" refers to a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to allow hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotides herein comprise a targeting sequence having a region complementary to an mRNA target sequence.

[0024] As used herein, "duplex" in relation to nucleic acid or oligonucleotide, such as sense strand or antisense strand, refers to the structure formed through the hydrogen bond of complementary base pairing of two antiparallel sequences of nucleotides under conditions suitable for promoting such structure.Duplex can be formed even if there is no complete complementarity between the two strands or if there are abasic nucleotides.Duplex numbers, for example, as shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4, correspond to the specific sense strand and antisense strand that comprise a given RNAi agent.

[0025] RNA interference is a specialized cellular process that utilizes RISC to degrade RNA in a sequence-dependent manner. As used herein, an "RNAi agent" includes either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion of the antisense strand is used by Argonaute 2 (Ago2) endonuclease to cleave the target mRNA, or (b) a single-stranded oligonucleotide having a single-stranded antisense strand, where the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease to cleave the target mRNA. In some embodiments, the RNAi agent includes a delivery moiety.

[0026] As used herein,

[0027] [ka] A bond illustrated as denotes a point of attachment as described therein. For example, if a general variable, such as X, is described as being attached to a point of attachment E as shown below, this is intended to indicate that X is attached to the atom at the point of attachment (see scheme below).

[0028] [ka]

[0029] As used herein, "treatment" or "treating" refers to any process that can slow, control, delay, or halt the progression of a disorder or disease disclosed herein, and does not necessarily indicate the complete disappearance of all disorder or disease symptoms. Treatment includes the administration of an RNAi agent or pharmaceutical composition thereof to treat a disease or condition in a mammal, including a human.

[0030] "Effective amount" refers to the amount (duration and administration means) necessary to achieve the desired therapeutic result. The effective amount of an RNAi agent can vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the RNAi agent to induce the desired response in the individual. An effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the RNAi agent.

[0031] Provided herein is an RNAi agent for reducing ANGPTL8 gene expression, the RNAi agent comprising a delivery moiety of Formula I conjugated to R, wherein R is double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand;

[0032] [ka] R is conjugated, optionally via a linker, to connection point E of Formula I, and the sense strand and antisense strand form a duplex region, wherein the antisense strand comprises a region of complementarity to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and the sense strand and antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

[0033] Also provided herein is an RNAi agent for reducing ANGPTL8 gene expression, the RNAi agent comprising a delivery moiety of Formula Ia conjugated to R, wherein R comprises an antisense strand and a sense strand;

[0034] [ka] R is optionally conjugated to Formula Ia via a linker, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a region complementary to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, and wherein the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages. Disclosed herein is an RNAi agent for reducing ANGPTL8 gene expression, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, and wherein the antisense strand comprises at least 15 nucleotides as described in the antisense strand sequence disclosed herein, and wherein the sense strand and / or the antisense strand each optionally comprise one or more modified nucleotides and / or modified internucleotide linkages. In a further embodiment, the antisense strand comprises at least 15 nucleotides of the antisense strand sequence of Table 2A, Table 2B, Table 3A, Table 3B, or Table 4. In a further embodiment, the RNAi agent reduces ANGPTL8 gene expression in cells expressing ANGPTL8 by about 50% or more compared to a control. In a further embodiment, the RNAi agent reduces ANGPTL8 gene expression by reducing ANGPTL8 mRNA transcript levels, ANGPTL8 protein levels, or both.

[0035] In a further embodiment, the antisense strand is 15-25 nucleotides in length and / or the sense strand is 15-25 nucleotides in length. In a further embodiment, the antisense strand is 18-23 nucleotides in length. In a further embodiment, the sense strand is 18-21 nucleotides in length.

[0036] In further embodiments, the RNAi agent comprises at least 18 contiguous nucleotides of the antisense strand sequence shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4.

[0037] In a further embodiment, the antisense strand of the RNAi agent is 23 nucleotides in length. In yet a further embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the sense strand and the antisense strand comprise a sequence selected from the sequences shown in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4.

[0038] The sense strand and antisense strand of the RNAi agent disclosed herein do not need to be completely complementary.Therefore, in the RNAi agent disclosed herein, the duplex region between the sense strand and the antisense strand comprises 0, 1, 2 or 3 mismatches between the sense strand and the antisense strand.In a further embodiment, the duplex region between the sense strand and the antisense strand comprises 0, 1, 2 or 3 mismatches between the sense strand and the antisense strand.

[0039] In further embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides, such as 2'fluoro-modified nucleotides or 2'-O-methyl-modified nucleotides. In yet further embodiments of the RNAi agent disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide. In further embodiments, each nucleotide is a 2'fluoro-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0040] In further embodiments, the antisense strand has a sequence set forth in Table 2A, Table 2B, or Table 4, or a sequence having at least 90% sequence identity thereto, or an antisense strand sequence in Table 3A or Table 3B, or a sequence having at least 90% sequence identity thereto. In other embodiments, the antisense strand sequence or sense strand sequence in Table 2A or Table 2B or Table 3A or Table 3B or Table 4 is independently at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical thereto.

[0041] In further embodiments of the RNAi agents disclosed herein, the antisense strand is 23 nucleotides in length, and each nucleotide of the antisense strand is a modified nucleotide, and the 2' fluoro-modified nucleotide may appear at a position different from that shown in the sequence of Table 2A, Table 2B, Table 3A, Table 3B, or Table 4. In one embodiment, the 2' fluoro-modified nucleotide is a. positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand, or b. positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand, or c. positions 2, 3, 8, 14, and 16 from the 5' end of the antisense strand, or d. positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand; or e. Positions 2, 6, 14, and 16 from the 5' end of the antisense strand exists in.

[0042] In a further embodiment, the nucleotide that is not a 2' fluoro-modified nucleotide is a 2'-O-methyl-modified nucleotide.

[0043] In further embodiments of the RNAi agent disclosed herein, the sense strand and the antisense strand each independently comprise one or more modified internucleotide bonds, and each modified internucleotide bond is a phosphorothioate bond.In further embodiments, the sense strand and the antisense strand each independently comprise four phosphorothioate bonds.In yet further embodiments, the two terminal nucleotides at the 5'-end and 3'-end of each of the sense strand and the antisense strand are phosphorothioate bonds.

[0044] In other embodiments, the 5' nucleotide of the antisense strand contains a naturally occurring OH group or is modified to contain a phosphate group or phosphate analog. As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of the oligonucleotide in place of the 5'-phosphate. The 5' phosphate analog can contain a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide can have a phosphate analog at the 4'-carbon position of the sugar of the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of an oxymethyl group is attached to the sugar moiety (e.g., at the 4'-carbon) or an analog thereof. See, e.g., WO 2018 / 045317. Other modifications of the 5' end of oligonucleotides have also been developed (see, e.g., WO 2011 / 133871, U.S. Pat. No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0045] In further embodiments, the 5'-terminal nucleotide of the antisense strand may contain a further modification, wherein the 5'-terminal nucleotide contains a vinyl phosphonate, phosphate, or hydroxyl group at its 5' end. In other embodiments, the recited phosphate group at the 5'-end of the recited SEQ ID NO: is removed and replaced with OH. In other embodiments, the recited phosphate group at the 5'-end of the recited SEQ ID NO: is replaced with 5' vinyl phosphonate.

[0046] In further embodiments, one, two, or three mismatches are introduced into the sense strand sequences of Table 2A, Table 2B, Table 3A, Table 3B. In further embodiments, one, two, or both terminal nucleotides at the 5' end of the antisense strand are altered.

[0047] In some embodiments of the RNAi agents herein, the antisense strand comprises a first nucleic acid sequence having at least 90% sequence identity to the antisense sequence corresponding to a duplex number in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity to the sense sequence corresponding to the same duplex number in Table 2A, Table 2B, Table 3A, Table 3B, or Table 4. For example, in one embodiment, the antisense strand comprises a first nucleic acid sequence having at least 90% sequence identity to the antisense sequence corresponding to duplex number 1 in Table 2A, i.e., a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 6, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity to the sense sequence corresponding to duplex number 1 in Table 2A, i.e., SEQ ID NO: 1. In further embodiments, the 5' phosphate of the antisense strand is further modified / replaced with a 5' vinyl phosphonate or an OH group.

[0048] In a further embodiment, the 5' terminal nucleotide of the antisense strand is substituted such that the final sequence contains a vinyl phosphonate, a phosphate group, or an OH group.

[0049] In other embodiments, disclosed herein are RNAi agents having a delivery moiety of formula I conjugated to R,

[0050] [ka] R is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides having complementarity to the ANGPTL8 mRNA target sequence of SEQ ID NO: 511, wherein the sense strand and antisense strand form a region of complementarity of at least 15 nucleotides, wherein the sense strand and antisense strand are each independently 18-23 nucleotides in length, optionally, the sense strand and antisense strand each independently comprise one or more modified nucleotides, and optionally, the sense strand and antisense strand each independently comprise one or more modified internucleotide linkages, and R is optionally conjugated to Formula I via a linker. In further embodiments, the sense strand or antisense strand is selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 disclosed herein. In other embodiments, the antisense or antisense strand of the RNAi agent has an antisense strand sequence and / or a sense strand sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding sequence selected from Table 2A, Table 2B, Table 3A, Table 3B, or Table 4 herein.

[0051] In other embodiments, the RNAi agents disclosed herein comprise a linker. In yet other embodiments, R is conjugated to Formula I via a linker. In further embodiments, the linker comprises a linker of Formula II having connection points A and B, or the linker comprises a linker of Formula III having connection points C and D,

[0052] [ka] the RNAi agent comprises Formula I conjugated at connection point E to Formula II at connection point A, wherein Formula II is conjugated to a phosphate group at connection point B, wherein the phosphate group is conjugated to R; or b. The RNAi agent comprises Formula I conjugated at connection point E to Formula III at connection point C, which is conjugated to a phosphate group at connection point D, which is further conjugated to R.

[0053] In other embodiments, the RNAi agent comprises a linker, and R is conjugated to Formula I via the linker, wherein the linker comprises Formula III having attachment points C and D,

[0054] [ka] The RNAi agent comprises Formula I conjugated at attachment point C to Formula III, which is conjugated to a phosphate group at attachment point D, which is further conjugated to R.

[0055] The sense and antisense strands of RNAi agents can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis using phosphoramidite chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphotriester chemistry, or enzymatic synthesis. Automated commercially available synthesizers, such as the MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems, can be used. Phosphorothioate bonds can be introduced using sulfurizing reagents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione). The synthesis of modified oligonucleotides using similar techniques and commercially available modified amidite and controlled-pore glass (CPG) products is well known.

[0056] In yet another embodiment, the RNAi agent can reduce expression of the ANGPTL8 gene in hepatocytes. In another embodiment, the RNAi agent disclosed herein is for use in therapy. In a further embodiment, the use is for the treatment of dyslipidemia, such as high plasma triglyceride levels. In another embodiment, the RNAi agent disclosed herein is for use in the treatment of cardiovascular disease. In other embodiments herein, the RNAi agent is for use in the prevention of cardiovascular events. In a further embodiment, the cardiovascular event is myocardial infarction. In another embodiment, the use is for reducing hospitalizations related to cardiovascular disease or cardiovascular events. In another embodiment, the use is for treating non-alcoholic steatohepatitis (NAFLD). In a further embodiment, the NAFLD is non-alcoholic steatohepatitis (NASH). In another embodiment, the use is for reducing inhibition of lipoprotein lipase (LPL). In a further embodiment, the use is for increasing the catabolism of triglyceride-rich lipoproteins. In other embodiments, the RNAi agent is for use in treating liver disease in patients who would benefit from reducing the expression level of ANGPTL8. In other embodiments, the use is for any of the foregoing treatments after statin use has failed to control one or more symptoms, e.g., after failure to reduce one or more of elevated total C, LDL-C, apoB, and / or after failure to increase HDLC. In other embodiments, the use is for any of the foregoing treatments in patients who are statin intolerant, and in further embodiments, the use is for lowering LDL-C in patients who are statin intolerant. In further embodiments, the use is for any of the foregoing uses after dietary changes have failed to control one or more symptoms. In yet other embodiments, the use is for any of the foregoing uses as an adjunct to diet.

[0057] RNAi agent can be formulated into pharmaceutical composition.Therefore, this paper discloses pharmaceutical composition comprising RNAi agent disclosed herein and one or more pharmaceutically acceptable excipients.Pharmaceutical composition can be prepared by the method well known in the art (for example, Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A.Loyd et al., Academic Press).

[0058] In another embodiment is a use of an RNAi agent herein or any of the uses listed in the previous paragraph for the manufacture of a medicament for the treatment of dyslipidemia.

[0059] In another embodiment, it is a method of treating a patient in need of treatment for dyslipidemia, comprising administering to the patient an RNAi agent disclosed herein or a pharmaceutical composition thereof. In another embodiment, it is a method of treating a patient in need of treatment for dyslipidemia, or any of the above-listed uses in the patient, or a pharmaceutical composition thereof. In another embodiment, it is a method of treating a patient unable to achieve lipid levels following statin and / or dietary therapy, comprising administering to the patient an RNAi agent disclosed herein or a pharmaceutical composition thereof.

[0060] The RNAi agent can be administered to the patient intravenously or subcutaneously.

[0061] RNAi dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0062] Dosage values ​​can vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0063] In another embodiment, a method for reducing ANGPTL8 expression in a cell includes contacting the cell with an RNAi agent disclosed herein and incubating the cell for a period of time sufficient to reduce the level of ANGPTL8 mRNA by at least 50% compared to untreated or control-treated cells.

[0064] Certain abbreviations are defined as follows: "1,2-DCE" refers to 1,2-dichloroethane, "DCM" refers to dichloromethane, "DIEA" refers to N,N-diisopropylethylamine, "DMF" refers to N,N-dimethylformamide, "DMAP" refers to 4-dimethylaminopyridine, "DMTCl" refers to 4,4'-dimethoxytrityl chloride, "DPP4" refers to dipeptidyl peptidase, and "EDC" refers to 1-ethylhexyl 1,2-dichloroethane. "Hal" refers to N-acetylgalactosamine; "Hal" refers to N-acetyl-3-(3-dimethylaminopropyl)carbodiimide; "EtAc" refers to ethyl acetate; "GalNAc" refers to N-acetylgalactosamine; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; and "HBTU" refers to O-(benzotriazol-1-yl)-N,N,N',"N'-tetramethyluronium hexafluorophosphate," "HOBt" refers to 1-hydroxybenzotriazole hydrate, "HPRT" refers to hypoxanthine-guanine phosphoribosyltransferase, "IPA" refers to isopropanol and isopropyl alcohol, "LDHA" refers to lactate dehydrogenase-A, "MeCN" refers to acetonitrile, "MeOH" refers to methanol and methyl alcohol, "MWCO (molecular weight cut-off)" refers to molecular weight cut-off, "NHS" refers to N-hydroxysuccinimide, "OD (optical density)" refers to optical density, "PBS (phosphate-buffered saline)" refers to phosphate-buffered saline, "PhSiH3" refers to phenylsilane, "PTS (portable endotoxin testing system)" refers to a portable endotoxin testing system, and "siRNA (small interfering ribonucleic acid)" refers to a nucleotide sequence. "TEA" refers to triethylamine, "TFA" refers to trifluoroacetic acid, "THF" refers to tetrahydrofuran, "TLC (thin line chromatography)" refers to thin line chromatography, and "TMP" refers to 2,2,6,6-tetramethylpiperidine.

[0065] Delivery moieties comprising Formula I can be made by the following non-limiting synthetic steps and schemes.

[0066] Scheme 1

[0067] [ka] Step A in Scheme 1 depicts the cyclization of compound (1) using trimethyl trifluoromethanesulfonate in a solvent such as 1,2-DCE to give compound (2). Step B depicts the addition of hex-5-en-1-ol to compound (2) using trimethylsilyl trifluoromethanesulfonate in a solvent such as 1,2-DCE to give compound (3). Step C depicts the oxidation of compound (3) using a suitable oxidizing agent such as sodium periodate with a catalyst such as ruthenium(III) chloride to give compound (4).

[0068] Scheme 2

[0069] [ka]

[0070] [ka] Step A in Scheme 2 illustrates the amide coupling of compound (5) with tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate using HBTU and HOBt in a solvent such as DMF with a suitable base such as DIEA to obtain compound (6). Step B illustrates the basic hydrolysis of compound (6) using a base such as aqueous NaOH in a THF and MeOH solvent system to obtain compound (7). Step C illustrates the amide coupling of compound (7) with allyl 11-aminoundecanoate hydrochloride using HATU in a solvent such as DMF with a suitable base such as DIEA to obtain compound (8). Step D illustrates the acidic deprotection of compound (8) with TFA in a solvent such as DCM to obtain compound (9). Step E illustrates the amide coupling of compound (9) with compound (4) using EDC and HOBt in a solvent such as DCM to obtain compound (10). Step F illustrates the deprotection of compound (10) with tetrakis(triphenylphosphine)palladium and PhSiH in a solvent such as DCM to give compound (11). Step F illustrates the coupling of compound (11) with NHS using EDC in a solvent such as DCM to give compound (12).

[0071] Scheme 3

[0072] [ka]

[0073] [ka] Steps A-C of Scheme 3 are essentially similar to steps C-E of Scheme 2, starting from compound 7 to give compounds 13, 14, and 15. Step D shows the hydrogenation of compound 15 using palladium on carbon in a solvent such as MeOH to give compound 16. Step E is essentially similar to the preparation of step G of Scheme 2 to give compound 17.

[0074] Scheme 4

[0075] [ka] Steps AI of Scheme 4 consist of a series of amide couplings and deprotections using methods essentially similar to those found in Schemes 2 and 3, starting from compound (18) to give compound (27).

[0076] Scheme 5

[0077] [ka] Steps AC of Scheme 5 show a method essentially similar to that found in Steps GI of Scheme 4, starting from compound (24) to give compound (30).

[0078] Scheme 6

[0079] [ka] Step A of Scheme 6 depicts the protection of compound 31 using DMTCl with a suitable base such as DIEA in a solvent such as DCM to give compound 32. Step B depicts the amide coupling of compound 32 with piperidin-4-ylmethanol using HBTU and HOBt with TMP in a solvent such as DCM to give compound 33. Step C depicts the deprotection of compound 33 with 20% piperidine in DMF to give compound 34.

[0080] Scheme 7

[0081] [ka] Step A of Scheme 7 is essentially similar to Step A of Scheme 2, and involves the coupling of compound 16 with compound 34 to give compound 35. Step B illustrates the addition of succinic anhydride to compound 35 with a base system of TEA and DMAP in a suitable solvent such as DCM to form compound 36. Step C illustrates the loading of compound 36 onto a resin with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and a base such as DIEA in a solvent system such as MeCN and DCM to give compound 37.

[0082] Preparation 1 Methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate

[0083] [ka] To a solution of methyl (5-acetamido-3,4,6-triacetoxy-tetrahydropyran-2-yl)acetate (9.00 g, 23.1 mmol) in 1,2-DCE (46 mL) is added trimethylsilyl trifluoromethanesulfonate (6.5 mL, 35 mmol). The mixture is heated to 50 °C and stirred for 18 h. After this time, the mixture is diluted with DCM (200 mL), washed with saturated aqueous NaHCO (200 mL) and saturated aqueous sodium chloride (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (6.434 g, 84%). ES / MS m / z 330 (M+H).

[0084] Preparation 2 (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate methyl

[0085] [ka] To a solution of methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate (30.43 g, 92.42 mmol) in 1,2-DCE (231 mL) is added hex-5-en-1-ol (22.2 mL, 185 mmol), followed by activated powdered 4 Å molecular sieves (15.6 g). The suspension is stirred at ambient temperature for 30 min, then trimethylsilyl trifluoromethanesulfonate (19 mL, 101.9 mmol) is added. The mixture is stirred at ambient temperature for 18 h. After this time, the solution is filtered through diatomaceous earth and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 30-100% EtOAc / hexane to give the title compound (34.76 g, 86%). ES / MS m / z 430.4 (M+H).

[0086] Preparation 3 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid

[0087] [ka] A solution of methyl (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate (34.76 g, 80.93 mmol) in MeCN (174 mL) and DCM (174 mL) is cooled to 0 °C. A solution of sodium periodate (22.4 g, 104.7 mmol) is added, and stirring is continued at 0 °C for 10 minutes. After this time, ruthenium(III) chloride (270 mg, 1.3 mmol) is added, and the mixture is stirred while warming to ambient temperature. After stirring for 2 hours, additional sodium periodate (66 g, 308.4 mmol) is added, and stirring is continued for 18 hours. After this time, the mixture is extracted with 3:1 CHCl:IPA (2 × 500 mL), washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM to give the title compound (29.75 g, 82%). ES / MS m / z 448.4 (M+H).

[0088] Preparation 4 Benzyl 6-aminohexanoate hydrochloride

[0089] [ka] To a suspension of 6-aminohexanoic acid (5.00 g, 38.1 mmol) in THF (38 mL) is added benzyl alcohol (47 mL, 453.7 mmol) and the mixture is cooled to 0 °C. Thionyl chloride (8.6 mL, 120 mmol) is added dropwise and the mixture is stirred for 18 hours while warming to ambient temperature. After this time, ether (166 mL) is added and the reaction vessel is transferred to a -20 °C freezer for 1 hour. After this time, the solid precipitate is collected by filtration to give the title compound (8.57 g, 81%). ES / MS m / z 222 (M+H).

[0090] Preparation 5 Benzyl 11-aminoundecanoate hydrochloride

[0091] [ka] The title compound is prepared from 11-aminoundecanoic acid in a manner essentially similar to the method of Preparation 4. ES / MS m / z 292.2 (M+H).

[0092] Preparation 6 Allyl 11-aminoundecanoate hydrochloride

[0093] [ka] 11-Aminoundecanoic acid (9.00 g, 44.7 mmol) in allyl alcohol (42 mL) is placed in a vessel and the mixture is cooled to 0 °C. Thionyl chloride (6.5 mL, 89.4 mmol) is added, and the mixture is stirred for 18 hours while warming to ambient temperature. After this time, the mixture is concentrated in vacuo, and ether (200 mL) is added to the residue to give a white suspension. The mixture is stirred at ambient temperature for 10 minutes, and the solid precipitate is collected by filtration to give the product (12.0 g, 97%). ES / MS m / z 242.2 (M+H).

[0094] Preparation 7 (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid

[0095] [ka] To a stirred solution of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-hydroxy-propanoic acid (40 g, 0.122 mol) in dry DCM (400 mL) is added DIEA (64 mL, 0.366 mol) under an inert atmosphere at 0° C. To this is slowly added a solution of DMTCl (49.6 g, 0.146 mol) in DCM (200 mL). The resulting reaction mixture is allowed to reach ambient temperature and stirred for 16 hours. After this time, the reaction mixture is diluted with water (12.5 vol) and extracted with DCM (25 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting crude product is washed with 10% EtOAc / hexanes (12.5 vol) and dried under vacuum to afford the title compound as a light brown solid (62 g, crude). This material was used in the next step without further purification. TLC indicated 5% MeOH / CHCl (R f is 0.5), and UV is 254 nM.

[0096] Preparation 8 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]carbamate

[0097] [ka] To a stirred solution of (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid (62 g, 0.103 mol) in DCM (750 mL) is slowly added HBTU (78.3 g, 0.206 mol), HOBt (27.9 g, 0.206 mol), and piperidin-4-ylmethanol (15.4 g, 0.134 mol), followed by TMP (15 mL, 0.113 mol) under an inert atmosphere at 0° C. The resulting reaction mixture is allowed to reach ambient temperature and stirred for 4 hours. After this time, the reaction mixture is diluted with water (8 vol) and extracted with DCM (15 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 20-40% EtOAc / hexanes and 1% MeOH / DCM to give the title compound (40 g, 52% over two steps). 1 H NMR(DMSO-d6)δ 7.88(br d,J=7.5Hz,2H),7.79-7.59(m,3H),7.45-7.12(m,13H),6.92-6.76(m,4H),4.79-4.44(m,2H),4.32(br d.

[0098] Preparation 9 (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidyl]propan-1-one

[0099] [ka] A solution of 20% piperidine in DMF (400 mL) is slowly added to 9H-fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]carbamate (40 g, 0.055 mol) under an inert atmosphere at 0 °C. The resulting reaction mixture is stirred at ambient temperature for 1 h. After this time, the mixture is diluted with water (15 vol) and extracted with EtOAc (30 vol). The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 1-8% MeOH / DCM to afford the title compound as an off-white solid (13 g, 47%). ES / MS m / z 1009.5 (2M+H).

[0100] Preparation 10 Methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate

[0101] [ka] To a flask containing S-4-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (7.00 g, 26.8 mmol) and HOBt (4.16 g, 30.8 mmol) is added DMF (179 mL) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (11.7 g, 30.9 mmol). DIEA (14 mL, 80.3 mmol) is added and the mixture is stirred at ambient temperature for 5 minutes. After this time, tert-butyl N-[2-[2-(ter Add t-butoxycarbonylamino)ethylamino]ethyl]carbamate (8.94 g, 29.5 mmol) in one portion and continue stirring at ambient temperature. After stirring for 18 h, the mixture is diluted with EtOAc (400 mL), washed with water (2 × 400 mL) and saturated aqueous sodium chloride solution (400 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 40–100% EtOAc / hexane to give the title compound (13.01 g, 89%). ES / MS m / z 547.40 (M+H).

[0102] Preparation 11 (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid

[0103] [ka] A flask is charged with methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate (13.01 g, 23.8 mmol), THF (120 mL), and MeOH (120 mL). 1N NaOH (71 mL, 71 mmol) is added, and the mixture is stirred at ambient temperature. After 1 hour, the mixture is concentrated in vacuo and redissolved in water (300 mL). 5N HCl (12 mL) is added to bring the pH to 4. The mixture is extracted with DCM (3 x 300 mL), and the combined organic layers are washed with saturated aqueous sodium chloride (1 L), dried over sodium sulfate, filtered, and concentrated to give the title compound (12.41 g, 98%). ES / MS m / z 531.60 (MH).

[0104] Preparation 12 Allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoate

[0105] [ka] A flask containing (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (500 mg, 0.94 mmol) and allyl 11-aminoundecanoate hydrochloride (313 mg, 1.13 mmol) was charged with DMF (6.25 mL) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (428 mL). g, 1.12 mmol) is added. After adding DIEA (0.5 mL, 3 mmol), the mixture is stirred at ambient temperature for 18 h. After this time, the mixture is diluted with EtOAc (200 mL), washed with water (3 x 200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 40-100% EtOAc / hexane to give the title compound (687 mg, 97%). 1 H NMR(DMSO-d6)δ7.78-7.64(m, 1H), 6.98-6.7(m, 2H), 5.96-5.84(m, 1H), 5.31-5.25(m, 1H), 5.23-5.17(m, 1H), 4.56-4.50(m, 2H), 3.88-3.67(m , 1H), 3.30-3.19(m, 4H), 3.11-2.91(m, 6H), 2.35-2.12(m, 4H), 1.88-1 .65(m, 2H), 1.58-1.47(m, 2H), 1.46-1.30(m, 30H), 1.30-1.18(m, 12H).

[0106] Preparation 13 Allyl (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)undecanoate

[0107] [ka] To a solution of allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoate (687 mg, 0.91 mmol) in DCM (15 mL) is added TFA (15 mL). The mixture is stirred at room temperature. After 1.5 h, the mixture is concentrated in vacuo. The residue is dissolved in MeOH and loaded onto an ion exchange cartridge. The cartridge is eluted with MeOH (150 mL) followed by 7N NH3 / MeOH (150 mL). The basic fractions are concentrated in vacuo to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0108] Preparation 14 Allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]undecanoate

[0109] [ka] A flask is charged with 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid (489 mg, 1.09 mmol) and allyl S-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)undecanoate (150 mg, 0.33 mmol). DCM (3.35 mL) is added, followed by 1-hydroxybenzotriazole monohydrate (164 mg, 1.07 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (206 mg, 1.07 mmol). The mixture is stirred at ambient temperature for 18 h. After this time, the solution is diluted with EtOAc (100 mL) and washed with saturated NaHCO3 (2 × 100 mL), saturated aqueous NH4Cl (100 mL), and saturated aqueous sodium chloride (100 mL). The organic layer is dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (424 mg, 74%). ES / MS m / z 872.80 (M+2H) / 2.

[0110] Preparation 15 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]undecanoic acid

[0111] [ka] To a solution of allyl 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate (354 mg, 0.20 mmol) in DCM (2 mL) was added tetrakis(triphenylphosphine)palladium (29 mg, 0.02 mmol), followed by PhSiH3 (51 µL, 0.41 mmol). The mixture was stirred at ambient temperature for 2 h and then diluted with saturated aqueous NaHCO3 (100 mL). 1 N NaOH (15 mL) was added to bring the pH to approximately 10. The aqueous solution is washed with DCM (3 x 100 mL) and then acidified with concentrated HCl (5 mL) and then 5N aqueous HCl (15 mL). The aqueous layer is extracted with DCM (100 mL), and the organic layer is dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-20% MeOH / DCM to give the title compound (151 mg, 44%). ES / MS m / z 852.60 (M+2H) / 2.

[0112] Preparation 16 (2,5-Dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0113] [ka] To a reaction vial, 11-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid (50 mg, 0.03 mmol), N-hydroxysuccinimide (5 mg, 0.04 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8 mg, 0.04 mmol) were added. DCM (0.3 mL) was added, and the mixture was stirred at ambient temperature. After 18 h, the mixture is loaded directly onto a silica gel cartridge and the crude mixture is purified by silica gel flash chromatography eluting with 0-10% MeOH / DCM to give the title compound (49 mg, 93%). ES / MS m / z 901.40 (M+2H) / 2.

[0114] Preparation 17 Benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoate

[0115] [ka] The title compound is prepared from (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid and benzyl 6-aminohexanoate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 736.40 (M+H).

[0116] Preparation 18 Benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoate tris(trifluoroacetic acid)

[0117] [ka] To a solution of benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoate (15.47 g, 21.02 mmol) in DCM (105 mL) is added TFA (16 mL, 210.2 mmol). The mixture is stirred at ambient temperature for 24 h. After this time, additional TFA (16 mL, 210.2 mmol) is added and stirring is continued for another 2 h. After this time, the mixture is concentrated in vacuo. The resulting residue is azeotroped with toluene (2 × 30 mL). The resulting oil is further dried in a vacuum oven at 40 °C for 4 h to give the title compound (28.08 g, 58% purity, 99+%, taking into account residual toluene). ES / MS m / z 436.40 (M+H). The compound is dissolved in 70 mL of DMF to make a 0.3 M solution which is used in the next step.

[0118] Preparation 19 Benzyl 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0119] [ka] The title compound is prepared from 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid and benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoate tristrifluoroacetate in a manner essentially similar to that of Preparation 10. ES / MS m / z 862 (M+2H) / 2.

[0120] Preparation 20 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]hexanoic acid

[0121] [ka] Palladium on carbon (1.90 g, 0.89 mmol, 5% by weight, 50% wet) was placed in a round-bottom flask, and the vessel was evacuated and refilled with nitrogen three times. A solution of benzyl 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate (15.41 g, 8.94 mmol) in MeOH (178 mL) was added via syringe. The flask was evacuated and refilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen for 18 h. After this time, the mixture is filtered through diatomaceous earth and the filtrate is concentrated in vacuo to give the title compound (13.85 g, 95%). ES / MS m / z 817.2 (M+2H) / 2.

[0122] Preparation 21 (2,5-Dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0123] [ka] The title compound is prepared from 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner essentially similar to that of Preparation 16. ES / MS m / z 866.20 (M+2H) / 2.

[0124] Preparation 22 Benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate

[0125] [ka] The title compound is prepared from tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate and (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid in a manner essentially similar to that of Preparation 12. ES / MS m / z 623.6 (M+H).

[0126] Preparation 23 Benzyl (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxopentanoate tris(trifluoroacetate) salt

[0127] [ka] The title compound is prepared from benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate in a manner essentially similar to that of Preparation 18. ES / MS m / z 323.2 (M+H).

[0128] Preparation 24 Benzyl (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoate

[0129] [ka] The title compound is prepared from 5-(tert-butoxycarbonylamino)pentanoic acid and benzyl (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoate tris(trifluoroacetate) salt in a manner essentially similar to that of Preparation 10. ES / MS m / z 920.6 (M+H).

[0130] Preparation 25 Benzyl (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetic acid) salt

[0131] [ka] The title compound is prepared from benzyl (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoate in a manner essentially similar to that of Preparation 18. ES / MS m / z 620.4 (M+H).

[0132] Preparation 26 Benzyl (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoate

[0133] [ka] The title compound is prepared from 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid and benzyl (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetate) salt in a manner essentially similar to that of Preparation 10. ES / MS m / z 954.80 (M+2H) / 2.

[0134] Preparation 27 (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoic acid

[0135] [ka] A round-bottom flask was charged with palladium on carbon (467 mg, 0.22 mmol, 5% by weight, 50% wet). The flask was evacuated and refilled with nitrogen three times. A solution of benzyl (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoate (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via syringe, followed by the addition of three drops of acetic acid. The flask was evacuated and refilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After 2 hours, the mixture is filtered through diatomaceous earth and the filtrate is concentrated in vacuo to give the title compound (3.99 g, 99+%). ES / MS m / z 909.6 (M+2H) / 2.

[0136] Preparation 28 Benzyl 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0137] [ka] The title compound is prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 6-aminohexanoate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1011.6 (M+2H) / 2.

[0138] Preparation 29 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]hexanoic acid

[0139] [ka] A round-bottom flask was charged with palladium on carbon (24 mg, 0.01 mmol, 5% by weight, 50% wet), the flask was evacuated, and backfilled with nitrogen. A solution of benzyl 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate (222 mg, 0.11 mmol) in MeOH (2.2 mL) was added via syringe, followed by the addition of 3 drops of acetic acid. The flask is evacuated and backfilled with 1 atm of hydrogen, and the mixture is stirred at ambient temperature under 1 atm of hydrogen. After 5 hours, the flask is purged with nitrogen, and the mixture is filtered through diatomaceous earth. The filtrate is concentrated in vacuo to give the title compound (180 mg, 85%). ES / MS m / z 966.2 (M+2H) / 2.

[0140] Preparation 30 (2,5-Dioxopyrrolidin-1-yl)6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoate

[0141] [ka] The title compound is prepared from 6-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid in a manner essentially similar to that of Preparation 16. ES / MS m / z 1014.6 (M+2H) / 2.

[0142] Preparation 31 Benzyl 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0143] [ka] The title compound is prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 11-aminoundecanoate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1046.6 (M+2H) / 2.

[0144] Preparation 32 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]undecanoic acid

[0145] [ka] Palladium on carbon (35 mg, 0.02 mmol, 5% by weight, 50% wet) was added to a round-bottom flask, which was then evacuated and refilled with nitrogen three times. A solution of benzyl 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate (285 mg, 80% purity, 0.11 mmol) was added via syringe. The vessel was evacuated and refilled with 1 atm of hydrogen, and the mixture was stirred at ambient temperature under 1 atm of hydrogen. After stirring for 3 hours, the flask is purged with nitrogen and the mixture is filtered through diatomaceous earth. The filtrate is concentrated to give the title compound (213 mg, 79% purity, 77%). ES / MS m / z 1001.20 (M+2H) / 2.

[0146] Preparation 33 (2,5-Dioxopyrrolidin-1-yl)11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoate

[0147] [ka] The title compound is prepared from 11-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]undecanoic acid in a manner essentially similar to that of Preparation 16. ES / MS m / z 1050(M+2H) / 2

[0148] Preparation 34 [5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentoxy]-3,4-diacetoxytetrahydropyran-2-yl]acetate methyl ester

[0149] [ka] The title compound is prepared from 6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoic acid and (2S)-2-amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidyl]propan-1-one in a manner essentially similar to that of Preparation 10. ES / MS m / z 1059.2 (M-2H) / 2.

[0150] Preparation 35 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propanoyl]-4-piperidyl]methoxy]-4-oxo-butanoic acid

[0151] [ka] [5-acetamido-6-[5-[2-[[(4S)-4-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl in DCM (11 mL) To a solution of methyl 2-[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxo-pentoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]acetate (1.194 g, 0.56 mmol), succinic anhydride (113 mg, 1.13 mmol), TEA (0.4 mL, 3 mmol), and DMAP (213 mg, 1.69 mmol) are added. The mixture is stirred at ambient temperature for 1 hour. After this time, the mixture is diluted with saturated NH4Cl (200 mL) and extracted with DCM (3 x 200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers are combined, dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue is purified by silica gel flash chromatography eluting with 0-40% MeOH / DCM, and the resulting product is dried in a vacuum oven at 40 °C for 3 h to give the title compound (1.081 g, 86%). ES / MS m / z 1109.60 (M-2H) / 2.

[0152] Preparation 36 Resin Loading

[0153] [ka] A solution of 4-[[1-[(2S)-2-[6-[[(2S)-2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[bis[2-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]hexanoylamino]-3-[bis(4-methoxyphenyl)-phenyl-methoxy]propanoyl]-4-piperidyl]methoxy]-4-oxo-butanoic acid (1.00 g, 0.61 mmol) in MeCN (6 mL) and DCM (1 mL) is transferred to a resin loading cartridge. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (386 mg, 0.97 mmol) and DIEA (0.25 mL, 0.48 mmol) were added to the vessel, and the cartridge was shaken at ambient temperature for 5 minutes. After this, 1000 Å LCAA controlled pore glass resin (5.39 g, 90 μmol / g loading, purchased from ChemGenes) was added, and the mixture was shaken at ambient temperature for 18 hours. After this, the cartridge was drained by suction, and the resin was washed with DCM (10 mL) by shaking for 10 minutes. The cartridge was drained, and the washing and draining procedure was repeated with 10% MeOH / DCM (10 mL) and EtO (10 mL). After draining, a solution of acetic anhydride (6.4 mL), pyridine (20 mL), and TEA (0.22 mL) is added, and the cartridge is shaken for 2 hours. After this, the cartridge is drained, and the above washing and draining procedure is repeated using DCM (10 mL), 10% MeOH / DCM (10 mL), and diethyl ether (10 mL). After draining, the resin is dried under vacuum for 30 minutes. The resin loading is measured using a standard trityl assay. The resin loading was calculated to be 34.7 μmol / g.

[0154] Preparation 37 Benzyl 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]acetate

[0155] [ka] The title compound is prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 2-(2-aminoethoxy)acetate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1005.2 (M+2H / 2).

[0156] Preparation 38 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]acetic acid

[0157] [ka] 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]benzyl acetate (0.120 mmol, 240 mg) is mixed with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture is hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 48 minutes, filtered through diatomaceous earth, and concentrated in vacuo to give the title compound as a gray solid (187 mg, 82%). ES / MS m / z 960.0 (M+2H / 2).

[0158] Preparation 39 (2,3,5,6-Tetrafluorophenyl)2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]acetic acid

[0159] [ka] To a mixture of 2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]acetic acid (0.096 mmol, 184 mg) and DIEA (0.765 mmol, 140 μL) in DCM (3.0 mL) was added dropwise (2,3,5,6-tetrafluorophenyl)-2,2,2-trifluoroacetic acid (0.383 mmol, 100 mg). The mixture was stirred at ambient temperature for 16 hours. The reaction mixture is directly purified by silica gel flash chromatography eluting with 0% to 50% MeOH / DCM to give the title compound as a tan solid (197 mg, 99%). ES / MS m / z 1034.0 (M+2H / 2).

[0160] Preparation 40 Benzyl 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetate

[0161] [ka] The title compound is prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]acetate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1049.0 (M+2H / 2).

[0162] Preparation 41 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid

[0163] [ka] 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]benzyl acetate (0.118 mmol, 247 mg) is mixed with 5% Pd / C (1.17 mmol, 124 mg) in MeOH (12.0 mL). The mixture is hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 1 hour, filtered through diatomaceous earth, and concentrated in vacuo to give the title compound as a gray solid (227 mg, 96%). ES / MS m / z 1004.0 (M+2H / 2).

[0164] Preparation 42 (2,3,5,6-Tetrafluorophenyl)2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxopentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid

[0165] [ka] To a mixture of 2-[2-[2-[2-[[(2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]acetic acid (0.111 mmol, 222 mg) and DIEA (0.883 mmol, 154 μL) in DCM (3.0 mL), (2,3,5,6-tetrafluorophenyl)-2,2,2-trifluoroacetic acid (0.443 mmol, 116 mg) was added dropwise. The mixture is stirred at ambient temperature for 16 hours. The reaction mixture is directly purified by silica gel flash chromatography eluting with 0% to 50% MeOH / DCM to give the title compound as a tan solid (174 mg, 73%). ES / MS m / z 1078.2 (M+2H / 2).

[0166] Example 1: Conjugation Protocol For the synthesis of the GalNAc-conjugated sense strand, the sense strand bearing a 3'C6-NH2 functional group was first synthesized using standard phosphoramidite chemistry. A stock solution of GalNAc ligand-NHS ester (10 mmol / L in acetonitrile, 1 equivalent) was prepared. Borate buffer (10% v / v, 20x) was added to the oligonucleotide C6-NH2 sense strand in an Eppendorf tube, followed by the addition of GalNAc ligand (5 equivalents). The mixture was shaken at ambient temperature for 16 hours. After this time, the mixture was transferred to a 15 mL Falcon tube, ammonium hydroxide (28% by weight) was added, and the mixture was shaken at ambient temperature for 2 hours. The ammonia was then removed in vacuo. The residue was purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20 mM NaH2PO4, Solvent B: 15% MeCN / 20 mM NaH2PO4, 1 M NaBr; 35-55% B over 5 CV at 8 mL / min, column temperature 60 °C. Desired fractions were pooled and desalted by Eppendorf centrifugation or spin filtration using a desalting column. After desalting, the material was collected and the OD and volume were measured to obtain the concentration.

[0167] Alternatively, conjugation to the 5' position of the sense strand by immobilizing the GalNAc ligand on a microporous polystyrene resin or controlled pore glass and synthesizing it using established solid-phase oligonucleotide synthesis methods with a 5'-CEβ-cyanoethyl) phosphoramidite.

[0168] Alternatively, the GalNAc ligand was converted to a suitable phosphoramidite and delivered to the 5' position of the sense strand using standard phosphoramidite chemistry.

[0169] Example 2: Annealing To generate sense and antisense siRNA duplexes, the following procedure was performed: To the Falcon tube containing the oligonucleotide sense-GalNAc conjugate, the corresponding antisense oligonucleotide (1 equivalent) was added, vortexed for 10 seconds, and then spin-filtered through a 100K MWCO Amicon filter unit to remove particulates. The filtrate was collected and concentrated in vacuo in a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2μ filter, and the OD and volume were measured to obtain the concentration.

[0170] Endotoxin testing was performed using Limulus amebocyte lysate on an Endosafe®-nexgen PTS device.

[0171] Table 1 - Exemplary molecules synthesized using the conjugation and annealing protocols described above. [Table 1]

[0172] Example 3: General Procedure for Oligo Synthesis Using GalNAc-Functionalized CPG Oligos were synthesized using phosphoramidite chemistry on a MerMade™ 12 instrument. The sense strand was synthesized from a pre-functionalized GalNAc solid support, and the antisense strand was synthesized using a standard support pre-loaded with the first nucleotide of the oligo sequence. The oligos were cleaved and deprotected using concentrated ammonium hydroxide solution (28% by weight) and purified by ion exchange chromatography using the conditions described above. Desalting, annealing, and endotoxin testing were performed.

[0173] Antisense oligonucleotide sequences were designed using 15 to 50 nucleotides of the antisense strand described herein, including those in Tables 2A, 2B and 3A, 3B.

[0174] Exemplary antisense strand sequences of 23 nucleotides in length are shown below in Tables 2A and 2B, which can optionally be further modified and synthesized and incorporated into RNAi agents as described herein.

[0175] Table 2A: Modified ANGPTL8 sequences for GalNAc-RNAi agents [Table 2]

[0176] Table 2B: Modified ANGPTL8 sequences for GalNAc-RNAi agents [Table 3]

[0177] Table 3A: Modified sense and antisense strands for GalNAc-ANGPTL8 [Table 4-1]

[0178] [Table 4-2]

[0179] [Table 4-3]

[0180] [Table 4-4]

[0181] [Table 4-5]

[0182] [Table 4-6]

[0183] [Table 4-7]

[0184] [Table 4-8] P or [Phos] indicates 5' phosphate m indicates a 2'O-methyl modified ribose on the listed nucleotide f indicates a 2'F modified ribose on the listed nucleotide * indicates a phosphorothioate bond (instead of a phosphodiester bond)

[0185] Table 3B: Modified sense and antisense strands for GalNAc-ANGPTL8 RNAi agents herein [Table 5-1]

[0186] [Table 5-2]

[0187] [Table 5-3]

[0188] [Table 5-4]

[0189] [Table 5-5]

[0190] [Table 5-6]

[0191] [Table 5-7] m indicates a 2'O-methyl modified ribose on the listed nucleotide f indicates a 2'F modified ribose on the listed nucleotide * indicates a phosphorothioate bond (instead of a phosphodiester bond)

[0192] Table 4: Sense and antisense strands of GalNAc-ANGPTL8 [Table 6-1]

[0193] [Table 6-2]

[0194] [Table 6-3]

[0195] [Table 6-4]

[0196] Human ANGPTL8 transcript NM_018687.7 SEQ ID NO: 511:

[0197] [Table 7]

[0198] Example 4: In vitro knockdown of human ANGPTL8 in AAV-ANGPTL8-humanized mouse primary hepatocytes (MPH) using GalNAc-conjugated ANGPTL8 siRNA Knockdown of ANGPTL8 expression by LYGal1-conjugated ANGPTL8 siRNA was assayed using the following procedure. Mouse primary hepatocytes (MPH) were freshly isolated from AAV-ANGPTL8-humanized mice and added to Corning 96-well plates at 15,000 cells / well, and siRNA was added directly to the wells. For single-point (SP) screening, 1 μM (1,000 nM) GalNAc-conjugated siRNA was used. To generate concentration / dose-response curves, final concentrations of GalNAc-conjugated siRNA were used: 1,000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM.

[0199] Treated cells were lysed, and RNA was isolated directly into 96-well plates using Quick-RNA96Kit (Zymo Research). Eluted RNA was used immediately or frozen and stored. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) in a thermocycler using the following steps: 37°C for 30 minutes, 95°C for 5 minutes, and a 4°C hold. Polymerase chain reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using the following cycle temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute.

[0200] Human ANGPTL8 levels were normalized to mouse Rplp0 (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression compared to vehicle-treated control cells. A four-parameter fit model using XLFit was used to calculate IC 50 values ​​were calculated.

[0201] Table 5 shows the results of single-dose screening in AAV-ANGPTL8-humanized mouse primary hepatocytes by free uptake with the indicated GalNAc-conjugated ANGPTL8 siRNA. Data were expressed as percent message knockdown relative to untreated cells. IC of top hits from single-point screening. 50 and percent maximum knockdown, followed by concentration / dose response curves are also included.

[0202] Table 5: IC with percent knockdown of single dose screening and maximum percent knockdown of top hits from single point screening in AAV-ANGPTL8 humanized mouse primary hepatocytes. 50 [Table 8-1]

[0203] [Table 8-2]

[0204] [Table 8-3]

[0205] Example 5: In vitro knockdown of human ANGPTL8 in Hep3B cells using GalNAc-conjugated ANGPTL8 siRNA Knockdown of ANGPTL8 expression by LYGal1-conjugated ANGPTL8 siRNA was assayed using the following procedure: 0.3 μl / well of transfection reagent RNAiMAX (Life Technologies) was mixed with siRNA in a Corning 96-well plate, followed by addition of Hep3B cells (ATCC) at 8,000 cells / well. To generate a concentration / dose-response curve, GalNAc-conjugated siRNA concentrations of 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.137, 0.046, 0.015, 0.005, and 0.0017 nM were used.

[0206] Treated cells were lysed, and RNA was isolated directly into 96-well plates using Quick-RNA96Kit (Zymo Research). The eluted RNA was used immediately or frozen. cDNA was synthesized using Fast Advanced RT Master Mix (Invitrogen) in a thermocycler using the following steps: 37°C for 30 minutes, 95°C for 5 minutes, and a 4°C hold. Polymerase chain reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using the following cycle temperatures and times: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute.

[0207] Human ANGPTL8 levels were normalized to human RPLP0 (Life Technologies) and represent the relative knockdown of human ANGPTL8 mRNA expression compared to vehicle-treated control cells. A four-parameter fit model using XLFit was used to calculate IC 50 values ​​were calculated.

[0208] Table 6 shows the IC calculated from the concentration / dose-response curves in Hep3B cells transfected with the indicated ANGPTL8 siRNAs. 50 Results are shown for percent maximum knockdown and percent maximum knockdown. In most cases, duplexes showed subnanomolar IC 50This resulted in a knockdown of over 90%.

[0209] Table 6: IC of ANGPTL8 message in Hep3B cells by transfection reagent RNAiMAX with the indicated ANGPTL8 siRNAs 50 and maximum knockdown percentage [Table 9]

[0210] Example 6: In vivo single dose screening GalNAc-siRNA (n = 16) was tested in male C57bl / 6 mice (Taconic Farms). Each siRNA was tested in a single study. Mice were administered an adeno-associated virus (AAV) vector containing a plasmid carrying the albumin promoter and the coding sequence of human ANGPTL8 (NM_018687.7) (Vector BioLabs) via retroorbital injection. 14 days after AAV administration, blood was collected from the mice via the retroorbital sinus. Serum was prepared from the blood, and triglycerides were measured using a COBAS clinical chemistry analyzer (Roche) and ANGPTL4 / 8 were measured by ELISA (Meso Scale Diagnostics). Mouse body weights were measured 22 days after AAV administration. Mice were assigned to groups (n = 6 / group) with similar body weights, serum triglyceride levels, and serum ANGPTL4 / 8 levels. Mice were subcutaneously administered either PBS or GalNac-siRNA test substance at a dose of 5 mg / kg. Seven days after subcutaneous injection, blood was collected from all mice, and serum was analyzed for triglycerides. Fourteen days after subcutaneous injection, mice were euthanized under isoflurane anesthesia, blood was collected, and serum was analyzed for triglycerides. Livers were collected from mice and frozen in liquid nitrogen. Triglycerides as a percentage change from time-matched PBS were calculated as ((triglycerides - triglycerides in PBS group) / (triglycerides in PBS group)). *The ratio was calculated as 100. Livers were homogenized in TriZol (Invitrogen) using Lysing Matrix D bead tubes on a FastPrep-24 (MP Bio). Chloroform was added, and the aqueous phase was mixed with ethanol to precipitate RNA. RNA was isolated on a column using the PureLink Pro96 Total RNA Purification Kit (Invitrogen) according to the manufacturer's protocol and quantified on a NanoDrop (ThermoFisher). Equal amounts (1 μg) of RNA were reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) on a Mastercycler Nexus (Eppendorf). The thermocycler settings were 25°C for 10 minutes, 37°C for 2 hours, and then 85°C for 5 minutes. The template cDNA was combined with Taqman Universal Master Mix and Assays on Demand primer / probe set, and RT-PCR was performed on a QuantStudio Pro7 (ThermoFisher) with the following parameters: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fold change (FC) was calculated as follows: The C value of mouse Rplp0 was subtracted from the C value of human ANGPTL8 to obtain a delta C value. Delta-delta C values ​​were then calculated by subtracting the delta C value of the untreated sample (average of the PBS controls) from the delta C value of each test sample. Fold change was calculated by taking the base-2 logarithm of the negative delta-delta C value. The fold change was multiplied by 100 to calculate the percent remaining mRNA. The data are shown in Table 7.

[0211] Table 7: C57 mouse hANGPTL8 AAV in vivo single dose screening [Table 10]

[0212] Example 7: In vivo durability 8 weeks GalNAc-siRNA (n = 11) was tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein A1 (Taconic Pharmaceuticals). The siRNAs were divided and tested in two studies (n = 7 and n = 4). Mice were administered two adeno-associated virus (AAV) vectors via retroorbital injection. One vector contained a plasmid with the albumin promoter and coding sequence for human ANGPTL8 (NM_018687.7) (Vector BioLabs). The second vector contained the mouse codon-optimized sequence for human ANGPTL3 (NP_055310.1) (Vector BioLabs). Blood was collected from mice 3–4 weeks after AAV administration. This was considered the baseline blood collection. Serum was prepared from blood, ANGPTL3 / 8 was measured by ELISA (Meso Scale Diagnostics), and triglycerides were measured as described. Mice were weighed and assigned to groups with similar body weight, serum triglyceride, and ANGPTL3 / 8 levels (n = 9 / group). Four to five weeks after AAV administration, mice were subcutaneously administered either PBS or the test substance, GalNac-siRNA, at doses of 1.75 and 10 mg / kg. Two weeks after siRNA administration, three mice from each group were euthanized under isoflurane anesthesia, blood was collected, and serum was analyzed for triglycerides. Livers were harvested from mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n = 6 / group) 2, 4, and 6 weeks after siRNA administration under isoflurane anesthesia. Serum was prepared from the blood, and triglycerides were measured. Eight weeks after siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and livers were collected from the mice. Livers were processed and residual mRNA was calculated as described in the in vivo single dose screen. Triglycerides as % change from PBS was calculated as described in the in vivo single dose screen.

[0213] [Table 11]

[0214] Example 8: In vivo durability 15 weeks (dose response (C57 mouse hANGPTL8 AAV)) GalNAc-siRNA (n=5) was tested in male mice transgenic for human cholesterol ester transfer protein (CETP) and human apolipoprotein A1 (Taconic Pharmaceuticals). Each siRNA was tested in a single study. Mice were administered two adeno-associated virus (AAV) vectors via retroorbital injection. One vector contained a plasmid with the albumin promoter and the coding sequence of human ANGPTL8 (NM_018687.7) (Vector BioLabs). The second vector contained the mouse codon-optimized sequence of human ANGPTL3 (NP_055310.1) (Vector BioLabs). Blood was collected from the mice 3 weeks after AAV administration. This was considered the baseline blood collection. Serum was prepared from the blood, and triglycerides and ANGPTL3 / 8 were measured. Mice were weighed and assigned to groups with similar body weights, serum triglycerides, and ANGPTL3 / 8 levels (n = 10 per group). Four weeks after AAV administration, mice were subcutaneously administered PBS or the test substance, GalNac-siRNA, at doses of 0.3, 1.75, and 10 mg / kg. Two weeks after siRNA administration, three mice from each group were euthanized under isoflurane anesthesia, blood was collected, and serum was analyzed for triglycerides. Livers were harvested from the mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n = 7 per group) under isoflurane anesthesia at 3, 6, 9, and 12 weeks after siRNA administration. Serum was prepared from the blood, and triglycerides were measured. Fifteen weeks after siRNA administration, the remaining mice were euthanized under isoflurane anesthesia. Blood and livers were harvested from the mice. Serum was prepared from the blood, and triglycerides were measured. Livers were processed and residual mRNA was calculated as described in the in vivo single dose screen. Triglycerides as % change from PBS was calculated as described in the in vivo single dose screen.

[0215] Table 12

Claims

1. 1. An RNAi agent for reducing ANGPTL8 gene expression, the RNAi agent comprising a delivery moiety of Formula I conjugated to R, wherein R is double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand; 【Chemistry 1】 wherein R is conjugated, optionally via a linker, to connection point E of Formula I, and wherein the sense strand and the antisense strand form a duplex region, and the antisense strand comprises any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or a sequence with 90% sequence identity thereto, or an antisense strand sequence set forth in Table 3A, Table 3B, and Table 4, or a sequence with 90% sequence identity thereto, and wherein the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and optionally one or more modified internucleotide linkages.

2. 2. The RNAi agent of claim 1, wherein Formula I is conjugated to the sense strand, optionally via a linker.

3. 3. The RNAi agent of claim 2, wherein Formula I is conjugated to the 3'-terminal nucleotide of the sense strand, optionally via a linker.

4. The RNAi agent of any one of claims 1 to 3, wherein the sense strand is 15 to 25 nucleotides in length.

5. The RNAi agent of any one of claims 1 to 4, wherein the antisense strand is 18 to 23 nucleotides in length.

6. The RNAi agent of any one of claims 1 to 5, wherein the sense strand is 18 to 21 nucleotides in length.

7. The RNAi agent of any one of claims 1 to 6, wherein the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.

8. The RNAi agent according to any one of claims 1 to 7, wherein the sense strand and the antisense strand form a complementary region at least 18 nucleotides in length.

9. 9. The RNAi agent of claim 1, wherein the duplex region between the sense strand and the antisense strand comprises 0, 1, or 2 mismatches between the sense strand and the antisense strand.

10. The RNAi agent of any one of claims 1 to 9, wherein the duplex region between the sense strand and the antisense strand comprises 0 mismatches between the sense strand and the antisense strand.

11. 11. The RNAi agent of any one of claims 1 to 10, wherein the antisense strand comprises 15 contiguous nucleotides of any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or the sequences of Table 3A, Table 3B, and Table 4.

12. 12. The RNAi agent of any one of claims 1 to 11, wherein the antisense strand comprises 18 contiguous nucleotides of any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or the sequences of Table 3A, Table 3B, and Table 4.

13. 13. The RNAi agent of any one of claims 1 to 12, wherein the sense strand is selected from the group consisting of SEQ ID NOs: 1 to 5, or a sequence having at least 90% sequence identity thereto, or a sense strand sequence shown in Table 3, or a sequence having 90% sequence identity thereto.

14. The RNAi agent of any one of claims 1 to 13, wherein the sense strand or the antisense strand each independently comprises one or more modified nucleotides.

15. 15. The RNAi agent of any one of claims 1 to 14, wherein each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide, and the modified nucleotide is independently a 2' fluoro-modified nucleotide residue or a 2'-O-methyl-modified nucleotide.

16. the antisense strand is 23 nucleotides in length, each nucleotide of the antisense strand is a modified nucleotide, and the position of the 2' fluoro-modified nucleotide is: a. positions 2, 3, 7, 14, and 16 from the 5' end of the antisense strand; or b. positions 2, 5, 7, 14, and 16 from the 5' end of the antisense strand, or c. positions 2, 3, 8, 14, and 16 from the 5' end of the antisense strand, or d. positions 2, 5, 8, 14, and 16 from the 5' end of the antisense strand, or e. positions 2, 6, 14, and 16 from the 5' end of the antisense strand; The RNAi agent of any one of claims 1 to 15, wherein the RNAi agent is modified to be present at a position selected from the group consisting of:

17. 17. The RNAi agent of any one of claims 1 to 16, wherein the sense strand and the antisense strand each independently comprise one or more modified internucleotide linkages, and each modified internucleotide linkage is a phosphorothioate linkage.

18. The RNAi agent of any one of claims 1 to 17, wherein the sense strand and the antisense strand each independently comprise four phosphorothioate linkages.

19. The RNAi agent of any one of claims 1 to 18, wherein the 5' terminal nucleotide of the antisense strand comprises an OH group, a phosphate group, a vinyl phosphonate, or a phosphate analog.

20. 20. The RNAi agent of claim 19, wherein the 5' terminal nucleotide of the antisense strand is further modified to replace the 5' phosphate group with an OH group.

21. 21. The RNAi agent of any one of claims 1 to 20, wherein R is conjugated to formula I via a linker.

22. R is conjugated to Formula I via a linker, wherein the linker comprises a linker of Formula II having connection points A and B, or the linker comprises a linker of Formula III having connection points C and D; 【Chemistry 2】 a. Formula I is conjugated at connection point E to connection point A of Formula II, and Formula II is conjugated at connection point B to a phosphate group or a phosphorothioate group, which is further conjugated to R; or b. The RNAi agent of any one of claims 1-21, wherein Formula I is conjugated at connection point E to connection point C of Formula III, and Formula III is conjugated to a phosphate group or a phosphorothioate group at connection point D, wherein the phosphate group or the phosphorothioate group is further conjugated to R.

23. R is conjugated to Formula I via a linker, said linker comprising Formula III having attachment points C and D; 【Transformation 3】 23. The RNAi agent of any one of claims 1-22, wherein Formula I is conjugated at connection point E to connection point C of Formula III, and Formula III is conjugated to a phosphate group or a phosphorothioate group at connection point D, wherein the phosphate group or the phosphorothioate group is further conjugated to R.

24. The RNAi agent of any one of claims 1 to 23, wherein the RNAi agent reduces expression of the ANGPTL8 gene in liver cells compared to a control agent.

25. The RNAi agent of any one of claims 1 to 24 for use in therapy.

26. 25. The RNAi agent of any one of claims 1 to 24 for use in the treatment of a disease or disorder selected from cardiovascular disease, cardiometabolic disease, diabetes, dyslipidemia, renal dysfunction, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), and obesity.

27. 27. The RNAi agent for use according to claim 26, wherein the disease or disorder is dyslipidemia.

28. 25. A pharmaceutical composition comprising the RNAi agent of any one of claims 1 to 24 and one or more pharmaceutically acceptable excipients.

29. 25. Use of the RNAi agent of any one of claims 1 to 24 in the manufacture of a medicament for the treatment of a disease or disorder selected from cardiovascular disease, diabetes, dyslipidemia, renal dysfunction, hypertension, non-alcoholic fatty liver disease such as non-alcoholic steatohepatitis (NASH), and obesity.

30. 30. The use according to claim 29, wherein the disease or disorder is dyslipidemia.

31. 25. A method of treating dyslipidemia in a patient in need thereof, comprising administering to the patient an RNAi agent according to any one of claims 1 to 24, or a pharmaceutical composition thereof.

32. 25. A method of reducing ANGPTL8 expression in a cell, comprising contacting the cell with the RNAi agent of any one of claims 1-24.

33. 33. The method of claim 32, wherein the method further comprises incubating the cells for a time sufficient to reduce the level of ANGPTL8 mRNA by at least 50% compared to untreated or control-treated cells.

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