Novel RNA therapeutics and their uses

Abstract

The present disclosure relates to novel therapeutic compounds known as RNAi agents that reduce the expression of the HMGCR receptor (expressed by the HMGCR gene), thereby reducing mRNA expression and protein expression. Such RNAi agents are useful in the treatment of diseases and disorders involving the regulation of HMGCR expression and function, such as diseases and disorders that are risk factors for ASCVD (e.g., dyslipidemia, e.g., hypercholesterolemia).

Description

[Background technology]

[0001] The present invention relates to novel therapeutic compounds known as RNAi agents that decrease the expression of HMGCR (expressed by the HMGCR gene), thereby decreasing the expression of HMGCR mRNA and HMGCR protein. Such RNAi agents are useful in the treatment of diseases or disorders involving the regulation of HMGCR expression and function (e.g., diseases or disorders known to be risk factors for atherosclerotic cardiovascular disease (ASCVD), such as dyslipidemia).

[0002] HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase or HMGCo reductase, is the rate-limiting enzyme for the synthesis of cholesterol, which plays an important role in cell construction and hormone production. HMGCR catalyzes the production of mevalonate, a precursor for cholesterol biosynthesis. Nonsterols and mevalonate-derived sterols regulate HMGCR through a negative feedback mechanism. In mammalian cells, HMGCR is normally inhibited by cholesterol derived from the internalization and degradation of low-density lipoprotein (LDL) via the LDL receptor.

[0003] Although plasma cholesterol may be elevated due to genetic predisposition, it is often the result of a poor diet high in fat and / or sugar and a sedentary lifestyle. Cholesterol can be deposited in arteries and is an important determinant of atherosclerosis and ischemia. Competitive inhibitors of HMGCR induce the expression of LDL receptors in the liver, which then increases the catabolism of plasma LDL and reduces the plasma concentration of cholesterol. However, statins have multiple side effects, including insufficient reduction of cholesterol and / or LDL. Therefore, more treatments are needed to lower cholesterol and treat diseases or disorders (e.g., dyslipidemia) that are known risk factors for (ASCVD). Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the disclosure describes an RNAi agent for reducing HMGCR 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 the connection point E of Formula I, optionally via a linker, and the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region complementary to the HMGCR mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each optionally comprise one or more modified nucleotides and 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 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.

[0007] In some embodiments, the sense strand or antisense strand comprises a sequence selected from Table 2, Table 3, Table 4A, and Table 4B as disclosed herein. In some embodiments, the sense strand and antisense strand comprise a sequence selected from Table 2, Table 3, Table 4A, and Table 4B as disclosed herein.

[0008] In some embodiments, R is conjugated to Formula I via a linker. In some 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,

[0009] [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 or phosphorothioate group at connection point B, and the phosphate or phosphorothioate group is conjugated to R; or b. Formula I is conjugated at connection point E to Formula III at connection point C, Formula III is conjugated to a phosphate or phosphorothioate group at connection point D, and the phosphate or phosphorothioate group is further conjugated to R.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition comprising an RNAi agent described herein and one or more pharmaceutically acceptable excipients.

[0011] In another aspect, the present disclosure provides a method for treating a disease or disorder associated with ASCVD. In some embodiments, the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the present disclosure provides a method for treating dyslipidemia, comprising administering to a patient an RNAi agent or pharmaceutical composition thereof as described herein.

[0012] In another aspect, the present disclosure provides an RNAi agent for use in treatment. In some embodiments, the present disclosure provides an RNAi agent for use in treating a disease or disorder associated with ASCVD. In some embodiments, the present disclosure provides an RNAi agent for use in treating a disease or disorder, wherein the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis.

[0013] In another aspect, the present disclosure provides the use of an RNAi agent for the manufacture of a medicament for use in treatment. In some embodiments, the present disclosure provides an RNAi agent for the manufacture of a medicament for use in the treatment of a disease or disorder associated with ASCVD. In some embodiments, the present disclosure provides an RNAi agent for the manufacture of a medicament for use in the treatment of a disease or disorder, wherein the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis. DETAILED DESCRIPTION OF THE INVENTION

[0014] Although HMGCR siRNA and ASO have been described, none have made progress in treating patients. Using the HMGCR RNAi agents herein to reduce HMGCR expression can be used to treat ASCVD-related diseases or disorders (e.g., dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis). 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 HMGCR siRNAs containing different delivery ligands, different sequences, or different modified sequences, or compared to treatment with a vehicle control. Other embodiments of the HMGCR 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 an independent advantage.

[0015] 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).

[0016] 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).

[0017] 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.

[0018] As used herein, "modified internucleotide linkage" means an internucleotide linkage that has one or more chemical modifications when compared to a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage may be a non-naturally occurring linkage.

[0019] 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. A modified nucleotide may be a non-naturally occurring nucleotide. A modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, a modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0020] 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), and computer software programs 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, although the fragment of nucleic acid sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a 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 nucleic acid base is present 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 of 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 percent of identical nucleotide residues 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, for example, Raghava, G., Barton, G. J. Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0021] 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).

[0022] 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.

[0023] As used herein, a "duplex" in reference to a nucleic acid or oligonucleotide, such as a sense strand or antisense strand, refers to a structure formed through hydrogen bonding of complementary base pairing of two antiparallel sequences of nucleotides under conditions suitable to promote such structure. A duplex can form even when there is no perfect complementarity between the two strands or when abasic nucleotides are present.

[0024] RNA interference is a specialized cellular process that utilizes RISC to degrade RNA in a sequence-dependent manner. As used herein, "RNAi agent" refers to an agent that includes either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion thereof 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 thereof) is used by Ago2 endonuclease to cleave the target mRNA. In some embodiments, the RNAi agent described herein also includes a delivery moiety.

[0025] As used herein,

[0026] [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 at 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).

[0027] [ka]

[0028] As used herein, "effective amount" refers to the amount (relative to the administration period and administration method) 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, and 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.

[0029] As used herein, "treatment" or "treating" refers to any process that may slow, control, retard, or halt the progression of a disorder or disease symptom disclosed herein, but 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] As used herein, the term "ASCVD-related disease or disorder" refers to any disease or disorder that is a risk factor for ASCVD.

[0031] Provided herein is an RNAi agent for reducing HMGCR 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 to connection point E of Formula I, optionally via a linker, and the sense strand and antisense strand form a duplex region, the antisense strand comprising a region of complementarity to the HMGCR mRNA target sequence of SEQ ID NO: 1, 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 HMGCR 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 conjugated to Formula Ia via a linker, and the sense strand and antisense strand form a duplex region, the antisense strand comprises a region of complementarity to the HMGCR mRNA target sequence of SEQ ID NO: 1, and the sense strand and antisense strand each optionally comprise one or more modified nucleotides and one or more modified internucleotide linkages.

[0035] Disclosed herein is an RNAi agent for reducing HMGCR 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, wherein the antisense strand comprises a region of complementarity of at least 15 nucleotides to the sequence set forth in SEQ ID NO: 1, and wherein the sense strand and / or the antisense strand each optionally comprises one or more modified nucleotides and / or modified internucleotide linkages. In a further embodiment, the antisense strand comprises at least 15 nucleotides of a sequence in Table 2. In a further embodiment, the antisense strand comprises at least 18 nucleotides of a sequence in Table 2. In a further embodiment, the RNAi agent reduces HMGCR gene expression in hepatocytes compared to a control. In a further embodiment, the RNAi agent reduces HMGCR gene expression in cells expressing HMGCR by about 50% or more compared to a control. In a further embodiment, the RNAi agent reduces HMGCR gene expression by reducing HMGCR mRNA transcript levels, HMGCR protein levels, or both.

[0036] In further embodiments, the antisense strand is 15-50 nucleotides in length and / or the sense strand is 15-50 nucleotides in length. In further embodiments, the sense and / or sense strands are independently 15-30 nucleotides in length. In further embodiments, the antisense strand is 18-23 nucleotides in length. In further embodiments, the sense strand is 18-21 nucleotides in length.

[0037] In further embodiments, the RNAi agent comprises an antisense strand comprising at least 15 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-387. In yet further embodiments, the antisense strand comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-387.

[0038] In other further embodiments, the antisense strand comprises at least 18 consecutive nucleotides of a sequence selected from the group consisting of 18 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-387.

[0039] 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 antisense strand comprises a sequence selected from the group consisting of 774-1159, or a sequence having at least 90% sequence identity thereto. In another embodiment, the sense strand and the antisense strand comprise a sequence selected from the sequences set forth in Table 3.

[0040] 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.

[0041] In a further embodiment, the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 388-773.

[0042] 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.

[0043] In further embodiments of the RNAi agents disclosed herein, the antisense strand is 23 nucleotides in length, and each nucleotide in the antisense strand is a modified nucleotide, and 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.

[0044] In a further embodiment, the nucleotide that is not a 2' fluoro-modified nucleotide is a 2'-O-methyl-modified nucleotide.

[0045] 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.

[0046] In other embodiments, the 5' nucleotide of the antisense strand comprises a phosphate group or a 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 comprise 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).

[0047] In further embodiments of the RNAi agents disclosed herein, the antisense strand is selected from the group consisting of SEQ ID NOs: 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 12 245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1 295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1 345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543,1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1592 3, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1 643, 1645, 1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691 , 1693, 1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 17 41, 1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 183 a sequence selected from the group consisting of: 9, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931;or a sequence having at least 90% sequence identity thereto, wherein the 5'-terminal nucleotide of the antisense strand comprises a 5' vinyl phosphonate, phosphate, or hydroxyl group. 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.

[0048] In further embodiments of the RNAi agents disclosed herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1932-2317, or a sequence having at least 90% sequence identity thereto.

[0049] In further embodiments of the RNAi agents disclosed herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1932-2317, or a sequence having at least 95% sequence identity thereto.

[0050] In further embodiments, the sense strand is selected from the group consisting of SEQ ID NOs: 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550,1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600 , 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650 , 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 170 0, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 175 0, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 18 00, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 18 50, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, or a sequence having at least 90% sequence identity thereto.

[0051] In still further embodiments of the RNAi agents disclosed herein, the sense strand and antisense strand are a pair of oligonucleotide sequences selected from Table 4A, Table 4B, or a sequence that is 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 to a sequence in Table 4A or Table 4B. In further embodiments, one, two, or three mismatches are introduced into the sense strand of the pair in Table 4A or Table 4B. In further embodiments, one, two, or both terminal nucleotides at the 5' end of the antisense strand are altered.

[0052] 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 4A or Table 4B, 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 4A or Table 4B. 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 387 in Table 4A, i.e., a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1161, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity to the sense sequence corresponding to duplex number 387 in Table 4A, i.e., SEQ ID NO: 1160. In further embodiments, the 5' phosphate of the antisense strand is further modified / replaced with a 5' vinyl phosphonate or an OH group.

[0053] The duplexes (eg, "duplex numbers") correspond to particular sense and antisense strands as indicated herein (see, eg, Tables 4A and 4B).

[0054] 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. For example, SEQ ID NOs: 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1342 3, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 147 437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529,1531、1533、1535、1537、1539、1541、1543、1545、1547、1549、1551、1553、1555、1557、1559、1561、1563、1565、1567、1569、1571、1573、1575、1577、1579、1581、1583、1585、1587、1589、1591、1593、1595、1597、1599、1601、1603、1605、1607、1609、1611、1613、1615、1617、1619、1621、1623、1625、1627、1629、1631、1633、1635、1637、1639、1641、1643、1645、1647、1649、1651、1653、1655、1657、1659、1661、1663、1665、1667、1669、1671、1673、1675、1677、1679、1681、1683、1685、1687、1689、1691、1693、1695、1697、1699、1701、1703、1705、1707、1709、1711、1713、1715、1717、1719、1721、1723、1725、1727、1729、1731、1733、1735、1737、1739、1741、1743、1745、1747、1749、1751、1753、1755、1757、1759、1761、1763、1765、1767、1769、1771、1773、1775、1777、1779、1781、1783、1785、1787、1789、1791、1793、1795、1797、1799、1801、1803、1805、1807、1809、1811、1813、1815、1817、1819、1821、1823、1825、1827、1829、1831、1833、1835、1837、1839、1841、1843、1845、1847、1849、1851、1853、1855、1857、1859、1861、1863、1865、1867、1869、1871、1873、1875、1877、1879、1881、1883、1885、1887、1889、1891、1893、1895、1897、1899、1901、1903、1905、1907、1909、1911、1913、1915、1917、1919、1921、1923、1925、1927、1929、For the antisense sequences of 1931, or sequences with at least 90% sequence identity thereto, the 5' phosphate group is replaced with an OH group.

[0055] In further embodiments of the RNAi agents disclosed herein, the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1932-2317, or a sequence having at least 90% sequence identity thereto.

[0056] In other embodiments, disclosed herein is an RNAi agent having a delivery moiety of Formula I conjugated to R,

[0057] [ka] R comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides having complementarity to the HMGCR mRNA target sequence of SEQ ID NO: 1, 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 conjugated to Formula I via a linker. In further embodiments, the sense strand or antisense strand is selected from Table 2, Table 3, Table 4A, or Table 4B 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 2, Table 3, Table 4A, or Table 4B herein.

[0058] In other embodiments, the RNAi agents disclosed herein comprise a linker. In further embodiments, R is conjugated to Formula I via a linker. In other further 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,

[0059] [ka] a. the RNAi agent comprises Formula I conjugated at attachment point A to Formula II, which is conjugated to a phosphate group at attachment point B, which is conjugated to R; or b. 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.

[0060] 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,

[0061] [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.

[0062] In yet other embodiments, the RNAi agent can reduce expression of the HMGCR gene in hepatocytes. In other embodiments, the RNAi agent disclosed herein is for use in therapy. In some embodiments, the use is for the treatment of a disease or disorder related to ASCVD. In further embodiments, the use is for the treatment of dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the use is for the treatment of dyslipidemia. In some embodiments, the use is for the treatment of primary dysbetalipoproteinemia. In some embodiments, the use is for the treatment of hypertriglyceridemia. In some embodiments, the use is for the treatment of atherosclerosis. In further embodiments, the dyslipidemia is hypercholesterolemia. In other embodiments, the use is for reducing the risk of one or more of myocardial infarction (MI), stroke, revascularization, and angina. In other embodiments, the use for reducing such risk is in adult patients who do not have cardiovascular heart disease (CHD) but have one or more risk factors for any one or more of the listed health events. In other embodiments, the use is for reducing the risk of MI and / or stroke in adult patients, for example, with type 2 diabetes, who do not have CHD but have one or more risk factors. In other embodiments, the use is for reducing the risk of one or more of non-fatal MI, fatal and non-fatal stroke, revascularization procedures, hospitalization for CHF, and angina pectoris in adult patients with CHD. In other embodiments, the use is for reducing elevated one or more of total C, LDL-C, apoB, and TG levels and / or increasing HDLC in adult patients with primary hyperlipidemia (heterozygous familial and non-familial) and mixed dyslipidemia. In another embodiment, the use is for reducing elevated triglycerides (TG) in adult patients with hypertriglyceridemia and primary dysbetalipoproteinemia.In another embodiment, the use is for reducing total C and LDL-C in patients with homozygous familial hypercholesterolemia (HoFH). In yet another embodiment, the use is for reducing one or more elevated total C, LDL-C, and apoB levels in pediatric patients aged 10 to 17 years with heterozygous familial hypercholesterolemia (HeFH), e.g., after failing an adequate trial of dietary therapy. In another embodiment, the use is for treating any of the foregoing after statin use has failed to control one or more symptoms, e.g., after failing to reduce elevated total C, LDL-C, or apoB, and / or after failing to increase HDLC. In another embodiment, the use is for treating any of the foregoing in patients who are statin intolerant, and in a further embodiment, the use is for lowering LDL-C in patients who are statin intolerant. In further embodiments, the use is for any of the aforementioned uses after dietary changes have failed to control one or more symptoms, hi other further embodiments, the use is for any of the aforementioned uses as an adjunct to diet.

[0063] RNAi agent can be formulated into pharmaceutical composition.Therefore, the present specification 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).

[0064] In another embodiment is a use of an RNAi agent herein or any of the uses listed in the preceding paragraph for the manufacture of a medicament for the treatment of dyslipidemia.

[0065] In another embodiment, it is a method of treating dyslipidemia in a patient in need thereof, comprising administering an RNAi agent disclosed herein or a pharmaceutical composition thereof. In another embodiment, it is a method of treating dyslipidemia in a patient in need thereof, or any of the uses listed above in a patient in need thereof, 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.

[0066] The RNAi agent may be administered to the patient intravenously or subcutaneously.

[0067] RNAi dosing regimens can be adjusted to provide the optimum desired response (e.g., a 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.

[0068] 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.

[0069] In another embodiment, a method for reducing HMGCR expression in a cell comprises contacting the cell with an RNAi agent disclosed herein and incubating the cell for a time sufficient to reduce the level of HMGCR mRNA by at least 50% compared to untreated or control-treated cells.

[0070] Certain abbreviations are defined as follows: "1,2-DCE" refers to 1,2-dichloroethane, "DCM" refers to dichloromethane, "N,N-diisopropylethylamine (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, "EDC" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "EtOAc" refers to ethyl acetate. 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" refers to molecular weight cut-off, "NHS" refers to N-hydroxysuccinimide, "optical density (OD)" refers to optical density, "PBS" refers to phosphate-buffered saline, "PhSiH3" refers to phenylsilane, "PTS" refers to portable endotoxin test system, and "siRNA" refers to small interfering ribonucleic acid. "TEA" refers to triethylamine, "TFA" refers to trifluoroacetic acid, "THF" refers to tetrahydrofuran, "TLC" refers to thin-line chromatography, and "TMP" refers to 2,2,6,6-tetramethylpiperidine.

[0071] Delivery moieties comprising Formula I can be made by the following non-limiting synthetic steps and schemes.

[0072] [ka]

[0073] Step A of 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).

[0074] [ka]

[0075] Step A of Scheme 2 illustrates the amide coupling of compound (5) with tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate using HBTU and HOBt with a suitable base such as DIEA in a solvent such as DMF to give 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 give compound (7). Step C illustrates the amide coupling of compound (7) with allyl 11-aminoundecanoate hydrochloride using HATU with a suitable base such as DIEA in a solvent such as DMF to give compound (8). Step D illustrates the acidic deprotection of compound (8) with TFA in a solvent such as DCM to give 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 give 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).

[0076] [ka]

[0077] Steps A-C of Scheme 3 are essentially similar to steps C-E of Scheme 2, starting with 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.

[0078] [ka]

[0079] Steps AI of Scheme 4 consist of a series of amide couplings and deprotections using methods essentially similar to those shown in Schemes 2 and 3, starting from compound 18, to give compound 27.

[0080] [ka]

[0081] Steps AC of Scheme 5 show essentially similar methods as shown in Steps GI of Scheme 4, starting from compound (24) to give compound (30).

[0082] [ka]

[0083] Step A of Scheme 6 shows 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 shows 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 shows the deprotection of compound 33 with 20% piperidine in DMF to give compound 34.

[0084] [ka]

[0085] 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 formation of compound 36 by adding succinic anhydride to compound 35 with a base system of TEA and DMAP in a suitable solvent such as DCM. Step C illustrates the loading of compound 36 onto the resin using 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.

[0086] Preparation 1 Methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate

[0087] [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 200 mL of saturated aqueous NaHCO3 and 200 mL of saturated aqueous sodium chloride, 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).

[0088] Preparation 2 (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate methyl

[0089] [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).

[0090] Preparation 3 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoic acid

[0091] [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).

[0092] Preparation 4 Benzyl 6-aminohexanoate hydrochloride

[0093] [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).

[0094] Preparation 5 Benzyl 11-aminoundecanoate hydrochloride

[0095] [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).

[0096] Preparation 6 Allyl 11-aminoundecanoate hydrochloride

[0097] [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).

[0098] Preparation 7 (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluoren-9ylmethoxycarbonylamino)propanoic acid

[0099] [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) at 0° C. under an inert atmosphere. To this is slowly added a solution of DMTCl (49.6 g, 0.146 mol) in DCM (200 mL). The resulting reaction mixture is warmed to 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 / hexane (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: 5% MeOH / CH2Cl2 (R f :0.5)UV, 254nM.

[0100] Preparation 8 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidyl]-2-oxo-ethyl]carbamate

[0101] [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 warm to 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.

[0102] Preparation 9 (2S)-2-Amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidyl]propan-1-one

[0103] [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).

[0104] Preparation 10 Methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate

[0105] [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-(tert Add 1-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 (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).

[0106] Preparation 11 (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid

[0107] [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).

[0108] Preparation 12 Allyl 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoate

[0109] [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 (42 To the resulting solution, add 1.8 mg (1.12 mmol). 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 / hexanes 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).

[0110] Preparation 13 Allyl (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)undecanoate

[0111] [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).

[0112] 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

[0113] [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.

[0114] 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

[0115] [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.

[0116] 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

[0117] [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.

[0118] Preparation 17 Benzyl 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]hexanoate

[0119] [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).

[0120] Preparation 18 Benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]hexanoate tris(trifluoroacetic acid)

[0121] [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 considering residual toluene, 99+%). 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.

[0122] 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

[0123] [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.

[0124] 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

[0125] [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.

[0126] 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

[0127] [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.

[0128] Preparation 22 Benzyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoate

[0129] [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).

[0130] Preparation 23 Benzyl (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxopentanoate tris(trifluoroacetate) salt

[0131] [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 the method of Preparation 18. ES / MS m / z 323.2 (M+H).

[0132] Preparation 24 Benzyl (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoate

[0133] [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).

[0134] Preparation 25 Benzyl (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoate tris(trifluoroacetic acid) salt

[0135] [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 the method of Preparation 18. ES / MS m / z 620.4 (M+H).

[0136] 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

[0137] [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.

[0138] 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

[0139] [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.

[0140] 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

[0141] [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 the method of Preparation 10. ES / MS m / z 1011.6 (M+2H) / 2.

[0142] 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

[0143] [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 h, 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.

[0144] 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

[0145] [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.

[0146] 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

[0147] [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-aminodecanoate hydrochloride in a manner essentially similar to that of Preparation 10. ES / MS m / z 1046.6 (M+2H) / 2.

[0148] 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

[0149] [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% pure, 0.11 mmol) was added via syringe. The vessel was evacuated and refilled with 1 atm of hydrogen, and the mixture was then 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.

[0150] 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

[0151] [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 the method of Preparation 16. ES / MS m / z 1050(M+2H) / 2

[0152] 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

[0153] [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.

[0154] 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

[0155] [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.

[0156] Preparation 36 Resin Loading

[0157] [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-butyric 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) are added to the vessel, and the cartridge is 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) is added, and the mixture is shaken at ambient temperature for 18 hours. After this, the cartridge is drained by suction, and the resin is washed with DCM (10 mL) by shaking for 10 minutes. The cartridge is drained, and the washing and draining procedure is 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.

[0158] 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

[0159] [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).

[0160] 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

[0161] [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).

[0162] 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

[0163] [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).

[0164] 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

[0165] [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).

[0166] 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

[0167] [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).

[0168] 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

[0169] [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) was added dropwise (2,3,5,6-tetrafluorophenyl)-2,2,2-trifluoroacetic acid (0.443 mmol, 116 mg). 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).

[0170] 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.

[0171] 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 5'-CEβ-cyanoethyl) phosphoramidites.

[0172] Alternatively, the GalNAc ligand was converted to a suitable phosphoramidite and delivered to the 5' position of the sense strand using standard phosphoramidite chemistry.

[0173] 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.

[0174] Endotoxin testing was performed using Limulus amebocyte lysate on an Endosafe®-nexgen PTS device.

[0175] [Table 1]

[0176] 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.

[0177] Antisense oligonucleotide sequences were designed using 15-50 nucleotides of the following HMGCR transcript (SEQ ID NO: 1), in which T nucleotides were replaced with U nucleotides, and one or more nucleotides and one or more internucleotide linkages were further modified, as needed, as described herein.

[0178] Homo sapiens HMGCR death receptor (HMGCR) transcript, SEQ ID NO: 1

[0179] [ka]

[0180] [ka]

[0181] [ka]

[0182] Exemplary antisense strand sequences of 18 nucleotides in length are shown below in Table 2, which may optionally be further modified and synthesized and incorporated into RNAi agents as described herein.

[0183] [Table 2-1]

[0184] [Table 2-2]

[0185] [Table 2-3]

[0186] [Table 2-4]

[0187] [Table 2-5]

[0188] [Table 2-6]

[0189] Table 2-7

[0190] Table 2-8

[0191] Table 2-9

[0192] Table 2-10

[0193] Table 2-11

[0194] Table 3-1

[0195] Table 3-2

[0196] Table 3-3

[0197] Table 3-4

[0198] Table 3-5

[0199] Table 3-6

[0200] Table 3-7

[0201] Table 3-8

[0202] Table 3-9

[0203] Table 3-10

[0204] Table 3-11

[0205] Table 3-12

[0206] Table 3-13

[0207] Table 3-14

[0208] Table 3-15

[0209] Table 3-16

[0210] Table 3-17

[0211] Table 3-18

[0212] Table 4A-1

[0213] Table 4A-2

[0214] Table 4A-3

[0215] Table 4A-4

[0216]

Table 4A-5

[0217] Table 4A-6

[0218] Table 4A-7

[0219] Table 4A-8

[0220] Table 4A-9

[0221] Table 4A-10

[0222] Table 4A-11

[0223]

Table 4A-12

[0224]

Table 4A-13

[0225] Table 4A-14

[0226] Table 4A-15

[0227] Table 4A-16

[0228] Table 4A-17

[0229]

Table 4A-18

[0230] Table 4A-19

[0231] Table 4A-20

[0232] Table 4A-21

[0233] Table 4A-22

[0234] Table 4A-23

[0235] Table 4A-24

[0236] Table 4A-25

[0237] Table 4A-26

[0238] Table 4A-27

[0239] [Table 4A-28]

[0240] [Table 4A-29]

[0241] [Table 4A-30]

[0242] [Table 4A-31]

[0243] [Table 4A-32]

[0244] [Table 4A-33]

[0245] [Table 4A-34]

[0246] [Table 4A-35]

[0247] [Table 4A-36] P 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)

[0248] [Table 4B-1]

[0249] [Table 4B-2]

[0250] [Table 4B-3]

[0251] [Table 4B-4]

[0252] [Table 4B-5]

[0253] [Table 4B-6]

[0254] [Table 4B-7]

[0255] [Table 4B-8]

[0256] [Table 4B-9]

[0257] [Table 4B-10]

[0258] Table 4B-11

[0259] Table 4B-12

[0260] Table 4B-13

[0261] Table 4B-14

[0262] Table 4B-15

[0263] Table 4B-16

[0264] Table 4B-17

[0265] Table 4B-18

[0266] Table 4B-19

[0267] Table 4B-20

[0268] Table 4B-21

[0269] Table 4B-22

[0270] Table 4B-23

[0271] Table 4B-24

[0272] Table 4B-25

[0273] Table 4B-26

[0274] Table 4B-27

[0275] Table 4B-28

[0276] Table 4B-29

[0277] Table 4B-30

[0278] [Table 4B-31]

[0279] [Table 4B-32]

[0280] [Table 4B-33]

[0281] [Table 4B-34]

[0282] [Table 4B-35]

[0283] [Table 4B-36] 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)

[0284] Example 4: In vitro knockdown of human HMGCR in Hep3B cells using cholesterol-conjugated HMGCR siRNA Knockdown of human HMGCR expression by cholesterol-conjugated HMGCR siRNA was assayed using the following procedure: On day 1, Hep3B cells (ATCC) were added to Corning 96-well plates at 5,000 cells per well in growth medium. On day 2, the culture medium was replaced with ACCELL medium (Dharmacon) and siRNA was added directly to the wells. For single-point (SP) screening, 1 μM (1,000 nM) cholesterol-conjugated siRNA was used. To generate concentration / dose-response curves, final concentrations of cholesterol-conjugated siRNA were used: 1,000, 200, 40, 8, 1.6, 0.32, and 0.064 nM.

[0285] Treated cells were lysed and gene expression was tracked using the TaqMan Fast Advanced Cells-to-Ct Kit (Invitrogen). Cell lysates were immediately used for cDNA synthesis using the Fast Advanced RT Master Mix (Invitrogen) in a thermocycler with the following steps: 37°C for 30 minutes, 95°C for 5 minutes, and a 4°C hold. Quantitative polymerase chain reaction (qPCR) was performed via TaqMan Gene Expression Assay (Invitrogen) with the following cycle temperatures and times: 50°C for 2 minutes, 95°C for 20 seconds, 40 cycles of 95°C for 1 second, and 60°C for 20 seconds.

[0286] Human HMGCR levels were normalized to human Rplp0 (Life Technologies) and represent the relative knockdown of human HMGCR mRNA expression compared to vehicle-treated control cells. IC50 values ​​were calculated using a four-parameter fit model using XLFit.

[0287] [Table 5-1]

[0288] Table 5-2

[0289] Table 5-3

[0290] Table 5-4

[0291] Table 5-5

[0292] Table 5-6

[0293] Table 5-7

[0294] Table 5-8

[0295] Table 5-9

[0296] Table 5-10

[0297] Table 5-11

[0298] Table 5 shows the results of single-dose screening in Hep3B cells by free uptake using the indicated cholesterol-conjugated HMGCR siRNA. Data are expressed as percent message knockdown relative to untreated cells. The IC50 and percent maximum knockdown of the top hits from the single-point screening are also included, followed by concentration / dose-response curves.

[0299] Example 5: In vitro knockdown of HMGCR in wild-type mouse primary hepatocytes (MPH) and Hep3B cells using GalNAc-conjugated HMGCR siRNA Knockdown of mouse HMGCR expression by LYGal1-conjugated HMGCR siRNA was assayed using the following procedure: Mouse primary hepatocytes (MPH) were freshly isolated from wild-type mice and added to Corning plates at 15,000 cells per well, and siRNA was added directly to the wells. For Hep3B (ATCC) cells, 0.3 μL / well of transfection reagent RNAiMAX (Life Technologies) was mixed with siRNA in the Corning plate, and cells were added at 20,000 cells per well. To generate a concentration / dose-response curve, GalNAc-conjugated siRNA concentrations of 1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM were used for MPH. For Hep3B, final concentrations of GalNAc-conjugated siRNA concentrations of 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.014, 0.005, 0.002, 0.0005, and 0.0002 nM were used.

[0300] 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.

[0301] Mouse or human HMGCR levels were normalized to mouse (for MPH) or human (for Hep3B) Rplp0 (Life Technologies) and represent the relative knockdown of mouse or human HMGCR mRNA expression compared to vehicle-treated control cells. IC50 values ​​were calculated using a four-parameter fit model using XLFit.

[0302] [Table 6] Table 6. In vitro knockdown of HMGCR in wild-type mouse primary hepatocytes (MPH) and Hep3B cells using GalNAc-conjugated HMGCR siRNA. Table 6 shows IC50 results using percent maximum knockdown of top hits from single-point screening in wild-type mouse primary hepatocytes and Hep3B cells by free uptake with the transfection reagent RNAiMAX using the indicated HMGCR siRNA. Data are expressed as percent HMGCR message knockdown relative to untreated cells.

[0303] Example 6: In vivo single dose screening mouse HMGCR KD GalNAc-siRNA was tested in male C57bl / 6 mice (n=7) (Taconic Farms). Mice were assigned to groups with similar body weights. 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. Fourteen days after subcutaneous injection, mice were euthanized under isoflurane anesthesia. Blood was collected by cardiac puncture. Livers were harvested from mice and frozen in liquid nitrogen. 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 (Thermo Fisher). 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 sets, and RT-PCR was performed on a QuantStudio Pro7 (Thermo Fisher) 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 mouse HMGCR to obtain the delta C value. The delta-delta C value was 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 changes were calculated by taking the log base 2 of the negative delta delta CT values.Percent mRNA knockdown (KD) was calculated by subtracting the fold change from that of the PBS group and then multiplying by -100. The data are shown in Table 7.

[0304] [Table 7]

[0305] Example 7: In vivo durable 8-week mouse HMGCR KD GalNAc-siRNA was tested in male C57 / BL6 mice (n=9) (Taconic Farms). Mice were weighed and assigned to groups with similar body weights. PBS or the test substance GalNAc-siRNA was subcutaneously administered to mice 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, and blood was collected. Livers were harvested from the mice and frozen in liquid nitrogen. Blood was collected from the remaining mice (n=6 / group) under isoflurane anesthesia at 2, 4, and 6 weeks after siRNA administration. Eight weeks after siRNA administration, the remaining mice (n=6) were euthanized under isoflurane anesthesia. Blood and livers were harvested from the mice. Livers were processed, and the percent KD of mRNA was calculated as described in the in vivo single-dose screening.

[0306] [Table 8]

[0307] Example 8: In vivo single dose screening human HMGCR KD using AAV8 GalNAc-siRNA was tested in male C57bl / 6 mice (n=7) (Taconic farms). siRNAs were tested in a single study. Mice were administered an adeno-associated virus (AAV) vector containing a plasmid carrying the TBGS1 promoter and the coding sequence and 3'UTR of human HMGCR (NM_000859.3) (Vector BioLabs) via retroorbital injection. Mouse body weights were measured approximately 4 weeks after AAV administration. Mice were assigned to groups with similar body weights. 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 the retroorbital sinus of all mice. 14 days after subcutaneous injection, mice were euthanized under isoflurane anesthesia. Blood was collected by cardiac puncture. Livers were harvested from mice and frozen in liquid nitrogen. Human HMGCR mRNA was quantified as described herein. All reagents mentioned in the following sections are from the QuantiGene Singleplex Assay Kit produced by Invitrogen. Approximately 10 mg of liver was weighed into a 96-well cluster tube plate. 300 μL of homogenization buffer with proteinase k was added to each liver sample and homogenized for 12 minutes in a Qiagen homogenizer. The plate was centrifuged at 3500 rpm for 10 minutes and then heated at 60°C for 30 minutes with a vortex step every 10 minutes. The samples were centrifuged again at 3500 rpm for 10 minutes and then diluted or used directly in the following steps. A working probe set for each gene of interest was prepared in a separate tube by combining the following reagents in the order listed and scaled according to the number of wells to be run in excess amounts required: nuclease-free water (25.4 μL), lysis mix (33.3 μL), blocking reagent (1 μL), QuantiGene Singleplex Probe Set (0.3 μL) per well. Capture plates were prepared by dispensing 60 μL of the working probe set into each well of the plate.The mGAPDH (SB-10001) and hHMGCR (SA-11011) probe sets were aliquoted separately onto plates, and then 60 μL of intact liver homogenate was added to the hHMGCR working set in the plate, while 60 μL of a 20-fold dilution of RNA isolate was added to the mGAPDH probe set pre-aliquoted onto the capture plate. The introduction of air bubbles was avoided, and the plate was not mixed. An adhesive seal was placed firmly onto the plate, and then the plate was incubated at 55±1°C for 20.5 hours to allow the probes to hybridize to the RNA targets. After 20.5 hours, 200 μL of 1x wash buffer was added to the capture plate, which was then inverted to remove the wash solution. The plate was then washed two more times with 300 μL of wash buffer for each wash. Next, 100 μL of preamplification solution was added to the plate. It was sealed and incubated at 55±1°C for 60 minutes. After 1 hour, the above washing procedure was repeated, and 100 μL of amplification solution was added to the plate. It was sealed and incubated at 55±1°C for 60 minutes. After 1 hour of incubation, a washing step was performed again, followed by the addition of 100 μL of labeled probe. It was sealed and incubated at 50±1°C for 60 minutes. One more washing step was performed, followed by the addition of 100 μL of substrate at room temperature and incubation for 5 minutes protected from light. The plate was then read on a luminometer with the integration time set to 0.2 seconds. Gene knockdown was calculated by first dividing the hHMGCR chemiluminescent signal by the mGAPDH signal. The fold change from the PBS (control) group was calculated by dividing all groups by the average signal from the control group. The % hHMGCR gene knockdown was then calculated by subtracting the average control fold change from all groups, followed by dividing all of those groups by the average signal from the control group.

[0308] [Table 9]

Claims

1. 1. An RNAi agent for reducing HMGCR 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】 R may be conjugated to the connection point E of formula I via a linker, wherein the sense strand and the antisense strand form a duplex region, the antisense strand comprises a region of complementarity to the HMGCR mRNA target sequence of SEQ ID NO: 1, and the sense strand and the antisense strand each may comprise one or more modified nucleotides and one or more modified internucleotide linkages. RNAi agents.

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

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

4. The RNAi agent of any one of claims 1 to 3, wherein the antisense strand is 15 to 50 nucleotides in length.

5. The RNAi agent of any one of claims 1 to 4, wherein the sense strand is 15 to 50 nucleotides in length.

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

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

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

9. The RNAi agent of any one of claims 1 to 8, wherein the complementary region is at least 18 nucleotides in length.

10. The RNAi agent of any one of claims 1 to 9, wherein the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 387.

11. The RNAi agent of any one of claims 1 to 10, wherein the antisense strand has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 774 to 1159, or a sequence having at least 90% sequence identity thereto.

12. The RNAi agent of any one of claims 1 to 11, wherein the sense strand is selected from the group consisting of SEQ ID NOs: 388 to 773, or a sequence having at least 90% sequence identity thereto.

13. The RNAi agent of any one of claims 1 to 12, wherein the sense strand or the antisense strand each independently comprises one or more modified nucleotides.

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, and the modified nucleotides are independently a 2' fluoro-modified nucleotide residue or a 2'-O-methyl-modified nucleotide.

15. The RNAi agent according to 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.

16. the antisense strand is 23 nucleotides in length, each nucleotide of the antisense strand is a modified nucleotide, and 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; c. positions 2, 3, 8, 14, and 16 from the 5' end of the antisense strand; 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

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 a phosphate group or a phosphate analog.

20. the antisense strand is selected from the group consisting of SEQ ID NOs: 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553,1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603 , 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645, 1647, 1649, 1651, 165 3, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693, 1695, 1697, 1699, 1701, 1702 03, 1705, 1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1 753, 1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851 , 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, or a sequence with at least 90% sequence identity thereto;The RNAi agent of any one of claims 1 to 19, wherein the 5'-terminal nucleotide of the antisense strand comprises a vinyl phosphonate, a phosphate, or a hydroxyl group.

21. the antisense strand is selected from the group consisting of SEQ ID NOs: 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553,1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603 , 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645, 1647, 1649, 1651, 165 3, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693, 1695, 1697, 1699, 1701, 1702 03, 1705, 1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1 753, 1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851 , 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, or a sequence with at least 95% sequence identity thereto;The RNAi agent of any one of claims 1 to 20, wherein the 5'-terminal nucleotide of the antisense strand comprises a vinyl phosphonate, a phosphate, or a hydroxyl group.

22. The sense strand is selected from the group consisting of SEQ ID NOs: 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 12 54, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1 304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1 354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552,1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602 , 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 165 2, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1701 02, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1 752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850 , 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, or a sequence having at least 90% sequence identity thereto;The RNAi agent according to any one of claims 1 to 21.

23. The sense strand is selected from the group consisting of SEQ ID NOs: 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 12 54, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1 304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1 354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552,1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602 , 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 165 2, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1701 02, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1 752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850 , 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, or a sequence having at least 95% sequence identity thereto;The RNAi agent according to any one of claims 1 to 22.

24. 24. The RNAi agent of any one of claims 1 to 23, wherein the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1932 to 2317, or a sequence having at least 90% sequence identity thereto.

25. 24. The RNAi agent of any one of claims 1 to 23, wherein the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 1932 to 2317, or a sequence having at least 95% sequence identity thereto.

26. 26. The RNAi agent of any one of claims 1-25, wherein the antisense strand of the RNAi agent comprises a first nucleic acid sequence having at least 90% sequence identity to the antisense sequence of any one of duplex numbers 387-772 in Table 4A, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity to the sense sequence of the same duplex in Table 4A.

27. 27. The RNAi agent of any one of claims 1-26, wherein the antisense strand of the RNAi agent comprises a first nucleic acid sequence having at least 90% sequence identity to the antisense sequence of any one of duplex numbers 773-1158 in Table 4B, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity to the sense sequence of the same duplex in Table 4B.

28. 28. The RNAi agent of claim 26 or 27, wherein the 5' terminal nucleotide of the antisense strand contains a vinyl phosphonate, a phosphate group, or an OH group.

29. 29. The RNAi agent of any one of claims 1 to 28, wherein R is conjugated to formula I via a linker.

30. R is conjugated to Formula I via a linker, wherein the linker comprises Formula II having connection points A and B, or the linker comprises 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 to a phosphate or phosphorothioate group at connection point B, and the phosphate or phosphorothioate group is conjugated to R; or b. The RNAi agent of any one of claims 1-29, wherein Formula I is conjugated at connection point E to connection point C of Formula III, and Formula III is conjugated to a phosphate or phosphorothioate group at connection point D, wherein the phosphate or phosphorothioate group is further conjugated to R.

31. R is conjugated to Formula I via a linker, said linker comprising Formula III having connection points C and D; 【Transformation 3】 31. The RNAi agent of any one of claims 1-30, 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.

32. 32. A pharmaceutical composition comprising the RNAi agent of any one of claims 1 to 31 and one or more pharmaceutically acceptable excipients.

33. 32. The RNAi agent of any one of claims 1 to 31 for use in therapy.

34. 32. The RNAi agent of any one of claims 1 to 31 for use in the treatment of a disease or disorder associated with ASCVD.

35. 35. The RNAi agent for use according to claim 34, wherein the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis.

36. 36. The RNAi agent for use according to claim 35, wherein the disease or disorder is dyslipidemia.

37. 37. The RNAi agent for use according to claim 36, wherein the dyslipidemia is hypercholesterolemia.

38. 32. Use of the RNAi agent of any one of claims 1 to 31 in the manufacture of a medicament for the treatment of a disease or disorder associated with ASCVD.

39. 39. The use of claim 38, wherein the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis.

40. 40. The use of claim 39, wherein the disease or disorder is dyslipidemia.

41. 41. The use of claim 40, wherein the dyslipidemia is hypercholesterolemia.

42. 32. A method of treating a disease or disorder associated with ASCVD in a patient in need thereof, comprising administering to said patient an RNAi agent or pharmaceutical composition thereof of any one of claims 1-31.

43. 43. The method of claim 42, wherein the disease or disorder is dyslipidemia, primary dysbetalipoproteinemia, hypertriglyceridemia, or atherosclerosis.

44. 43. The method of claim 42, wherein the disease or disorder is dyslipidemia.

45. 45. The method of claim 44, wherein the dyslipidemia is hypercholesterolemia.

46. 32. A method of reducing HMGCR expression in a cell, comprising contacting said cell with an RNAi agent according to any one of claims 1 to 31.

47. 47. The method of claim 46, wherein the method further comprises incubating the cells for a time sufficient to reduce the level of HMGCR mRNA by at least 50% compared to untreated or control-treated cells.

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