Novel RNA therapeutics and uses thereof

Abstract

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

Description

Background of the Invention

[0002] This invention relates to novel therapeutic compounds known as RNAi agents that reduce the expression of HMGCR (expressed by the HMGCR gene), thereby reducing the expression of HMGCR mRNA and HMGCR protein. Such RNAi agents can be used to treat diseases or conditions involving the regulation of HMGCR expression and function (e.g., diseases or conditions known as risk factors for atherosclerotic cardiovascular disease (ASCVD), such as dyslipidemia).

[0003] HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase or HMGCo reductase, is the rate-limiting enzyme in cholesterol synthesis and plays a crucial role in cell structure and hormone production. HMGCR catalyzes the production of mevalonate, a precursor in cholesterol biosynthesis. Non-steroidal and sterols derived from mevalonate regulate HMGCR via a negative feedback mechanism. In mammalian cells, HMGCR is typically suppressed by cholesterol, which is derived from low-density lipoprotein (LDL) and internalized and degraded via LDL receptors.

[0004] Elevated plasma cholesterol can be caused by genetic factors, but it is more often a result of a poor diet high in fat and / or sugar, as well as a sedentary lifestyle. Cholesterol can deposit in arteries and is a major determinant of atherosclerosis and ischemia. Competitive inhibitors of HMGCR induce the expression of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL and lowers plasma cholesterol concentrations. However, statins have multiple side effects, including the inability to adequately lower cholesterol and / or LDL. Accordingly, more treatments are needed to lower cholesterol and treat diseases or conditions with risk factors known as autoimmune cardiovascular disease (ASCVD) (e.g., dyslipidemia). Invention Overview

[0006] In one aspect, this disclosure describes an RNAi reagent for reducing HMGCR gene expression, said RNAi reagent comprising a delivery portion of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand:

[0007]

[0008] Optionally, R is conjugated to the linker site E of Formula I via a linker, wherein the sense strand and antisense strand form a double-stranded region, and wherein the antisense strand includes a region complementary to the HMGCR mRNA target sequence of SEQ ID NO:1, and wherein each of the sense strand and antisense strand optionally includes one or more modified nucleotides and inter-nucleotide bonds of one or more modified nucleotides. In some embodiments, Formula I is optionally conjugated to the sense strand via a linker. In some embodiments, Formula I is optionally conjugated to the 3' terminal nucleotide of the sense strand via a linker.

[0009] In some embodiments, the antisense strand is 15 to 50 nucleotides long. In some embodiments, the sense strand is 15 to 50 nucleotides long. In some embodiments, the antisense strand is 18 to 23 nucleotides long. In some embodiments, the sense strand is 18 to 21 nucleotides long. In some embodiments, the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

[0010] In some embodiments, the sense or antisense strand comprises sequences selected from Tables 2, 3, 4A, and 4B disclosed herein.

[0011] In some embodiments, R is coupled to Formula I via a connector. In some embodiments, the connector comprises a Formula II connector having connection points A and B, or a Formula III connector having connection points C and D, wherein:

[0012]

[0013]

[0014] a. Formula I at connection point E is conjugated with Formula II at connection point A, and Formula II is conjugated with a phosphate group or a thiophosphate group at connection point B, and the phosphate group or thiophosphate group is conjugated with R; or

[0015] b. Formula I at connection point E is conjugated with Formula III at connection point C, and Formula III is conjugated with a phosphate group or a thiophosphate group at connection point D, and the phosphate group or the thiophosphate group is further conjugated with R.

[0016] In another aspect, this disclosure provides pharmaceutical compositions comprising the RNAi reagent described herein and one or more pharmaceutically acceptable excipients.

[0017] In another aspect, this disclosure provides methods for treating diseases or conditions associated with ASCVD. In some embodiments, the disease or condition is dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, this disclosure provides methods for treating dyslipidemia comprising administering to a patient an RNAi reagent or a pharmaceutical composition thereof as described herein.

[0018] In another aspect, this disclosure provides RNAi reagents for treatment. In some embodiments, this disclosure provides RNAi reagents for treating diseases or conditions associated with ASCVD. In some embodiments, this disclosure provides RNAi reagents for treating diseases or conditions, wherein said diseases or conditions are dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis.

[0019] In another aspect, this disclosure provides the use of RNAi reagents in the manufacture of pharmaceutical agents for treatment. In some embodiments, this disclosure provides RNAi reagents for the manufacture of pharmaceutical agents for the treatment of diseases or conditions associated with ASCVD. In some embodiments, this disclosure provides RNAi reagents for the manufacture of pharmaceutical agents for the treatment of diseases or conditions, wherein said diseases or conditions are dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis. Invention Details

[0021] HMGCR siRNAs and ASOs have been described, but none have yielded significant therapeutic benefits in patients. Using the HMGCR RNAi reagent described herein to reduce HMGCR expression could be used to treat diseases or conditions associated with ASCVD (e.g., dyslipidemia, primary dysbeta-lipoproteinemia, hypertriglyceridemia, or atherosclerosis). For example, compared to other liver-targeting siRNAs, such as HMGCR siRNAs containing different delivery ligands, different sequences, or different modified sequences, or compared to treatments controlled with a mediator, such siRNAs may exhibit one or more of the following: improved knockdown in the liver; improved tissue exposure; improved exposure in hepatocytes; improved durable response; improved pharmacokinetic profile; fewer off-target effects; and / or improved toxicity profile. Other embodiments of the HMGCR RNAi reagent described herein may include one or more of the following: fewer side effects compared to statins or other standards of care; improved toxicity profile; improved safety profile; improved tolerability or adherence; and / or improved liver function testing. Other siRNAs described herein may have other benefits, such as in combination with any of the foregoing or as independent benefits, including improved and / or simplified synthesis, synthetic processes with fewer degradation products; or any combination thereof.

[0022] The RNAi reagent described herein comprises a sense strand and an antisense strand, each of which is an oligonucleotide. In some embodiments, the RNAi reagent described herein also comprises a delivery portion. As used herein, "nucleotide" means an organic compound having a nucleoside (nucleobase such as adenine, cytosine, guanine, thymine, or uracil; and a pentose such as ribose or 2'-deoxyribose) and a phosphate group. A "nucleotide" can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0023] As used herein, “oligonucleotide” means a short nucleic acid compound (e.g., less than about 100 nucleotides in length). Oligonucleotides may be single-stranded (ss) or double-stranded (ds). Oligonucleotides may or may not have double-stranded regions. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (DsiRNA), or antisense oligonucleotides (ASO).

[0024] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and containing a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide with one or more modifications or substitutions of atoms other than hydrogen at the 2' position, including modifications or substitutions of nucleobases, sugars, or phosphate groups.

[0025] As used herein, "modified internucleotide bond" means an internucleotide bond with one or more chemical modifications compared to a reference internucleotide bond having a phosphodiester bond. Modified internucleotide bonds can be non-naturally occurring bonds.

[0026] As used herein, a “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared to a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. The modified nucleotide may be a non-naturally occurring nucleotide. The modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleotide, and / or phosphate groups. Additionally or alternatively, the modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide.

[0027] The term "percentage sequence identity" for a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleosides in a candidate sequence that are identical to those in the reference nucleic acid sequence after optimal alignment of the sequence and, where necessary, the introduction of gaps or overhangs to achieve maximum percentage sequence identity. Alignments used to determine percentage nucleic acid sequence identity can be performed in various ways within the art, such as using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supplement 30, Section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, ClustalW2.0, or Clustal X2.0, or Megalign (DNASTAR) software. In one embodiment herein, sequence identity is calculated using ClustalW2.0 or Clustal X2.0. In another embodiment, sequence identity is calculated using ClustalW2.0. 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 across the full length of the sequences being compared. The percentage of “sequence identity” can be determined by comparing two best-aligned sequences within a comparison window, where the nucleic acid sequence fragments in the comparison window may contain additions or deletions (e.g., vacancies 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 in both sequences where the same nucleotide, nucleoside, or nucleotide appears to arrive at a matching position, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The output is the percentage identity of the subject sequence relative to the query sequence. In some embodiments, the percentage sequence identity is calculated using PID3, which is the percentage of identical nucleotide residues between the two strands, calculated as the number of identical nucleotide residues divided by the total number of nucleotides in the shortest sequence of the two sequences, multiplied by 100. See, eg, Raghava, G., Barton, GJ Quantification of the variation in percentage identity for protein sequence alignments. BMC Bioinformatics 7, 415 (2006).

[0028] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or stereochemical 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 may include a phosphatase-resistant bond. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). The oligonucleotide may have a phosphate analog at the 4' carbon position of the sugar at the 5' terminal nucleotide (referred to as a “4'-phosphate analog”). An example of a 4'-phosphate analog is oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications for the 5' end of oligonucleotides have been developed (see, for example, International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0029] As used herein, a “complementary region” means 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 nucleotide sequences under appropriate hybridization conditions (e.g., in phosphate-buffered saline, in cells, etc.). In some embodiments, the oligonucleotides herein include a targeting sequence having a region complementary to the mRNA target sequence.

[0030] As used herein, the term "double helix" for nucleic acids or oligonucleotides, such as sense or antisense strands, refers to a structure formed by hydrogen bonds through complementary base pairing of two antiparallel nucleotide sequences under suitable conditions. Double helixes can still form even when there is no perfect complementarity between the two strands, or when debased nucleotides are present.

[0031] RNA interference is a specific cellular process that utilizes RISC to degrade RNA in a sequence-dependent manner. As used herein, “RNAi reagent” means a reagent comprising: (a) a double-stranded oligonucleotide having a sense strand (passenger strand) and an antisense strand (guide strand), wherein the antisense strand or a portion thereof is used by the Argonaute 2 (Ago2) endonuclease to cleave target mRNA; or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion thereof) is used by the Ago2 endonuclease to cleave target mRNA. In some embodiments, the RNAi reagent described herein also includes a delivery portion.

[0032] As used in this article, as The bonds shown indicate the connection points as described herein. For example, if a general variable such as X is stated as being attached at connection point E as shown below, this is intended to show the atomic bonding of X to the connection point (see the scheme below).

[0033]

[0034] As used herein, “effective amount” refers to the amount necessary to achieve the desired therapeutic outcome (for the time period and method of administration). The effective amount of an RNAi agent can vary depending on factors such as an individual’s disease state, age, sex, and weight, as well as the ability of the RNAi agent to elicit the desired response in the individual. An effective amount is also the amount in which any toxic or adverse effects of the RNAi agent are outweighed by the beneficial therapeutic effect.

[0035] As used herein, “treatment” or “treating” refers to all processes in which the progression of the symptoms or diseases disclosed herein may be slowed, controlled, delayed, or stopped, or symptoms of the symptoms or diseases may be improved, without necessarily indicating the complete elimination of all symptoms or diseases. Treatment includes the administration of RNAi reagents or pharmaceutical compositions thereof for the treatment of diseases or conditions in mammals, including humans.

[0036] As used in this article, the term "disease or condition associated with ASCVD" refers to any disease or condition that is a risk factor for ASCVD.

[0037] This article provides an RNAi reagent for reducing HMGCR gene expression, wherein the RNAi reagent comprises a delivery portion of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) containing an antisense strand and a sense strand:

[0038]

[0039] R is optionally conjugated to the connection point E of Formula I via a linker, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the antisense strand includes a region complementary to the HMGCR mRNA target sequence of SEQ ID NO:1, and wherein each of the sense strand and the antisense strand optionally includes one or more modified nucleotides and one or more modified nucleotides bonded together.

[0040] Also provided herein is an RNAi reagent for reducing HMGCR gene expression, wherein the RNAi reagent comprises a delivery portion of formula Ia conjugated to R, wherein R comprises an antisense strand and a sense strand:

[0041]

[0042] Wherein R is conjugated to formula Ia via a linker, wherein the sense strand and antisense strand form a double-stranded region, and wherein the antisense strand contains a region complementary to the HMGCR mRNA target sequence of SEQ ID NO:1, and wherein each of the sense strand and antisense strand optionally contains one or more modified nucleotides and one or more modified nucleotides bonded together.

[0043] This document discloses an RNAi reagent for reducing HMGCR gene expression, wherein the RNAi reagent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the antisense strand comprises a region complementary to the sequence shown in SEQ ID NO:1 for at least 15 nucleotides, and wherein the sense strand and / or antisense strand each optionally comprises one or more modified nucleotides and / or modified internucleotide bonds. In a further embodiment, the antisense strand comprises at least 15 nucleotides of the sequence in Table 2. In a further embodiment, the antisense strand comprises at least 18 nucleotides of the sequence in Table 2. In a further embodiment, the RNAi reagent reduces HMGCR gene expression in hepatocytes compared to a control. In a further embodiment, the RNAi reagent reduces HMGCR gene expression in cells expressing HMGCR by about 50% or more compared to a control. In a further embodiment, the RNAi reagent reduces HMGCR gene expression by reducing the level of HMGCR mRNA transcript, the level of HMGCR protein, or both.

[0044] In a further embodiment, the antisense strand is 15 to 50 nucleotides long, and / or the sense strand is 15 to 50 nucleotides long. In a further embodiment, the sense strand and / or the sense strand is independently 15 to 30 nucleotides long. In a further embodiment, the antisense strand is 18 to 23 nucleotides long. In a further embodiment, the sense strand is 18 to 21 nucleotides long.

[0045] In a further embodiment, the RNAi reagent comprises an antisense strand containing at least 15 adjacent nucleotides selected from the sequences of SEQ ID NO:2 to 387. In a still further embodiment, the antisense strand comprises at least 18 adjacent nucleotides selected from the sequences of SEQ ID NO:2 to 387.

[0046] In other further embodiments, the antisense strand comprises at least 18 adjacent nucleotides of a sequence consisting of at least 18 adjacent nucleotides selected from the sequences of SEQ ID NO:2 to 387.

[0047] In a further embodiment, the antisense strand of the RNAi reagent is 23 nucleotides long. In a still further embodiment, the sense strand is 21 nucleotides long. In another embodiment, the antisense strand comprises a sequence selected from 774 to 1159, or a sequence having at least 90% sequence identity with it. In yet another embodiment, both the sense strand and the antisense strand comprise sequences selected from the sequences shown in Table 3.

[0048] The sense and antisense strands of the RNAi reagents disclosed herein are not required to be perfectly complementary. Accordingly, in the RNAi reagents disclosed herein, the bistranded region between the sense and antisense strands comprises 0, 1, 2, or 3 mismatches between the sense and antisense strands. In a further embodiment, the bistranded region between the sense and antisense strands consists of 0, 1, 2, or 3 mismatches between the sense and antisense strands.

[0049] In a further embodiment, the sense strand comprises a sequence selected from SEQ ID NO:388 to 773.

[0050] In a further embodiment, the sense strand and antisense strand each independently comprise one or more modified nucleotides, such as 2'-fluorine-modified nucleotides or 2'-O-methyl-modified nucleotides. In a further embodiment of the RNAi reagent disclosed herein, each nucleotide of the sense strand and each nucleotide of the antisense strand is a modified nucleotide. In a further embodiment, each nucleotide is a 2'-fluorine-modified nucleotide or a 2'-O-methyl-modified nucleotide.

[0051] In a further embodiment of the RNAi reagent disclosed herein, the antisense strand is 23 nucleotides in length, each nucleotide of the antisense strand is a modified nucleotide, and the 2' fluorine-modified nucleotide is present.

[0052] a. Positions 2, 3, 7, 14, and 16 from the 5' end of the antisense chain; or

[0053] b. Positions 2, 5, 7, 14, and 16 from the 5' end of the antisense chain; or

[0054] c. Positions 2, 3, 8, 14, and 16 from the 5' end of the antisense chain; or

[0055] d. Positions 2, 5, 8, 14, and 16 from the 5' end of the antisense chain; or

[0056] e. Positions 2, 6, 14, and 16 from the 5' end of the antisense chain.

[0057] In a further embodiment, the nucleotide that is not 2'-fluorine modified is a 2'-O-methyl modified nucleotide.

[0058] In a further embodiment of the RNAi reagent disclosed herein, the sense strand and antisense strand each independently comprise one or more modified nucleotide internucleotide bonds, and each modified nucleotide internucleotide bond is a phosphate thioester bond. In a further embodiment, the sense strand and antisense strand each independently comprise four phosphate thioester bonds. In a still further embodiment, the two terminal nucleotides at the 5' and 3' ends of each of the sense strand and antisense strand are phosphate thioester bonds.

[0059] In other embodiments, the 5' nucleotide of the antisense strand comprises a phosphate group or a phosphate analog. As used herein, "phosphate analog" means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is positioned at the 5' terminal nucleotide of the oligonucleotide in place of the 5'-phosphate. The 5' phosphate analog may include a phosphatase-resistant bond. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). The oligonucleotide may have a phosphate analog at the 4' carbon position of the sugar at the 5' terminal nucleotide (referred to as a "4'-phosphate analog"). An example of a 4'-phosphate analog is oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4' carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications for the 5' end of oligonucleotides have been developed (see, for example, International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0060] In a further embodiment of the RNAi reagent disclosed herein, the antisense strand comprises a sequence selected from the following: SEQ ID NO:1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1 183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1 207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1 231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1 255, 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, 1 351, 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, 15411543、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、1931, or a sequence having at least 90% sequence identity with it, wherein the 5' terminal nucleotide of the antisense strand comprises a 5' vinylphosphonate, phosphate, or hydroxyl group. In other embodiments, the phosphate group listed at the 5' end of SEQ ID NO: is removed and replaced with OH. In other embodiments, the phosphate group listed at the 5' end of SEQ ID NO: is replaced with a 5' vinylphosphonate.

[0061] In a further embodiment of the RNAi reagent disclosed herein, the antisense strand comprises a sequence selected from SEQ ID No:1932-2317 or a sequence having at least 90% sequence identity with it.

[0062] In a further embodiment of the RNAi reagent disclosed herein, the antisense strand comprises a sequence selected from SEQ ID No:1932-2317 or a sequence having at least 95% sequence identity with it.

[0063] In a further embodiment, the sense strand comprises a sequence selected from: SEQ ID NO:1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 11 82, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 120 6. 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 123 0, 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, 1 352, 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, 144 8, 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, 159 4, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1 644, 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, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 17 42, 1744, 1746, 1748, 1750, 1752, 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, 184 0, 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 sequences having at least 90% sequence identity with them.

[0064] In a further embodiment of the RNAi reagent disclosed herein, the sense strand and antisense strand are a pair of oligonucleotide sequences selected from Tables 4A and 4B, or sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the sequences in Tables 4A or 4B. In a further embodiment, one, two, or three mismatches are introduced into the sense strand of the pair in Tables 4A or 4B. In a further embodiment, one, two, or both terminal nucleotides at the 5' end of the antisense strand are modified.

[0065] In some embodiments of the RNAi reagent described herein, the antisense strand comprises a first nucleic acid sequence having at least 90% sequence identity with the antisense sequence corresponding to the double-stranded number in Table 4A or 4B, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity with the sense sequence corresponding to the same double-stranded number in Table 4A or 4B. For example, in one embodiment, the antisense strand comprises a first nucleic acid sequence having at least 90% sequence identity with the antisense sequence corresponding to double-stranded number 387 in Table 4A, i.e., a first nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 1161, and the sense strand comprises a second nucleic acid sequence having at least 90% sequence identity with the sense sequence corresponding to double-stranded number 387 in Table 4A, i.e., SEQ ID NO: 1160. In a further embodiment, the 5' phosphate of the antisense strand is further modified / replaced and is a 5' vinylphosphonate or OH group.

[0066] The dopes shown in this article (e.g., “Dope number:”) (see, for example, Tables 4A and 4B) correspond to specific meaningful and antisense chains.

[0067] In a further embodiment, the 5' terminal nucleotide of the antisense strand is substituted such that the final sequence contains a vinylphosphonate, a phosphate group, or an OH group. For example, for the following antisense sequence: SEQ ID NO 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, 1 253, 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, 13 45, 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, 143 7, 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、In 1931, or a sequence with at least 90% sequence identity, the 5' phosphate group is replaced by an OH group.

[0068] In a further embodiment of the RNAi reagent disclosed herein, the antisense strand comprises a sequence selected from SEQ ID No:1932-2317 or a sequence having at least 90% sequence identity with it.

[0069] In other embodiments, this document discloses RNAi reagents having a delivery portion of Formula I conjugated with R:

[0070]

[0071] R comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 adjacent nucleotides complementary to the HMGCR mRNA target sequence of SEQ ID NO:1, and wherein the sense strand and the antisense strand form a complementary region of at least 15 nucleotides, and wherein the length of each of the sense strand and the antisense strand is independently 18 to 23 nucleotides, and optionally wherein each of the sense strand and the antisense strand independently comprises one or more modified nucleotides, and optionally wherein each of the sense strand and the antisense strand independently comprises one or more modified nucleotide bonds, and wherein R is conjugated to Formula I via a linker. In a further embodiment, the sense strand or the antisense strand is selected from Tables 2, 3, 4A, or 4B disclosed herein. In other embodiments, the antisense strand of the RNAi reagent or the antisense strand sequence and / or sense strand sequence having 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 with the corresponding sequences selected from Tables 2, 3, 4A, or 4B herein.

[0072] In other embodiments, the RNAi reagent disclosed herein comprises a adapter. In a further embodiment, R is conjugated to Formula I via a adapter. In other further embodiments, R is conjugated to Formula I via a adapter. In a further embodiment, the adapter comprises a adapter of Formula II having connection points A and B, or the adapter comprises a adapter of Formula III having connection points C and D, wherein:

[0073]

[0074]

[0075] a. The RNAi reagent comprises Formula I conjugated to Formula II at linker A, and Formula II conjugated to a phosphate group at linker B, with the phosphate group conjugated to R; or

[0076] b. The RNAi reagent comprises Formula I conjugated to Formula III at linker C, and Formula III conjugated to a phosphate group at linker D, and the phosphate group is further conjugated to R.

[0077] In other embodiments where the RNAi reagent includes a linker, R is conjugated to Formula I via the linker, and the linker is a Formula III linker having connection points C and D:

[0078]

[0079] Furthermore, the RNAi reagent comprises Formula I conjugated to Formula III at link point C, and Formula III conjugated to a phosphate group at link point D, and the phosphate group is further conjugated to R.

[0080] In other embodiments, the RNAi reagent reduces HMGCR gene expression in hepatocytes. In other embodiments, the RNAi reagent disclosed herein is used for treatment. In some embodiments, it is used to treat diseases or conditions associated with ASCVD. In further embodiments, it is used to treat dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, it is used to treat dyslipidemia. In some embodiments, it is used to treat primary dyslipoproteinemia. In some embodiments, it is used to treat hypertriglyceridemia. In some embodiments, it is used to treat atherosclerosis. In a further embodiment, the dyslipidemia is hypercholesteremia. In other embodiments, it is used to reduce the risk of one or more of myocardial infarction (MI), stroke, revascularization, and angina. In other embodiments, the use of reducing such risk is in adult patients without cardiovascular heart disease (CHD) but with one or more risk factors for any one or more of the aforementioned health events. In other embodiments, the use is to reduce the risk of MI and / or stroke in adult patients, for example, those with type 2 diabetes and no CHD, but with one or more risk factors. In other embodiments, the use is to reduce the risk of one or more of nonfatal MI, fatal and nonfatal stroke, revascularization, hospitalization for CHF, and angina in adult patients with CHD. In other embodiments, the use is to reduce elevated levels of one or more of total C, LDL-C, apo B, and TG in adult patients with primary hyperlipidemia (heterozygous familial and nonfamilial) and mixed dyslipidemia, and / or increase HDLC. In other embodiments, the use is to reduce elevated triglycerides (TG) in adult patients with hypertriglyceridemia and primary dyslipoproteinemia. In other embodiments, the use is to reduce total C and LDL-C in patients with homozygous familial hypercholesterolemia (HoFH). In other embodiments, the use is, for example, to reduce elevated levels of one or more of total C, LDL-C, and apo B in pediatric patients aged 10 to 17 years with heterozygous familial hypercholesterolemia (HeFH) after adequate trial failure of dietary therapy. In other embodiments, the use is for treating any of the foregoing after statin use has failed to control one or more symptoms, such as failing to reduce elevated total C, LDL-C, apo B, and / or failing to increase HDLC. In other embodiments, the use is for treating any of the foregoing in patients intolerant to statins; in a further embodiment, the use is for reducing LDL-C in patients intolerant to statins.In a further embodiment, the use is for any of the foregoing uses after dietary changes have failed to control one or more symptoms. In other further embodiments, the use is as an adjunct to diet therapy for any of the foregoing uses.

[0081] RNAi reagents can be formulated into pharmaceutical compositions. Accordingly, this document discloses pharmaceutical compositions comprising the RNAi reagents disclosed herein and one or more pharmaceutically acceptable excipients. Pharmaceutical compositions can be prepared using methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[0082] In other embodiments, the RNAi reagent described herein may be used to manufacture a medicament for treating dyslipidemia or any of the uses described in the preceding paragraph.

[0083] Other embodiments include methods for treating dyslipidemia in patients with this need, comprising administering the RNAi reagent disclosed herein or a pharmaceutical composition thereof. Other embodiments include methods for treating dyslipidemia in patients with this need, or any of the uses described above, or pharmaceutical compositions thereof. Other embodiments include methods for treating patients who fail to achieve lipid levels after statin therapy and / or dietary therapy, comprising administering the RNAi reagent disclosed herein or a pharmaceutical composition thereof to the patient.

[0084] RNAi reagents can be administered to patients intravenously or subcutaneously.

[0085] RNAi dosing regimens may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several fractionated doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation.

[0086] Dosage values ​​may vary depending on the type and severity of the condition to be relieved. It should be further understood that, for any given subject, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0087] Another embodiment is a method for reducing HMGCR expression in cells, which includes contacting cells with the RNAi reagent disclosed herein and incubating the cells for a period of time sufficient to reduce HMGCR mRNA levels by at least 50% compared to untreated or control-treated cells.

[0088] Some abbreviations are defined as follows: "1,2-DCE" refers to 1,2-dichloroethane; "DCM" refers to dichloromethane; "DIEA" refers to N,N-diisopropylethylamine; "DMF" refers to N,N-dimethylformamide; "DMAP" refers to 4-dimethylaminopyridine; "DMTCl" refers to 4,4'-dimethoxytriphenylmethylchloro; "DPP4" refers to dipeptidyl peptidase; "EDC" refers to 1-ethyl-3-(3-dimethyl)-2-ethyl-3-(2 ... (Aminopropyl)carbodiimide; "EtOAc" refers to ethyl acetate; "GalNAc" refers to N-acetylgalactosamine; "HATU" refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; "HBTU" refers to O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate; "HOBt" refers to 1-hydroxy Benzotriazole 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 cutoff; "NHS" refers to N-hydroxysuccinimide; "OD" refers to optical density; "PBS" refers to phosphate-buffered saline; "PhSiH3" refers to phenylsilane; "PTS" refers to portable endotoxin testing system; "siRNA" refers to small interfering ribonucleic acid; "TEA" refers to triethylamine; "TFA" refers to trifluoroacetic acid; "THF" refers to tetrahydrofuran; "TLC" refers to thin-layer chromatography; and "TMP" refers to 2,2,6,6-tetramethylpiperidine.

[0089] The delivery portion of Formula I may be prepared by the following non-limiting synthetic steps and schemes.

[0090] Option 1

[0091]

[0092] Scheme 1, step A, describes the cyclization of compound (1) in a solvent such as 1,2-DCE using trimethyl trifluoromethanesulfonate to give compound (2). Step B shows the addition of hex-5-en-1-ol to compound (2) in a solvent such as 1,2-DCE to give compound (3). Step C shows the oxidation of compound (3) with a suitable oxidant such as sodium periodate and a catalyst such as ruthenium(III) chloride to give compound (4).

[0093] Option 2

[0094]

[0095] Scheme 2, step A, shows the amide coupling between compound (5) and N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate tert-butyl in a solvent such as DMF using HBTU and HOBt along with a suitable base such as DIEA to give compound (6). Step B depicts the alkaline hydrolysis of compound (6) in a THF and MeOH solvent system using a base such as aqueous NaOH to give compound (7). Step C shows the amide coupling between compound (7) and 11-aminoundecanoate allyl hydrochloride in a solvent such as DMF using HATU along with a suitable base such as DIEA to give compound (8). Step D shows the acidic deprotection of compound (8) with TFA in a solvent such as DCM to give compound (9). Step E shows the amide coupling between compound (9) and compound (4) in a solvent such as DCM using EDC and HOBt to give compound (10). Step F illustrates the deprotection of compound (10) in a solvent such as DCM with tetrakis(triphenylphosphine)palladium and PhSiH3 to give compound (11). Step F also describes the coupling of compound (11) with NHS in a solvent such as DCM using EDC to give compound (12).

[0096] Option 3

[0097]

[0098] Scheme 3, steps AC are substantially similar to those steps in Scheme 2, steps CE, starting with compound (7) to give compounds (13), (14) and (15). Step D describes the hydrogenation of compound (15) in a solvent such as MeOH using palladium on carbon to give compound (16). Step E is substantially similar to the preparation of step G in Scheme 2 to give compound (17).

[0099] Option 4

[0100]

[0101] Scheme 4, step AI, consists of a series of amide couplings and deprotections, using methods substantially similar to those found in Schemes 2 and 3, starting with compound (18) to give compound (27).

[0102] Option 5

[0103]

[0104] Scheme 5, step AC describes a method that is essentially similar to those found in Scheme 4, step GI, starting with compound (24) to give compound (30).

[0105] Option 6

[0106]

[0107] Scheme 6, step A describes the protection of compound (31) in a solvent such as DCM using DMTCl along with a suitable base such as DIEA to give compound (32). Step B shows the amide coupling between compound (32) and piperidine-4-ylmethanol in a solvent such as DCM using HBTU and HOBt along with TMP to give compound (33). Step C shows the deprotection of compound (33) with a 20% DMF solution of piperidine to give compound (34).

[0108] Option 7

[0109]

[0110] Scheme 7, step A is essentially similar to Scheme 2, step A, to give compound (35) by coupling of compounds (16) and (34). Step B shows the formation of compound (36) by adding succinic anhydride to compound (35) in a suitable solvent, such as DCM, with a base system containing TEA and DMAP. Step C describes the loading of compound (36) together with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate and a base, such as DIEA, onto a resin in a solvent system such as MeCN and DCM to give compound (37).

[0111] Preparation 1

[0112] Methyl (6,7-diacetoxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazol-5-yl)acetate

[0113]

[0114] 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), trimethylsilyl trifluoromethanesulfonate (6.5 mL, 35 mmol) was added. The mixture was heated to 50 °C and stirred for 18 hours. Afterward, the mixture was diluted with DCM (200 mL), washed with saturated NaHCO3 (200 mL) and saturated sodium chloride aqueous solution (200 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 0–10% MeOH / DCM to give the title compound (6.434 g, 84%). ES / MS m / z 330 (M+H).

[0115] Preparation 2

[0116] Methyl (5-acetamido-3,4-diacetoxy-6-hex-5-enoxy-tetrahydropyran-2-yl)acetate

[0117]

[0118] 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), hex-5-en-1-ol (22.2 mL, 185 mmol) was added, followed by activation as a powder. Molecular sieve (15.6 g). The suspension was stirred at ambient temperature for 30 min, then trimethylsilyl trifluoromethanesulfonate (19 mL, 101.9 mmol) was added. The mixture was stirred at ambient temperature for 18 h. After this, the solution was filtered through diatomaceous earth and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 30–100% EtOAc / hexane to give the title compound (34.76 g, 86%). ES / MS m / z 430.4 (M+H).

[0119] Preparation 3

[0120] 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyvalerate

[0121]

[0122] 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) was cooled to 0 °C. A solution of sodium periodate (22.4 g, 104.7 mmol) was added, and stirring was continued at 0 °C for 10 minutes. After this, ruthenium(III) chloride (270 mg, 1.3 mmol) was added, and the mixture was stirred while heating to ambient temperature. After stirring for 2 hours, additional sodium periodate (66 g, 308.4 mmol) was added, and stirring was continued for 18 hours. After this, the mixture was extracted with 3:1 CH3Cl:IPA (2 × 500 mL), washed with a saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 0–40% MeOH / DCM to give the title compound (29.75 g, 82%). ES / MS m / z 448.4 (M+H).

[0123] Preparation 4

[0124] 6-Aminohexanoate benzyl ester hydrochloride

[0125]

[0126] Benzyl alcohol (47 mL, 453.7 mmol) was added to a THF (38 mL) suspension of 6-aminohexanoic acid (5.00 g, 38.1 mmol), and the mixture was cooled to 0 °C. Thionyl chloride (8.6 mL, 120 mmol) was added dropwise, and the mixture was stirred for 18 hours while being heated to ambient temperature. After this, diethyl ether (166 mL) was added, and the reaction vessel was transferred to a freezer at -20 °C for 1 hour. The solid precipitate was then collected by filtration to give the title compound (8.57 g, 81%). ES / MSm / z 222(M+H).

[0127] Preparation 5

[0128] Benzyl 11-aminoundecanoate hydrochloride

[0129]

[0130] The title compound was prepared from 11-aminoundecanoic acid, in a manner substantially similar to that used in preparation 4. ES / MS m / z 292.2 (M+H).

[0131] Preparation of 6

[0132] 11-Aminoundecanoic acid allyl ester hydrochloride

[0133]

[0134] A solution of 11-aminoundecanoic acid (9.00 g, 44.7 mmol) in allyl alcohol (42 mL) was placed in a container and the mixture was cooled to 0 °C. Thionyl chloride (6.5 mL, 89.4 mmol) was added, and the mixture was stirred for 18 hours while being heated to ambient temperature. Afterward, the mixture was concentrated under vacuum, and diethyl ether (200 mL) was added to the residue to obtain a white suspension. The mixture was stirred at ambient temperature for 10 minutes, and the solid precipitate was collected by filtration to obtain the product (12.0 g, 97%). ES / MSm / z 242.2 (M+H).

[0135] Preparation 7

[0136] (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluorene-9-ylmethoxycarbonylamino)propionic acid

[0137]

[0138] Under an inert atmosphere and at 0°C, DIEA (64 mL, 0.366 mol) was added to an anhydrous DCM (400 mL) stirred solution of (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-hydroxy-propionic acid (40 g, 0.122 mol). A DCM (200 mL) solution of DMTCl (49.6 g, 0.146 mol) was then slowly added. The resulting reaction mixture was allowed to reach ambient temperature and stirred for 16 hours. After this, the reaction mixture was diluted with water (12.5 v / v) and extracted with DCM (25 v / v). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was washed with 10% EtOAc / hexane (12.5 v / v) and dried under vacuum to give the title compound as a light brown solid (62 g, crude product). This material proceeded to the next step without any further purification. TLC: 5% MeOH / CH2Cl2 (Rf: 0.5) UV, 254nM.

[0139] Preparation of 8

[0140] 9H-fluorene-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]carbamate

[0141]

[0142] Under an inert atmosphere and at 0 °C, 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), were slowly added to a stirred solution of (2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-(9H-fluorene-9-ylmethoxycarbonylamino)propionic acid (62 g, 0.103 mol) in DCM (750 mL). The resulting reaction mixture was allowed to reach ambient temperature and stirred for 4 hours. After this, the reaction mixture was diluted with water (8 volumes) and extracted with DCM (15 volumes). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 20–40% EtOAc / hexane and 1% MeOH / DCM to give the title compound (40 g, 52% in two steps). 1HNMR(DMSO-d6)δ7.88(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,J=11.4Hz,2H),4.20(br s,2H),3.71(s,6H),3.21(br s,4H),2.99-2.79(m,1H),2.69(br s,2H),1.81-1.43(m,3H),1.08-0.73(m,2H).

[0143] Preparation 9

[0144] (2S)-2-amino-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-1-[4-(hydroxymethyl)-1-piperidinyl]prop-1-one

[0145]

[0146] Under an inert atmosphere and at 0 °C, a 20% DMF solution of piperidine (400 mL) was slowly added to 9H-fluorene-9-ylmethyl N-[(1S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxo-ethyl]carbamate (40 g, 0.055 mol). The resulting reaction mixture was stirred at ambient temperature for 1 hour. Afterward, the mixture was diluted with water (15 v / v) and extracted with EtOAc (30 v / v). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 1–8% MeOH / DCM to give the title compound as a grayish-white solid (13 g, 47%). ES / MSm / z 1009.5 (2 M + H).

[0147] Preparation 10

[0148] Methyl (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxovalerate

[0149]

[0150] To a flask containing (S)-4-((tert-butyloxycarbonyl)amino)-5-methoxy-5-oxovaleric acid (7.00 g, 26.8 mmol) and HOBt (4.16 g, 30.8 mmol), DMF (179 mL) and (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (11.7 g, 30.9 mmol) were added. DIEA (14 mL, 80.3 mmol) was added and the mixture was stirred at ambient temperature for 5 minutes. Following this, a portion of N-[2-[2-(tert-butyloxycarbonyl)ethylamino] was added. [Ethyl]carbamate tert-butyl ester (8.94 g, 29.5 mmol) was added and stirred continuously at ambient temperature. After stirring for 18 hours, the mixture was diluted with EtOAc (400 mL), washed with water (2 × 400 mL) and saturated sodium chloride aqueous solution (400 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 40–100% EtOAc / hexane to give the title compound (13.01 g, 89%). ES / MSm / z 547.40 (M+H).

[0151] Preparation 11

[0152] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid

[0153]

[0154] Methyl (2S)-5-[bis[2-(tert-Butoxycarbonylamino)ethyl]amino]-2-(tert-Butoxycarbonylamino)-5-oxovalerate (13.01 g, 23.8 mmol), THF (120 mL), and MeOH (120 mL) were placed in a flask. 1N NaOH (71 mL, 71 mmol) was added, and the mixture was stirred at ambient temperature. After 1 hour, the mixture was concentrated under vacuum and redissolved in water (300 mL). 5N HCl (12 mL) was added to bring the pH to 4. The mixture was extracted with DCM (3 × 300 mL), and the combined organic layers were washed with a saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated to give the title compound (12.41 g, 98%). ES / MSm / z 531.60 (MH).

[0155] Preparation 12

[0156] 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]allyl undecanoate

[0157]

[0158] Add DMF (6.25 mL) and (1-[bis(dimethylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxovalerate (500 mg, 0.94 mmol) and 11-aminoundecanoate allyl ester hydrochloride (313 mg, 1.13 mmol) to a flask containing (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (428 mg, 1.12 mmol). After the addition of DIEA (0.5 mL, 3 mmol), the mixture was stirred at ambient temperature for 18 hours. Following this, the mixture was diluted with EtOAc (200 mL), washed with water (3 × 200 mL) and a saturated sodium chloride aqueous solution (200 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluting with 40–100% EtOAc / hexane to give the title compound (687 mg, 97%). 1H 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).

[0159] Preparation 13

[0160] (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)allyl undecanoate

[0161]

[0162] TFA (15 mL) was added to a DCM (15 mL) solution of 11-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]undecanoic acid allyl ester (687 mg, 0.91 mmol). The mixture was stirred at ambient temperature. After 1.5 hours, the mixture was concentrated under vacuum. The residue was aspirated into MeOH and applied to an ion exchange reagent cartridge. The cartridge was eluted with MeOH (150 mL) followed by 7NNH3 / MeOH (150 mL). The basic fraction was concentrated under vacuum to give the title compound (410 mg, 99%). ES / MS m / z 456.4 (M+H).

[0163] Preparation 14

[0164] 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]allyl undecanoate

[0165]

[0166] 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyvalerate (489 mg, 1.09 mmol) and (S)-11-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxovalerate)undecanoate allyl ester (150 mg, 0.33 mmol) were added to a flask. DCM (3.35 mL) was 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 was stirred at ambient temperature for 18 hours. After this, the solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO3 (2 × 100 mL), saturated NH4Cl aqueous solution (100 mL), and saturated sodium chloride aqueous solution (100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 0–10% MeOH / DCM to give the title compound (424 mg, 74%). ES / MSm / z 872.80(M+2H) / 2.

[0167] Preparation 15

[0168] 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

[0169]

[0170] To a DCM solution (2 mL) of 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 allyl ester (354 mg, 0.20 mmol) was added, followed by PhSiH3 (51 μL, 0.41 mmol). The mixture was stirred at ambient temperature for 2 hours, after which it was diluted with a saturated aqueous solution of NaHCO3 (100 mL). 1 N NaOH (15 mL) was added to bring the pH to approximately 10. The aqueous solution was washed with DCM (3 × 100 mL) and then acidified with concentrated HCl (5 mL), followed by 5N HCl aqueous solution (15 mL). The aqueous layer was extracted with DCM (100 mL), and the organic layer was dried over sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by rapid silica gel chromatography eluted with 0–20% MeOH / DCM to give the title compound (151 mg, 44%). ES / MSm / z 852.60(M+2H) / 2.

[0171] Preparation of 16

[0172] (2,5-Dioxopyrrolidone-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

[0173]

[0174] Add 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) to a reaction vial. Add DCM (0.3 mL) and stir the mixture at ambient temperature. After 18 hours, the mixture was loaded directly onto a silica gel cartridge, and the crude mixture was purified by rapid silica gel chromatography eluted with 0–10% MeOH / DCM to give the title compound (49 mg, 93%). ES / MSm / z 901.40(M+2H) / 2.

[0175] Preparation of 17

[0176] 6-[[(2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]benzyl hexanoate

[0177]

[0178] The title compound was prepared from (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxovaleric acid and benzyl 6-aminohexanoate hydrochloride, in a manner substantially similar to that used in the preparation of compound 10. ES / MS m / z 736.40 (M+H).

[0179] Preparation of 18

[0180] 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-pentanoyl]amino]benzyl hexanoate tri(trifluoroacetic acid)

[0181]

[0182] TFA (16 mL, 210.2 mmol) was added to a DCM (105 mL) solution of benzyl hexanoate (15.47 g, 21.02 mmol). The mixture was stirred at ambient temperature for 24 h. After this, another 16 mL of TFA (210.2 mmol) was added, and stirring continued for another 2 h. After this, the mixture was concentrated under vacuum. The resulting residue was azeotropically reacted with toluene (2 × 30 mL). The resulting oil was further dried in a vacuum oven at 40 °C for 4 h to give the title compound (28.08 g, 58% purity, 99+% for residual toluene). ES / MSm / z 436.40 (M+H). The compound was dissolved in 70 mL of DMF to prepare a 0.3 M solution for the next step.

[0183] Preparation of 19

[0184] 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]benzyl hexanoate

[0185]

[0186] The title compound was prepared from 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyvalerate and benzyl 6-[[(2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-valerate]amino]hexanoate trifluoroacetic acid, in a manner substantially similar to that used in the preparation of compound 10. ES / MS m / z 862(M+2H) / 2.

[0187] Preparation 20

[0188] 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

[0189]

[0190] Palladium on carbon (1.90 g, 0.89 mmol, 5% by mass, 50% wet weight) was placed in a round-bottom flask, and the container was evacuated and backfilled with nitrogen three times. A solution of 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]benzyl hexanoate (15.41 g, 8.94 mmol) in MeOH (178 mL) was added via syringe. The flask was evacuated and backfilled with 1 atm hydrogen, and the mixture was stirred at 1 atm hydrogen at ambient temperature for 18 hours. The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to give the title compound (13.85 g, 95%). ES / MSm / z 817.2(M+2H) / 2.

[0191] Preparation 21

[0192] (2,5-Dioxopyrrolidone-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

[0193]

[0194] The title compound was 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 substantially similar to that used in the preparation of 16. ES / MS m / z 866.20(M+2H) / 2.

[0195] Preparation 22

[0196] (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid benzyl ester

[0197]

[0198] The title compound was prepared from tert-butyl N-[2-[2-(tert-butoxycarbonylamino)ethylamino]ethyl]carbamate and (4S)-5-benzyloxy-4-(tert-butoxycarbonylamino)-5-oxovaleric acid, in a manner substantially similar to that used in the preparation of compound 12. ES / MS m / z 623.6 (M+H).

[0199] Preparation 23

[0200] (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxo-valerate benzyl ester tri(trifluoroacetic acid) salt

[0201]

[0202] The title compound was prepared from (2S)-5-[bis[2-(tert-butoxycarbonylamino)ethyl]amino]-2-(tert-butoxycarbonylamino)-5-oxo-valerate benzyl ester, in a manner substantially similar to that used in the preparation of 18. ES / MS m / z 323.2 (M+H).

[0203] Preparation 24

[0204] (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoic acid benzyl ester

[0205]

[0206] The title compound was prepared from 5-(tert-butoxycarbonylamino)valerate and (2S)-2-amino-5-[bis(2-aminoethyl)amino]-5-oxovalerate benzyl ester tri(trifluoroacetic acid) salt, in a manner substantially similar to that used in the preparation of compound 10. ES / MS m / z 920.6 (M+H).

[0207] Preparation 25

[0208] (2S)-2-(5-aminopentanoylamino)-5-[bis[2-(5-aminopentanoylamino)ethyl]amino]-5-oxo-pentanoic acid benzyl ester tri(trifluoroacetic acid) salt

[0209]

[0210] The title compound was prepared from (2S)-5-[bis[2-[5-(tert-butoxycarbonylamino)pentanoylamino]ethyl]amino]-2-[5-(tert-butoxycarbonylamino)pentanoylamino]-5-oxo-pentanoic acid benzyl ester, in a manner substantially similar to that used in the preparation of 18. ES / MS m / z 620.4 (M+H).

[0211] Preparation 26

[0212] (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 benzyl ester

[0213]

[0214] The title compound was prepared from 5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyvalerate and (2S)-2-(5-aminovaleroylamino)-5-[bis[2-(5-aminovaleroylamino)ethyl]amino]-5-oxovalerate benzyl ester tri(trifluoroacetic acid) salt, in a manner substantially similar to that used in the preparation of compound 10. ES / MSm / z 954.80(M+2H) / 2.

[0215] Preparation 27

[0216] (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

[0217]

[0218] Palladium on carbon (467 mg, 0.22 mmol, 5% by mass, 50% wet weight) was placed in a round-bottom flask, which was then evacuated and backfilled with nitrogen three times. A solution of (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 benzyl ester (4.19 g, 2.20 mmol) in MeOH (44 mL) was added via syringe, followed by three drops of acetic acid. The flask was evacuated and backfilled with 1 atm hydrogen, and the mixture was stirred at 1 atm hydrogen at ambient temperature. After 2 hours, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to give the title compound (3.99 g, 99+%). ES / MSm / z 909.6(M+2H) / 2.

[0219] Preparation 28

[0220] 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]benzyl hexanoate

[0221]

[0222] The title compound was 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 substantially similar to that used to prepare 10. ES / MSm / z 1011.6(M+2H) / 2.

[0223] Preparation 29

[0224] 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

[0225]

[0226] Palladium on carbon (24 mg, 0.01 mmol, 5% by weight, 50% wet weight) was placed in a round-bottom flask, which was then evacuated and backfilled with nitrogen. A solution of 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]amino]benzyl hexanoate (222 mg, 0.11 mmol) in MeOH (2.2 mL) was added via syringe, followed by three drops of acetic acid. The flask was evacuated and backfilled with 1 atm hydrogen, and the mixture was stirred at ambient temperature under 1 atm hydrogen. After 5 hours, the flask was purged with nitrogen and the mixture was filtered through diatomaceous earth. The filtrate was concentrated under vacuum to give the title compound (180 mg, 85%). ES / MSm / z 966.2(M+2H) / 2.

[0227] Preparation 30

[0228] (2,5-Dioxopyrrolidone-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

[0229]

[0230] The title compound was 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 substantially similar to that used in the preparation of 16. ES / MS m / z 1014.6(M+2H) / 2.

[0231] Preparation 31

[0232] 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]benzyl undecanoate

[0233]

[0234] The title compound was prepared from (2S)-2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]-5-[bis[2-[5-[5-[3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]pentanoylamino]ethyl]amino]-5-oxo-pentanoic acid and benzyl 11-aminoundecanoate hydrochloride, in a manner substantially similar to that used to prepare 10. ES / MS m / z 1046.6(M+2H) / 2.

[0235] Preparation of 32

[0236] 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

[0237]

[0238] Add palladium on carbon (35 mg, 0.02 mmol, 5% by mass, 50% wet weight) to a round-bottom flask, and evacuate the flask and backfill with nitrogen three times. Add a solution of 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]benzyl undecanoate (285 mg, 80% purity, 0.11 mmol) via syringe. Evacuate the container and backfill with 1 atm hydrogen, and then stir the mixture at ambient temperature under 1 atm hydrogen. After stirring for 3 hours, the flask was purged with nitrogen and the mixture was filtered through diatomaceous earth. The filtrate was concentrated to give the title compound (213 mg, 79% purity, 77%). ES / MSm / z 1001.20(M+2H) / 2.

[0239] Preparation of 33

[0240] (2,5-Dioxopyrrolidone-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

[0241]

[0242] The title compound was 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 substantially similar to that used in the preparation of 16. ES / MSm / z1050(M+2H) / 2

[0243] Preparation of 34

[0244] [5-acetamino-6-[5-[2-[(4S)-4-[5-[3-acetamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]-5-[[6-[[(1S)-1-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-2-[4-(hydroxymethyl)-1-piperidinyl]-2-oxoethyl]amino]-6-oxohexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentanoylamino]ethyl]amino]ethylamino]-5-oxopentanoyl]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate]

[0245]

[0246] The title compound was 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)-phenylmethoxy]-1-[4-(hydroxymethyl)-1-piperidinyl]prop-1-one, in a manner substantially similar to that used to prepare compound 10. ES / MS m / z 1059.2(M-2H) / 2.

[0247] Preparation of 35

[0248] 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)-phenylmethoxy]propionyl]-4-piperidinyl]methoxy]-4-oxobutyric acid

[0249]

[0250] [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-piperidinyl]-2-oxo-ethyl]amino]-6-oxo-hexyl]amino]-5-oxo-pentanoyl]-[2-[5-[3-acetamido] Methyl acetate (1.194 g, 0.56 mmol) of acetylamino-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) was mixed with succinic anhydride (113 mg, 1.13 mmol), TEA (0.4 mL, 3 mmol), and DMAP (213 mg, 1.69 mmol) in 11 mL of DCM. The mixture was stirred at ambient temperature for 1 hour. Afterward, the mixture was diluted with saturated NH4Cl (200 mL) and extracted with DCM (3 × 200 mL) and 3:1 CHCl3:IPA (200 mL). The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under vacuum. The residue obtained was purified by rapid silica gel chromatography eluted with 0–40% MeOH / DCM, and the product was dried in a vacuum oven at 40 °C for 3 h to give the title compound (1.081 g, 86%). ES / MSm / z 1109.60(M-2H) / 2.

[0251] Preparation of 36

[0252] Resin loading

[0253]

[0254] 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)-phenylmethoxy]propionyl]-4-piperidinyl]methoxy]-4-oxobutyric acid (1.00 g, 0.61 mmol) in MeCN (6 mL) and DCM (1 mL) was transferred to a resin-loaded drug solution cartridge. Add 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (386 mg, 0.97 mmol) and DIEA (0.25 mL, 0.48 mmol) to the container, and shake the container at ambient temperature for 5 minutes. After this, add... LCAA-controlled pore size glass resin (5.39 g, 90 μmol / g loading, purchased from ChemGenes) was mixed and shaken at ambient temperature for 18 hours. Afterward, the reagent cartridge was evacuated by suction, and the resin was washed with DCM (10 mL) by shaking for 10 minutes. The reagent cartridge was evacuated, and the washing and evacuation procedure was repeated with 10% MeOH / DCM (10 mL) and Et₂O (10 mL). After evacuation, acetic anhydride (6.4 mL), pyridine (20 mL), and TEA (0.22 mL) solution were added, and the reagent cartridge was shaken for 2 hours. Afterward, the reagent cartridge was evacuated, and the washing and evacuation procedure was repeated with DCM (10 mL), 10% MeOH / DCM (10 mL), and diethyl ether (10 mL). After evacuation, the resin was dried under vacuum for 30 minutes. The resin loading was determined using a standard triphenylmethyl assay. The resin loading was calculated to be 34.7 μmol / g.

[0255] Preparation of 37

[0256] 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

[0257]

[0258] The title compound was 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 2-(2-aminoethoxy)benzyl acetate hydrochloride, in a manner substantially similar to that used in the preparation of 10. ES / MS m / z 1005.2 (M+2H / 2).

[0259] Preparation of 38

[0260] 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

[0261]

[0262] 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) was combined with a 12.0 mL solution of MeOH in 5% Pd / C (1.17 mmol, 124 mg). The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 48 min, filtered through diatomaceous earth, and concentrated under vacuum to give the title compound as a gray solid (187 mg, 82%). ES / MSm / z 960.0(M+2H / 2).

[0263] Preparation 39

[0264] (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-oxo-pentanoyl]amino]ethoxy]acetate

[0265]

[0266] (2,3,5,6-Tetrafluorophenyl)2,2,2-trifluoroacetate (0.383 mmol, 100 mg) was added dropwise to a solution 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). The mixture was stirred at ambient temperature for 16 hours. The reaction mixture was purified directly by rapid silica gel chromatography eluting with 0% to 50% MeOH / DCM to give the title compound (197 mg, 99%) as a brown solid. ES / MS m / z 1034.0 (M+2H / 2).

[0267] Preparation of 40

[0268] 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

[0269]

[0270] The title compound was 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 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]benzyl acetate hydrochloride, in a manner substantially similar to that used in the preparation of 10. ES / MS m / z 1049.0 (M+2H / 2).

[0271] Preparation 41

[0272] 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

[0273]

[0274] A solution of 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) was combined with a 12.0 mL solution of 5% Pd / C (1.17 mmol, 124 mg) in MeOH. The mixture was hydrogenated on a Parr shaker (ambient temperature, 10 psi) for 1 hour, filtered through diatomaceous earth, and concentrated under vacuum to give the title compound as a gray solid (227 mg, 96%). ES / MSm / z 1004.0 (M+2H / 2).

[0275] Preparation 42

[0276] (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-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]acetate

[0277]

[0278] (2,3,5,6-Tetrafluorophenyl)2,2,2-trifluoroacetate (0.443 mmol, 116 mg) was added dropwise to a solution 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). The mixture was stirred at ambient temperature for 16 hours. The reaction mixture was purified directly by rapid silica gel chromatography eluting with 0% to 50% MeOH / DCM to give the title compound (174 mg, 73%) as a brown solid. ES / MS m / z 1078.2 (M+2H / 2).

[0279] Example 1: Joining Scheme

[0280] To synthesize the GalNAc-conjugated sense chain, a sense chain with a 3'C6-NH2 functional group was first synthesized using standard phosphoramidite chemistry. A stock solution of the GalNAc ligand-NHS ester was prepared (10 mmol / L acetonitrile solution; 1 equivalent). Borate buffer (10% v / v; 20x) was added to the oligonucleotide C6-NH2 sense chain in an Eppendorf tube, followed by the addition of the GalNAc ligand (5 equivalents). The mixture was allowed to vibrate at ambient temperature for 16 hours. After this, the mixture was transferred to a 15 mL FAlcon tube, ammonium hydroxide (28% by mass) was added, and the mixture was allowed to vibrate at ambient temperature for 2 hours. Ammonia was then removed under vacuum. The residue was purified by ion-exchange chromatography. Conditions: Solvent A: 15% MeCN / 20mM NaH2PO4, Solvent B: 15% MeCN / 20mM NaH2PO4, 1M NaBr; 35%-55% B at 8 mL / min within 5 CV, column temperature 60°C. The desired fractions were combined and desalted using a rotary filtration system or a desalting column on an Eppendorf centrifuge. After desalting, the material was recovered, and OD and volume were measured to obtain the concentration.

[0281] Alternatively, GalNAc ligands can be immobilized on microporous polystyrene resin or glass with controllable pore size and synthesized using 5'-CEβ-cyanoethyl)phosphoramide via an established solid-phase oligonucleotide synthesis method, conjugated to the 5' position of the sense chain.

[0282] Alternatively, the GalNAc ligand can be converted to a suitable phosphoramide and delivered to the 5' position of the sense chain using standard phosphoramide chemistry.

[0283] Example 2: Annealing

[0284] To generate siRNA duplexes with sense and antisense strands, the following procedure was performed: The corresponding antisense oligonucleotide (1 equivalent) was added to a Falcon tube containing the oligonucleotide sense-GalNAc conjugate, and the tube was vortexed for 10 seconds, followed by rotary filtration through a 100 kM WCO Amicon filter to remove particulates. The filtrate was recovered and concentrated under vacuum on a Genevac evaporator. The residue was reconstituted in 1x PBS, filtered through a 0.2 μL filter, and the OD and volume were measured to obtain the concentration.

[0285] Using horseshoe crab amoeboids cell lysates in Endotoxin testing is performed on the PTS instrument.

[0286] Table 1 - Exemplary molecules synthesized using the above-described conjugation and annealing schemes.

[0287]

[0288] Example 3:

[0289] A general procedure for oligonucleotide synthesis using GalNAc-functionalized CPG.

[0290] Using phosphoramide chemistry in MerMade TM Oligonucleotide synthesis was performed on an instrument. Sense strands were synthesized using a prefunctionalized GalNAc solid support, and antisense strands were synthesized using a standard support containing the first nucleotide of a pre-loaded oligonucleotide sequence. The oligonucleotides were cleaved and deprotected using concentrated ammonium hydroxide solution (28% by mass) and purified by ion-exchange chromatography under the conditions described above. Desalting, annealing, and endotoxin tests were performed.

[0291] The sequences of antisense oligonucleotides were designed using 15 to 50 nucleotides of the following HMGCR transcript (SEQ ID NO:1), wherein T nucleotides were replaced by U nucleotides, and one or more nucleotides and one or more nucleotide bonds were optionally further modified as described herein.

[0292] Homo sapiens HMGCR cell death receptor (HMGCR) transcript,

[0293] SEQ ID NO:1

[0294]

[0295]

[0296]

[0297] Table 2 below shows an exemplary antisense strand sequence of 18 nucleotides in length, which may optionally be further modified and synthesized and incorporated into RNAi reagents as described herein.

[0298] Table 2. Antisense 18-mer of HMGCR RNAi reagent

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] Table 3. Exemplary full-length sense and antisense strands of HMGCR RNAi reagents

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337] Table 4A. HMGCR-GalNAc RNAi reagent, modified sense and antisense strands

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] P indicates 5' phosphate

[0371] m indicates the ribose with a 2'O-methyl modification on the listed nucleotides.

[0372] f indicates the ribose with 2'F modification on the listed nucleotides.

[0373] *Indicates the thiophosphate bond (instead of the phosphate diester bond).

[0374] Table 4B. HMGCR RNAi reagents, modified sense and antisense strands

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408] m indicates the ribose with a 2'O-methyl modification on the listed nucleotides.

[0409] f indicates the ribose with 2'F modification on the listed nucleotides.

[0410] *Indicates the thiophosphate bond (instead of the phosphate diester bond).

[0411] Example 4:

[0412] Human HMGCR was knocked down in Hep3B cells in vitro using cholesterol-conjugated HMGCR siRNA.

[0413] The knockdown of human HMGCR expression via cholesterol-conjugated HMGCR siRNA was determined using the following procedure: On day 1, Hep3B cells (ATCC) were added to Corning 96-well plates at 5,000 cells / well. On day 2, the medium was replaced with ACCELL Media (Dharmacon), and the siRNA was added directly to the wells. For single-point (SP) selection, 1 μM (1,000 nM) of cholesterol-conjugated siRNA was used. To generate concentration / dose-response curves, final concentrations of cholesterol-conjugated siRNA of 1000, 200, 40, 8, 1.6, 0.32, and 0.064 nM were used.

[0414] The treated cells were lysed using the TaqMan Fast Advanced Cells-to-Ct Kit (Invitrogen) and subsequently used for gene expression. Cell lysates were immediately used for cDNA synthesis using the Fast AdvancedRT MasterMix (Invitrogen) and a thermal cycler with the following steps: 37°C for 30 min, 95°C for 5 min, and a hold at 4°C. Quantitative polymerase chain reaction (qPCR) was performed via the TaqMan Gene Expression Assay (Invitrogen) using 40 cycles at the following temperatures and times: 50°C for 2 min, 95°C for 20 s, 95°C for 1 s, and 60°C for 20 s.

[0415] Human HMGCR levels were normalized to human Rplp0 (Life Technologies) and represent a relative knockdown of human HMGCR mRNA expression compared to control cells treated with the vector. IC50 values ​​were calculated using an XLFit 4-parameter fitting model.

[0416] Table 5. Percentage inhibition of human HMGCR expression in Hep3B cells

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427] Table 5 shows the results of single-dose screening in Hep3B cells with indicated cholesterol-conjugated HMGCR siRNA via free uptake. Data are presented as percentage knockdown relative to untreated cells. Also included are IC50 values ​​for the highest hit and maximum percentage knockdown from the single-point screening, followed by the concentration / dose response curves.

[0428] Example 5:

[0429] In vitro in vitro assays using GalNAc-conjugated HMGCR siRNA in wild-type mouse primary hepatocytes (MPH) and Hep3B cells. Knock down HMGCR

[0430] The knockdown of mouse HMGCR expression via LYGal1-conjugated HMGCR siRNA was determined using the following procedure: Primary mouse hepatocytes (MPHs) were freshly isolated from wild-type mice and added to Corning plates at 15,000 / well, with siRNA added directly to the wells. For Hep3B (ATCC) cells, siRNA was mixed with 0.3 μL / well of the transfection reagent RNAiMAX (Life Technologies) in Corning plates, followed by the addition of 20,000 / wells of cells. To generate concentration / dose-response curves, final concentrations of GalNAc-conjugated siRNA (1000, 333, 111, 37, 12, 4, 1.37, 0.46, 0.15, 0.05, and 0.017 nM) were used for MPHs. For Hep3B concentration / dose response curves, the final concentrations of GalNAc-conjugated siRNA were calculated using 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.

[0431] Cells were lysed, and RNA was directly isolated into 96-well plates using the Quick-RNA 96 Kit (Zymo Research). The eluted RNA was used immediately or frozen for storage. cDNA was synthesized using the Fast Advanced RT Master Mix (Invitrogen) and the following steps in a thermal cycler: 37°C for 30 min, 95°C for 5 min, and incubation at 4°C. Polymerase chain reaction (PCR) was performed via TaqMan RT PCR (Life Technologies) using 40 cycles at the following temperatures and times: 50°C for 2 min, 95°C for 10 min, 95°C for 15 sec, and 60°C for 1 min.

[0432] Mouse or human HMGCR levels were normalized against mouse (for MPH) or human (for Hep3B) Rplp0 (Life Technologies) and represent the relative knockdown of mouse or human HMGCR mRNA expression compared to control cells treated with the vector. IC50 values ​​were calculated using an XLFit 4-parameter fitted model.

[0433] Table 6. In vitro knockdown of HMGCR in wild-type mouse primary hepatocytes (MPH) and Hep3B cells using GalNAc-conjugated HMGCR siRNA

[0434]

[0435]

[0436] Table 6 shows the results of the highest hit IC50 with the largest percentage knockdown using the indicated HMGCR siRNA from single-point screening via free uptake in wild-type mouse primary hepatocytes and via transfection with RNAiMAX in Hep3B cells. Data are expressed as a percentage of HMGCR knockdown relative to untreated cells.

[0437] Example 6: In vivo single-dose screening of HMGCR KD in mice

[0438] GalNAc-siRNA was tested in male C57bl / 6 mice (n=7) (Taconic Farms). Mice were assigned to groups with similar body weights. PBS or GalNac-siRNA test material was administered subcutaneously to mice at a dose of 5 mg / kg. Blood was collected from all mice 7 days after subcutaneous injection. Mice were euthanized under isoflurane anesthesia 14 days after subcutaneous injection. Blood was collected via cardiac puncture. Liver was removed from the mice and frozen in liquid nitrogen. The liver was homogenized in TriZol (Invitrogen) using Lysing Matrix D beads 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 using NanoDrop (Thermo Fisher). An equal volume (1 μg) of RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) on a Mastercycler Nexus (Eppendorf). The thermal cycler was set to 25°C for 10 min, 37°C for 2 h, and then 85°C for 5 min. The template cDNA was mixed with the Taqman Universal MasterMix and AssaysonDemand primer / probe set and RT-PCR was performed on a QuantStudio Pro7 (Thermo Fisher) with the following parameters: 50°C for 2 min, 95°C for 10 min, then 95°C for 15 s and 60°C for 1 min, for 40 cycles. Fold change (FC) was calculated as follows: the ΔCT value was obtained by subtracting the CT value of mouse Rplp0 from the CT value of mouse HMGCR. The ΔΔCT value was then calculated by subtracting the ΔCT value of the untreated sample (the average of the PBS control) from the ΔCT value of each test sample. The fold change was calculated by taking the base-2 logarithm of the negative ΔΔCT value. The percentage mRNA knockdown (KD) was calculated by subtracting the fold change from the fold change in the PBS group and then multiplying by -100. The data are shown in Table 7.

[0439] Table 7. In vivo single-dose screening of HMGCR KD in mice

[0440]

[0441]

[0442] Example 7: In vivo durability of HMGCR KD in 8-week-old mice

[0443] GalNAc-siRNA was tested in male C57 / BL6 mice (n=9) (Taconic Farms). Mouse body weight was measured, and mice were assigned to groups with similar body weights. GalNac-siRNA or the test sample was administered subcutaneously 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. Liver samples were collected from the mice and frozen in liquid nitrogen. At 2, 4, and 6 weeks after siRNA administration, blood was collected from the remaining mice (n=6 / group) under isoflurane anesthesia. Eight weeks after siRNA administration, the remaining mice (n=6) were euthanized under isoflurane anesthesia. Blood and liver samples were collected from the mice. Liver samples were processed, and the percentage KD of mRNA was calculated as described in the in vivo single-dose screening.

[0444] Table 8: In vivo durability of HMGCR KD in 8-week-old mice

[0445] Double chain number: Dosage mg / kg 2-week %KD 8-week %KD D:388 0.3 37 -1 D:388 1.75 39 -9 D:388 10 47 -41 D:735 0.3 7 6 D:735 1.75 20 4 D:735 10 28 -44 D:734 0.3 -18 0 D:734 1.75 -1 38 D:734 10 18 45 D:444 0.3 -38 2 D:444 1.75 -36 -21 D:444 10 -64 -30 D:448 0.3 -30 2 D:448 1.75 -4 5 D:448 10 -64 -45 D:442 0.3 -29 -14 D:442 1.75 -35 -34 D:442 10 -44 -33 D:706 0.3 26 -12 D:706 1.75 -47 11 D:706 10 -57 -43 D:707 0.3 -36 6 D:707 1.75 -55 -5 D:707 10 -76 -38

[0446] Example 8: Human HMGCR KD in vivo screening with a single dose of AAV8

[0447] GalNAc-siRNA was tested in male C57bl / 6 mice (n=7) (Taconic Farms). The siRNA was tested in a single study. Mice were administered via retro-orbital injection of an adeno-associated virus (AAV) vector containing a plasmid (VectorBioLabs) with the TBGS1 promoter and the coding sequence for human HMGCR (NM_000859.3) and its 3' UTR. Mouse body weight was measured approximately 4 weeks after AAV administration. Mice were assigned to groups with similar body weights. The GalNAc-siRNA test item was administered subcutaneously to mice at a dose of 5 mg / kg using PBS or GalNAc-siRNA. Seven days after subcutaneous injection, blood was collected from the retro-orbital sinus from all mice. Fourteen days after subcutaneous injection, mice were euthanized under isoflurane anesthesia. Blood was collected via cardiac puncture. Liver samples were collected 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 prepared by Invitrogen. Weigh approximately 10 mg of liver into a 96-well cluster tube plate. Add 300 μL of homogenization buffer containing proteinase K to each liver sample and homogenize on a Qiagen homogenizer for 12 min. Centrifuge the plate at 3500 rpm for 10 min, then heat at 60 °C for 30 min with vortexing every 10 min. Centrifuge the sample again at 3500 rpm for 10 min, then dilute or use directly in subsequent steps. Prepare a working probe set for each gene of interest by mixing the following reagents in separate tubes in the listed order and scaled according to the number of wells to be run, with the required overfill: nuclease-free water (25.4 μL), lysis mixture (33.3 μL), blocking agent (1 μL), QuantiGene Singleplex Probe Set (0.3 μL) / well. The capture plate was prepared by dispensing 60 μL of working probe sets into each well. Probe sets for mGAPDH (SB-10001) and hHMGCR (SA-11011) were separately aliquoted into the plate. Then, 60 μL of pure liver homogenate was added to the hHMGCR working set in the plate, and 60 μL of a 20-fold diluted RNA isolate was added to the previously aliquoted mGAPDH probe set. Air bubbles were avoided, and the plate was not mixed. The adhesive seal was firmly placed on the plate, and it was incubated at 55 ± 1 °C for 20.5 h to allow the probes to hybridize with the RNA target. After 20.5 h, 200 μL of 1X wash buffer was added to the capture plate, and the plate was inverted to remove the wash buffer. The plate was then washed twice more, using 300 μL of wash buffer for each wash.Next, 100 μL of pre-amplification solution was added to the plate. It was sealed and incubated at 55 ± 1 °C for 60 minutes. After 1 hour, the 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, the washing step was performed again, and then 100 μL of labeled probe was added. It was sealed and incubated at 50 ± 1 °C for 60 minutes. The washing step was performed again, and then 100 μL of substrate was added at room temperature and incubated for 5 minutes in the dark. The plate was then read on a photometer with an integration time of 0.2 seconds. Gene knockdown was calculated by first dividing the hHMGCR chemiluminescence signal by the mGAPDH signal. The fold change compared to the PBS (control) group was calculated by dividing all groups by the average signal from the control group. Then, %hHMGCR gene knockdown was calculated by subtracting the average fold change from the control group from all groups, followed by dividing all these groups by the average signal from the control group.

[0448] Table 9: In vivo screening of human HMGCR KD using a single dose of AAV8

[0449] Double chain number: Dosage mg / kg 2-week %KD D:448 5 -62.5 D:442 5 -66.4 D:707 5 -59.6 D:735 5 -33.6 D:705 5 -58.0 D:731 5 -64.1 D:745 5 -55.2 D:486 5 -54.8 D:665 5 -63.9

Claims

1. An RNAi reagent for reducing HMGCR gene expression, wherein the RNAi reagent comprises a delivery portion of Formula I conjugated to R, wherein R is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand: R is optionally conjugated to the connection point E of Formula I via a linker, wherein the sense strand and the antisense strand form a double-stranded region, and wherein the antisense strand includes a region complementary to the HMGCR mRNA target sequence of SEQ ID NO:1, and wherein each of the sense strand and the antisense strand optionally includes one or more modified nucleotides and one or more modified nucleotides bonded together.

2. The RNAi reagent according to claim 1, wherein formula I is optionally conjugated to the sense strand via a linker.

3. The RNAi reagent according to claim 2, wherein formula I is optionally conjugated to the 3' terminal nucleotide of the sense strand via a linker.

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

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

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

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

8. The RNAi reagent according to any one of claims 1 to 7, wherein the antisense strand is 23 nucleotides long and the sense strand is 21 nucleotides long.

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

10. The RNAi reagent according to any one of claims 1 to 9, wherein the antisense strand comprises a sequence selected from SEQ ID NO: 2 to 387.

11. The RNAi reagent according to any one of claims 1 to 10, wherein the antisense strand has a nucleotide sequence selected from SEQ ID NO: 774 to 1159, or a sequence having at least 90% sequence identity with it.

12. The RNAi reagent according to any one of claims 1 to 11, wherein the sense strand is selected from SEQ ID NO:388 to 773, or a sequence having at least 90% sequence identity with it.

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

14. The RNAi reagent according to any one of claims 1 to 13, wherein the sense strand or antisense strand each independently comprises one or more modified nucleotides, and the modified nucleotides are independently 2'-fluorine modified nucleotide residues or 2'-O-methyl modified nucleotides.

15. The RNAi reagent according to any one of claims 1 to 14, wherein each nucleotide of the sense strand and each nucleotide of the antisense strand are modified nucleotides.

16. The RNAi reagent according to any one of claims 1 to 15, wherein the antisense strand is 23 nucleotides in length, and wherein each nucleotide of the antisense strand is a modified nucleotide, and the 2' fluorine-modified nucleotide is present. a. Positions 2, 3, 7, 14, and 16 from the 5' end of the antisense chain; or b. Positions 2, 5, 7, 14, and 16 from the 5' end of the antisense chain; or c. Positions 2, 3, 8, 14, and 16 from the 5' end of the antisense chain; or d. Positions 2, 5, 8, 14, and 16 from the 5' end of the antisense chain; or e. Positions 2, 6, 14, and 16 from the 5' end of the antisense chain.

17. The RNAi reagent according to any one of claims 1 to 16, wherein the sense strand and the antisense strand each independently comprise one or more modified nucleotide bonds, and wherein each modified nucleotide bond is a phosphate thioester bond.

18. The RNAi reagent according to claims 1 to 17, wherein the sense strand and the antisense strand each independently comprise four phosphate thioester bonds.

19. The RNAi reagent according to 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 RNAi reagent according to any one of claims 1 to 19, wherein the antisense strand comprises a sequence selected from: SEQ ID NO:1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1 183, 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, 1 349, 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, 144 3, 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、1931, or a sequence having at least 90% sequence identity with it, wherein the 5' terminal nucleotide of the antisense strand contains a vinylphosphonate, phosphate, or hydroxyl group.

21. The RNAi reagent according to any one of claims 1 to 20, wherein the antisense strand comprises a sequence selected from: SEQ ID NO:1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1 183, 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, 1 349, 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, 144 3, 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、1931, or a sequence having at least 95% sequence identity with it, wherein the 5' terminal nucleotide of the antisense strand contains a vinylphosphonate, phosphate, or hydroxyl group.

22. The RNAi reagent according to any one of claims 1 to 21, wherein the sense strand comprises a sequence selected from: SEQ ID NO:1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1 182, 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, 1 348, 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, 144 2, 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、1700、1702、1704、1706、1708、1710、1712、1714、1716、1718、1720、1722、1724、1726、1728、1730、1732、1734、1736、1738、1740、1742、1744、1746、1748、1750、1752、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 that has at least 90% sequence identity with it.

23. The RNAi reagent according to any one of claims 1 to 22, wherein the sense strand comprises a sequence selected from: SEQ ID NO:1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1 182, 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, 1 348, 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, 144 2, 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、1700、1702、1704、1706、1708、1710、1712、1714、1716、1718、1720、1722、1724、1726、1728、1730、1732、1734、1736、1738、1740、1742、1744、1746、1748、1750、1752、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 that has at least 95% sequence identity with it.

24. The RNAi reagent according to any one of claims 1 to 23, wherein the sense strand comprises a sequence selected from SEQ ID NO:1932-2317, or a sequence having at least 90% sequence identity with it.

25. The RNAi reagent according to any one of claims 1 to 23, wherein the sense strand comprises a sequence selected from SEQ ID NO:1932-2317, or a sequence having at least 95% sequence identity with it.

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

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

28. The RNAi reagent according to claim 26 or 27, wherein the 5' terminal nucleotide of the antisense strand comprises a vinylphosphonate, a phosphate group, or an OH group.

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

30. The RNAi reagent according to any one of claims 1 to 29, wherein R is conjugated to Formula I via a adapter, and wherein the adapter comprises Formula II having connection points A and B, or the adapter comprises Formula III having connection points C and D, and wherein: a. Formula I at connection point E is conjugated with Formula II at connection point A, and Formula II is conjugated with a phosphate group or a thiophosphate group at connection point B, and said phosphate group or thiophosphate group is conjugated with R; or b. Formula I at connection point E is conjugated with Formula III at connection point C, and Formula III is conjugated with a phosphate group or a thiophosphate group at connection point D, and the phosphate group or thiophosphate group is further conjugated with R.

31. The RNAi reagent according to any one of claims 1 to 30, wherein R is conjugated to formula I via a adapter, and wherein the adapter comprises formula III having connection points C and D: Formula I at connection point E is conjugated with Formula III at connection point C, and Formula III is conjugated with a phosphate group or a thiophosphate group at connection point D, and the phosphate group or thiophosphate group is further conjugated with R.

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

33. The RNAi reagent according to any one of claims 1 to 31, for use in treatment.

34. The RNAi reagent according to any one of claims 1 to 31, for the treatment of diseases or conditions associated with ASCVD.

35. The RNAi reagent for the stated purpose according to claim 34, wherein the disease or condition is dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis.

36. The RNAi reagent for the stated purpose according to claim 35, wherein the disease or condition is dyslipidemia.

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

38. Use of the RNAi reagent according to any one of claims 1 to 31 in the manufacture of a medicament for treating diseases or conditions associated with ASCVD.

39. The use according to claim 38, wherein the disease or condition is dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis.

40. The use according to claim 39, wherein the disease or condition is dyslipidemia.

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

42. A method of treating a patient with an ASCVD-related disease or condition, comprising administering to the patient an RNAi reagent or a pharmaceutical composition thereof according to any one of claims 1 to 31.

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

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

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

46. ​​A method for reducing HMGCR expression in cells, comprising contacting the cells with an RNAi reagent according to any one of claims 1 to 31.

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

Images